Borrelia burgdorferi sensu lato and - szu · pathogens Borrelia afzelii and Borrelia garinii were...

Charles University in PragueFaculty of Science

Borrelia burgdorferi sensu lato andAnaplasma phagocytophilum in the

Czech Republic

RNDr. Katerina Kybicova

Prague 2010

Study program: Molecular and Cellular Biology, Genetics and Virology

Laboratory: Laboratory of Lyme Borreliosis,Centre of Epidemiology and Microbiology,National Institute of Public Health

Author: RNDr. Katerina Kybicova

Supervisor: RNDr. Dagmar Hulınska, CSc.

This thesis is presented in partial fulfillment of the requirements for the degree of Doctorof Philosophy at the Faculty of Science, Charles University in Prague, Czech Republic.I declare, that I have not presented this work or an important part thereof to obtain thesame or another academic title.

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Abstract

The aim of this thesis was to assess the occurrence of two tick-borne bacteria,Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum, in ticks, wild anddomestic animals in the Czech Republic. In ticks, similar prevalence of both bac-teria was observed. In rodents, the majority of infections were caused by B. afzeliiwhile the infection with B. burgdorferi s. s. was also quite frequent. Infection withB. burgdorferi s. l. was more common in bank voles than in wood or yellow-neckedmice. The prevalence of anti-Borrelia antibodies was higher in wood or yellow-necked mice than in bank voles. A. phagocytophilum was in a higher percentage ofcases in the deer family and hares as compared to foxes and boars. We observeda similar prevalence of anaplasmosis in all domestic animals tested. We demon-strated that symptomatic dogs had a higher chance to be infected with A. pha-gocytophilum than asymptomatic dogs. Our findings suggest that the exposure toB. burgdorferi s. l. and A. phagocytophilum is common in vectors, reservoirs andhosts in the Czech Republic.

Molecular and serological techniques for detection of these pathogens are alsodescribed in this thesis, including conventional PCR, nested PCR, real-time PCRwith DNA quantification and melting curve analysis, RFLP analysis of the 5S-23SrDNA intergenic spacer and direct sequencing of the 16S rDNA. We found thatreal-time PCR is a very fast method but it cannot distinguish within the group ofBorrelia genospecies and that of A. phagocytophilum variants.

Finally, clinical signs and diagnostic findings for three examples cases of Bor-relia infection in dogs are presented. We showed that borrelial infection must beconsidered not only in cases with febrile and orthopaedic signs but also for manyother clinical syndromes.

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Acknowledgements

I would like to thank Dr. D. Hulınska for supervising me during my PhD studiesand Doc. A. Nemec for frequent consultations, many helpful comments and forreviewing the final text of this thesis and included articles.

I would like to thank Dr. P. Schanilec and Dr. S. Spejchalova for canine sam-ples, Dr. M. Pejcoch for rodent capture and Dr. I. Pavlasek for wild animal sam-ples.

This work would not have been possible without the help of my colleaguesMgr. G. Lipinova, Dr. Z. Kurzova, and Dr. L. Uherkova, who provided technicalassistance with serological laboratory examinations. I further acknowledge thehelp of Dr. M. Musılek with sequencing, Dr. M. Maly with statistical analysis,and Dr. E. Kodytkova with reviewing the articles.

Finally, I would like to thank my family for their patience and support, espe-cially my husband Dr. J. Kybic for helpful comments and for reviewing the textof this thesis and all included articles.

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Contents

1 Introduction 11.1 Lyme borreliosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.1 Borrelia burgdorferi sensu lato . . . . . . . . . . . . . . . . . . 21.1.2 Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1.3 Reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.1.4 Domestic animals as hosts . . . . . . . . . . . . . . . . . . . . 51.1.5 Laboratory diagnostic . . . . . . . . . . . . . . . . . . . . . . 7

1.2 Anaplasmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.2.1 Anaplasma phagocytophilum . . . . . . . . . . . . . . . . . . . 81.2.2 Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.2.3 Reservoirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.2.4 Domestic animals as hosts . . . . . . . . . . . . . . . . . . . . 101.2.5 Laboratory diagnostic . . . . . . . . . . . . . . . . . . . . . . 11

1.3 Aims of this thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2 Overview and results 132.1 Molecular and serological evidence of Borrelia burgdorferi sensu lato

in wild rodents in the Czech Republic. . . . . . . . . . . . . . . . . . 132.2 Detection of Anaplasma phagocytophilum and Borrelia burgdorferi

sensu lato in dogs in the Czech Republic. . . . . . . . . . . . . . . . 142.3 Clinical and Diagnostic Features in Three Dogs Naturally Infected

with Borrelia spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.4 Detection of Anaplasma phagocytophilum in animals by real-time

polymerase chain reaction. . . . . . . . . . . . . . . . . . . . . . . . . 152.5 Summary of the results . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3 Molecular and serological evidence of Borrelia burgdorferi sensu lato inwild rodents in the Czech Republic. 17

4 Detection of Anaplasma phagocytophilum and Borrelia burgdorferisensu lato in dogs in the Czech Republic. 27

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Contents

5 Clinical and Diagnostic Features in Three Dogs Naturally Infected withBorrelia spp. 37

6 Detection of Anaplasma phagocytophilum in animals by real-time poly-merase chain reaction. 57

7 Discussion 69

8 Conclusions 77

References 79

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Chapter 1

Introduction

This thesis studies the occurrence of two tick-borne bacteria, Borrelia burgdorferisensu lato (s. l.) and Anaplasma phagocytophilum, in ticks and wild and domesticanimals in the Czech Republic. This contributes to the understanding of the nat-ural cycle of these bacteria. So far only a limited data has been available in theCzech Republic, especially concerning animal hosts. We also describe the molec-ular and serological techniques for detection of these pathogens. Finally, we giveclinical signs and diagnostic findings for three examples cases of Borrelia infectionin dogs.

We will now describe these pathogens, their vectors and hosts, and the dis-eases they cause, mainly focusing on the situation in Europe.

1.1 Lyme borreliosis

Lyme borreliosis is a multi-organ disease of mammals in the northern hemi-sphere. The Lyme disease is named after Lyme, a town in Connecticut, USA,where the first cases of this disease were reported in 1975 (Steer et al. 1977). Beforethat, at the end of the 19th century, a chronic skin disorder had been described inEurope and was later named acrodermatis chronica atropicans (ACA) (Buchwald1883). At the beginning of the 20th century in Sweden, Dr. Afzelius presenteda patient with skin lesion, which he called Erythema migrans (EM) (Afzelius1910). It is known that in humans, both ACA and EM are among clinical symp-toms of the Lyme disease. Other clinical symptoms include Borrelial lympho-cytoma, neurological manifestations, joint manifestations, and cardiac manifes-tations. Lyme borreliosis is categorized as a zoonotic disease in humans becausethe infection is maintained in nature by animals. Humans are dead-end hosts andare not involved in natural circulation of B. burgdorferi s. l. However, humans canbe infected and subsequently can fall ill (Humair and Gern 2000, Gern 2009).

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Chapter 1. Introduction

1.1.1 Borrelia burgdorferi sensu lato

Lyme disease agent was discovered by Willy Burgdorfer in North America in1982 and was isolated and cultured from the midgut of Ixodes ricinus ticks, andfrom patients with Lyme disease in North America and in Europe (Burgdorfer etal. 1982, Barbour 1984). This spirochete was identified as a new species amongborreliae and named Borrelia burgdorferi (Johnson et al. 1984). In 1998, humanpathogens Borrelia afzelii and Borrelia garinii were described in Europe and Asia(Baranton et al. 1998) but there were not found in North America. Borrelia burgdor-feri s. l. includes at least 15 genospecies, four of them are known to be pathogenicfor humans: Borrelia burgdorferi sensu stricto (s. s.), Borrelia afzelii, Borrelia gariniiand Borrelia spielmanii (Johnson et al. 1984, Baranton et al. 1992, Canica et al. 1993,Wang et al. 1999, Baranton and Martino 2009, Rudenko et al. 2009a, Rudenko et al.2009b). Occasionally, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii werealso detected in patients (Picken et al. 1996, Rijpkema et al. 1997, Collares-Pereiraet al. 2004, Fingerle et al. 2008, Hulınska et al. 2009, Nau et al. 2009, Rudenko etal. 2009c).

B. burgdorferi s. l. has been reported in all Europe from at least 30 countries(Hubalek and Halouzka 1997, Hubalek 2009). Although there is much that is notyet known about the distribution of the various genospecies in Europe, currentknowledge suggests that B. garinii and B. afzelii are the most frequent and mostwidely distributed species whereas B. burgdorferi s. s. is less frequent (Piesmanand Gern 2004, Gern 2009).

Borrelia is a spirochete, a narrow, long, helical bacterium, measuring 15 to30 µm in length by 0.2 to 0.3 µm in width. This spirochete has a fragile outermembrane surrounding the protoplasmic cylinder consisting of a peptidoglycanlayer (Burgdorfer et al. 1982). Borrelia spp. circulate between ticks and a widevariety of vertebrates, including different mammals, some bird species and fewreptiles (Humair and Gern 2000, Piesman and Gern 2004, Gern 2009).

1.1.2 Vectors

The main Borrelia vectors are ticks. a vector must be shown to be capable of get-ting infected and later infecting naive hosts. Ticks usually get infected by feed-ing as larvae, moult to the next life stage and then infect their next feeding host(Humair and Gern 2000, Gray et al. 2002).

The epidemiologically most important ticks transmitting B. burgdorferi s. l.to humans are Ixodes persulcatus (northern middle Asia), Ixodes scapularis (east-ern North America), Ixodes pacificus (western North America) and Ixodes ricinus(Europe) (Parola and Raoult 2001). In Europe in particular, three tick species havebeen described as being vectors of B. burgdorferi s. l.: I. ricinus, Ixodes hexagonus

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and Ixodes uriae.All are three tick stages (larva, nymph and adult female) feed on different

hosts. Although adult male Ixodes sometimes ingest fluids from hosts, they donot ingest significant amounts of blood and their role as vectors is probablyinsignificant, however this has not been thoroughly evaluated (Piesman and Gern2004). Tick vectors acquire borrelia while feeding on infected reservoir hosts orby cofeeding (Gern 2009). Borrelia remains inactive in the midguts of the tickswhile they moult. In the next developmental stage the spirochetes disseminaterapidly to all other body organs, including salivary glands, wherefrom the Bor-relia is transferred to vertebrates. If the Borrelia load in a tick is high, or for someBorrelia species, the spirochetes may be present in salivary glands and hence betransferred to the host even earlier (Gern 2009, Hubalek 2009). Transovarial trans-mission in ticks is rare (Humair and Gern 2000, Gray et al. 2002).

Ticks are dependent upon the availability of suitable host individuals for eachlife stage. They also need a microclimate with high relative humidity during theoff-host phases. Host-seeking is a seasonal activity and is strongly dependenton environmental factors. Proliferation of B. burgdorferi s. l. requires the simul-taneous occurrence of ticks and vertebrate populations capable of transmittingborreliae (Humair and Gern 2000, Gray et al. 2002).

The reported prevalence of B. burgdorferi s. l. in ticks I. ricinus in Europe varyfrom 0 to 11% for larvae, from 2 to 43% for nymphs and from 3 to 58% for adults(Hubalek and Halouzka 1998, Hulınska et al. 2007).

Seven Borrelia genospecies have been found associated with I. ricinus inEurope: B. burgdorferi s. s., B. garinii, B. afzelii, B. valaisiana, B. lusitaniae, B. bis-settii and B. spielmanii (Johnson et al. 1984, Baranton et al. 1992, Canica et al. 1993,Le Fleche et al. 1997, Wang et al. 1997, Lencakova et al. 2006, Gern 2009).

Since many Borrelia species may circulate in an area, mixed infection in tickscan be observed (Basta et al. 1999). Ticks and their hosts may be co-infected withmultiple genospecies of B. burgdorferi s. l. In central Europe, for example, I. ricinuspopulation harbour B. afzelii, B. garinii, B. burgdorferi s. s. and B. valaisiana to vary-ing degrees. Identification of mixed infection in ticks is possible with polymerasechain reaction (PCR).

1.1.3 Reservoirs

The animal reservoirs of Borrelia burgdorferi s. l. are numerous, mainly wild smallmammals and birds. The reservoirs do not manifest signs of the disease as a resultof infection. Xenodiagnosis is the classical method for determining the host infec-tivity of a particular vertebrate species. It is very difficult to determine the wholespectrum of reservoir hosts or the relative capacity of one or several vertebrate

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species (Humair and Gern 2000, Gray et al. 2002, Gern 2009).In North America, Borrelia spirochetes were detected in a variety of mam-

malian and bird species (Anderson 1988, Anderson 1989). The white-footedmouse Peromyscus leucopus is the most competent reservoir host (Levine et al.1985, Donahue et al. 1987, Mather et al. 1989). The while-tailed deer Odocoileusvirginianus play an important role in the Lyme disease natural cycle in NorthAmerica (Piesman et Gern 2004).

Small mammals

I. ricinus feeds a large variety of vertebrate hosts, but only a few dozen hosts havebeen currently identified as reservoirs for B. burgdorferi s. l. in Europe (Gern etal. 1998, Gern 2009). Small mammals are frequent hosts of larval and nymphaltick stages. Several species of mice, voles, rats and shrews have been shown tobe competent reservoirs of B. burgdorferi s. l. in Europe (Gern et al. 1998). In par-ticular, there is evidence that the mice Apodemus flavicollis, A. sylvaticus, A. agrar-ius and the vole, Clethrionomys glareolus, act as reservoirs for B. burgdorferi s. l.(Aeschlimann et al. 1986, Matuschka et al. 1992, de Boer et al. 1993, Humair etal. 1993a, 1999, Gern et al. 1994, Kurtenbach et al. 1994, 1995, 1998b, Talleklintand Jaenson 1994, Hu et al. 1997, Richter et al. 1999, Hanincova et al. 2003, Gern2009). It was observed in Germany and France that dormice Glis glis and gar-den dormice Eliomys quercinus are reservoir hosts for Borrelia spp. (Matuschka etal. 1994a, 1999). Only a few studies mentioned B. burgdorferi s. l. in shrews, Sorexminutus and S. araneus (Humair et al. 1993a, Talleklint and Jaeson, 1994). Similarly,the vole Microtus agrestis (Talleklint and Jaeson, 1994), black rats Rattus rattus, andNorway rats R. norvegicus (Matuschka et al. 1994b, 1996) may also serve to infectI. ricinus ticks.

Other rodents, such as grey squirrels Sciurus carolinensis in the UK (Craine etal. 1997) and red squirrels S. vulgaris in Switzerland (Humair and Gern 1998) actas reservoirs of B. burgdorferi s. l. Several studies demonstrated that the Europeanhedgehog Erinaceus europaneus also perpetuates B. burgdorferi s. l. in Ireland, Ger-many and Switzerland (Gray et al. 1994, Liebisch et al. 1996, Gern et al. 1997).

Birds

The role of birds in the maintenance of B. burgdorferi s. l. is recognized (Humair2002). The first report in Europe of B. burgdorferi s. l. in I. ricinus ticks feeding onbirds was published by Humair et al. 1993b. Olsen et al. 1993 demonstrated theexistence of a transmission cycle of B. burgdorferi s. l. in seabird colonies amongrazorbills Alca torda. Spirochetes were reported in I. ricinus ticks collected frommigratory birds in Sweden and Switzerland (Olsen et al. 1995, Poupon et al. 2006).

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Later, few studies clearly defined the reservoir role of birds: on passerine bird,blackbird Turdus merula (Humair et al. 1998, Taragelova et al. 2008) and on galli-naceous bird, the pheasant Phasianus colchicus (Kurtenbach et al. 1998a).

There are differences in the relationship between different reservoir hosts anddifferent Borrelia sp. Rodents are mainly associated with B. afzelii, but also withB. burgdorferi s. s. and with B. garinii OspA serotype 4 whereas other B. gariniiserotypes are associated with birds (Kurtenbach et al. 2002). B. valaisiana has beendescribed only in birds and never in rodents (Humair et al. 1998, Kurtenbach etal. 1998b).

Candidate reservoirs

Lizards can be important hosts for I. ricinus larvae and nymphs and also playa role in the natural cycle of B. lusitaniae in Europe (Majlathova et al. 2006, Fold-vari et al. 2009).

Lagomorphs play a role in the support of the enzootic cycle of B. burgdor-feri s. l. The brown hare Lepus europaeus and the varying hare L. timidus (Jaensonand Talleklint, 1996) and European rabbit Oryctolagus cuniculus (Matuschka et al.2000) contribute to the maintenance of B. burgdorferi s. l. in nature.

The red fox Vulpes vulpes is embroiled in the maintenance of Borrelia in nature(Liebisch et al. 1998)

Sheep was found to be a reservoir of B. burgdorferi s. l. in the UK, but itwas found that ticks might have been infected by cofeeding (Ogden et al. 1997,Travnıcek et al. 2002). Host did not necessarily become infected, but neighboringticks serve to infect each other while feeding on the host.

1.1.4 Domestic animals as hosts

Besides humans, the clinical form of borreliosis occurs in domestic animals, espe-cially dogs, horses and cattle (Burgess et al. 1987, Greene et al. 1991, Cohen et al.1992). Most data on Lyme borreliosis in domestic animals concern dogs andhorses, with isolated reports of infection in cattle, sheep and cats. Anti-borrelialantibodies have been detected in a wide range of domestic animal species. Itis likely that most infections are subclinical and self-limiting. It is also possiblethat infections are missed or misdiagnosed because of the non-specifity of clini-cal symptoms.

Lyme borreliosis in dogs

Canine Lyme borreliosis was first reported in the USA in 1984 (Lissman et al.1984). Typical signs are lameness combined with malaise, fatigue, listlessness,

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inappetence and fever. Canine borreliosis most commonly affects the limb joints(Skotarczak et Wodecka, 2003, Skotarczak et al. 2005), with clinical manifestationssuch as arthritis and arthralgia (Jacobson et al. 1996). Even though the signs arethe same as in humans, they are more difficult to detect in dogs and develop inrelatively few of them (Levy and Magnarelli, 1992).

Diagnosis of canine borreliosis requires presence of typical clinical signstogether with antibodies to B. burgdorferi s. l. or PCR detection and evidence ofexposure to ticks. Positive serology is not sufficient to diagnose borreliosis sincemost infection are subclinical, though serosurveys have shown that prevalenceof B. burgdorferi s. l. antibodies is usually higher in symptomatic dogs comparedwith healthy ones (Hovius 1999).

Lyme borreliosis in horses

Equine borreliosis was reported in endemic areas in the USA a few years afteridentification of B. burgdorferi s. l. (Burgess et al. 1986, Burgess and Mattison 1987).Serosurveys have shown high seroprevalence in USA and in Europe (Magnarelliet al. 2000, Egenvall et al. 2001, Stefancıkova et al. 2008). Clinical signs consist ofmalaise, fever, lameness and swollen joints (Burgess et al. 1986).

Lyme borreliosis in cattle

Borreliosis in cattle was reported in the USA in the 1980s (Burgess et al. 1987,Burgess, 1988). Clinical symptoms were lameness, weight loss and abortion.Lyme borreliosis in cattle is probably an infrequent disease that is difficult to diag-nose due to the non-specific nature of the symptoms and there are also doubtsabout the specificity of the serology (Gray et al. 2002, Stefancıkova et al. 2002).Cattle are not regarded as reservoir hosts of B. burgdorferi s. l. (Gern et al. 1998)and the incidence of infected ticks on cattle pastures is probably due to the pres-ence of other hosts in the same habitat (Gray et al. 1995).

Lyme borreliosis in cats

Lyme borreliosis has not been reported in cats exposed to natural infection. How-ever, anti-Borrelia antibodies have been detected (Magnarelli et al. 1990, May et al.1994). Cats may be affected when they are exposed to a heavy tick challenge(Burgess 1992).

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Lyme borreliosis in sheep

B. burgdorferi s. l. infection of sheep rarely results in a disease; sheep have evenbeen identified as reservoirs in the UK (see section 1.1.3.3). There are severalreports of the detection of anti-borrelial antibodies in blood of sheep (Hovmarket al. 1986, Ciceroni et al. 1996, Travnıcek et al. 2002). There is some evidence thatadult ticks may become infected as the result of the transfer of the pathogens frominfected to uninfected nymphal tick co-feeding on sheep (Ogden et al. 1997)

1.1.5 Laboratory diagnostic

Detection of infection in vertebrates can be based on host seroconversion, detec-tion of borrelial DNA in host fluids or tissue, or recovery of live borreliae fromcultured host tissue. Host seroconversion demonstrates that the vertebrate wasexposed to spirochetes but does not confirm that a viable infection was estab-lished. Likewise, detection of borrellial DNA in host tissue or fluids, althoughallowing detection of the genospecies involved, is not necessarily indicative ofa viable infection. It is very useful to have standardized and dependable tests toascertain borrelial infection, with ability to distinguish between active and inac-tive infection (Gray et al. 2002, Piesman and Gern 2004).

Methods for detection of B. burgdorferi s. l. are direct and indirect. Directmethods detect the bacteria or its parts. The most often used ones are cultiva-tion, microscopy and molecular methods. Indirect methods mainly serologicalare based on detecting bacteria antibodies.

Indirect laboratory methods detect only certain parts of borrelial cell such asthe outer surface and other proteins. Examples are immunofluorescence assay(IFA), enzyme-linked immunosorbent assay (ELISA), and western blot test (WB)(Piesman and Gern 2004). Official recommendation for human serology (fromCenters for Disease Control) is to use a two-step procedure for detection of Lymedisease – first, to perform a sensitive screening test, such as ELISA and, if theresult is positive or equivocal, a WB test to confirm the results (Shapiro 2008).

IFA may indicate living borreliae by showing the typical helical morphologyof the spirochaete. It is also possible to combine culture and IFA to obtain clearevidence of living borreliae.

Borrelia infection should be confirmed by isolation of B. burgdorferi s. l. fromthe tissue and body fluids involved. Although B. burgdorferi s. l. grows relativelywell under laboratory conditions, spirochetes are not easily recovered from clin-ical specimens other than skin biopsy samples. Culture is rather costly and timeconsuming to be used for routine detection of borreliae. However, it providesa large number of cells that facilitates subsequent identification and characteriza-tion e.g. by a polymerase chain reaction (PCR) (Gray et al. 2002).

