University of Veterinary Medicine Hannover Department of ...€¦ · University of Veterinary...

University of Veterinary Medicine Hannover Department of Pathology

Aimara Bello

Casuistic evaluation of necropsied ratites

(Struthioniformes) in northwestern Germany

- a retrospective study -

Hannover 2016

Attachment 4a: § 9 paragraph 1 of the Doctoral Regulations

University of Veterinary Medicine Hannover

Casuistic evaluation of necropsied ratites

(Struthioniformes) in northwestern Germany

- a retrospective study -

INAUGURAL –DISSERTATION

in partial fulfillment of the requirements of the degree of

Doctor of Veterinary Medicine

-Doctor medicinae veterinariae –

(Dr. med. vet.)

Submitted by

Aimara Bello

Caracas, Venezuela

Hannover 2016

Academic supervision: 1. Prof. Dr. med.vet. W. Baumgärtner. Department of

Pathology TiHo

1. Referee: Prof. Dr. med.vet. W. Baumgärtner

2. Referee: Prof. Dr. med. vet. S. Rautenschlein

Day of the oral examination: 31.10.2016

Results of the Doctoral Thesis were partially presented in the following events:

Authors: A. Bello, S. Lapp, P. Wohlsein, W. Baumgärtner

Title (German): Retrospektive Untersuchung der Erkrankungs- und Todesursachen von 66

Laufvögeln (Struthioniformes).

Format: Oral presentation

Event: 57. Jahrestagung der Fachgruppe Pathologie der Deutschen Veterinärmedizinischen

Gesellschaft, 08. – 09.03.2014. Fulda, Germany.

Published in: Tierärztliche Praxis G 42 (2014) S. A9

To my family

“It is only with the heart that one can see rightly; what is essential is invisible to the eye.”

― Antoine de Saint-Exupéry, The Little Prince

T a b l e o f c o n t e n t s

Table of Contents

Chapter 1: Introduction 9

Chapter 2: Literature overview 10

2.1 Zoological classification 10

2.2 Most common diseases in ratites: overview 12

2.2.1 Diseases of the skin 12

2.2.2 Diseases of the musculoskeletal system 13

2.2.3 Diseases of the lymphoid and hematopoietic system 13

2.2.4 Diseases of the cardiovascular system 14

2.2.5 Diseases of the respiratory system 14

2.2.6 Diseases of the alimentary system 15

2.2.7 Diseases of liver and pancreas 16

2.2.8 Diseases of the urogenital system 17

2.2.9 Diseases of nervous system 17

2.2.10 Diseases of eyes and ocular adnexae 18

2.2.11 Systemic diseases 18

2.2.12 Diseases of the yolk sac 19

Chapter 3: Materials and Methods 20

3.1 Study design 20

3.2 Histopathology 20

3.3 Immunohistochemistry 21

Chapter 4: Results 22

Spontaneous diseases of captive ratites (Struthioniformes) in northwestern Germany:

a retrospective study 22

Chapter 5: Discussion

5.1 General aspects 62

5.2 Musculoskeletal system 62

5.3 Alimentary system 64

5.4 Cardiovascular system 66

5.5 Nervous system 68

T a b l e o f c o n t e n t s

5.6 Respiratory system 69

5.7 Urogenital system 70

5.8 Eyes and ocular adnexae 70

5.9 Systemic diseases 70

5.10 Conclusion 71

Chapter 6: Summary 72

Chapter 7: Zusammenfassung 73

Chapter 8: References 75

Chapter 9: Acknowledgements 102

List of abbreviations

A. Aspergillus

ABC Avidin–biotin-peroxidase complex

BDV Borna disease virus

C. Clostridium

CNS Central nervous system

DNA Deoxyribonucleic acid

EEE Eastern Equine Encephalitis

DIC Disseminated intravascular coagulopathy

E. Escherichia

EHV Equine herpesvirus

FOS Fading ostrich syndrome

GI Gastro-intestinal

M. Mycobacterium

ND Newcastle disease

PCR Polymerase Chain Reaction

PrP Prion protein

S. Struthiolipeurus

TBE Tick-borne encephalitis

TSE Transmissible spongiform degeneration

WEE Western Equine Encephalitis

C h a p t e r 1 : I n t r o d u c t i o n

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Chapter 1: Introduction

Ratites belong to the order Struthioniformes, and common ratite species include

ostriches (e. g. North African ostrich [Struthio camelus]), emus (Dromaius novaehollandiae),

rheas (e. g. Rhea americana), cassowaries (Casuarius casuarius), and kiwis (Apteryx

australis) (FOLCH 1992; STEWART 1994; TULLY 2009). In zoological collections, ratites

are very popular animals but in recent decades there has been a worldwide increase in farming

ostriches, emus and rheas because of their assumed healthy red meat or their versatile use of

the skin in the leather industry (TULLY 2009). Ratites represent an alternative livestock in

some countries (COOPER 2004) with the potential to expand in small areas that are

unproductive for other farm work (STEWART 1994). Ratite producers around the world are

currently developing a niche for the meat, hide and byproducts of these avian species.

Among the ratites, diseases of ostriches are best documented because of the

development of a breeder industry (KUMMROW 2015). However, compared to poultry, there

are differences in disease susceptibilities and relative prevalences. In addition, clinical and

morphological characteristics of many infectious diseases are also shared by psittacines,

waterfowl as well as other common companion and aviary birds (STEWART 1994).

Due to the increase and development of the ostrich and other ratites farming in

Germany since the end of the 1980s and the scarcity of literature on ratites disorders in

Germany, this retrospective study focuses on the analysis of gross and histopathological

findings and highlights rare salient lesions in different age groups of ratites that originated

from zoos and farm in northwestern Germany.