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Detection of borrelial genetic material by PCR has been acclaimed for its highsensitivity and specificity. However, the value of detection of borreliae by thismethod in tissues and fluids from vertebrates has not yet been unequivocallyestablished. They demonstrate infection but not the presence of living agents,although the detection of RNA by PCR may indicate a living organism since RNAdegrades very rapidly (Dumler 2003).

1.2 Anaplasmosis

The second tick-borne disease addressed in this work is Anaplasmosis. Com-pared to borreliosis, there are less published reports on Anaplasma because it wasdiscovered much later. Human granulocytic anaplasmosis (HGA) was first iden-tified in 1990 in USA, in patient who died with a severe febrile illness two weeksafter a tick bite (Chen et al. 1994). In Europe, antibodies against Anaplasma phago-cytophilum were described in 1995 in the Swiss population (Brouqui et al. 1995).First documented HGA case in Europe was reported by Petrovec et al. 1997 inSlovenia. Anaplasmosis in domestic ruminants is also called tick-borne fever andhas been mentioned in 1932 (Gordon et al. 1932). HGA agent is maintained innature in a tick-ruminant-rodent cycle. Humans are involved only as accidental“dead-end” hosts (Blanco and Oteo 2002).

The agent A. phagocytophilum, may cause infection in several animal speciesincluding human. Anaplasmosis may cause high fever, inappetence, malaise,cytoplasmatic inclusions in granulocytic neutrophils, neutropenia and thrombo-cytopenia (Rikihisa 1991, Greig et al. 1996, Egenvall et al. 1997, Engvall and Egen-vall 2002). Anaplasmosis is seldom fatal unless there are complications by otherinfection. In humans, clinical manifestations range from a mild self-limited flu-like illness to a life-threatening infection. Most human infection probably resultsin minimal or no clinical manifestation (Dumler et al. 2005).

1.2.1 Anaplasma phagocytophilum

A. phagocytophilum was described in 1994 in the USA (Bakken et al. 1994, Chenet al. 1994). The agent was recognized by molecular amplification and DNAsequencing and was initially named Human granulocytic ehrlichiosis (HGE)agent. Recently, the families Rickettsiaceae and Anaplasmataceae were completerevised and these bacteria: Ehrlichia phagocytophilum, E. equi and the HGE agentwere reclassified as a single species, Anaplasma phagocytophilum (Dumler et al.2001).

A. phagocytophilum has been detected in ticks and mammals in many Euro-pean countries (Strle 2004). Seroprevalence rates in European countries range

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from zero to up to 28% of the examined human population (Strle 2004).A. phagocytophilum is small (0.2 – 1.0 µm) obligate intracellular bacteria with

a gram-negative cell wall (Walker and Dumler 1996, Parola and Raoult 2001).This bacterium resides in an early endosome in granulocytic neutrophils, whereAnaplasma obtains nutrients for fission and grows into a cluster called a morulae(Lin and Rikihisa 2003).

1.2.2 Vectors

The main vector of A. phagocytophilum in Europe is Ixodes ricinus (Parola andRaoult 2001, Strle, 2004). The prevalence of A. phagocytophilum infection in I. rici-nus in Europe varies from area to area and between development stages of thetick (Lillini et al. 2006). Occurrence in nymphs has been found to vary between0.25-25% (Walker et al. 2001). Prevalence is usually higher in adult ticks than innymphs and ranges from zero to 30% (Pusterla et al. 1999a, Liz et al. 2000, Strle2004). Tick is infected after feeding on an infected host. The bacterium is passedtransstadially but not transovarially (Dumler et al. 2001).

A. phagocytophilum has been associated with other ticks such as Haemaphysalispunctata (Barandika et al. 2008), I. persulcatus (Alekseev et al. 1998), I. trianguliceps(Ogden et al. 1998a) and Riphicephalus sanguineus (Alberti et al. 2005).

In the USA, A. phagocytophilum has been often associated with I. scapularis andwith I. pacificus, and these may serve as the primary vectors (Barlough et al. 1997a,Chang et al. 1998).

1.2.3 Reservoirs

There is field evidence that sheep are natural hosts for A. phagocytophilum in theUK (Ogden et al. 1998a,b, Ogden et al. 2002). In the USA rodents, particularlywhite-footed mice P. leucopus (Bunnell et al. 1998), and white-tailed deer O. vir-ginianus (Belongia et al. 1997) are involved as natural reservoirs for A. phagocyto-philum.

Small mammals

Wild rodents in Europe have been suggested to be competent reservoirs forA. phagocytophilum (Ogden et al. 1998a, Liz et al. 2000). Long-tailed mice A. sylvati-cus, yellow-necked mice A. flavicollis, common shrew S. araneus, and bank volesC. glareolus were found to be likely natural reservoir for A. phagocytophilum (Lizet al. 2000, Liz 2002).

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Candidate reservoirs

Migrating birds may be important in dispersal of A. phagocytophilum infectedI. ricinus in Europe (Alekseev et al. 2001, Bjoersdorff et al. 2001, de la Fuente et al.2005a).

A study from Slovenia revealed by PCR that red deer Cervus elaphus androe deer Capreolus capreolus are infected with A. phagocytophilum in about 86% ofcases, and the prevalence of IFA antibodies was found to be 35% and 94%, respec-tively (Petrovec et al. 2002). Red foxes V. vulpes and wild boar Sus scrofa were alsofound to be PCR positive (Petrovec et al. 2003). Infection by Anaplasma has beenalso identified in European bison (Grzeszcuk et al. 2003), donkey (de la Fuenteet al. 2005b), and moose (Jenkins et al. 2001). Antibodies have been detected inhare (Groen et al. 2002) and Eurasian lynx (Ryser-Degiorgis et al. 2005).

1.2.4 Domestic animals as hosts

A. phagocytophilum is known to cause granulocytic anaplasmosis in humans(Petrovec et al. 1997) and domestic animals such as horses (Bjoersdorff 1990, Eng-vall et al. 1996, Engvall and Egenvall 2002), dogs (Bellstrom 1989, Engvall et al.1996, Engvall and Egenvall 2002, Skotarczak 2003, Lester et al. 2005, Poitout et al.2005), cats (Bjoersdorff et al. 1999), cattle (Engvall et al. 1996), and llamas (Bar-lough et al. 1997b). A. phagocytophilum has been found to persist in sheep (Stuenet al. 1998).

Anaplasmosis signs in domestic animals include fever, fatigue, inappetence,lethargy, lameness, gastrointestinal and central nervous system signs (Rikihisa1991, Greig et al. 1996, Egenvall et al. 1997, Engvall and Egenvall 2002). Therelated hematologic and biochemical abnormalities are anemia, thrombocytope-nia, lymphopenia and elevated serum alkaline phosphatase activity (Greig et al.1996, Goldman et al. 1998).

Anaplasmosis in dogs

A. phagocytophilum often causes chronic disease in dogs with non-specific clini-cal findings. Clinical and haematological findings in dogs are fever, inappetence,joint swelling and pain, lameness, stiffness, neurologic inflammation, thrombo-cytopenia and neutropenia. Canine anaplasmosis was reported from many Euro-pean countries (Sweden, Greece, Italy, Slovenia, Austria, Poland, Switzerland,and Czech Republic) (Lillini et al. 2006).

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Anaplasmosis in horses

A. phagocytophilum in horses was reported in many European countries (GreatBritain, Denmark, Sweden, Switzerland, France, Germany, Czech Republic andItaly). It was detected by serological and molecular methods, and also by posi-tive findings in buffy coat smears. Hematological findings on Anaplasma-positivehorses showed thrombocytopenia and leukocytosis (Bjoersdorff 1990, Bjoersdorffet al. 2002, Lillini et al. 2006, Zeman and Jahn 2009).

Anaplasmosis in ruminants

In domestic ruminants A. phagocytophilum causes a disease known as a tick-bornefever (TBF). A. phagocytophilum in sheep causes very high fever, reduced milkyield and abortion in pregnant animals. Laboratory findings are neutropenia andcytoplasmatic inclusion with more than 95% of neutrophils infected (Garcia-Perezet al. 2003).

1.2.5 Laboratory diagnostic

The majority of Anaplasma infection is identified by indirect laboratory methods.The most frequent used method is the IFA test. While this test is often used, reac-tivity in samples with anticytoplasmic antibodies or with other autoimmune anti-bodies may confound interpretation, and the correlation of results from differ-ent laboratories and different commercial tests may be difficult (Blanco and Oteo2002).

A. phagocytophilum is visible as a cluster of small cocci in cytoplasm of neu-trophils in the peripheral blood on a Wright- or Giemsa-stained smear. The char-acteristic cytoplasmatic inclusion in neutrophils can be detected in between 25and 80% of patients during the active stage of HGA (Dumler and Brouqui 2004).

This bacterium is an obligate intracellular parasite that can only be cultivatedin cell lines derived from bone marrow myeloid progenitors, for example thehuman HL-60 promyolocytic leukemia cell line (Goodman et al. 1996). Cultiva-tion may require several days to several weeks. Morphologic identification in cul-ture still requires confirmation for example by PCR (Dumler and Brouqui 2004).

Detection of Anaplasma genetic material by PCR demonstrates the presenceof A. phagocytophilum nucleic acid in peripheral blood but not the presence ofliving agents. HGA is one of the rare infectious diseases that was first confirmedbased upon molecular tests rather than culture or serology (Chen et al. 1994). Thefirst tests used were based on an amplification of the highly conserved 16S rRNAgene, followed by a second stage that utilized primers suspected to anneal onlyto A. phagocytophilum rrs-specific sequence (Dumler et Brouqui 2004).

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1.3 Aims of this thesis

• Determine the prevalence of B. burgdorferi s. l. and A. phagocytophilum in vec-tors, confirmed and candidate reservoirs, and hosts in the Czech Republic.Compare the results with other European studies.

• Compare the prevalence of B. burgdorferi s. l. and A. phagocytophilum in dif-ferent rodent species, detected by molecular and serological methods.

• Determine Borrelia genospecies occurring in their important reservoir hosts,rodents, using different molecular methods.

• Study the prevalence of B. burgdorferi s. l. and A. phagocytophilum in domesticanimals, confirm whether there is a difference between symptomatic andasymptomatic hosts. Study and describe borreliosis symptoms.

• Compare A. phagocytophilum variants occurring in wild and domestic ani-mals and ticks by direct sequence analysis.

• Determine, implement and test suitable diagnostic methods for detection ofborreliosis and anaplasmosis from blood and tissue samples.

• Implement molecular detection methods for B. burgdorferi s. l. and A. phago-cytophilum using real-time PCR and evaluate its advantages and disadvan-tages with respect to other molecular methods.

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Chapter 2

Overview and results

The main part of this thesis consists of four papers which have been publishedin peer-reviewed international journals and that we are including verbatim. Wegive here a brief summary of each paper and its relevance.

2.1 Molecular and serological evidence of Borreliaburgdorferi sensu lato in wild rodents in theCzech Republic.

The aim of the first paper (Kybicova et al. 2008) was to determine the frequencyand spatial distribution of the Borrelia species in wild rodents in the Czech Repub-lic. In total, 293 muscle tissue samples and 106 sera from 293 wild rodents cap-tured in North Bohemia and North-East and South Moravia were examined forthe presence of Borrelia spp. and antibodies. Apart from a small sample prelim-inary study (Hulınska et al. 2002), the prevalence has not yet been determinedusing PCR methods and only serological data have been available (Vostal andZakovska 2003).

Infection with B. burgdorferi s. l. was found in 16.4% of the muscle samples.The most abundant genospecies was B. afzelii (11.3%), followed by B. burgdorferis. s. (4.8%) and B. garinii (0.7%). Borrelia infection was more frequently observedin C. glareolus than in Apodemus spp. Sera were analyzed using an ELISA test,yielding the total seropositivity rates of 24.5% for anti-Borrelia IgM antibodies and25.5% for IgG antibodies. Total seroprevalence was higher in Apodemus spp. thanin C. glareolus. Our data indicate that in the Czech Republic small wild rodentscan serve as hosts for B. burgdorferi s. s. as well as for B. afzelii.

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Chapter 2. Overview and results

2.2 Detection of Anaplasma phagocytophilum andBorrelia burgdorferi sensu lato in dogs in theCzech Republic.

The second paper (Kybicova et al. 2009) presents molecular, serological and clin-ical findings for dogs that were naturally infected with A. phagocytophilum orB. burgdorferi s. l. in the Czech Republic. In total, blood samples from 296 dogsand 118 engorged ticks were examined. Dogs are important domestic hosts ofA. phagocytophilum and B. burgdorferi s. l. Data on vector-borne infections in dogscan provide important information for the potential of human infection in a par-ticular geographic location (Duncan et al. 2005). Data on the prevalence of infec-tion with A. phagocytophilum and B. burgdorferi s. l. in dogs in the Czech Republicwas missing, except for a serology study by Pejchalova et al. 2006 and a few casereports.

Ten (3.4%) dogs were PCR-positive for A. phagocytophilum. Morulae of A. pha-gocytophilum in granulocytes were found in two of these dogs. Nine of the PCR-positive dogs had clinical signs related to anaplasmosis. Statistically significantdifferences in the PCR detection rates were found between breeds and betweensymptomatic and asymptomatic dogs. Infection with B. garinii was detectedby PCR in a dog with meningoencephalitis. DNA of A. phagocytophilum andB. burgdorferi s. l. (B. garinii or B. afzelii) was detected in 8.5% and 6.8% of ticks,respectively. IgG seropositivity to A. phagocytophilum was 26%. Significant differ-ences were found with respect to breed and gender. IgM and IgG antibodies toB. burgdorferi s. l. were detected in 2.4% and 10.3% of dogs, respectively. Our find-ings suggest that the exposure to B. burgdorferi s. l. exists in dogs in the CzechRepublic and exposure to A. phagocytophilum is common.

2.3 Clinical and Diagnostic Features in Three DogsNaturally Infected with Borrelia spp.

The third paper (Schanilec et al. 2010) presents clinical and neurological signs,laboratory abnormalities, serologic and/or molecular findings in three dogs fromthe region of Brno in the Czech Republic. All dogs were naturally infected withBorrelia burgdorferi sensu lato. The evidence of borrelial infection was proved byserial blood sampling for IgM and IgG anti-borrelial antibodies and plasma PCR.

All three dogs showed neurological signs (two of them had meningoen-cephalomyelitis, one had a seizure connected with a progressive renal disease).Their history, clinical signs, diagnostic procedures and treatment are described.

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Two of the infected dogs died and only one with meningoencephalomyelitis sur-vived. This paper shows that borrelial infection must be considered, not onlyin cases with febrile and orthopaedic signs but also in many other clinical syn-dromes.

2.4 Detection of Anaplasma phagocytophilum inanimals by real-time polymerase chain reaction.

The aim of the fourth paper (Hulınska et al. 2004) is the detection of A. phagocy-tophilum in wild and domesticated animals and the identification of phylogeneticrelationships of different variants of this bacterium. Six published conventionalmethods targeting 16S rDNA fragments were adapted for a real-time polymerasechain reaction, using the LightCycler real-time PCR technique.

Initial screening of samples from 419 animals found 37 Anaplasma positives,later confirmed with several different primers and a TaqMan probe. The nucleicacid of Anaplasma sp. was detected in a higher percentage of cases in members ofthe deer family, hares, bank voles and mice (12.5–15%) than in foxes, boars, cows,and horses (around 4–6%).

At the time of writing there were only a small number of studies on animalreservoirs for A. phagocytophilum in Europe in the literature. To the best of ourknowledge, the direct PCR sequence analysis from samples of a large numberof wild animals was reported here for the first time. We succeeded in properlyselecting primers for direct sequencing that differentiate well between the dif-ferent variants of A. phagocytophilum. To analyze the relationships between thesevariants, we sequenced the PCR products from our samples and formed a phylo-genetic tree using as a reference the sequence of Ehrlichia equi. Mutual identity ofthe sequencing ranged from 99% to 100%.

2.5 Summary of the results

Prevalence of B. burgdorferi s. l. and A. phagocytophilum detected by molecular(PCR) and serological (ELISA and IFA) methods in vectors, reservoirs, candidatereservoirs and domestic hosts is shown in Table 2.1. For more details please seethe articles mentioned in the previous section.

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Table 2.1: Summary of detected prevalences. B. b. and A. p. stand for Borrelia bur-gorferi sensu lato and Anaplasma phagocytophylum, respectively.

PCR SerologyB. b. A. p. B. b. A. p.

Vectorsticks from rodents 9.8%ticks from dogs 6.8% 8.5%

Reservoirsbank voles 25.3% 13.3%

IgM 16.1%IgG 21.4%

mice 8.1% 15.0%IgM 26.7%IgG 31.1%

Candidate

reservoirs

deer family 12.9%hares 12.5%foxes 4.0%boars 4.4%

Domestic

hosts

cows 5.5%horses 5.0%

dogs 0.3% 3.4%IgM 2.4%

IgG 25.9%IgG 10.3%

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Chapter 3

Molecular and serological evidenceof Borrelia burgdorferi sensu lato inwild rodents in the Czech Republic.

Kybicova, K., Kurzova, Z. and Hulınska, D.: “Molecular and sero-logical evidence of Borrelia burgdorferi sensu lato in wild rodents inthe Czech Republic.” Vector-Borne and Zoonotic Diseases, October2008;8(5), pages 645–652.

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VECTOR-BORNE AND ZOONOTIC DISEASESVolume 8, Number , 2008© Mary Ann Liebert, Inc.DOI: 10.1089/vbz.2007.0249

Molecular and Serological Evidence of Borrelia burgdorferisensu lato in Wild Rodents in the Czech Republic

K. KYBICOVÁ, Z. KURZOVÁ, and D. HULÍNSKÁ

ABSTRACT

The aim of the present study was to determine the frequency and spatial distribution of the Borrelia species inwild rodents in the Czech Republic. In total, 293 muscle tissue samples and 106 sera from 293 wild rodents cap-tured in North Bohemia and North-East and South Moravia were examined for the presence of Borrelia spp. andantibodies. Muscle samples were investigated with real-time polymerase chain reaction (PCR) with a recA primerset, with DNA quantification and melting curve analysis, and with restriction fragment length polymorphism(RFLP) analysis of the 5S–23S rDNA intergenic spacer. Infection with Borrelia burgdorferi sensu lato was foundin 16.4% of the muscle samples. The most abundant genospecies was Borrelia afzelii (11.3%), followed by Borre-lia burgdorferi sensu stricto (4.8%) and Borrelia garinii (0.7%). Borrelia infection was more frequently observed inClethrionomys glareolus than in Apodemus spp. Sera were analyzed with an enzyme-linked immunosorbent as-say (ELISA) test, yielding the total seropositivity rates of 24.5% for anti-Borrelia IgM antibodies and 25.5% for IgGantibodies. Total seroprevalence was higher in Apodemus spp. than in C. glareolus. In conclusion, our data indi-cate that in the Czech Republic small wild rodents can serve as hosts for B. burgdorferi s. s. as well as for B. afzelii.Key Word: Borrelia

1

INTRODUCTION

LYME BORRELIOSIS IS A MULTI-ORGAN disease ofmammals in the northern hemisphere. It is

caused by Borrelia burgdorferi sensu lato (s. l.),which currently includes 11 genospecies, threeof which are known to be pathogenic: Borreliaburgdorferi sensu stricto (s. s.), Borrelia afzelii,Borrelia garinii (Johnson et al. 1984; Baranton etal. 1992; Canica et al. 1993). Occasionally, Bor-relia valaisiana, Borrelia lusitaniae, Borrelia biset-tii, and Borrelia spielmanii were detected in pa-tients (Picken et al. 1996; Ripkiema et al. 1997;Wang et al. 1999; Collares-Pereira et al. 2004).Nevertheless, the association of these and otherBorrelia species with Lyme disease has not yetbeen confirmed.

Studies of the ecology of Lyme borreliosishave demonstrated that the persistence of Bor-

relia in endemic areas depends on the presenceof reservoir hosts (Gern et al. 1998; Humair etal. 1998). In North America, Borrelia spiro-chetes have been detected in a variety of mam-malian and bird species (Anderson 1988,1989). The white-footed mouse Peromyscus leu-copus is the most competent reservoir host(Levine et al.1985; Donahue et al.1987; Matheret al.1989). In Europe, wild rodents were stud-ied in various enzootic areas (de Boer et al.1993; Humair et al. 1993; Tallekint and Jaen-son 1994; Kurtenbach et al. 1995), and severalrodent species have been suggested as naturalreservoirs for B. burgdorferi s. l. (Matuschka etal. 1992, 1997), especially the woodmouseApodemus sylvaticus, the yellow-necked mouseApodemus flavicollis (Aeschlimann et al. 1986),and the bank vole Clethrionomys glareolus(Hovmark et al.1988).

National Reference Laboratory for Lyme Borreliosis, National Institute of Public Health, Prague, Czech Republic.

Chapter 3: Borrelia burgdorferi s. l. in wild rodents

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Molecular identification methods have madeit possible to determine specific associations be-tween hosts and Borrelia genospecies. In smallrodents, B. afzelii dominated in Switzerland(Humair et al.1995; Hu et al.1997), whereas inthe United Kingdom, B. burgdorferi s. s. pre-vailed (Kurtenbach et al.1998b). Generally, themost prevalent genospecies in rodents in Eu-rope appears to be B. afzelii, followed by B.burgdorferi s. s., while B. garinii is rare. B. afzeliiseems to be preferentially transmitted by ro-dents (Hu et al.1997; Humair et al.1995, 1998,1999). Most strains of B. garinii and B. valaisianadisplay resistance to the bactericidal activity ofavian complement and are therefore consid-ered bird-associated Borrelia ecotypes (Kurten-bach et al. 1998a; Hanincova et al. 2003). How-ever, some subtypes of B. garinii can also infectEuropean rodents (Richter et al. 1999; Gray etal. 2000; Huegli et al. 2002).

The aim of the present study was to assessthe prevalence of B. burgdorferi s. l. infectionsin wild rodents in the Czech Republic, and todetermine the genospecies within B. burgdorferis. l. associated with these hosts. Apart from asmall sample preliminary study (Hulinska etal. 2002), the prevalence has not yet been de-termined using polymerase chain reaction(PCR) methods, and only serological data havebeen available (Vostal and Zakovska 2003).

MATERIALS AND METHODS

Locations studied

Rodents were collected in May and July 2003and between October and December 2004 inwooded areas (mixed forest) and nearby fieldsin three different areas of the Czech Republic:area 1, the terrain surrounding Decn, a town in North Bohemia (50°43N, 14°7E; altitude300–500 m); area 2, the Vsetín highlands inNorth-East Moravia (49°22N, 18°14E; altitude500–600 m); and area 3, the terrain surround-ing Brno, a large city in South Moravia (49°3N,16°38E; altitude 100–300 m) (Fig. 1). The aver-age temperature and average rainfall were16.8°C and 70 mm in May 2003, 20°C and 66mm in July 2003, 10.2°C and 73 mm in October2004, 3.6°C and 95 mm in November 2004, and0.1°C and 19 mm in December 2004.

Rodent capture

Rodents were captured alive with iron boxtraps or dead with collapsible traps. The trapswere spaced 5 m apart, baited with hay, grains,and pieces of vegetables, and exposedovernight. In laboratory each animal was clas-sified into species, weighed, measured andthen euthanized. Samples from muscle tissue(from all study areas) and blood sera (from ar-

KYBICOVÁ ET AL.2

FIG. 1. Geographical representation of three localities in the Czech Republic from which rodent samples were obtained.


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eas 1 and 2) were extracted and stored in 1.5ml microcentrifuge tubes at 20°C prior toDNA extraction and the enzyme-linked im-munosorbent assay (ELISA) test.

DNA extraction

Total DNA was isolated with the DNeasyTissue Kit (Qiagen, Hilden, Germany). Ap-proximately 20 mm3 of tissue was transferredto a tube containing 180 L of lysis buffer and20 L of proteinase K and lysed at 56°C untilthe tissue was completely lysed (2–3 h). DNAwas extracted according to the manufacturer’sinstructions and stored at 20°C until PCR am-plification was performed.

PCR and real-time PCR amplification

All samples were screened by standard PCRwith the LD primer set targeting the 16S rDNAgene (Marconi et al.1992). The PCR amplifica-tion was performed in a Peltier cycler (MJ Re-search, MA, USA). The reaction mixture con-sisted of 5 L of DNA extract as a template and1 M solution of each primer (Generi Biotech,Czech Republic) in a total volume of 25 L HotStart Master mix (Qiagen, Hilden, Germany).Cycling conditions involved an initial 15minute denaturation at 95°C, followed by 37cycles, each consisting of a 1 minute denatura-tion at 94°C, a 30 second annealing at 52°C, anda 1.5 minute extension at 72°C. These 37 cycleswere followed by a 7 minute extension at 72°C.The PCR products were separated by elec-trophoresis in 1% agarose gel and stained withethidium bromide and visualized by UV tran-silluminator. LD primers produce a 357 bp am-plicon.

The initially positive samples were furthertested by real-time PCR (LightCycler, RocheMolecular Biochemicals, Mannheim, Germany)with melting curve analysis. The reaction mix-ture consisted of 2 L of DNA extract as a tem-plate and a 5 M solution of each primer(Generi Biotech, Czech Republic) in a total vol-ume of 20 L of master mix (Roche MolecularBiochemicals, Mannheim, Germany), contain-ing Taq DNA polymerase, SYBR-Green I, de-oxynucleoside triphosphate mix, and 3 mMMgCl2. The samples were tested with primersnTM17F and nTM17R (Morrison et al. 1999) to

amplify a 222-bp sequence from the recA genewith the following cycling conditions: an ini-tial denaturation of 5 minutes at 95°C, 40 cy-cles consisting of a 5 second denaturation at95°C, 5 second annealing at 60°C, and a 11 sec-ond extension at 72°C. After the final PCR cy-cle, the PCR products were denaturated at95°C, annealed at 60°C, and then slowly heatedto 95°C.

We performed DNA relative quantificationfor the recA primers. The standard curve andthe amplification plot were derived from a ten-fold dilution series of the positive control B.afzelii strain Kc90. The concentration of BorreliaDNA in the positive control was determinedspectrophotometrically.

RFLP analysis

For further characterization, positive DNAsamples were tested by RFLP analysis with 5S(rrfA)-23S (rrlB) rDNA intergenic spacerprimers (222 255 bp) (Derdáková et al. 2003)under the following cycling conditions: an ini-tial denaturation of 15 minutes at 95°C, five cy-cles of 94°C for 15 seconds, 61°C (for the firstcycle with the temperature decreasing by0.2°C/cycle) for 25 seconds, and 72°C for 30seconds, followed by five cycles of 94°C for 15seconds, 60°C (for the first cycle with the tem-perature decreasing by 0.4°C/cycle) for 25 sec-onds, and then 30 cycles of 94°C for 15 seconds,58°C for 30 seconds, and 72°C for 30 seconds,followed by a 10 minute extension at 72°C. Thereaction mixture consisted of 2.5 L of DNAextract as a template and a 1 M solution ofeach primer (Generi Biotech, Czech Republic)in a total volume of 25 L of Hot Start MasterMix (Qiagen, Hilden, Germany). DNAs of B.afzelii strain Kc90, B. garinii strain M192, B.burgdorferi s. s. strain B31, and B. valaisianastrain E117 were used as positive controls. Foreach positive sample, 10–20 L of amplifiedDNA was digested at 65°C for 2 h in a solutioncontaining 5 U of Tru 9l (10,000 U/mL) and 1buffer M (Roche Molecular Biochemicals,Mannheim, Germany). The restricted DNAfragments were electrophoresed on a 3%agarose gel for 2 hours at 150 V, stained withethidium bromide, and visualized with a UVtransilluminator.

BORRELIA BURGDORFERI SENSU LATO IN WILD RODENTS 3


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ELISA

The sera were examined by a modifiedELISA as described elsewhere (Stefancikova etal. 2004). The whole cell lysate of the B. afzeliistrain Kc90 was used as an antigen for detec-tion of anti-Borrelia antibodies. Mouse sera withabsorbance values of less than 0.4 were used asnegative controls. Sera from naturally infectedrodents, which were positive in repeated titra-tions, served as positive controls. All these ro-dents were also PCR positive. The cut-off wasdetermined as 3 standard deviations above themean optical density of the negative controls(Magnarelli et al. 1988; Stefancikova et al. 2004).

Statistical analysis

Statistical significance of differences (at a levelof p 0.05) in the prevalence among areas, ro-dent species, and Borrelia genospecies was ana-lyzed by analysis of variance (ANOVA) with arepeated measures post-hoc test.

RESULTS

Wild rodents

A total of 293 wild rodents were trapped;they were from the following species: Apode-mus flavicollis (yellow-necked mouse, 105 indi-viduals), Apodemus sylvaticus (wood mouse,42), Clethrionomys glareolus (bank vole, 95),Sorex araneus (common shrew, 10), Pitymys sub-terraneus (earth vole, 6), Microtus arvalis (com-

mon vole, 33) and Microtus agrestis (field vole,2). In total, 293 muscle tissue samples and 106sera samples (from areas 1 and 2) were tested.

Detection of B. burgdorferi s. l. in rodents byareas and species

Tissue samples were screened for B. burgdor-feri s. l. by PCR. The rate of infection with Bor-relia burgdorferi s. l. was 16.4% (48 of 293 sam-ples were positive). The positive samples wereanalyzed by real-time PCR and PCR-RFLPgenotyping. Three genospecies were detected:B. afzelii, B. burgdorferi s. s., and B. garinii. B.afzelii was the most abundant (11.3%), followedby B. burgdorferi s. s. (4.8%); B. garinii was rare(0.7%) (Table 1).

We found the highest percentage of infectedrodents (17.1%; 15 of 82 animals) in area 1,where three genospecies were present: B. afzelii(12 animals), B. garinii (2 animals), and onemixed infection with B. afzelii and B. burgdor-feri s. s. In area 2, the positivity rate was 13.8%(11/80), and all infections were caused by B.afzelii. In area 3, Borrelia DNA was detected in16.8% of animals (22/131), with 9 rodents in-fected by B. afzelii and 13 infected by B. burgdor-feri s. s. The differences between areas were notstatistically significant.

The positivity rate was significantly higher(p 0.05) in C. glareolus (25.3%; 24/95) than inA. flavicollis (7.6%; 8/105), and A. sylvaticus(9.5%; 4/42). The rates of other infected rodentswere as follows: M. arvalis, 21.2% (7/33); P. sub-terraneus, 50% (3/6); M. agrestis, 100% (2/2),

KYBICOVÁ ET AL.4

TABLE 1. DISTRIBUTION OF B. BURGDORFERI S.L. GENOSPECIES DETECTED BY POLYMERASE

CHAIN REACTION (PCR) METHODS, BY RODENT SPECIES AND BY AREAS

No. ofexamined No. (%) of

Number of rodents infected with

Rodent animals positive B.a. B.g. B.b.s.s. B.a. B.b.s.s.

Clethrionomys glareolus 95 24 (25.3) 17 2 5 0Apodemus flavicollis 105 8 (7.6) 1 0 5 0Apodemus sylvaticus 42 4 (9.5) 3 0 2 1Sorex araneus 10 0 (0) 0 0 0 0Pitymys subterraneus 6 3 (50) 3 0 0 0Microtus arvalis 33 7 (21.2) 6 0 1 0Microtus agrestis 2 2 (100) 2 0 0 0AreasArea 1 82 15 (18.3) 12 2 0 1Area 2 80 11 (13.8) 11 0 0 0Area 3 131 22 (16.8) 9 0 13 0Total 293 48 (16.4) 32 2 13 1

B.a., Borrelia afzelii; B.g., B. garinii; B.b.s.s., B. burgdorferi s.s.


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S. araneus, and 0% (0/10). All three Borreliagenospecies were detected in C. glareolus: B.afzelii in 17 animals, B. burgdorferi s.s. in 5 ani-mals, and B. garinii in 2 animals. In A. flavicol-lis, B. afzelii and B. burgdorferi s. s. were detectedin 3 and 5 animals, respectively. A. sylvaticuswas positive once for B. afzelii and twice for B.burgdorferi s. s., and one animal showed coin-fection with B. afzelii and B. burgdorferi s. s. Inall other infected rodent species, only B. afzeliiwas detected, except for M. arvalis, with one an-imal infected by B. burgdorferi s. s. (Table 1).

Detection of B. burgdorferi s. l. in rodents byRT-PCR and RFLP

DNAs from Borrelia controls and 48 positivesamples were subjected to the recA gene Light-Cycler PCR and melting curve analysis. WithuTM17F and nTM17R used as primers, meanmelting temperatures for controls strains of B.afzelii, B. burgdorferi s. s., and B. garinii were 83.5°,84.6°, and 80.8°C, respectively. All unspecificproducts melted at temperatures below 80°C. Theestimated concentration of B. burgdorferi s. l. DNAin rodent samples ranged between 4.2 and 42pg/L. This corresponds to 20 200 spirochetesper nanogram of the original tissue, using the av-erage nucleotide MW 330 pg/pmol and the Bor-relia DNA length of 1.5 Mb (Lederer et al. 2005).

The RFLP restriction pattern of B. afzeliiis 105 bp, 70 bp, 68 bp, and 20 bp; that ofB. burgdorferi s. s. is 105 bp, 70 bp, 38 bp, and 29 bp; and that of B. garinii is 105bp, 97 bp, and 80 bp (Fig. 2). The RFLPstudy confirmed the findings of real-timePCR and could distinguish between B. afzeliiand B. burgdorferi s. s. We also confirmed themixed infection with B. afzelii and B. burgdor-feri s. s.

Serological findings

We analyzed 106 rodent sera: 34 from A. flav-icollis, 11 from A. sylvaticus, 56 from C. glareo-lus, 2 from P. subterraneus, and 3 from M.agrestis. The total seropositivity rates were24.5% (26/106) for anti-Borrelia IgM antibodiesand 25.5% (27/106) for anti-Borrelia IgG anti-bodies. As for the rodent species, the highestprevalence of IgM and IgG antibodies was ob-served in Apodemus spp. (26.7% and 31.1%, re-spectively), followed by C. glareolus (16.1% and21.4%, respectively) (Table 2). All animals ofthe P. subterraneus and M. agrestis species wereIgM positive, with P. subterraneus showing IgGpositivity only once (1/3). Seroprevalence inother rodent species was not determined be-cause of the small number of samples. In area1 there were 14 IgM, and 15 IgG positive sam-


FIG. 2. RFLP profiles for the 5S-23S rDNA intergenic spacer of samples for rodents and positive controls. Lanes 11,12, and 13 in both panels contain positive controls for B. afzelii, B. garinii, B. burgdorferi s. s., respectively. In the left-hand panel in lanes 1, 2, 5, 6–10 there are positive samples from rodents corresponding to B. afzelii, in lane 3 there isa positive sample from a rodent with B. garinii, and lane 4 contains a co-infection with B. afzelii and B. burgdorferi s. s. In the right-hand panel in lanes 14–25 there are positive samples corresponding to B. burgdorferi s. s. Lane M1contains a PCR 20Bp Low Ladder Marker (Sigma) and lane M2 contains a Wide Range DNA Marker (Sigma).


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ples, in area 2 there were 12 IgM and 12 IgGpositive samples. The difference between areas1 and 2 was not statistically significant.

DISCUSSION

Many species have been reported to serveas competent reservoirs of B. burgdorferi s. l. inEurope, in particular rodents of the generaClethrionomys and Apodemus (Matuschka et al.1992; Hu et al. 1997; Humair et al. 1999; Han-incova et al. 2003). In the present study, wefound Borrelia infection to be more common inC. glareolus than in Apodemus spp. (Zore et al.1999). The most frequently captured rodentspecies were A. flavicollis and C. glareolus, themost abundant sylvan rodents in the CzechRepublic and its neighboring states (Hanin-cova et al. 2003).

B. afzelii, B. burgdorferi s. s., and B. garinii weredetected in the present study. To our bestknowledge, this is the first report of the pres-ence of B. burgdorferi s. s. in rodents in the CzechRepublic. In our data, the majority of infectionswere caused by B. afzelii (Humair et al. 1995,1998, 1999; Hu et al. 1997; Hanincova et al.2003). A possible explanation is a reduced andshorter-lived infectivity of B. burgdorferi s. s. incomparison with B. afzelii, which is betteradapted to rodent hosts (Richter et al. 2004).However, B. burgdorferi s. s. was detected insmall rodents in the United Kingdom, Poland,Ireland, and Switzerland (Kurtenbach et al.1998b; Gray et al. 1999; Humair et al. 1999;Michalik et al. 2005). B. burgdorferi s. s. seemsto be less specialized and may be maintainedby both avian and rodent hosts (Kurtenbach etal. 1998a). We have demonstrated one case of

coinfection with B. afzelii and B. burgdorferi s. s.in A. sylvaticus (Zore et al. 1999).

The presence of Borrelia was previously stud-ied in ear biopsies and internal organs of smallmammals (spleen, heart, liver, and urinarybladder) (Humair et al. 1993; Zore et al. 1999;Hanincova et al. 2003; Christova et al. 2005). Asear biopsies may not reveal the full diversity ofthe infection, (Richter et al. 1999; Hanincova etal. 2003), we have used muscle samples, ob-serving an infection rate of 16.4% (Kurtenbach1998b; Christova et al. 2005).

Real-time PCR analysis of the recA gene(Fraser et al.1997) is a rapid method for detec-tion of B. burgdorferi s. l., with sensitivity simi-lar to that of nested PCR (Pietila et al. 2000;Wang et al. 2003). In the present study, the con-cordance between real-time PCR and RFLPanalysis was 97.6%. The melting curve analy-sis permits differentiation of B. garinii from B.afzelii and B. burgdorferi s. s., but it does notmake it possible to distinguish between B.afzelii and B. burgdorferi s. s., as the melting tem-perature difference can be as small as 1°C(Mommert et al. 2001; Wang et al. 2003; Casatiet al. 2004). The RFLP method not only can con-firm the real-time PCR results but also is ableto distinguish between B. afzelii and B. burgdor-feri s. s. (Derdáková et al. 2003).

Seropositivity of wild rodents indicates pre-vious exposure to infected ticks (Stefancikovaet al. 2004). The prevalence of anti-Borrelia an-tibodies was higher in Apodemus spp. than inC. glareolus (Aeschlimann et al. 1986; Vostal andZakovska 2003; Stefancikova et al. 2004). Thedifference may be a result of either higher in-festation of Apodemus spp. than C. glareolus byIxodes ricinus ticks (Hanincova et al. 2003;Michalik et al. 2005) or interspecies variability

KYBICOVÁ ET AL.6

TABLE 2. PRESENCE OF ANTI-BORRELIA IGM AND IGG ANTIBODIES BY RODENT SPECIES

No. ofexamined No. of IgM Prevalence No. of IgG Prevalence

Rodent animals positive (%) positive (%)

Clethrionomys glareolus 56 9 16.1 12 21.4Apodemus flavicollis 34 10 29.4 12 35.3Apodemus sylvaticus 11 2 18.2 2 18.2Pitymys subterraneus 2 2 100.0 0 0.0Microtus agrestis 3 3 100.0 1 33.3Total 106 26 24.5 27 25.5


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in the immune response (Kurtenbach et al.1998a).

In summary, we found that muscle tissuefrom approximately one-sixth of the 293 ani-mals studied was infected with B. burgdorferis.l. Most infections were caused by B. afzelii, al-though infection with B. burgdorferi s. s. wasalso quite frequent. Infection with B. burgdor-feri s. l. was more common in bank voles thanin wood mice or yellow-necked mice. Theprevalence of anti-Borrelia antibodies washigher in wood mice and yellow-necked micethan in bank voles. We conclude that smallwild rodents in the Czech Republic can serveas hosts for B. burgdorferi s. l..

ACKNOWLEDGMENTS

The authors thank M. Pejcoch for rodent cap-ture, G. Lipinova for technical assistance, andA. Nemec and J. Kybic for helpful comments.This work was supported in part by grantNR/8263-3 from the Internal Grant Agency ofthe Ministry of Health of the Czech Republic.

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Address reprint requests to:Katerina.Kybicová

National Institute of Public HealthSrobarova 48

10042 Prague 10Czech Republic

E-mail: [email protected]

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Chapter 4

Detection of Anaplasma phagocyto-philum and Borrelia burgdorferi sensulato in dogs in the Czech Republic.

Kybicova, K. Schanilec, P., Hulınska D., Uherkova, L., Kurzova, Z.,and Spejchalova, S.: “Detection of Anaplasma phagocytophilum andBorrelia burgdorferi sensu lato in dogs in the Czech Republic.” Vector-Borne and Zoonotic Diseases, December 2009;9(6), pages 655–661.

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VECTOR-BORNE AND ZOONOTIC DISEASESVolume , Number , 2008© Mary Ann Liebert, Inc.DOI: 10.1089/vbz.2008.0127

Detection of Anaplasma phagocytophilum and Borreliaburgdorferi Sensu Lato in Dogs in the Czech Republic

Katerina Kybicová,1 Pavel Schánilec,2 Dagmar Hulínská,1 Lenka Uherková,1

Zuzana Kurzová,1 and Sandra Spejchalová2

Abstract

The aim of this study is to present molecular, serologic, and clinical findings for dogs that were naturally in-fected with Anaplasma phagocytophilum or Borrelia burgdorferi sensu lato (s. l.) in the Czech Republic. This datacan provide information relevant to human infection. In total, blood samples from 296 dogs and 118 engorgedticks were examined. Samples were tested for A. phagocytophilum using polymerase chain reaction (PCR) am-plification, nested PCR, and direct sequencing of the 16S rDNA, and for B. burgdorferi s. l. using PCR amplifi-cation of the 16S rDNA and restriction fragment length polymorphism analysis of the 5S-23S rDNA intergenicspacer. In addition, blood samples were screened for antibodies to these bacteria. Ten (3.4%) dogs were PCR-positive for A. phagocytophilum. Morulae of A. phagocytophilum in granulocytes were found in two of these dogs.Nine of the PCR-positive dogs had clinical signs related to anaplasmosis. Statistically significant differences inthe PCR detection rates were found between breeds and between symptomatic and asymptomatic dogs. In-fection with Borrelia garinii was detected by PCR in a dog with meningoencephalitis. DNA of A. phagocytophilumand B. burgdorferi s. l. (B. garinii or Borrelia afzelii) was detected in 8.5% and 6.8% of ticks, respectively. Im-munoglobulin (Ig) G seropositivity to A. phagocytophilum was 26%. Significant differences were found with re-spect to breed and gender. IgM and IgG antibodies to B. burgdorferi s. l. were detected in 2.4% and 10.3% ofdogs, respectively. Our findings suggest that the exposure to B. burgdorferi s. l. exists in dogs in the Czech Re-public, and exposure to A. phagocytophilum is common. Key Words: Anaplasma—Arbovirus(es)—Borrelia—Di-agnostics—Ixodes—Lyme disease—Tick(s). Vector Borne Zoonotic Dis. 0, 000–000.

1

Introduction

DOGS ARE IMPORTANT domestic hosts of Anaplasma phago-cytophilum and Borrelia burgdorferi sensu lato (s. l.). Data

on vector-borne infections in dogs can provide important in-formation for the potential of human infection in a particu-lar geographic location (Duncan et al. 2005). Both A. phago-cytophilum and B. burgdorferi s. l. are transmitted by ticks ofthe genus Ixodes (Parola and Raoult 2001).

A. phagocytophilum is a Gram-negative obligate intercellu-lar rickettsial bacterium found in neutrophil granulocytes(Parola and Raoult 2001). A. phagocytophilum is known tocause granulocytic anaplasmosis in humans and domesticanimals such as dogs, horses, cattle, sheep, goats, llamas, andcats (Engvall et al. 1996, Barlough et al. 1997, Bjöersdorff etal. 1999, Engvall et al. 2002, Skotarczak 2003, Lester et al.2005, Poitout et al. 2005, Lillini et al. 2006). Anaplasmosis

signs include fever, fatigue, inappetence, lethargy, lameness,and gastrointestinal and central nervous system signs (Rik-ihisa et al. 1991, Greig et al. 1996, Egenvall et al. 1997, Eng-vall et al. 2002). The related hematologic and biochemical ab-normalities are anemia, thrombocytopenia, lymphopenia,and elevated serum alkaline phosphatase activity (Greig etal. 1996, Goldman et al. 1998). Epidemiologic studies of A.phagocytophilum in dogs using polymerase chain reaction(PCR) and serologic methods are available in the literaturefor various countries. Only case reports are available in theCzech Republic. Two cases of granulocytic anaplasmosis indogs in the Czech Republic were described by Huml et al.(1996), one case by Melter et al. (2007), and four cases by Spe-jchalova et al. (2008). A. phagocytophilum was identified alsoin horses and cows by Hulinská et al. (2004).

Lyme disease (borreliosis) is a zoonotic, tick-borne diseasecaused by a spirochete B. burgdorferi s. l., which actively mi-

1National Institute of Public Health, National Reference Laboratory for Lyme Borreliosis, Prague, Czech Republic.2Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic.

Chapter 4: Detection of A. phagocytophilum and B. burgdorferi in dogs

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grates in body tissues. The clinical form of borreliosis occursin humans and domestic animals, especially dogs, horses,and cattle (Burgess et al. 1987, Greene et al. 1991, Cohen etal. 1992). Canine borreliosis most commonly affects the limbjoints (Skotarczak and Wodecka 2003, Skotarczak et al. 2005),with clinical manifestations such as arthritis and arthralgia(Jacobson et al. 1996). Other associated signs are malaise,fever, inappetence, fatigue, and lameness. Even though thesigns are the same as in humans, they are more difficult todetect in dogs and develop in relatively few of them (Levyand Magnarelli 1992). Serologic reactivity to B. burgdorferi s.l. in dogs was analyzed in the Czech Republic (Pejchalová etal. 2006). B. burgdorferi s. l. infection prevalence in questingticks from Czech Republic were found by Pejchalová et al.(2007) to be 12.1%. A. phagocytophilum prevalence in ticks(14.7%) was suggested by Sikutová et al. (2007); however,less specific primers EHR521/747 were used.

Data on the prevalence of infection with A. phagocy-tophilum and B. burgdorferi s. l. in dogs in the Czech Repub-lic is lacking, except for the above-mentioned serologicstudy. In the present study, we attempt to gather molecular,serologic, and clinical findings for dogs that were naturallyinfected with A. phagocytophilum or B. burgdorferi s. l. in theCzech Republic.

Material and Methods

Study sites and animals

All dogs examined in the Faculty of Veterinary Medicine,University of Veterinary and Pharmaceutical Sciences, Brno,Czech Republic and in the Veterinary Clinic in Jablonec nadNisou, Czech Republic between November 2005 and Octo-ber 2007 were included in the study. We also included dogsof the clinic students and employees and 39 hunting dogsexamined for a different study. The 292 dogs came from var-ious locations in the Czech Republic, principally from South(202) and North Moravia (32) and from North Bohemia (62).None had traveled outside of the country during the 6months prior to presentation. Data on the age, gender, breed,geographical origin, health status, and purpose (workingdog or pet dog) were recorded.

The dogs were divided into two groups. Group A consistedof dogs showing clinical signs attributable to infection with A.phagocytophilum or B. burgdorferi s. l. The inclusion criteria forgroup A was the presence of any the following signs: apathy,fever, lameness, lethargy, inappetence, or gastrointestinal andcentral nervous system disorders. Dogs of group B werehealthy or with a diagnosis or signs not attributable to thestudied infections such as trauma or epilepsy. Samples fromdogs were collected on the day of presentation to the clinic.Blood was drawn from the jugular or cephalic vein. Buffy-coatblood smears were made and immediately stained by theGiemsa method. Ethylenediaminetetraacetic acid-anticoagu-lated whole blood and serum samples were taken from eachdog. Attached engorged ticks were removed using forceps,identified by species and life stage, and stored individually inmicrocentrifuge tubes at 2 to 8°C prior to DNA extraction.

DNA extraction

Total DNA was isolated from blood samples with a HighPure PCR Template Preparation Kit (Roche, Mannheim, Ger-

many): 200 L of blood was transferred to a tube containing200 L of lysis buffer and 40 L of proteinase K, mixed, lysedat 56°C for 1 h, and continued according to manufacturer’sprotocol. Purified DNA was stored at 20°C before using itas a template for PCR amplification.

Ticks were washed in phosphate-buffered saline solutionbefore DNA extraction using a DNeasy Tissue Kit (Qiagen,Hilden, Germany). Ticks were transferred to a tube contain-ing 180 L of lysis buffer and 20 L of proteinase K, crushedwith a sterile scalpel, mixed, and incubated at 56°C for atleast 2 h.

PCR amplification

All samples were analyzed by standard PCR with the Ehr521-790 primer set (for Anaplasma) (Pancholi et al. 1995) andthe LD primer set (for Borrelia) (Marconi and Garon 1992),both targeting the 16S rDNA. PCR amplification was per-formed in a Peltier Cycler (MJ Research, Waltham, MA,USA). The reaction mixture consisted of 2.5 L of DNA ex-tract as a template and 1 M solution of each primer(Biotech, Czech Republic) in a total volume of 25 L HotStart Master Mix (Qiagen, Hilden, Germany). The DNA wasamplified with the Ehr 521–790 primer set as follows: aninitial 15-min denaturation at 95°C and then 40 cycles of 45sec at 95°C, 45 sec at 60°C, and 45 sec at 72°C. A final ex-tension was done for 7 min at 72°C. Cycling conditions forthe LD primer set were described previously (Kybicová etal. 2008). The PCR products were separated by elec-trophoresis in 1% agarose gel and stained with ethidiumbromide. Previously extracted DNA of B. garinii strain310M was used as a positive control. Purified water servedas a negative control.

Nested PCR for Anaplasma detection

The samples found PCR positive for Anaplasma wereretested by nested PCR with two sets of primers targetingalso the 16S rDNA gene (ge3a, ge10r and ge9f, ge2) (Mas-sung et al. 1998). It has been demonstrated that nested PCRwith these primers and standard PCR with the Ehr 521–790primers have the same detection limits (Massung and Slater2003). The primary reaction mixture used 2.5 L of DNA ex-tract and 0.5 M solution of each primer in a total volumeof 25 L Hot Start Master Mix. Cycling conditions involvedan initial 15 min denaturation at 95°C, 40 cycles of 30 sec at94°C, 30 sec at 55°C, and 60 sec at 72°C and a final extensionof 5 min at 72°C. The reaction mixture for the nested ampli-fications used 0.5 L of the primary PCR product as the tem-plate and 0.2 M solution of each primer in a total volumeof 25 L. The nested cycling conditions were the same asthose for the primary amplification, except that only 30 cy-cles were run. DNA of A. phagocytophilum of an infected dogwas used as a positive control.

DNA sequencing

To prove that the positive PCR results were truly due toA. phagocytophilum, all positive PCR products from nestedPCR amplification were directly analyzed on a CEQ 2000XLsequencer (Beckman Coulter, Buckinghamshire, UK). Size oftargeted DNA sequence was 497 bp without primer-anneal-ing areas.

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Restriction fragment length polymorphism analysis forBorrelia detection

The Borelia-positive DNA samples were tested by restric-tion fragment length polymorphism (RFLP) analysis with 5S(rrfA)-23S (rrlB) rDNA intergenic spacer primers (222–255bp) (Derdakova et al. 2003) using the same procedure as inKybicová et al. (2008).

Serology, indirect immunofluorescence assay, andenzyme-linked immunosorbent assay

Indirect immunofluorescence assay (IFA) for the detectionof canine immunoglobulin (Ig) G antibodies against A. phago-cytophilum (Fuller Laboratories, Fullerton, CA) was used ac-cording to the manufacturer’s instructions. Examination ofthe slides was performed using a fluorescence microscope at400-fold magnification. The fluorescence intensity at a dilu-tion of 1:640 was used as the cutoff level. A positive reactionwas manifested by apple green fluorescence of inclusionbodies (morulae).

The sera were also examined by an enzyme-linked im-munosorbent assay (ELISA) (Test-line, Brno, Czech Repub-lic) for detection of specific antibodies against B. burgdorferis. l. in dogs in the IgG and IgM class (against antigens: OspA,OspC, p41, p100) according to the manufacturer’s instruc-tions, with serum dilution 1:400.

Buffy coat smears

Buffy coat smears were stained with Giemsa and observedunder 1000-fold magnification. For each Anaplasma-positivedog found by PCR, one buffy coat smear was examined forthe presence of morulae.

Statistical analysis

All statistical tests were performed with respect to breed,age group, purpose (working dog or family dog), gender,and group A/B (symptomatic/asymptomatic). For statisti-cal analysis, the dogs were divided into five age groups (1,2–4, 5–7, 8–10, 11 years) according to their age after the on-set of clinical signs. Breeds were separated according to FCIgroups (Federation cynologique internationale, www.fci.be).To obtain a sufficiently large sample in each group for sta-tistical analysis, cross-breeds were considered jointly withFCI group V, and also group VI with group VII, and groupIX with group X. The Fisher exact test was used to comparePCR, IFA, and ELISA results. The chi-square test was usedfor analysis of differences between antibody titers. Multi-variate analysis using logistic regression was used for anal-ysis of PCR and IFA results with respect to all parameterstogether except breeds, because of a limited number of sam-ples. Values of p 0.05 were considered significant, andodds ratios and 95% confidence intervals are reported. Cal-culations were realized in the Stata program (StataCorp, Col-lege Station, TX).

Results

Dogs

The study group included 296 dogs, 131 females and 165males, mean age 6.5 years, age range 2 months to 16 years.

Their distribution by age, gender, breed, and purpose isshown in Table 1. The group was divided into two sub-groups: group A, 141 symptomatic dogs, and group B, 155asymptomatic dogs. Most frequent hematologic abnormali-ties in group A (symptomatic) were anemia, thrombocy-topenia, and leukocytosis. In group B, 55 dogs were clini-cally healthy and 100 had a neurologic (epilepsy) oroncologic diagnosis, unrelated to infection. None of the dogsof group B showed hematologic or biochemical abnormali-ties.

Molecular detection of A. phagocytophilum

DNA of A. phagocytophilum was detected by PCR in 10(3.4%) of 296 dogs. Of these dogs, nine belonged to group A(9/141, 6.4%) and one to group B (1/155, 0.6%) (Table 2); thedifference between groups was statistically significant (oddsratio 11.8, confidence interval 1.44-96.9, p 0.02 by logisticregression). Six infected dogs were terriers and four were re-trievers (FCI groups III and VIII), with the difference be-tween breeds being significant (p 0.001). Eight of the 10PCR-positive dogs were diagnosed between April and July,one dog was diagnosed in August, and one in September.

Clinical sings and diagnosis of the nine PCR positive dogsin group A were fever (3), apathy (2), inappetence (1), py-ometra (1), gastroenteritis (2), and seizure (2). Hematologicabnormalities included anemia (4), trombocytopenia (2),leukocytosis (2), neutropenia (2), neutrofilia (3), lymphope-nia (2), monocytosis (1), eosinopenia (1), and leukopenia (1).Biochemical abnormalities such as elevated alkaline phos-phatase (3) or alanine aminotransferase (1), hyperbilirubine-

ANAPLASMA PHAGOCYTOPHILUM AND BORRELIA BURGDORFERI IN DOG 3

TABLE 1. GENDER, AGE, BREED, AND PURPOSE

OF EXAMINED DOGS

Group A Group B Total(n 141) (n 155) (n 296)

GenderFemale (intact) 50 50 100Female (spayed) 15 16 31Male (intact) 71 86 157Male (castrated) 5 3 8

Age (y)1 15 16 312–4 36 30 665–7 37 43 808–10 35 39 7411 18 27 45

BreedFCI I 11 9 20FCI II 19 21 40FCI III 22 11 33FCI IV 10 17 27FCI V cross-breeds 22 25 47FCI VI FCI VII 6 29 35FCI VIII 39 32 71FCI IX FCI X 12 11 23

PurposeWorking dog 71 99 170Family dog (pet) 70 56 126

Group A is symptomatic dogs and group B is asymptomatic dogs.FCI, Federation cynologique internationale.


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mia (1), hypoproteinemia (1), hyperglycemia (1) were ob-served.

Sequencing of the 10 positive PCR products revealed thegene sequence of A. phagocytophilum. The sequences of theisolates were submitted to the NCBI database with the Gen-Bank accession numbers: EU847526 to EU847535.

Molecular detection of B. burgdorferi s. l.

DNA of B. burgdorferi s. l. was only found in one dog ofgroup A, an 8-year-old labrador retriever. The dog was di-agnosed with lymphocytic meningoencephalitis in April2006. We found no biochemical and hematologic abnormal-ities in blood, except for a slight anemia. Pleocytosis withsmall and activated lymphocytes was detected in the cere-brospinal fluid. Using RFLP, the agent was identified as Bor-relia garinii.

Ticks

A total of 118 engorged I. ricinus adult ticks (106 females,12 males) were collected from 67 dogs of groups A and B (42and 76 ticks, respectively) and individually screened for thepresence of DNAs of B. burgdorferi s. l. and A. phagocy-

tophilum. Ten ticks (8.5%), all females, were infected with A.phagocytophilum. Three of these 10 positive ticks were col-lected from two Anaplasma-positive dogs. Borrelial DNA wasfound in eight ticks (6.8%), seven females and one male. Allinfected ticks originated from Borrelia-negative dogs. B.garinii was detected in five ticks and Borrelia afzelii in threeticks, using RFLP.

Serologic findings

Using IFA, IgG antibodies to A. phagocytophilum were de-tected in 77 dogs, which corresponds to an overall sero-prevalence rate of 25.9%. There was no significant differencebetween groups A and B with 39 (27.5%) and 38 (24.7%)seropositive dogs, respectively. Similarly, there was no sig-nificant difference between groups A and B with respect toantibody titers. Very high antibody titers (1280) were sim-ilarly frequent in both groups, found in 17 of 141 group Adogs and in 17 of 155 group B dogs. Five of the 10 PCR-pos-itive dogs were seropositive, with antibody titers of 1:2560(n 1), 1:1280 (n 2), and 1:640 (n 2). There was a statis-tically significant difference in seropositivity between breeds(p 0.002). The highest seropositivity rates were found inFCI groups II and VIII; males showed higher seropositivity

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TABLE 2. PCR, IFA, AND ELISA POSITIVITY RESULTS WITH RESPECT TO THE PRESENCE

OF SIGNS, GENDER, AGE, BREED, AND DOG PURPOSE

Anaplasmaphagocytophilum Borrelia burgdorferi sensu lato

No. of No. of No. of No. of No. ofPCR- IFA PCR- ELISA ELISA

positive (IgG)-positive positive (IgM)-positive (IgG)-positivedogs dogs dogs dogs dogs

Group A (symptomatic) 9 39 1 4 13Group B (asymptomatic) 1 38 0 3 17Gender

Female (intact) 4 16 0 1 12Female (spayed) 0 5 0 0 3Male (intact) 6 52 1 6 15Male (castrated) 0 4 0 0 0

Age (y)1 1 5 0 0 12–4 5 12 0 5 65–7 3 25 0 1 118–10 1 22 1 1 911 0 13 0 0 3

BreedFCI I 0 3 0 0 1FCI II 0 14 0 0 4FCI III 6 5 0 1 2FCI IV 0 4 0 1 2FCI V cross-breeds 0 13 0 1 5FCI VI FCI VII 0 10 0 0 4FCI VIII 4 28 1 3 12FCI IX FCI X 0 0 0 1 0

PurposeWorking dog 8 50 1 4 18Family dog (pet) 2 27 0 3 12

Total 10 77 1 7 30

PCR, polymerase chain reaction; IFA, immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay; FCI, FCI, Federationcynologique internationale.


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rates than females (odds ratio 2.8, confidence interval 1.52-5.13, p 0.001 by logistic regression, Table 2).

Using ELISA, IgM and IgG antibodies to B. burgdorferi s.l. were detected in 7 and 30 dogs, respectively (Table 2). Theone PCR-positive dog was found negative in both IgM andIgG. Thus the overall seroprevalence rates were 2.4% and10.3%, respectively. There was no significant difference inseroprevalence between groups A and B or with respect toother studied parameters. The number of dogs with IgG an-tibodies to both A. phagocytophilum and B. burgdorferi s. l. was13 (4.4%).

Buffy coat smears

By examination of buffy coats, morulae of A. phagocy-tophilum (Fig. 1) were detected in peripheral blood neutrophilgranulocytes of 2 of the 10 PCR-positive dogs of group A,mostly at a rate of one morula per infected neutrophil.

Discussion

The seropositivity rates in the examined dogs indicate nat-ural exposure to A. phagocytophilum and B. burgdorferi s. l. Wefound a 25.9% positivity of IgG antibodies against A. phago-cytophilum. For comparison, the reported canine seropreva-lence rates were 17.7% for granulocytic Ehrlichia in Sweden(Egenvall et al. 2000b) and 7.5% for A. phagocytophilum inSwitzerland (Pusterla et al. 1998). In Germany, antibodies toA. phagocytophilum were found in 43.2% of examined dogs(Jensen et al. 2007), in Israel in 9% (Levi et al. 2006), and inthe United States, the canine seroprevalence rates were be-tween 9.4% (Magnarelli et al. 1997) and 29% (Beall et al. 2008).The canine seroprevalence was reported to be 11.5% and15.5% in Spain (Solano-Gallego et al. 2006, Amusategui et al.2008) and 34.4% in Italy (Torina and Caracappa 2006). Allstudies used IFA, except Beall et al. (2008), who used ELISA.The above differences may have resulted from the use of dif-

ferent selection criteria for examined dogs. In the presentstudy, no difference in seropositivity was found betweensymptomatic (27.5%) and asymptomatic (24.5%) dogs. Sim-ilar findings have been reported in Germany and the UnitedStates (Jensen et al. 2007, Beall et al. 2008). In our study,seropositivity in dogs without clinical signs can be explainedby persistent antibodies as reported in chronically infectedanimals (Egenvall et al. 2000a). Seropositivity in manyhealthy dogs indicates that antibodies to A. phagocytophilumin dogs can persist (Madigan et al. 1990, Magnarelli et al.1997, Engvall et al. 2002).

In the present study, DNA of A. phagocytophilum was de-tected in 3.4% (10/296) of dogs. Similar PCR positivity rates(i.e., 6.3%, 5.5%, and 9.5%) have been reported in Germany(Jensen et al. 2007), Italy (Torina and Caracappa 2006), andthe United States (Beall et al. 2008), respectively. One out of155 healthy dogs was PCR positive for A. phagocytophilum,which is similar to the study of Beall et al. (2008) in the UnitedStates (7/222 healthy dogs). Morulae in peripheral bloodgranulocytes were observed in only two of 10 PCR positivedogs, which corresponds to the findings of Jensen et al. (2007)(2/6). The inclusion bodies produced by A. phagocytophilumappear in granulocytes and can be demonstrated by mi-croscopy only at the peak of the acute infection, which usu-ally lasts only a few days (Engvall et al. 2002).

We found 2.4% IgM and 10.3% IgG canine seropositivityrates for B. burgdorferi s. l. Serologic detection of B. burgdor-feri s. l. in dogs in the Czech Republic performed in 2006 (Pe-jchalova et al. 2006) showed a seroprevalence rate of 6.5%.The reported figures vary widely from 0.6% and 6.9% inSpain (Solano-Gallego et al. 2006, Amusategui et al. 2008*),3.9% in Sweden (Egenvall et al. 2000b*), and 1.85% in Canada(Gary et al. 2006) to 11% in the United States (Beall et al.2008), approximately 20% in France, the United Kingdom,and Denmark (Doby et al. 1988*, May et al. 1991, Hansenand Dietz 1989*), 40.2% in Poland (Skotarczak et al. 2005),


FIG. 1. Morulae of A. phagocytophilum in neutrophil granulocytes of a dog.

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and 50% in Slovakia (Stefanciková et al. 1998). Most of thestudies were based on ELISA, except for those marked by anasterisk (*), which were based on IFA. As for A. phagocy-tophilum, the differences may have resulted from differentdog selection criteria. Differences in canine seroprevalencerates for tick-borne diseases can arise from variability in tickdensities or proportion of infected ticks.

In the present study, we detected DNA of B. burgdorferi s.l. in peripheral blood of only one PCR-positive dog. This lowyield can be explained by the transient nature of the pres-ence of the spirochetes in the blood (Straubinger et al. 1997,Chang et al. 2001). Using PCR, borrelial DNA can only bedetected in blood in the early stage of infection (Skotarczakand Wodecka 2003). Once the microorganism is dissemi-nated in the body, a variety of organs can be affected, espe-cially the skin, joints, heart, and central and peripheral ner-vous systems (Straubinger et al. 1997).

We found DNA of A. phagocytophilum and B. burgdorferi s.l. in 8.5% and 6.8% of ticks, respectively. Anaplasma-positiveticks on positive dogs might have become positive duringfeeding. Similar findings have been reported in Poland(Zygner et al. 2008). We found no coinfection of Anaplasmaand Borrelia in dogs or in ticks.

In conclusion, our findings suggest that exposure to A.phagocytophilum is common in dogs in the Czech Republic.This study demonstrated that symptomatic dogs from Northand South Moravia and from North Bohemia had higherchance to be infected with A. phagocytophilum that asympto-matic dogs. On the other hand, no correlation was found be-tween clinical signs of anaplasmosis or borreliosis and pos-itive antibody titers to A. phagocytophilum or B. burgdorferi s.l., respectively.

Acknowledgments

The authors thank M. Maly[acute] for statistical analysis, M.Musílek for sequencing, A. Nemec and J. Kybic for helpfulcomments, and E. Kodytková for reviewing the manuscript.

Disclosure Statement

No competing financial interests exist.

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Address reprint requests to:Katerina Kybicová

National Institute of Public HealthSrobarova 48

10042 Prague 10, Czech Republic

E-mail: [email protected].


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Chapter 5

Clinical and Diagnostic Features inThree Dogs Naturally Infected withBorrelia spp.

Schanilec, P., Kybicova, K., Agudelo, C. and Treml, F.: “Clinical andDiagnostic Features in Three Dogs Naturally Infected with Borreliaspp.”, Acta Veterinaria Brno, 2010, In press.

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Chapter 5: Clinical and Diagnostic Features in Dogs

Clinical and Diagnostic Features in ThreeDogs Naturally Infected with Borrelia spp.

Pavel Schanilec1, Katerina Kybicova2, Carlos F.Agudelo1 and Frantisek Treml3

1 Clinic of Dogs and Cats Diseases, Faculty of Veterinary Medicine, University ofVeterinary and Pharmaceutical Sciences Brno, Czech Republic2 National Institute of Public Health, National Reference Laboratory of Lyme Borreliosis,Prague, Czech Republic3 Department of Infectious Diseases and Veterinary Epidemiology, Faculty of VeterinaryMedicine, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic

Abstract

The aim of this study is to present clinical and neurological signs, labora-tory abnormalities, serologic and/or molecular findings in three dogs from theregion of Brno in Czech Republic. All dogs were naturally infected with Borreliaburgdorferi sensu lato. The evidence of borrelial infection was proved by serialblood sampling for IgM and IgG anti-borrelial antibodies or plasma PCR. Thedogs manifested corresponding clinical signs and one or more of the followingcriteria were fulfilled: (1) 4-fold or greater increase or decrease in B. burgdorferis. l. IgM or IgG antibodies serial titres in acute and convalescent stage of infec-tion, (2) a shift from positive IgM to IgG antibodies titres, (3) decrease of IgMwith concurrent increase of IgG antibodies in serial titres, (4) detection of borrelialDNA by PCR. Other possible tick-borne infections were excluded. All three dogsshowed neurological signs (two of them meningoencephalomyelitis, one seizureconnected with progressive renal disease). Their history, clinical signs, diagnos-tic procedures and treatment are described. Two of dogs died and only one withmeningoencephalomyelitis survived. This article showed that borrelial infectionmust be considered, not only in cases with febrile and orthopaedic signs but alsoin many other clinical syndromes.

Keywords: borrelial infection, meningoencephalomyelitis, PCR, seizure,renal disease

Introduction

Borreliosis is a zoonotic tick-borne disease caused by a Gram-negative spiro-chete Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) which includes complex of

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genospecies, three of which are considered to be pathogenic in dogs: B. burgdor-feri sensu stricto (B. burgdorferi s. s.), Borrelia garinii (B. garinii) and Borrelia afzelii(B. afzelii) (Hovius et al. 1999a; Hovius 2005; Greene and Straubinger 2006). Themain European vector is the tick Ixodes ricinus. Many species of mammals andbirds were recognized as a reservoir of B. burgdorferi s. l. (Gern et al. 1998; Hulin-ska et al. 2002; Piesman and Gern 2004). The clinical form of borreliosis occursin human, domestic animals, especially dogs, horses and cattle (Burges et al.1987; Greene et al. 1991; Cohen et al. 1992; Skotarczak et al. 2005; Kybicova etal. 2009). The close contact between dogs and humans, the common environ-ment and the fact that borreliosis is emerging even in the cities can be consid-ered as indicators for the outbreaks detection (Stefancıkova et al 1998; Goossenset al. 2001). Clinical signs depend on the individual host response and varywidely developing in relatively few individuals (Levy and Dreesen 1992; Levyand Magnarelli 1992). Both in humans and dogs, the condition can cause derma-tological, musculoskeletal, neurological, renal and cardiovascular signs (Greeneet al. 1991; Azuma et al. 1993; Straubinger et al. 1997; Straubinger 2000; Straub-inger et al. 2000; Skotarczak and Wodecka 2003; Skotarczak et al. 2005; Greeneand Straubinger 2006). The diagnosis is based on the combination of several fac-tors: epidemiology-epizootological information, clinical signs, serological testsand PCR (Straubinger 2000; Straubinger et al. 2000; Skotarczak and Wodecka2003; Skotarczak et al. 2005; Pejchalova et al. 2006; Kybicova et al. 2009). The sero-prevalence for B. burgdorferi s. l. in dogs was assessed in many European coun-tries, e.g. in Slovakia (Stefancikova et al. 1998), Poland (Skotarczak et al. 2005),and Sweden (Egenvall et al. 2000). Specific antibodies to B. burgdorferi s. l. weredetected in 6.5% of the population in the Czech Republic and the seroprevalenceranged between 0.0% and 28.6% (Pejchalova et al. 2006). In a recent study alsoin the Czech Republic the seropositivity of ELISA in IgM and IgG was 2.4% and10.3%, respectively (Kybicova et al. 2009). In the Czech Republic DNA of B. gariniiwas detected in a blood sample of a dog only in one case (Kybicova et al. 2009).The aim of this work is to present three cases of dog patients with a variety ofclinical signs and positive detection of borrelial infection by serological tests orPCR.

Materials and Methods

All patients were presented to the emergency service of the Clinic of Dog andCat Diseases of the University of Veterinary and Pharmaceutical Sciences Brno(CDCD-VFU) and hospitalized there. All dogs were regularly vaccinated withpolyvalent vaccines and dewormed but none of them was vaccinated againstborreliosis. All dogs suffered from severe tick infestation and all dogs came from

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Brno/South Moravia, an endemic area of tick-borne encephalitis and borreliosis(Klimes et al. 2001; Pejchalova et al. 2006).

Laboratory criteria for assessment of borrelial diagnosis

Patients were diagnosed with borrelial infection if they manifested correspond-ing clinical signs and one or more of the following criteria were fulfilled: (1) 4-foldor greater increase or decrease in B. burgdorferi s. l. IgM or IgG antibodies serialtitres in acute and convalescent stage of infection, (2) a shift from positive IgMto IgG antibodies titres, (3) decrease of IgM with concurrent increase of IgG anti-bodies in serial titres, (4) detection of borrelial DNA by PCR.

CSF examination, haematology and serum biochemistry

Complete CSF analysis was made within 30 minutes from collection (Bagley2003; Bagley and Bohn 2003; Bohn and Bagley 2003). Cell counts were per-formed using the Fuchs-Rosenthal counting chamber. Blood and cerebrospinalfluid (CSF) smears were stained with Hemacolor (Merck KGaA, Darmstadt, Ger-many). Routine haematological examination and selected biochemical values wasperformed.

Serology and PCR

The sera were examined by the enzyme immunoassay (EIA) for detection of bor-relial IgG and IgM antibodies (Test-line, Brno, Czech Republic), by EIA for thedetection of tick-borne encephalitis virus (TBEV) Ig antibodies (Test-line, Brno,Czech Republic) and by A. phagocytophilum immunofluorescence (IFA) canine IgGantibody test (Fuller Laboratories, Fullerton, CA, USA), all according to manu-facturer’s instructions. The DNA samples were isolated from blood and CSF andwere analyzed by standard PCR (Kybicova et al. 2009). Positive DNA sampleswere retested by restriction fragment length polymorphism (RFLP) analysis with5S (rrfA)-23S (rrlB) rDNA intergenic spacer primers (Derdakova et al. 2003) as in(Kybicova et al. 2008).

Case studies

Case 1 (5–7/2002)

A five-month-old dog, 13 kg, intact male Nova Scotia Duck Tolling Retriever waspresented with a 2 weeks history of inability to move, general hyperesthesia and

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spasticity of the muscles of the head and neck. The previous veterinarian col-lected blood samples (day -14) for borreliosis (Table 2) because found the dogpyretic (3 previous days) and treated him with high doses of co-amoxicillin (30mg kg−1 PO BID) and non-steroidal drugs. The condition of the dog improvedover the next 3 days. a week later, the dog started refusing to lie down; show-ing kyphosis, often remaining in a sitting position and having inability to makehead and neck ventroflexion. At admission (day 0), the dog showed generalizedhyperesthesia including vocalization and inability to move. The mental statusalternated from depression and stupor to hysteria, accompanied by episodes ofopisthotonus and myoclonia of both forelimbs. The cervical muscles were hyper-tonic and strong pain was elicited with mild manipulation of the neck. Cra-nial nerves examination revealed moderate decreased trigeminal (CN V) sen-sation and facial (CN VII) reactivity on the left side with absence of reactionsfor the same nerves on the right side. Spinal reflexes showed hyperreflexia inall of four limbs. The dog remained preferentially on the right lateral recum-bency. CBC (Table 1) and antinuclear antibody test (ANA test) were sampled.Standard fluid support and treatment with co-amoxicillin (25 mg kg−1 IV TID)was given, together with Diazepam (single dose 1.5 mg kg−1 IV slowly) fol-lowed by phenobarbital (2 mg kg−1 IV BID -TID) to control mental status. Duringthe first two days of hospitalization the neurological status worsened, includ-ing spastic tetraparesis, tonic seizures with mild manipulation and permanentposture in right lateral recumbency. Reactivity of cranial nerves changed: CNV and CN VII function changed to hyperreactivity on the left side with severehyporeflexia on the right side. Furthermore, direct and indirect pupilary reflexeswere decreased on the right side while normal on the left. Additional findingsincluded severe hyperesthesia in the orbital and cervical areas and bilateral ble-pharospasm. a multifocal lesion involving meninges, cortex, brainstem and cer-vical spinal cord was suspected and collection and examination of CSF was indi-cated. CSF analysis showed Pandy reaction (++) and white blood cell count of 40cells l−1. Cytology showed 60% of small lymphocytes, 13% of activated lympho-cytes, 13% of monocytes, 13% activated monocytes up to macrophages and spo-radically polymorphonuclears (PMN). Biochemistry showed proteinorhachia (TP0.81g l−1). The glucose level was normal (4.17 mmol l−1). Aerobic and anaerobiccultures of CSF were negative. These findings were compatible with lymphocyticmeningoencephalitis suggesting an infective etiology.

The clinical and neurological status slowly improved during the following 4days. On day 6, the patient revealed mild disorientation, generalized symmet-rical ataxia with hyperreflexia of all four limbs, lower neck and head positionwith moderate cervical discomfort. The general hyperesthesia and the pupilarand ocular changes persisted. The dog was able to eat and drink. Blood was

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sampled for TBE (negative) and borreliosis (Table 2). Dog was discharged andowners were instructed to provide nursing care. Treatment continued with co-amoxicillin (25 mg kg-1 PO BID) for the following 14 days and the dose ofphenobarbital tapered for the following 3 days. A week later patient’s condi-tion gradually worsened (lethargy, anorexia and hyperthesia) and the owner byown initiative gave him prednisone (1 mg kg−1 PO for three days) and by day21 visited CDCD-VFU facilities. The dog was lethargic, febrile (40.3 C), tachy-cardic (150 beats min−1), severely hyperesthetic with vision deficits on the right.Owner declined hospitalization. Treatment was changed to azithromycin (20 mgkg−1 PO SID). From Day 25, the antibiotic was substituted with cefotaximum(85 mg kg−1 IV TID). Serum samples were submitted for antiborrelial antibodies(Table 2). Up to Day 34, according to the owner’s information, the dog seemedbetter. On day 37, the patient was re-hospitalized due to the relapse of the clini-cal signs (hyperesthesia, hyperextension of the pelvic limbs and cervical discom-fort). On day 38, CBC and biochemistry were taken (Table 1). Serum samplesfor borreliosis (Table 2) and anaplasmosis also were submitted. However, duringthe following day the patient’s condition deteriorated and the owners requestedeuthanasia. Postmortem CSF analysis showed Pandy reaction (++) and whiteblood cell count of 110 cells l−1. Cytology showed 25% of small lymphocytes,43% of activated lymphocytes up to plasma cells, 21% of PMN, 11% monocytesup to macrophages. CSF biochemistry showed proteinorhachia (0.97 g l−1) anddecreased glycorhachia (1.95 mmol l−1, reference range: 2.7–4.2 mmol l−1). Cul-tures and borrelial PCR were also negative.

At necropsy, macroscopically there was thickening of dura mater on the leftcerebral hemisphere and multifocally through the spinal cord. Histopathologyshowed chronic meningoencephalomyelitis. Multiple malatic lesions were seenin the brain and spinal cord with atrophy of ventral spinal horns and their corre-sponding spinal roots. Additional findings were acute hepatitis, chronic diffusegranulomatous and necrotic myocarditis.

Case 2 (7–9/2003)

A five-year-old, 34 kg, intact male Labrador Retriever was evaluated due toa history of two days of generalized short episodes of tonic-clonic seizures withunconsciousness. Initial clinical examination (day 0) showed mild lethargy andslight generalized ataxia. CBC and biochemistry were taken (Table 1). Serologyfor borreliosis was positive (IgM 2.32 - positive; IgG 0.94 - dubious). The nextday the dog was hospitalized. Ultrasound examination showed bilateral hypere-chogenity of the kidneys suggesting renal disease (RD), glomerulonephritis (GN)or pyelonephritis (PN). Urine was collected by cystocentesis and showed alka-

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Table 1: Results of haematology and biochemistry

Parameter Reference range /

Units

Case 1

day 1

Case 1

day 38

Case 2

day 0

Case 2

day 44

Case 3

day 1

Case 3

day 10ERY 5.5-8.5 / 1012 l-1 4.08 3.61 4.5 4.71 5.18 5.68HB 120-180 / g l-1 100 83 107 119 115 124HT 0.37-0.55 / l l-1 0.28 0.24 0.33 0.34 0.35 0.39TRC 200-500 / 109 l-1 232 186 - - 227 365LEU 6-17 / 109 l-1 36.2 23.9 8.6 9.6 8.7 7.1Bands 0-1 / 109 l-1 1.09 1.43 0.34 0.1 0.17 0PMN 3-11.4 / 109 l-1 31.8 19.8 5.59 7.68 5.48 2.13 LY 1-4.8 / 109 l-1 2.53 2.39 2.15 0.58 2.09 4.12 MONO 0-1.3 / 109 l-1 0.36 0.24 0 0 0.78 0.71 EO 0-0.75 / 109 l-1 0.6 3.61 0.52 0.1 0.17 0.14 TP 55-75 / g l-1 - 51.2 67.8 57.7 63.1 -ALB 23-34 / g l-1 - 21.6 30 28 30.7 -GLO 30 – 47 / g l-1 - 29.6 37.8 29.7 32.4 -A/G 0.7-1.1 - 0.72 0.79 0.94 0.95 -TB 0-7 / μmol l-1 - 5.6 - 1.8 - -CRE 30-120 / μmol l-1 - 118.6 503 859.1 65.0 -U 2.5-7 / mmol l-1 - 9.18 39.8 82.4 3.37 -GLU 3.3-7 mmol l-1 - 5.17 4.85 8.04 NT -ALP 0.1-1.5 / μkat l-1 - 3.14 0.3 0.5 0.60 -ALT ≤ 1 / μkat l-1 - 2.4 1.6 2.3 0.74 -Ca 2-3 / mmol l-1 - 2.5 1.64 1.31 2.14 -P 0.9-2.5 / mmol l-1 - 2.01 3.36 5.04 1.6 -Na 135-155 / mmol l-1 - 151 152 145 143.0 -K 3.6-5.6 / mmol l-1 - 5.3 5.39 5.28 4.24 -

Legend: ERY – erythrocytes, HB – hemoglobin, HT – hematocrit , TRC – trombo-cytes, LEU – leucocytes count, PMN – polymorphonuclears, LY – lymphocytes,MONO – monocytes, EO – eosinophils, TP – total proteins, ALB – albumin, GLO– globulin, TB – total bilirubin, CRE – creatinine, U – urea., GLU – glucose, ALP– alkaline phosphatase , ALT – alanine aminotransferase, Ca – calcium, P – phos-phorus, Na – sodium, K – potassium.

Table 2: Results of the serological examination for Borrelia sp. (Case 1)

Index of

positivity

Day -14 Day 6 Day 25 Day 34 Day 38

IgM (result) 0.34 (-) 1.54 (+) 2.26 (+) 2.20 (+) 3.87 (+)IgG (result) 0.14 (-) 0.17 (-) 0.27 (-) 0.31 (-) 0.70 (-)

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Table 3: Results of the serological examination for Borrelia sp. (Case 3)

Index of

positivity

Day 2 Day 10 Day 33 Day 422

IgM (result) 0.5 (-) 0.634 (-) 1.044 (+/-) 0.517 (-)IgG (result) 0.3 (-) 0.562 (-) 0.716 (-) 0.869 (+/-)PCR B.g. (+) negative negative negative

liuria, hyperstenuria, cylindruria (granular casts), crystalluria (ammonium urate1/field), also occasional bacteriuria and mild proteinuria (+) were found. Urinaryprotein/creatinine ratio (Up/Uc ratio) was 3.6 (normal range: ? 1). The dog wastreated initially with saline 0.9%. TBE, anaplasmosis and leptospirosis (L. ictero-haemohrragiae and L. grippotyphosa) serologies were negative. On Day 4, ownerdeclined further hospitalization. The patient was discharged with doxycycline(10 mg kg−1 PO BID) initially for one week. Owner missed recommended follow-ups and came on day 44. According to him the dog was doing better exceptingfor mild exercise intolerance. CBC and biochemistry were checked (Table 1). Bor-relial antibodies were measured (IgM 1.47 - positive; IgG 3.52 - positive). Ownerdid not return for the next follow-ups and by telephonic communication after 2months he reported that the dog had died.

Case 3 (4/2006–8/2007)

An eight-year-old, 40 kg, intact male Labrador Retriever was referred with 1 dayhistory of reluctance to move and progressive ataxia, which worsened to tetra-paresis (day 0). The last 2 weeks the dog was being treated because a phlegmonon the right forelimb by the referring veterinarian a received one week unknownantibiotic treatment. The day before admission he started treating him with mar-bofloxacin. At admission (day 1) the dog seemed to be in good body condition,he had hyperemic mucous membranes, hot spot in the neck area lasting 4 days,mild antebrachial edema on the right forelimb, enlargement of the left axilarlymph node and bradycardia (60 beats min−1). Neurologically, the patient wasdepressed, tetraparetic with a lateralized generalized ataxia to the right, pleu-rothotonus to the right, and also showed signs of cervical discomfort especiallywith ventroflexion of the head. Although the dog fell down to the right, he wasable to walk few steps without help. Cranial nerves examination showed diffusehypersensitivity in the facial areas. The spinal reflexes were normal to increased.CBC and the biochemistry were taken (Table 1). An electrocardiography (ECG)revealed bradycardia, 1st degree atrioventricular block and prolonged QT inter-

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Figure 5.1: RFLP profiles for the 5S-23S rDNA intergenic spacer of samples fromdog and positive controls. Lanes 2, 3, and 4 contain positive controls for B. afzelii,B. garinii, B. burgdorferi s. s., respectively. In lane 1 there is positive sample fromdog Case 4 corresponding to B. garinii. Lanes M1 contain a Wide Range DNAMarker (Sigma) and lane M2 contains a PCR 20Bp Low Ladder Marker (Sigma).

val. CSF evaluation revealed Pandy reaction (++), and white blood cell count of99 cells/l. Cytology showed 70% of small lymphocytes, 9% of middle lympho-cytes up to plasmocytes, 20% of MONO and 1% PMN. Biochemistry showedproteinorhachia (1.32 g µl−1) and normal glucose value (3.3 mmol µl−1). Aero-bic and anaerobic cultures and also borrelial PCR were negative. Phenobarbital(2 - 4 mg kg−1 IV TID and then BID) was administered and the treatment withmarbofloxacin (2 mg kg−1 IV BID) was continued. Standard fluid support wasgiven. On day 2, ANA test, Borrelia, Anaplasma and TBE serology were negative,however the PCR from blood for borrelial DNA was positive (Figure 5.1).

On day 3, the neurological status worsened almost to stupor and the cervi-cal discomfort increased; there were also hypomotility of the tongue, severe ble-pharospasmus on the right side and intermittent neck myoclonus. Manitol wasadded to the therapy (1g kg−1 during 20 minutes, single dose). On day 4, thedog started to eat small amounts of meal but showing intention tremor and dys-metria of the head and neck, also he was able to alternate recumbency. Fromday 6, the dog was able to stand up and was able to go few steps with support.The marbofloxacin was changed to enrofloxacin (5 mg kg−1 SC BID). On day 10,

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CBC, biochemistry (Table 1), Borrelia serology (Table 3) were taken and the dogwas discharged with mild ataxia and recommended nursing care. On day 33, thedog showed only lameness on the left forelimb and both hindlimbs. Generalizedataxia was elicited after exercise. Re-examination of CBC, biochemistry (both inreference range), tests for TBE (negative) and B. burgdorferi s. l. (Table 3) weredone. The last follow-up, on day 422, the owners complained about a 4-monthhistory of faecal and urinary incontinence. Neurologic evaluation revealed symp-toms corresponding with syndrome of cauda equina. Lumbar X-rays were nor-mal. Borrelial serology and PCR were negative.

Results and Discussion

All presented cases showed historical, clinical and diagnostic findings thatstrongly indicated borrelial infection. In all dogs the clinical signs started betweenApril and July. It correlates with seasonal activities of ticks and dogs (Hovius etal. 1999b; Hovius et al. 1999c; Steere 2001; Bhide et al. 2004; Pejchalova et al. 2006;Kybicova et al. 2009). Clinical illness in infected dogs occurs 2 to 6 months aftertick exposure. In humans as well as in animals the clinical symptoms are dividedin three stages. Stage I (early localized infection) can be in humans presented aserythema migrans. This stage is not seen in dogs. Stage II (early disseminatedinfection) can affect all organ systems, and can manifest itself by musculoskeletalpains, variable neurological symptoms, cardiovascular and renal symptoms. Dur-ing this stage we can often see clinical symptoms and detect antibodies dynamics.a general malaise often precedes or combines with a lameness episode and can beaccompanied with moderate to high fever, anorexia and listlessness. Stage III (latepersistent infection) can cause chronic symptoms, recurrent arthritis and chronicneurological disorders. (Baranton 2002; Hovius 2002; Higgins 2004; Hovius 2005;Greene and Straubinger 2006).

Diagnosis borreliosis is obtained by the presence or absence of the followingfactors: 1) The presence of typical clinical symptoms; 2) exclusion of differentialdiagnosis; 3) apparent reaction to antibiotic; 4) evident contact with a tick or liv-ing in an endemic area; 5) the presence of antibodies in the blood serum. The lattercriterion has been a serious diagnostic indication, however, seropositivity deter-mined from a single sample cannot differentiate between past and present expo-sure, and cannot on its own constitute a basis for diagnosis of an active infection(Egenvall et al. 2001; Bhide et al. 2004; Skotarczak et al. 2005). ELISA is consideredto be sufficiently specific to be used separately for determining the dynamics ofthe antibody response to B. burgdorferi s. l. and can react in early stage of illness(Hovius et al. 1999b).

The timing of serologic examination is important in determination whether

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active or past infection is responsible for the seropositivity. Early serodiagnosticresults are usually negative because the immune response to Borreliae developsgradually. Antiborrelial antibodies IgM are produced 1 to 2 weeks after infection(Hovius et al. 1999b), correlate with onset of clinical illness, and remain elevatedfor 2 months (Greene and Straubinger 2006). IgG-ELISA positive titres developwithin 4 to 6 weeks (Appel et al. 1993), culminate at 3 months and last 1 to 2 yearsafter exposure (Straubinger et al. 2000; Goossens et al. 2001). Simultaneous mea-surements of IgG and IgM and paired-sample titres are recommended for detec-tion of borrelial infection. False-negative antibody tests results are rare (Greeneand Straubinger 2006) so clinical syndromes together with PCR and serial serol-ogy indicate a very likely borrelia induced disease. The clinical and laboratoryfindings of borrelial infection are described in more detail for each of the casesdue to the particular variations between subjects: In Case 1, the proof of infectionwith Borrelia spp. was based on an increase of more than 10-fold in IgM antibodiesin 52 days (Table 2). IgM increased from negative up to high positive titres. IgGwas always negative. Titres of antiborrelial antibodies were examined five times.In Case 2, antiborrelial antibodies were examined only twice, on Day 0 and 44.a shift from IgM to IgG with a decrease in IgM was detected. In Case 3, the proofof presence of borrelial infection was based on of positive PCR sera (B. garinii)and clinical, neurological, CSF and ECG findings. Sera testing for antiborrelialantibodies (four times) showed only a weak response at day 33rd (Table 3). Sincethe dog was treated two weeks before presentation, probably with antibiotics thataffected Borreliae, it is very possible that his immune response was delayed orreduced. The later PCR detection could be explained by insufficient initial antibi-otic treatment (dose, duration), repetitive proliferation of the surviving spiro-chetes or re-infection (Straubinger et al. 1997). The immune response and PCRfindings suggested that all three cases showed signs of early disseminated stageof borrelial infection. Variable neurological signs are described in dogs (Azumaet al. 1993; Azuma et al. 1994; Baranton 2002; Hovius 2002; Higgins 2004; Hovius2005; Greene and Straubinger 2006). Both dogs with meningoencephalomyelitis(Cases 1, 3) were CSF evaluated and the findings corresponded to a suspectedcentral nervous system (CNS) inflammation because of protein-cytological asso-ciation. Serial sampling of CSF in Case 1 showed increased activation of mononu-clear cells, their increased number and no signs of other bacterial presence.

CSF findings are not pathognomic only for borrelial infection (Adam 1999).For both dogs the PCR of CSF was negative. It corresponds with other findingsby many authors because of Borreliae are very easily eliminated by the immunesystem and/or by penetrating antibiotics. Different results are described in the lit-erature concerning culture and PCR detection Borreliae in nervous system (Appelet al. 1993; Hovius et al. 1999c; Chang et al. 2001; Straubinger 2000). Histological

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findings in Case 1 correspond with neurological and CSF findings.In Case 2, a neurological signs were reported. The origin of seizures remained

unclear. They can be part of nephro-encephalopathy, because of azotaemia orCNS borrelial infection cause irritation (Azuma et al. 1993; Azuma et al. 1994).Interestingly, Chang et al. 2001 described that experimentally infected dogs devel-oped mild focal meningitis, encephalitis, and perineuritis but without neurolog-ical signs. These symptoms in Case 2 could probably have been caused by silentborrelial CNS infection with a transient relapse. The progressive renal failure, themoderate to severe azotemia and the significant proteinuria were detected alongwith IgM/IgG shift (Grauer et al. 1988; Greene and Straubinger 2006; Gerber etal. 2007). a possible preponderance of Labrador and golden retrievers to renalinvolvement has been described. The same authors claim that the duration ofclinical renal illness is 24 hours to 8 weeks (Greene and Straubinger 2006).

The ECG changes and post-mortem findings in Cases 1 and 3, make us thinkthat the incidence of cardiac manifestations in borreliosis could be higher thanpreviously thought. Humans have a cardiac involvement in Lyme disease in 8-10% of adults (Nagi et al. 1996; Bateman and Sigal 2000; Nalmas et al. 2007).The natural incidence of Lyme carditis in veterinary medicine has not been yetestimated. The observed cardiovascular syndromes include presence of arrhyth-mia (variable degrees of atrioventricular block, supraventricular and ventriculararrhythmia Levy and Duray 1988; Nalmas et al. 2007), myocarditis, pericarditis,dilated cardiomyopathy and coronary artery disease (Gasser et al. 1999). Gen-erally, a positive Lyme titer and accompanying cardiac signs are present andthis was demonstrated in our patients. The overall prognosis of Lyme carditisis good, although recovery may be delayed and late the progressive effects ofinflammation and toxins can cause heart failure (Nagi et al. 1996; Gasser et al.1999; Cepelova 2008). The chronic diffuse granulomatous and necrotic myocardi-tis found in Case 1 could suggest the cystic form of the disease (Giudice et al.2003).

CBC revealed persistent mild hypochromic anaemia in two cases (Case 1, 2)and borderline anaemia in the Case 3. Our findings are similar to those men-tioned e.g. by Breitschwerdt et al. 1994, however the majority of authors did notrecorded any specific CBC changes (Greene and Straubinger 2006). Anaemia canbe caused in acute stage of bacteraemia or inflammation because of bone mar-row suppression or due to direct penetration of B.burgdorferi s. l. to bone marrow.PCR findings of Borreliae in bone marrow were described by Hovius et al. 1999c.In the Case 1, anaemia could have been caused because the dog was young andfast growing. Moderate leukocytosis observed in Case 1 is a nonspecific sign ofinflammation; it is not possible to rule out a concurrent disease that may explainthis finding.

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The moderate panhypoproteinemia in the Case 1 and elevated ALT (Cases 1,2) can be a nonspecific effect of the systemic inflammation. It could be causedby fasting, but also can be suggested as a consequence of acute hepatitis causedby Borreliae or concurrent disease. In one case acute hepatitis was verified bynecropsy. The higher prevalence of Borrelia in liver samples in symptomatic dogsis also described (Hovius et al. 1999c; Greene and Straubinger 2006). ElevatedALP in the Case 1 could be caused by the same reason but also very probablywas caused with fast growth of bones. The moderate to severe azotemia, hyper-phosphatemia and hypocalcemia in Case 2 are signs of progressive renal diseaseand were already described above.

Commonly, concurrent tick-borne diseases can occur in infested animals(Egenvall et al. 2000; Klimes et al. 2001; Higgins 2004; Kybicova et al. 2009). Seraof all three dogs were evaluated for borreliosis (see above), TBE and anaplasmo-sis with negative results, thus ruling out this possibility. The dog with signs ofRD (Case 2) was also negative for leptospirosis. Two dogs (Cases 1, 3) were alsotested for ANA antibodies with negative result. Lupus erythematosus is a dis-ease that can cause a lot of clinical signs (including neurological) and the testshould be done as a routine in patients showing systemic or organ inflammatorydiseases. Ceftriaxone, cefotaxime, azithromycin, amoxicilin and doxycycline arethought as the most effective antimicrobial drugs against Borrelia when appliedat early clinical stage of infection. In Case 1, co-amoxicilin, azithromycin and cefo-taxime were administered, but the use of corticosteroids could have been a triggerfor reactivation of a poorly controlled infection. The penetration of antibiotics toCNS also depends on the integrity of blood-brain barrier. It is probable that co-amoxicilin could be less effective in CNS. In Case 2, the owner aborted the admin-istration of doxycycline, which might therefore not have been sufficiently effec-tive. Quinolones are ineffective in treating borreliosis. There were used in Case3 because the results of PCR were known too late (a month after sampling) andthe owner declined further therapy. We suggest that supportive complementarynursing care and cage rest of patients are among the mainstays of the complextherapy of encephalitis. As showed in Case 3, it could be important for surviv-ing despite the wrong antibiotic treatment. Duration of cage rest depends on theseverity of the illness. In Cases 1 and 2, the owner finished hospitalization pre-maturely and the patients did not get adequate rest.

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Klinicke prıznaky a diagnosticke postupy u trı psuprirozene infikovanych Borrelia spp.

Cılem studie je popsat klinicke a neurologicke prıznaky, laboratornı, serologickea/nebo molekularnı nalezy u trı psu z okolı Brna (Ceska republika). Vsichnipsi byli prirozene infikovani Borrelia burgdorferi sensu lato a infekce bylapotvrzena opakovanym vysetrenım IgM a IgG boreliovych protilatek nebopomocı PCR. Psi s klinickymi prıznaky splnovali jedno nebo vıce z naslednychkriteriı: (1) ctyrnasobne nebo vyssı zvysenı nebo snızenı titru IgM nebo IgGprotilatek proti B. burgdorferi s. l. pri opakovanych vysetrenıch v akutnı neborekonvalescentnı fazi infekce, (2) zmena z pozitivnıch IgM do pozitivnıch IgGtitru, (3) soucasne snızenı IgM a zvysenı IgG protilatek, (4) nalez boreliove DNAmetodou PCR. Dalsı mozne infekce prenaseny klıstaty byly vylouceny. Vsichnitri psi vykazovali neurologicke prıznaky (dva meningoencefalitidu a jeden krecespojene s progresivnım onemocnenım ledvin). Je popsana anamneza, klinickeprıznaky, diagnosticke postupy a jejich lecba. Dva z pacientu uhynuli a jedenpacient s meningoencefalitidou prezil. Tento clanek ukazuje, ze boreliova infekcemusı byt zvazovana i v prıpadech jinych prıznaku nez jsou febrilnı stavya ortopedicke prıznaky.

Acknowledgements

The authors thank V. Valkova and J. Kybic for reviewing the manuscript.

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Stefancıkova A, Tresova G, Petko B, Skardova I, Sesztakova E 1998: ELISA comparison ofthree whole-cell antigens of Borrelia burgdorferi sensu lato in serological study of dogsfrom area of Kosice, eastern Slovakia. Ann Agric Environ Med 5: 25-30

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Chapter 6

Detection of Anaplasma phagocyto-philum in animals by real-timepolymerase chain reaction.

Hulınska, D, Langrova, K, Pejcoch, M, Pavlasek, I.: “Detectionof Anaplasma phagocytophilum in animals by real-time polymerasechain reaction.” APMIS, April–May 2004;112(4-5), pages 239–247.

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APMISSP155 P155 14-04-04 09:52:15 PDF – disk/mona / a206155/apm580

APMIS 112: 00–00, 2004 Copyright C APMIS 2004Printed in Denmark . All rights reserved

ISSN 0903-4641

Detection of Anaplasma phagocytophilum in animalsby real-time polymerase chain reaction

DAGMAR HULINSKA,1 KATERINA LANGROVA,1 MILAN PEJCOCH2 and IVAN PAVLASEK3

1National Institute of Public Health, National Reference Laboratory for Lyme Disease, Prague, CzechRepublic, 2National Institute of Public Health, National Reference Laboratory for Hantaviridae, Prague,Czech Republic, 3Veterinary Service Laboratory, National Veterinary Institute, Prague, Czech Republic

Hulınska D, Langrova K, Pejcoch M, Pavlasek I. Detection of Anaplasma phagocytophilum in animalsby real-time polymerase chain reaction. APMIS 2004;112:00–00.

The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animalsand to identify the phylogenetic relationships of different strains of this bacterium. We adapted sixpublished conventional methods targeting 16S fragments for real-time polymerase chain reaction.Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed withseveral different primers and a TaqMan probe. We also performed DNA quantification and meltingcurve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases inmembers of the deer family, hares, bank voles and mice (12.5∂15%) than in foxes, boars, cows, andhorses (around 4∂6%). We also performed blood analysis of cows, horses, mice, and ticks removedfrom animals, evaluating the presence of antibodies against granulocytic Anaplasma sp. Finally, wesubjected 11 randomly selected PCR amplified products to direct sequencing and we constructed thecorresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the humangranulocytic ehrlichiosis agent. Mutual identity of the sequencing ranged from 99% to 100%.

Key words: Anaplasma phagocytophilum; animals; real-time PCR; melting temperature; gene se-quences.

Dagmar Hulınska, National Reference Laboratory for Lyme Disease, Division of Microbiology andEpidemiology, National Institute of Public Health, Srobarova 48, 10042 Prague 10, Czech Republic,e-mail: dhulin/szu.cz

We investigated the frequency and distributionof Anaplasma phagocytophilum (formerlyknown as Ehrlichia phagocytophila), includinghuman granulocytic ehrlichiosis (HGE) agentand Ehrlichia equi-related variants such aswhite-tailed deer agent (1, 3), in wild and dom-esticated animals on horse and cow pasturesand animal enclosures in the same regions in the

Received November 11, 2003.Accepted March 22, 2004.

The Project was financially supported by the CzechMinistry of Health Grant Agency (IGA) (grants NI6880-3 and L31/93: 23795).

1

Czech Republic that we have studied previously(2). In the literature there are only a small num-ber of studies on animal reservoirs for A. phago-cytophilum in Europe. Knowledge of the pres-ence of the human granulocytic ehrlichiosisagent (HGE) and the human monocyticehrlichiosis agent (HME) in humans and ani-mals is based on polymerase chain reaction(PCR) studies (1–3, 5–8, 10). The most frequentnatural reservoirs for granulocytic ehrlichia A.phagocytophila seem to be Apodemus flavicollismice and Clethrionomys glareolus voles inSwitzerland (10) and white-tailed deer in theUnited States (3, 24). The observation that Slo-venian roe deer and red deer (13) are infected

Chapter 6: Detection of A. phagocytophilum by RT PCR

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HULINSKA et al.

with A. phagocytophilum in 86% of cases as de-termined by PCR is rather surprising consider-ing the studies mentioned above.

Interpretation of differences between PCR re-sults from different countries may be problem-atic. Massung et al. (14) showed variation in thesensitivity as well as specificity of PCR assaysfor A. phagocytophilum. Assay Ehr 521–790 (5),which amplifies nucleic acid (DNA) from the in-fected animals, was recommended for screening(15) and quantification.

We adapted conventional PCR methods (1,3–9) so that they were more standardized andfaster, using the LightCycler real-time PCRtechnique (RT-PCR) (Roche DiagnosticsGmbH, Mannheim, Germany). This enabled usto analyze more samples (8) from more animalsand to validate our previous findings (2) of hu-man granulocytic ehrlichiosis agent in patientsand animals.

There is some disagreement regarding thenaming of the bacterium. Recently publishedstudies use a new nomenclature Anaplasmaphagocytophila (11) and designate Ehrlichia equiand the HGE agent as subjective synonyms ofEhrlichia phagocytophila. Massung et al. (14)call this bacterium A. phagocytophilum, whileothers (13, 19) call it A. phagocytophila.

To the best of our knowledge, the direct PCRsequence analysis from samples of a large num-ber of wild animals is reported here for the firsttime. We succeeded in properly choosing theprimers for the direct sequencing that differen-tiate well between the different variants of A.phagocytophilum. To analyze the relationshipsbetween these variants, we sequenced the PCRproducts from our samples and formed a phylo-genetic tree using as a reference the sequence ofE. equi. We evaluated the similarity between oursequences and sequences reported in the Na-tional Center for Biotechnology Information(NCBI) BLAST network service.

MATERIALS AND METHODS

We processed samples from three groups of animals.Group 1 – mice and bank voles, Group 2 – cows andhorses, Group 3 – wild game animals. From all threegroups we also collected ticks. The animals of allthree groups were obtained from two localities: first,in the neighborhood of Napajedla, in Moravia, and

2

second, close to Chlumec in east Bohemia; on horse-grounds and in enclosures.

A total of 40 yellow-necked mice (Apodemus flav-icollis) and 15 bank voles (Clethrionomys glareolus)were trapped on horse-ground in both localities dur-ing the years 2002–2003. The animals were euthanat-ized and their blood and tissue samples from the earand spleen were stored individually, frozen at ª20 æCprior to DNA extraction.

Blood samples from 55 cows and 40 horses fed onthe same horse-ground were collected in plastic tubeswith or without EDTA (2) and were kept frozen atª20 æC.

A panel of tissue samples from wild game ani-mals – 69 wild boars (Sus scrofa), 25 red foxes (Vulpesvulpes), 40 roe deer (Capreolus capreolus), 15 fallowdeer (Dama dama), 15 red deer (Cervus elaphus), 8European hares (Lepus europaeus), and 15 mouflonsheep (Ovis musimon) is presented in Table 1 (thatalso includes domestic animals, mice and bank voles).Most samples were collected during the hunting sea-son in October and November 2002–2003 or duringindividual hunts in enclosures near the horse-groundsin both localities. Hunters collaborated with the Na-tional Veterinary Service Laboratory and were askedto obtain samples of spleen, heart and blood from theanimals, collecting them separately. Hunters placedsamples in sterile containers and kept them in icebags until shipped to the veterinary laboratory. Weexcised a 4–5 mm3 piece of tissue from the interiorof each organ and placed it in a numbered 1.5 mlmicrocentrifuge tube, refrigerated it or immediatelyprocessed it for molecular diagnostic assays.

Ticks Ixodes ricinus were recovered from infectedanimals, comprising nine roe deer, two fallow deer,two hares, two red foxes and six boars shot in theMoravian locality, and also from six mice, two bankvoles, four cows and six horses on horse-ground inthe Bohemian locality. The majority of these tickswere mainly adults and females, 26 out of 82 werenot engorged, 33 were partially engorged, and the restwere fully engorged. Ticks were kept frozen untilDNA extraction.

Serum specimens and indirect fluorescence assay(IFA)

A total of 135 blood specimens from 40 horses, 55cows and 40 mice were initially screened by IFA forHGE and E. equi (MRL Diagnostics, USA) as wedescribed previously (2). Fluorescein isothiocyanate-conjugated goat anti-horse, anti-bovine and anti-mouse immunoglobulin G (Sigma) was used at a1:200 dilution as secondary antibody. Serum titrationstarted at a 1:20 dilution. Interpretation of declaredIgG serum: endpoint titers of 1:64 and greater wereconsidered positive.

DNA extractionHigh Pure PCR template preparation kit (Roche

Diagnostics GmbH, Mannheim, Germany) was used


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TABLE 1. Total number of samples extracted from different species and corresponding number of Anaplasmaphagocytophila-positive samples using six different primer pairs

Name of Total Number of positive Infected Number of positive animals detected byanimals number animals in first in first RT-PCR with different primers

of screening with primers screening Ep.50r TaqMan ECC HE3-R GER3animals Ehr790–Ehr521 (%) Ep.145f Ep80 ECB 3-IP2 GER4

C. glareolus 15 2 13,33 2 1 2 2 2A. flavicollis 40 6 15,00 5 3 6 5 5S. scrofa 69 3 4,35 3 3 4 3 3V. vulpes 25 1 4,00 ND ND 1 1 1C. capreolus 40 5 12,50 5 4 6 5 5D. dama 15 2 13,33 1 2 2 2 2C. elaphus 15 2 13,33 ND ND 1 1 1L. europaeus 8 1 12,50 1 0 1 1 1O. musimon 15 2 13,33 2 1 2 2 2Cow 55 3 5,45 2 1 3 3 3Horse 40 2 5,00 2 1 3 2 2I. ricinus 82 8 9,76 7 8 7 8 8Total hosts 419 37 8,83NDΩnot done.

for the isolation of nucleic acids (DNA) from bloodand tissue samples. Tissue samples (predominantlysamples of spleen and heart from hunting animals,ear and spleen from mice) were mechanically homo-genized with a micro-pestle before addition of 200 mltissue lysis buffer, 40 ml of proteinase K (20 mg/ ml)and 5 ml of lysozyme (10 mg/ ml). After incubationat 55 æC for 3 h, the extraction protocol was followedas recommended by the supplier. We used alkalinelysis to extract DNA from the blood and tick samples(2).

DNA amplification and analysisSix conventional PCR methods using different

primer pairs were adapted for real-time PCR (RT-PCR). Samples from 419 animals were first screenedfor ehrlichial nucleic acid (DNA) by RT-PCR ampli-fication with a group-specific primer set Ehr521–

TABLE 2. The six variants of the real-time PCR procedure adapted from conventional PCR (references in thelast column)

Gene Primer pair Annealing No. of Specificity Mean Tm No. of Referencein æC cycles SD∫(æ C) base pairAp Ec Eq Bb

16S Ehr521-Ehr790 60 40 π π π ª 87.3∫0.40 293 (3, 5)16S Ep.50r-Ep.145f 62 40 π π π ª 81.7∫0.37 110 (8)16S TaqMan-Ep80 62 40 π ª π ª 106 (8)16S ECC – ECB 48 45 π π π π 88.6∫0.20 520 (1, 6)16S HE3-R – 3-IP2 55 40 π ª π ª 86.5∫0.27 239 (7, 9)16S GER3 – GER4 58 40 π ª ª ª 83.5∫0.30 150 (4)We show the gene target, the primer pair used, the annealing temperature, the number of amplification cycleschosen, and the results of the measurements: the melting temperature and its standard deviation, as well as theamplicon length found by gel electrophoresis.Ap: Anaplasma phagocytophylum, Ec: Ehrlichia chaffeensis, Eq: Ehrlichia equi, Bb: Borrelia burgdorferi sensulato.

3

Ehr790 as previously described (3, 5). The initiallyRT-PCR-positive samples were retested with a primerset Ep.50r-Ep.145f (8), also with the TaqMan Ep80probe (8), and with primer sets ECC-ECB (1, 6),nested HE3-R paired with E.equi3-IP2 (7, 9), andGER3–GER4 (4) to confirm the Anaplasma positiv-ity of the samples and to quantify the specific DNA.The complementary DNA to Anaplasma sp. wasamplified with these primer sets (Table 2) using astandardized real-time PCR (RT-PCR) protocol withthe LightCycler FastStart DNA Master SYBR GreenI kit and with the LightCycler FastStart DNA Mas-ter Hybridization Probes kit with the TaqMan probe(both from Roche Diagnostics, Mannheim, Ger-many). Primers were prepared by Generi Biotech Ltd.(DNA Synthesis Service, Czech Republic) and theTaqMan probe by TIB Molbiol (DNA Synthesis Ser-vice, Roche Diagnostics, Berlin, Germany).


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HULINSKA et al.

The 18-ml reaction mix in a glass capillary con-tained 2 ml of FastStart Master SYBR Green I mix,different volumes of primers corresponding to 5 mMof each, 3 mM MgCl2, and sterilized water for RT-PCR (Roche). We added 2 ml of template DNA fromthe unknown samples, the same volume of negativecontrol (water) or positive control (DNA from HGE,cultured from patients as we described previously (2))to the 18 ml reaction mix. The amplification programstarted with denaturation at 95 æC for 10 min. An-nealing temperatures and numbers of cycles used areshown in Table 2. Fluorescence was measured at theend of each extension step. Melting curves were ac-quired by heating at 20 æC/s to 95 æC, cooling to 55–62 æC (depending on the primers) for 20 s, and thenslowly heating at 0.1 or 0.2 æC/s to 95 æC with continu-ously measured fluorescence.

A modification of the nested primer RT-PCR (20)was used with the HE3-R inner primer paired withthe E. equi 3-IP2 primer (7, 9). The reaction volumeof 10 ml contained 1.5 ml of FastStart Master SYBRGreen I buffer, 0.5 ml of FastStart polymerase, 1.8 mlof MgCl2 corresponding to the final concentration of4 mM, 1 ml of each primer (5 mM). Two ml of thetemplate DNA was added. The capillary was spundown, overlaid with 5 ml of silicone oil, and spunagain. Subsequently, 10 ml of the second-round mix-ture containing 1 ml of each inner primer (10 mM)was added. The first round PCR was performed asdenaturation at 95 æC for 6 min and 40 cycles at95 æC for 5 s, 48 æC for 10 s, and 72 æC for 20 s. Thecooled capillaries were then spun down. Second am-plification started at 95 æC for 2 min with 40 cycles at95 æC for 5 s, 55 æC for 5 s, and 72 æC for 15 s. Fluor-escence melting curve analysis was performed asmentioned above.

Specific TaqMan Ep80 probe was used for detec-tion of ehrlichial amplicons as recommended by Pus-terla et al. (8). We performed electrophoresis of 7 mlof the products in 2 ml loading buffer (Sigma) in 1–2% agarose gel with a low size marker (Sigma,D7058).

We grew granulocytic A. phagocytophilum – HGEagent in HL-60 cell cultures as we described in (2).We washed the cells (0.5 ml of culture at a density of2¿105 cells/ml) with PBS, extracted the DNA, andperformed serial dilutions with factor three to makethe quantification. We measured the melting curveanalysis from five capillaries, obtaining the meanvalues Tm and their standard deviation for eachprimer pair following the supplied instructions (Ro-che manual). Student’s t-test was used to analyze stat-istical significance of the Tm differences, with p0.05.

DNA sequencingThe direct sequence analysis was made from 11

products amplified with a GER3–GER4 primer pairand from 15 products amplified with the ECC-ECBprimer pair by the dideoxy chain termination pro-cedure using the CEQ 2000 XL automatic sequencer

4

as we described in (2). Nucleotide sequence homo-logy searches were made through the NCBI BLASTnetwork service. The best corresponding sequences toour acquired ones had accession numbers AF 136712for E. phagocytophila, strain Frankonia II; AF136713 for strain Frankonia I, AF136714 for strainBaden; AF 482761, AF172167 for E. equi; AF189153for HGE; AF241532 for strain from a llama (Lamaglama).

Sequences were aligned and analyzed using theprogram CLUSTALW (http://ebi.ac.uk/clustalw/).The phylogenetic tree was generated with the soft-ware TREECON, using the Kimura algorithm to cal-culate the genetic distances with 1000 bootstrap repli-cates and the neighbor-joining method to infer thetree topology.

RESULTS

This study validated and completed our pre-vious report (2) on the occurrence of humangranulocytic ehrlichiosis (HGE) agent in theCzech Republic by using a more standardizedLightCycler RT-PCR assay. The limits of detec-tion of 16S rRNA assays were two infected cellsas tested by use of the A. phagocytophilum tem-plate DNA from our HGE culture.

A total of 419 animals were first screened forehrlichial DNA by RT-PCR amplification witha group-specific primer set Ehr521–Ehr790.Standard curve and amplification plot derivedfrom a threefold dilution series of the HGE con-trol from the screening test with Ehr521–Ehr790primers are shown in Fig. 1. The control DNAsamples of HGE had concentrations from 0.1pg/ml to 11 pg/ml. It helped us to determine thatthe unknown DNA concentration in animalsamples ranged from 0.1∂1.2 pg/ml (see Fig. 2).

Thirty-seven (8.83%) of the initially positivesamples out of the 419 were retested with fivedifferent primer sets which we adapted for RT-PCR. Results are shown in Table 1. The meltingtemperatures obtained and the cycling con-ditions used are shown in Table 2.

The percentage of infected animals is sum-marized in Table 1. Note especially the highnumber of cases among the deer family(12.5∂13.3%), hares (12.5%), and mice andbank voles (15% and 13.3%, respectively). Thesensitivity of detection using the six differentmethods was similar (Table 1); there was gener-ally less detection with the TaqMan Ep80 probe(only 24 Anaplasma-positive animals) than


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Fig. 1. The inset shows the standard RT-PCR curve (cycle number versus logarithm of concentration). Themain graph contains the corresponding amplification plot (fluorescence versus cycle number). From left toright, the curves were obtained from dilutions of a specific ehrlichial DNA with concentrations 11 pg/ml; 3.66pg/ml; 1.22 pg/ml; 0.41 pg/ml; 0.14 pg/ml. The horizontal line is a negative control. Small regression error indicatesgood accuracy and reproducibility of the method.

Fig. 2. Amplification plot was created from serially diluted control of HGE (as in Fig. 1) and typical samplesfrom infected animals of various species. It indicates that the unknown concentration of DNA in animalsamples ranged from 0.1 to 1.2 pg/ml.

when using the primers alone or using thenested RT-PCR. The Anaplasma DNA wasfound in fewer cases in boars (4.3%), which werefrequently infected with other bacteria (Borreliasp., Brachyspira hyodysenteriae, Pseudomonasfragi, etc.). Samples from red foxes were mostlynegative for ehrlichial DNA; only one fox waspositive. Domestic cows and horses were in-fected in about 5% of cases. Only 9.7% of theexamined ticks were found positive. Out of the17 adults and several nymphs of ticks from 6

5

trapped infected mice and 2 infected bank voles,only 3 ticks were infected. Another four infectedticks were collected from deer and one tick wascollected from a horse. The tick samples showthe same Tm as the samples from infected ani-mals and the positive HGE control. Samplesfrom different tissues of the same infected ani-mal were always all infected.

The melting curve analysis with the primerpair Ehr521–Ehr790 used in screening was per-formed on 37 positive samples. In all of them


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HULINSKA et al.

TmΩ87.3∫0.40 was found. This is consistentwith the Tm from the HGE control sample (seeFig. 3). The Tm temperatures obtained for theother primer pairs are reported in Table 2. Fig.4 shows the melting curves for the Ep.50r-Ep.145f primers for the animal samples and theHGE control.

The specificity of the primers with respect toAnaplasma phagocytophylum, Ehrlichia chaf-feensis, E. equi and Borrelia burgdorferi sensulato is shown in Table 2. The primer ECC-ECBalso reacted with B. burgdorferi sensu lato;

Fig. 3. Melting curves from RT-PRC with primers Ep521–790 from the first screening. Peaks corresponding tosamples from infected animals and positive control are located at temperature TmΩ87.30∫0.40 æC. Peaks atlower temperature (around 83 æC and less) come from non-infected animals and the negative control and corre-spond to primers/dimers.

Fig. 4. Melting curves for RT-PCR with primers Ep.50r-Ep.145f. We observe that the amplicons obtained withthese primers from the infected animals and from the HGE control both have melting temperature TmΩ81.70∫0.37 æC.

6

primers Ehr521–Ehr790, Ep.50r-Ep.145f andECC-ECB reacted with E. chaffeensis.

The gel electrophoresis measurements ident-ify the length of the Ehr521–Ehr790 ampliconas 293 bp for the positive samples, which corre-sponds to the HGE control. This check was per-formed after all the RT-PCR amplifications andthe lengths determined are shown in Table 2.

Blood samples of 40 horses, 55 cows, and 40mice were screened for specific antibodiesagainst A. phagocytophilum in indirect immuno-fluorescence antibody (IFA) tests. Antibodies


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?????

Fig. 5. Example phylogenetic tree showing relationships between direct sequencing results for 11 selectedsamples amplified using GER3-GER4 primers on 16S DNA. Sequence from Ehrlicha equi, 100% homologousto HGE, was used as reference. Each sequence is identified by the sample number and the animal species itwas extracted from. The horizontal bar represents a 0.5% sequence divergence. Bootstrap values are shown atbranch points.

were found in two horses, three cows and sixmice. These animals were also found positivewith the screening RT-PCR. Three cows and twohorses had positive IFA tests with anti-HGEand anti-E. equi antigens.

Direct microscopic examinations of Giemsa-stained buffy coat blood smears of one infectedcow showed intragranulocytic inclusions (mor-ulae) in some cells. No morulae were found inblood samples from PCR-positive horses andmice.

We performed the sequence analysis of a 520bp (with the ECC-ECB primer pair) and 150 bp(with the GER3–GER4 primer pair) phylogen-etically informative region in the 5ø end of the16S r RNA gene. The E. equi samples from twohorses (100% homologous with HGE) (11) were99.5% homologous to the selected samples fromother animals (two roe deer, one red deer, onefallow deer, one mouflon, one red fox, one boar,one bank vole and one tick). The sequencesfrom these animal samples were 99.6∂99.8%homologous to the A. phagocytophilum se-quence from the NCBI BLAST database. Therewere 2∂6 nucleotide differences between theanimal pathogen amplicon sequences and thepublished sequence for HGE.The correspond-ing phylogenetic tree obtained using the soft-ware package CLUSTALX and TREECON is

7

shown in Fig. 5. The tree shows that the ehrlich-ial agents in the wild and domesticated animalsfrom the Czech Republic belong to the A. phag-ocytophilum group. Four A. phagocytophilum16S rRNA genogroup variants were sequenced:variant Baden, variant Frankonia I, variantFranconia II, and variant Llama ehrlichia fromLama glama (22).

DISCUSSION

The reported prevalences of infection of I. ric-inus ticks with A. phagocytophilum and HGEwere 0.8% in Switzerland (10, 16), 3.2% inSlovenia (12), 3.1 and 9.2% on the east and westcoasts of Sweden (4), and 24.4% in a region ofItaly. These prevalences correspond with the re-sults from our study – we proved DNA of A.phagocytophilum in 9.76% of cases in ticks col-lected on animals in east Bohemia and in Mora-via. The amount of Anaplasma DNA in ticksdepends on many factors: the tick’s nutritionstatus, the stage of the tick, the time of year, thegeographical region, and the type of hosts.

Our findings of 13.3∂15% infection withehrlichial DNA in samples from bank voles C.glareolus and yellow-necked mice A. flavicollisare comparable with findings in Switzerland


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HULINSKA et al.

(10) and in the United Kingdom (19). We foundthat the percentage of Anaplasma-positive ani-mals was significantly higher in mice (15%) thanin bank voles (13.3%). Bank voles and micewere known to host larval I. ricinus tick. Wefound 17 adult ticks and several nymphs on 6infected mice and 2 infected bank voles, butonly 3 of the ticks hosted A. phagocytophilum.

Our findings confirm that wild animals suchas deer and mice were infected with a similargenotype of A. phagocytophilum, as were ticks,cows and horses (9). We detected DNA of A.phagocytophilum in a higher percentage of casesin members of the deer family, hares, bank volesand mice (12.5∂15%) than in foxes, boars, cowsand horses (around 4∂6%). Our results fromRT-PCR are comparable with our last examina-tion of the HGE agent made with conventionalPCR (2) and with observations in Europe (10)and in the United States (3, 5, 7), where HGEis endemic.

Reaction results with a TaqMan probe Ep80and melting temperatures with five differentprimer pairs were similar for HGE and E. equiand confirmed their 100% homology (11). Thesensitivity of detection of specific ehrlichialDNA in samples of infected animals using thesix different methods was similar. We found thatin naturally infected animals there was generallyless detection with the Taq Man Ep80 probethen when using the primers alone, contrary tothe results for animals infected purposefully (8).

Differences in the Tm value between HGEand variants of A. phagocytophilum in differentRT-PCR assays were so small that they couldnot be used for distinguishing these variants.However, our results indicate that the fluor-escence melting curve analysis of PCR productscan be used for rapid detection of these agents(16).

The direct sequence analysis of the 16S rRNAcan be used for the detection of closely relatedAnaplasma species in humans and in animals(17, 24). The study from Slovenia indicates (12)that the groESL gene should be sequenced inorder to answer the question about reservoirhosts (18, 19). Sequence comparison of the 16SrRNA gene is recognized as one of the powerfulmethods for determining the phylogenetic re-lationships of bacteria as shown by Wen et al.(17). In the nested RT-PCR reaction the ECC-ECB primer pair produced a 390 bp amplicon

8

and the GER3–GER4 primer pair produced a150 bp amplicon, containing the species-specificsignature sequences. The choice of these am-plicons for direct sequencing allowed us to dif-ferentiate between the bacterial species. Thephylogenetic tree that we constructed classifiesthe ehrlichial agents obtained from animals thatwere most closely related to the A. phagocyto-philum genogroup.

Some authors (10, 12, 17) showed a high de-gree of homology of partial 16S rRNA gene se-quences of granulocytic ehrlichia A. phagocyto-philum from ticks, rodents and deer with theHGE agent isolated from humans. Others (11,14, 15, 21) found four variants differing in twonucleotides from the HGE or E. equi. They re-ported that more than one strain of A. phagocy-tophilum exists both in the United States and inEurope. We found differences between se-quences of ehrlichial DNA extracted from adeer, boar, red fox, bank vole, tick and twohorses of only 2∂6 nucleotides.

We thank the 15 hunters for providing part of thesample material, J. Votypka, Dr. J. Plch and Dr. Z.Kurzova for technical assistance with PCR and IFAtests, Dr. C. Vlcek for assistance with the sequencingmachine, Ing. Brada for help with statisticalmethods.

REFERENCES

1. Dawson JE, Stallknecht DE, Howerth EW,Warner C, Biggie K, Davidson WR, LockhartJM, Nettles VF, Olson JG, Childs JE. Suscepti-bility of white-tailed deer (Odocoileus virginianus)to infection with Ehrlichia chaffeensis, the etiol-ogic agent of human ehrlichiosis. J Clin Micro-biol 1994;32:2725–8.

2. Hulinska D, Votypka J, Plch J, Vlcek E, ValesovaM, Bojar M, Hulinsky V. Molecular and micro-scopical evidence of Ehrlichia spp. and Borreliaburgdorferi sensu lato in patients, animals andticks in the Czech Republic. Microbiologica2002;25:437–48.

3. Belongia EA, Reed KD, Mitchell PD, KolbertCP, Persing DH, Gill JS, Kazmierczak JJ. Preva-lence of granulocytic Ehrlichia infection amongwhite-tailed deer in Wisconsin. J Clin Microbiol1997;35:1465–8.

4. Goodman JL, Nelson C, Vitale B, Madigan JE,Dumler JS, Kurtti TJ, Munderloh UG. Directcultivation of the causative agent of human gra-nulocytic ehrlichiosis. N Engl J Med 1996;334:209–15.


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5. Jackson CA, Lovrich SD, Agger WA, CallisterSM. Reassessment of a midwestern Lyme diseasefocus for Borrelia burgdorferi and the humangranulocytic ehrlichiosis agent. J Clin Microbiol2002;40:2070–3.

6. Harrus S, Wanner T, Aizenberg I, Foley JE, Po-land AM, Bark H. Amplification of ehrlichialDNA from dogs 34 months after infection withE. canis. J Clin Microbiol 1998;36:73–6.

7. Kordick SK, Breitschwerdt EB, Hegarty BC,Southwick KL, Colitz CM, Hancock SI, BradleyJM, Rumbough R, et al. Coinfection withmultiple tick-borne pathogens in a WalkerHound kennel in North Carolina. J Clin Micro-biol 1999;37:2631–8.

8. Pusterla A, Huder JB, Leutenegger CHM, BraunU, Madigan JE, Lutz H. Quantitative real-timePCR for detection of members of the Ehrlichiaphagocytophila genogroup in host animals andIxodes ricinus ticks. J Clin Microbiol 1999;37:1329–31.

9. Barlough JE, Madigan JE, DeRock E, BigorniaL. Nested polymerase chain reaction for detec-tion of Ehrlichia equi genomic DNA in horsesand ticks (Ixodes pacificus). Vet Parasitol 1996;63:319–29.

10. Liz JS, Anderes L, Sumner JW, Massung RF,Gern L, Rutti B, Brossard M. PCR detection ofgranulocytic Ehrlichiae in Ixodes ricinus ticksand wild small mammals in western Switzerland.J Clin Microbiol 2000;38:1002–7.

11. Dumler JS, Barbet AF, Bekker CPJ, Dash GA,Palmer GH, Ray SC. Reorganization of generain the families Rickettsiaceae and Anaplasmata-ceae in the order Rickettsiales, unification ofsome species of Ehrlichia with Anaplasma, Cow-dria with Ehrlichia and Ehrlichia with Neorick-ettsia, description of six new species combi-nations and designation of Ehrlichia equi andHGE agent as subjective synonyms to Ehrlichiaphagocytophila. Int J Syst Evol Microbiol 2001;51:2145–65.

12. Petrovec M, Sumner JW, Nicholson WL, ChildsJE, Strle F, Barlic J, Lotric-Furlan S, Avsic Zup-anc T. Identity of ehrlichial DNA sequences de-rived from Ixodes ricinus ticks with those ob-tained from patients with human granulocyticehrlichiosis in Slovenia. J Clin Microbiol 1999;37:209–10.

13. Petrovec M, Bidovec A, Sumner JW, et al. Ticks,reservoir hosts and the ecology of Anaplasmaphagocytophila in Slovenia. Infection with Ana-plasma phagocytophila in cervicids from Slovenia.Evidence of two genotypic lineages. Wien KlinWochenschr 2002. In Strle F. Human granulo-cytic ehrlichiosis (HGE) – an emerging infectionin Europe. IX Int Conf on Lyme Borreliosis andother tick-borne diseases. New York, USA Aug-ust 18–22; 2002:157–9.

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14. Massung RF, Manuel MJ, Owens JH, Allan A,Courtney JW, Stafford KC, Mather TN. Geneticvariants of Ehrlichia phagocytophila Rhode Is-land and Connecticut. Emerg Infect Dis 2002;8:467–72.

15. Massung RF, Slater KG. Comparison of PCRassays for detection of the agent of human gra-nulocytic ehrlichiosis, Anaplasma phagocytophil-um. J Clin Microbiol 2003;41:717–22.

16. Wicki R, Sauter P, Metter C, Natsch A, EnzlerT, Pusterla A, Kuchnert P, Egli G, Bernasconi M,Lienhard R. Swiss army survey in Switzerland todetermine the prevalence of Francisella tular-ensis, members of the Ehrlichia phagocytophilagenogroup, Borrelia burgdorferi sensu lato andtick-borne encephalitis virus in ticks. Eur J ClinMicrobiol Infect Dis 2000;19:427–32.

17. Wen B, Jian R, Zhang Y, Chen R. Simultaneousdetection of Anaplasma marginale and a newEhrlichia species closely related to Ehrlichia chaf-feensis by sequence analyses of 16S ribosomalDNA in Boophilus microplus ticks from Tibet. JClin Microbiol 2002;40:3286–90.

18. Daniels TJ, Battaly GR, Liveris D, Falco RC,Schwartz I. Avian reservoirs of the agent of hu-man granulocytic ehrlichiosis. Emerg Infect Dis2002;8:1524–6.

19. Bown KJ, Begon M, Bennett M, Woldehiwed Z,Ogden NH. Seasonal dynamics of Anaplasmaphagocytophila in a rodent-tick (Ixodes triangul-iceps) system, United Kingdom. Emerg InfectDis 2003;9:63–9.

20. Berg J, Nagl V, Muhlbauer G, Stekel H. Rapid-cycle PCR in temporarily compartmentalizedcapillaries: two-round PCR in a single capillaryprevents product carry-over. BioTechniques2000;29:680–4.

21. Baumgarten BU, Rollinghoff M, Bogdan CH.Prevalence of Borrelia burgdorferi and granulo-cytic and monocytic Ehrlichiae in Ixodes ricinusticks from Southern Germany. J Clin Microbiol1999;37:3448–51.

22. Barlough JE, Madigan JE, Turoff DR, CloverJR, Shelly SM, Dumler JS. An Ehrlichia strainfrom a llama (Lama glama) and Llama-associ-ated ticks (Ixodes pacificus). J Clin Microbiol1997;35:1005–7.

23. Munderloh UG, Madigan JE, Dumler JS, Good-man JL, Hayes SF, Barlough JE, Nelson CM,Kurtti TJ. Isolation of the equine granulocyticehrlichiosis agent Ehrlichia equi, in tick cell cul-ture. J Clin Microbiol 1996;34:664–70.

24. Dawson JE, Warner CK, Baker V, Ewing SA,Stallknecht DE, Davidson RL. Ehrlichia like 16SrDNA sequence from wild white-tailed deer(Odocoileus virginianus). J Parasitol 1996;82:52–8.


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Chapter 7

Discussion

We studied the occurrence of two tick-borne bacteria, B. burgdorferi s. l. and A. pha-gocytophilum in their main vector in the Czech Republic, tick I. ricinus. Ticks werecollected from domestic hosts - dogs, horses and cows - as well as from wild ani-mals - deer, hares, foxes, boars, mice and bank voles.

In ticks collected from dogs in north Bohemia and Moravia, we found (Kybi-cova et al. 2009) the DNA of A. phagocytophilum and B. burgdorferi s. l. in 8.5% and6.8% of cases, respectively. Anaplasma positive ticks on positive dogs might havebeen infected by feeding. Similar findings have been reported in Poland (Zygneret al. 2008). We found no coinfection of Anaplasma and Borrelia in these ticks.

In ticks collected on wild animals, cows and horses in east Bohemia andMoravia, the DNA of A. phagocytophilum was detected in 9.8% of cases (Hulınskaet al. 2004). The reported prevalences of infection of I. ricinus ticks with A. phago-cytophilum were 1.1 1.4% in Switzerland (Liz et al., 2000, Wicki et al., 2000) and3.2% in Slovenia (Petrovec et al., 1999). Our results are similar and correspondalso to our result on ticks collected on dogs.

Bank voles and mice are known to host larval I. ricinus ticks. We found 17adult ticks and several nymphs on 6 infected mice and 2 infected bank voles, butonly three of the ticks hosted A. phagocytophilum. The amount of Anaplasma andBorrelia DNA in ticks depends on many factors: the tick’s nutrition status, thestage of the tick, the time of year, the geographical region, and the type of hosts.

We have described the prevalence of A. phagocytophilum and B. burgdorferis. l. infections in the most important reservoir hosts in the Czech Republic, wildrodents, and determined the genospecies of B. burgdorferi s. l. associated withthese hosts.

Our findings (Hulınska et al. 2004) of 13.3% infection with A. phagocytophilumDNA in samples from bank voles C. glareolus and 15% for yellow-necked miceA. flavicollis are comparable with findings in Switzerland (Liz et al., 2000) and in

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Chapter 7: Discussion

the UK (Bown et al., 2003). We found that the percentage of Anaplasma positiveanimals was higher in mice (15%) than in bank voles (13.3%).

Many species have been reported to serve as competent reservoirs ofB. burgdorferi s. l. in Europe, in particular rodents of the genera Clethrionomys andApodemus (Matuschka et al. 1992, Hu et al. 1997, Humair et al. 1999, Hanincovaet al. 2003). We found Borrelia infection to be more common in C. glareolus thanin Apodemus spp. (Kybicova et al. 2008, Zore et al. 1999). The most frequentlycaptured rodent species were A. flavicollis and C. glareolus, the most abundantsylvan rodents in the Czech Republic and neighboring states (Hanincova et al.2003). B. afzelii, B. burgdorferi s. s. and B. garinii were detected. To the best of ourknowledge, this is the first report of the presence of B. burgdorferi s. s. in rodentsin the Czech Republic. In our data, the majority of infections were caused byB. afzelii (Humair et al. 1995, Hu et al. 1997, Humair et al. 1998, Humair et al.1999, Hanincova et al. 2003). A possible explanation is a reduced and shorter-lived infectivity of B. burgdorferi s. s. in comparison with B. afzelii which is betteradapted to rodent hosts (Richter et al. 2004). However, B. burgdorferi s. s. wasdetected in small rodents in the UK, Poland, Ireland and Switzerland (Kurten-bach et al. 1998b, Humair et al. 1999, Gray et al. 2000, Michalik et al. 2005).B. burgdorferi s. s. seems to be less specialized and may be maintained both byavian and rodent hosts (Kurtenbach et al. 1998a). We have demonstrated one caseof coinfection with B. afzelii and B. burgdorferi s. s. in A. sylvaticus, similarly as inZore et al. 1999.

The presence of Borrelia was previously studied from ear biopsies and inter-nal organs of small mammals (spleen, heart, liver, and urinary bladder) (Humairet al. 1993a, Zore et al. 1999, Hanincova et al. 2003, Christova et al. 2005). As earbiopsies may not reveal the full diversity of the infection, (Richter et al. 1999,Hanincova et al. 2003), we have used muscle samples, observing an infection rateof 16.4% (Kurtenbach 1998b, Christova et al. 2005).

Seropositivity of wild rodents indicates previous exposure to infected ticks(Stefancıkova et al. 2004). The prevalence of anti-Borrelia antibodies was higher inApodemus spp. than in C. glareolus (Aeschlimann et al. 1986, Vostal and Zakovska2003 and Stefancıkova et al. 2004). The difference may be a result of either higherinfestation of Apodemus spp. than C. glareolus by Ixodes ricinus ticks (Hanincovaet al. 2003, Michalik et al. 2005) or by interspecies variability of the immuneresponse (Kurtenbach et al. 1998a).

We have also investigated the occurrence of A. phagocytophilum in possiblereservoir hosts in the Czech Republic, wild animals such as roe deer, fallow deer,red deer, mouflon sheep, European hare, red fox and wild boar. We have foundthe DNA of A. phagocytophilum in a higher percentage of cases in members ofthe deer family and hares (12.5–13.3%), as compared to foxes and boars (4% and

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4.35%) (Hulınska et al. 2004). Similarly, in Spain, roe deer was infected in 18%of cases (de la Fuente et al. 2008). In Poland, roe deer were infected in 31.94% ofcases (Adamska and Skotarczak 2007). In Slovakia, the deer family was infectedin about 50% of cases and none of the wild boars was PCR positive (Stefanidesovaet al. 2007). In Slovenia, somewhat higher percentage of Anaplasma positive casesin deer, 86% (Petrovec et al. 2002), red foxes V. vulpes and wild boar Sus scrofawere reported (Petrovec et al. 2003).

Besides vectors and reservoirs, we have been also interested in the occur-rence of borreliosis and anaplasmosis in hosts — domestic animals, namely dogs,cows, and horses.

We have detected the DNA of A. phagocytophilum in 5% and 5.45% of cases, inhorses and cows, respectively (Hulınska et al. 2004). Similarly results we obtainedfor Anaplasma in dogs (Kybicova et al. 2009). In Italy, horses were reported to beinfected by Anaplasma in 8.14% of cases (Passamonti et al. 2010).

We have found the DNA of A. phagocytophilum in 3.4% (10 out of 296) of dogs(Kybicova et al. 2009). Similar PCR positivity rates, i.e. 6.3%, 5.5%, and 9.5% havebeen reported in Germany (Jensen et al. 2007), Italy (Torina et al. 2006), and USA(Beall et al. 2008), respectively. One out of our 155 healthy dogs was PCR posi-tive for A. phagocytophilum which is a similar proportion to the study of Beall et al.2008 in the USA (7 out of 222 healthy dogs). Morulae in peripheral blood granulo-cytes were observed in only two of the 10 PCR positive dogs, which correspond tothe findings of Jensen et al. 2007 (2/6). The inclusion bodies produced by A. pha-gocytophilum appear in granulocytes and can be observed by microscopy only atthe peak of the acute infection, which usually lasts only a few days (Engvall et al.2002).

We detected DNA of B. burgdorferi s. l. in peripheral blood of only one PCRpositive dog. This low yield can be explained by the transient nature of the pres-ence of the spirochetes in the blood (Straubinger et al. 1997, Chang et al. 2001).Using PCR, borrelial DNA can only be detected in blood in the early stage ofinfection (Skotarczak et Wodecka 2003). Once the microorganism is disseminatedin the body, a variety of organs can be affected, especially the skin, joints, heartand central and peripheral nervous systems (Straubinger et al. 1997).

The seropositivity rates in the examined dogs indicate natural exposure toA. phagocytophilum and B. burgdorferi s. l. We found a 25.9% positivity of IgGantibodies against A. phagocytophilum. For comparison, the reported canine sero-prevalence rates were 17.7% for granulocytic Ehrlichia in Sweden (Egenvall et al.2000a) and 7.5% for A. phagocytophilum in Switzerland (Pusterla et al. 1998). InGermany, antibodies to A. phagocytophilum were found in 43.2% of examineddogs (Jensen et al. 2007), in Israel in 9% (Levi et al. 2006), and in the USA, thecanine seroprevalence rates were between 9.4% (Magnarelli et al. 1997) and 29%

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(Beall et al. 2008). The canine seroprevalence was reported to be 11.5% and 15.5%in Spain (Solano-Gallego et al. 2006, Amusategui et al 2008) and 34.4% in Italy(Torina et al. 2006). All studies used IFA, except Beall et al. 2008, who used ELISA.The variability of reported seroprevalences may have resulted from the use of dif-ferent selection criteria for examined dogs. We found no difference in seropositiv-ity between symptomatic (27.5%) and asymptomatic (24.5%) dogs. Similar find-ings have been reported in Germany and USA (Jensen et al. 2007, Beall et al.2008). The seropositivity in dogs without clinical signs can be explained by thepersistence of antibodies as observed in chronically infected animals (Egenvallet al. 2000b). Seropositivity in many healthy dogs also indicates that antibodiesto A. phagocytophilum in dogs can persist (Madigan et al. 1990, Magnarelli et al.1997, Engvall et al. 2002).

We found 2.4% IgM and 10.3% IgG canine seropositivity rates for B. burgdorferis. l. Serological detection of B. burgdorferi s. l. in dogs in the Czech Republicperformed in 2006 (Pejchalova et al. 2006) showed a seroprevalence rate of 6.5%.The reported figures in other countries vary widely from 0.6% and 6.9% in Spain(Solano-Gallego et al. 2006, Amusategui et al. 2008*), 3.9% in Sweden (Egenvallet al. 2000a*), and 1.85% in Canada (Gary et al. 2006) to 11% in the USA (Beall et al.2008), approximately 20% in France, the United Kingdom and Denmark (Dobyet al. 1988*, May et al. 1991, Hansen and Dietz 1997*), 40.2% in Poland (Skotarczaket al. 2005) and 50% in Slovakia (Stefancıkova et al. 1998). Most of the studies werebased on ELISA, except for those marked by an asterisk (*), which were basedon IFA. As mentioned above for A. phagocytophilum, the differences may haveresulted from different dog selection criteria. Differences in canine seroprevalencerates for tick-borne diseases can also arise from variability in tick densities orproportion of infected ticks.

We also describe three cases of dogs naturally infected with borreliosis(Schanilec et al. 2010). All presented cases showed clinical and diagnostic featuresthat strongly indicated borrelial infection. In all dogs, the clinical signs startedbetween April and July. This corresponds with seasonal activities of ticks anddogs (Hovius et al. 1999a, Hovius et al. 1999b, Steere 2001, Bhide et al. 2004,Pejchalova et al. 2006, Kybicova et al. 2009). Clinical illness in infected dogs occurs2 to 6 months after tick exposure.

Borreliosis is diagnosed based on the presence or absence of the following fac-tors: 1) typical clinical symptoms; 2) exclusion of differential diagnosis; 3) reactionto antibiotics; 4) contact with a tick or living in an endemic area; 5) the presenceof antibodies in the blood serum. The last criterion is often used as a diagnos-tic indication. However, seropositivity determined from a single sample cannotdifferentiate between past and present exposure, and should not on its own con-stitute a basis for diagnosis of an active infection (Egenvall et al. 2000a, Bhide

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et al. 2004, Skotarczak et al. 2005).ELISA is considered to be sufficiently specific to be used for determining the

dynamics of the antibody response to B. burgdorferi s. l.; it can detect an early stageof the illness (Hovius et al. 1999a). A correct timing of serologic examinations isimportant in order to determine whether active or past infection is responsible forthe seropositivity. Early serodiagnostic results are usually negative because theimmune response to borreliae develops gradually. Antiborrelial antibodies IgMare produced one to two weeks after the infection (Hovius et al. 1999a), correlatewith the onset of the clinical illness, and remain elevated for two months (Greeneand Straubinger 2006). IgG–ELISA positive titres develop within 4 to 6 weeks(Appel et al. 1993), culminate at 3 months and last 1 to 2 years after exposure(Straubinger et al. 2000, Goossens et al. 2001). Simultaneous measurements ofIgG and IgM and paired-sample titres are recommended for detection of borrelialinfection. False-negative antibody tests results are rare (Greene and Straubinger2006). Clinical syndromes together with PCR and serial serology indicate a verylikely borrelia induced disease. In all three of our dog case studies (Schanilec et al.2010), the immune response and PCR findings indicated an early disseminatedstage of a borrelial infection.

In Case 2 (Schanilec et al. 2010), neurological signs were reported but the ori-gin of the seizures remained unclear. They could have been caused by nephro- orencephalopathy, because of azotemia or CNS borrelial infection cause irritation(Azuma et al. 1993, Azuma et al. 1994). The symptoms could have been causedby silent borrelial CNS infection with a transient relapse. Progressive renal fail-ure, moderate to severe azotemia and significant proteinuria were detected alongwith IgM/IgG shift (Grauer et al. 1988, Greene and Straubinger 2006, Gerber et al.2007). A possible increased sensitivity of labradors and golden retrievers to renalinvolvement has been described in the literature.

The ECG changes and post-mortem findings in Cases 1 and 3 make us thinkthat the incidence of cardiac manifestations of borreliosis in dogs could be higherthan previously thought. The observed cardiovascular syndromes include a pres-ence of arrhythmia (variable degrees of atrioventricular block, supraventricu-lar and ventricular arrhythmia) (Levy and Duray 1988, Nalmas et al. 2007),myocarditis, pericarditis, dilated cardiomyopathy and coronary artery disease(Gasser et al. 1999). The overall prognosis of Lyme carditis is good, althoughrecovery may be delayed and later on the effects of inflammation and toxinscan cause heart failure (Nagi et al. 1996, Gasser et al. 1999, Cepelova 2008). Thechronic diffuse granulomatous and necrotic myocarditis found in Case 1 suggeststhe cystic form of the disease (Giudice et al. 2003).

A complete blood count (CBC) revealed persistent mild hypochromic anaemiain two cases (Case 1, 2) and borderline anaemia in the Case 3. Anaemia can appear

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in the acute stage of bacteraemia or inflammation because of bone marrow sup-pression or due to direct penetration of B. burgdorferi s. l. to bone marrow. Themoderate panhypoproteinemia in the Case 1 and elevated alanine aminotrans-ferase (ALT) (Cases 1, 2) can be a nonspecific effect of the systemic inflammation.In one case acute hepatitis was verified by necropsy.

We suggest that supportive complementary nursing care and cage rest of thedog patients are among the mainstays of the complex therapy.

We have used molecular methods for detection of these two tick-borne bacte-ria, B. burgdorferi s. l. and A. phagocytophilum. We tested different primer sets tar-geting 16S rDNA, recA gene for rapid detection in real-time PCR. We used RFLPanalysis targeting 5S-23S intergenic spacer to differentiate Borrelia genospecies.To analyze the phylogenetic relationships between variants of A. phagocytophilum,we sequenced the PCR products of the 16S rDNA from our samples and createda phylogenetic tree.

Real-time PCR (RT-PCR) analysis of the recA gene (Fraser et al. 1997) is a rapidmethod for detection of B. burgdorferi s. l. with similar sensitivity as nested PCR(Pietila et al. 2000, Wang et al. 2003). We found the concordance between real-time PCR and RFLP analysis to be 97.6% (Kybicova et al. 2008). The melting curveanalysis permitted to differentiate B. garinii from B. afzelii and B. burgdorferi s. s.,but not to distinguish between B. afzelii and B. burgdorferi s. s., since the meltingtemperature difference can be as small as 1C (Mommert et al. 2001, Wang et al.2003, Casati et al. 2004). The RFLP method can confirm the real-time PCR resultsand is also able to distinguish between B. afzelii and B. burgdorferi s. s. (Derdakovaet al. 2003).

Our results (Hulınska et al. 2004) by RT-PCR are comparable with previ-ous examination of the HGA agent made with conventional PCR (Hulinskaet al., 2002) and with observations in Europe (Liz et al., 2000) and in the UnitedStates (Belongia et al., 1997), where HGA is endemic. The sensitivity of detec-tion of specific Anaplasma DNA in samples of infected animals using the sixdifferent RT-PCR methods was similar. We found that in naturally infected ani-mals, there was generally less detection with the TaqMan Ep80 probe then whenusing the primers alone, contrary to the results on animals infected purpose-fully (Pusterla et al., 1999b). Differences in the melting curve analysis betweenHGA and variants of A. phagocytophilum in different RT-PCR assays were so smallthat they could not be used for distinguishing these variants, similar to Borreliagenospecies. However, our results indicate that the fluorescence melting curveanalysis of PCR products can be used for a rapid detection of these agents (Wickiet al, 2000).

The direct sequence analysis of the 16S rDNA can be used for the detection ofclosely related Anaplasma species in humans and in animals (Chen et al., 1994, Liz

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et al., 2000). Sequence comparison of the 16S rDNA gene is recognized as one ofthe most powerful method for determining the phylogenetic relationships of bac-teria as showed by Wen et al., 2002. Our findings (Hulınska et al. 2004) confirmthat wild animals like deer and mice were infected with a similar variant of A. pha-gocytophilum as were ticks, cows and horses (Barlough et al., 1996). In the nestedRT-PCR reaction the ECC-ECB primer pair produced a 520bp amplicon and theGER3-GER4 primer pair produced a 150 bp amplicon, containing the species-specific signature sequences. The choice of these amplicons for direct sequencingallowed differentiating between the bacterial variants. The phylogenetic tree thatwe constructed classifies the bacteria obtained from wild and domestic animalsand tick as being closely related to the A. phagocytophilum genogroup.

Some authors (Petrovec et al., 1999, Liz et al., 2000, Wen et al., 2002) showeda high degree of similarity of partial 16S rDNA gene sequences of granulocyticA. phagocytophilum from ticks, rodents and deer with the HGA agent isolatedfrom humans. Others (Massung et al., 2002, Baumgarten et al., 1999) found fourvariants differing in two nucleotides from the HGA or E.equi. They reported thatmore than one strain of A. phagocytophilum exists both in the United States, andin Europe. We found differences between sequences of A. phagocytophilum DNAextracted from a deer, boar, red fox, bank vole, tick and two horses in only 2–6nucleotides.

In summary, our findings suggest that the exposure to B. burgdorferi s. l.a A. phagocytophilum is common in vectors, reservoirs and hosts in the CzechRepublic. In vectors (ticks), we detected similar prevalence of both bacteria.

We also found a similar level of prevalence of Borrelia and Anaplasma in reser-voir hosts (rodents). The majority of infections were caused by B. afzelii, while theinfection with B. burgdorferi s. s. was also quite frequent. Infection with B. burgdor-feri s. l. was more common in bank voles than in wood or yellow-necked mice.The prevalence of anti-Borrelia antibodies was higher in wood or yellow-neckedmice than in bank voles. We conclude that small wild rodents can serve as hostsfor B. burgdorferi s. l. and A. phagocytophilum in the Czech Republic.

We determined the occurrence of A. phagocytophilum in possible reservoirhosts. A. phagocytophilum was in a higher percentage of cases in the deer familyand hares, as compared to foxes and boars.

Finally, we detected similar prevalence of anaplasmosis in domestic animals,such as cows, horses and dogs. We demonstrated that symptomatic dogs hadhigher chance to be infected with A. phagocytophilum than asymptomatic dogs.

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Chapter 8

Conclusions

• The prevalence of both B. burgdorferi s. l. and A. phagocytophilum in ticks col-lected on animals was 6.8% and 9%, respectively. The prevalence of A. pha-gocytophilum in ticks from wild and domestic animals was very similar.

• In rodents, both B. burdorferi s. l. and A. phagocytophilum were found in 16.4%and 14.5% of cases, respectively. This corresponds to other results in Europereported in the literature.

• In wild rodents, the majority of infections were caused by B. afzelii, whilethe infection with B. burgdorferi s. s. was also quite frequent. To our bestknowledge, this is the first report of the presence of B. burgdorferi s. s. inrodents in the Czech Republic.

• Infection with B. burgdorferi s. l. was more common in bank voles than inwood or yellow-necked mice. However the prevalence of anti-Borrelia anti-bodies was higher in wood or yellow-necked mice than in bank voles.

• The Anaplasma positivity detected by PCR was higher in yellow-neckedmice than in bank voles.

• The infection rate of the deer family (12.5–15%), which is a possible reser-voir for A. phagocytophilum, was found to be much lower than reported else-where in the literature.

• The prevalence of A. phagocytophilum in all tested domestic hosts (cows,horses, and dogs) was similar, 3.4–5.5%.

• We found that the exposure to A. phagocytophilum is common in dogs in theCzech Republic.

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Chapter 8: Conclusions

• Symptomatic dogs had a higher chance to be infected with A. phagocytophi-lum than asymptomatic dogs.

• On the other hand, no significant correlation was found between clinicalsigns of anaplasmosis or borreliosis and positive antibody titers to A. pha-gocytophilum or B. burgdorferi s. l., respectively.

• The onset of clinical signs of borreliosis in dogs (April–July) correspondswith seasonal activity of ticks. The dogs infected by borreliosis suf-fered from neurological problems, renal failure, cardiovascular syndromes,inflammation, and anemia.

• Two of three infected dogs died, despite of antibiotic treatment, probablybecause the medical followup was prematurely terminated.

• Normally, borreliosis is associated with fever and orthopedic problems.However, two of the dogs did not suffer from fever and no dogs sufferedfrom orthopedic problems.

• Wild animals (foxes, deer, boars, hares and rodents), domestic animals(cows and horses), and ticks collected from them were infected with sim-ilar variants of the same A. phagocytophilum genotype. The similarity wasconfirmed by a phylogenetic tree constructed from direct sequence analysisof the 16 rDNA fragments.

• We have used real-time PCR to determine the presence of B. burgdorferi s. l.in rodent muscle tissue. It is a very fast method but cannot distinguishbetween Borrelia genospecies. For this purpose we have successfully usedRFLP analysis.

• We successfully adapted six published standard detection methods usingPCR for real-time PCR, obtaining an important improvement in speed.However, real-time PCR was not capable of distinguishing the A. phago-cytophilum variants.

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