C h a p t e r 2 : L i t e r a t u r e o v e r v i e w

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Chapter 2: Literature overview

2.1 Zoological classification

Ratites are classified into four suborders within the order Struthioniformes regarding

the different geographic distributions: Struthiones (Africa), Rhea (South America), Cassuarii

(Australia, New Guinea), and Apteryges (New Zealand) (FOLCH 1992). Struthiones include

a single family, Struthionidae, with one species, Struthio, and four recognized subspecies, the

North African ostrich (Struthio camelus [S. c.] camelus) (Fig. 1), the Somali ostrich (S. c.

domesticus), the Massai ostrich (S. c. massaicus), and the South African ostrich (S. c.

australis).

Figure 1. North African ostrich (Struthio camelus camelus) (Courtesy of I. Hoppe)


11

There are two species in the family of Rheaidae within the suborder Rheae including

the common or greater rhea (Rhea americana) (Fig. 2) and the Darwin´s or Lesser rhea

(Pterocnemia pennata) (FOLCH 1992).

Figure 2. Common or greater rhea (Rhea Americana) (Courtesy of I. Hoppe)

The families Dromaiidae (emus) and Casuariidae (cassowaries) are closely related

and pooled in the common suborder Casuarii. Whereas emus (Dromaius novaehollandiae)

(Fig. 3) represent the only species, cassowaries constitute three distinct species (Southern

cassowary, Casuarius [C.] casuarius; Dwarf cassowary, C. bennetty; Northern cassowary, C.

unappendiculatus), with numerous subspecies (FOLCH 1992). Apteryges includes one

family, Apterygidae, with three species (Brown Kiwi, Apteryx [A.] australis, Little Spotted

Kiwi, A. owenii and Great Spotted Kiwi, A. haastii) (KUMMROW 2015).


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Figure 3. Emu (Dromaius novaehollandiae) (Courtesy of I. Hoppe)

2.2 Most common diseases in ratites: overview

Diseases of ratites, especially ostriches, are reviewed by several authors (STEWART

1994; TULLY and SHANE, 1996; HUCHZERMEYER 1998; SHANE 1998;

HUCHZERMEYER 1999; VERWOERD 2000; HUCHZERMEYER 2002; COOPER et al.

2004; TULLY 2009; BLACK and GLATZ, 2011; KUMMROW 2015).

2.2.1 Diseases of the skin

Skin peeling, loss of feathers and dermolytic blistering similar to a genetically related

mechanobullous skin disease like epidermolysis bullosa are described in juvenile ostriches

(PERELMAN et al. 1995). Feather picking occurs commonly in adult ostriches and emus kept

in small, barren paddocks possibly caused by malnutrition or environmental stress

(STEWART 1994; SAMSON 1997). Ingestion of parsley may result in photosensitivity

(KUMMROW, 2015).

Viral diseases of the skin include ostrich pox virus infection that is transmitted by

direct contact or via mosquitos (COOPER et al. 2004) and affects preferentially juvenile

individuals (ALLWRIGHT et al. 1994; RAIDAL et al. 1996; PERELMAN et al. 1988).

Mycotic infections of the skin with Trichophyton sp. and Microsporum sp. are observed

occasionally (ONDERKA and DOORNENBAL 1992; HUCHZERMEYER 1998).

Several ectoparasites affect ratites including feather lice (Struthiolipeurus [S.]

struthionis, S. rheae, S. nandu, S. stresemanni, Meinertzhageniella lata, Meinertzhageniella


13

schubarti, Dahlemhornia asymmetrica), thribs (Limothrips denticornis), and quill mites

(Gabucinia sp., Struthiopterolichus sp., Dermoglyphus parchycnemis) causing pruritus,

feather and skin damage (PONCE GORDO et al. 2002; COOPER 2005; NEMEJC and

LUKESOVA 2012). Hippoboscid flies (Struthiobosca struthionis) may irritate the animals

and act as disease vector (NEMEJC and LUKESOVA 2012). Various species of ticks

(Hyalomma sp., Amblyomma sp., Haemaphysalis sp., Rhipicephalus sp., Argas sp., Otobius

sp.) are known to infect ratites (CRAIG and DIAMOND 1996; KUMMROW 2015). A

fibroma is described in the skin of an ostrich (COOPER et al. 2010).

2.2.2 Diseases of the musculoskeletal system

Degenerative myopathy occurs particularly in young ratites and includes exertional

myopathy, selenium or vitamin E deficiency, furazolidone and ionophore intoxications (VAN

HEERDEN et al. 1983; VORSTER 1984; GREGORY et al. 1992; SPEER 1996; BAIRD et

al. 1997; MUSHI et al. 1998; WOTTON and HEWITT 1999; SMITH et al. 2005; MENON et

al. 2014). Prolonged recumbency may lead to muscle necrosis and tendon contraction

(STEWART 1994). Intramuscular injection of irritant substances, particularly certain

antibiotics, may result in severe myositis (HUCHZERMEYER 1999).

Deformities of legs appear at different stages of growth and include tibiotarsal or

tarso-metatarsal rotation, rolled toes, slipped tendons (perosis), and bowed legs (TERZICH

and VANHOOSER 1993; SPEER 1996; SAMSON 1997; SQUIRE and MORE 1998;

HUCHZERMEYER 2002; KUMMROW 2015). Retarded growth may be caused by vitamin

B (pantothenic acid) and A deficiency (SAMSON 1997; KUMMROW 2015). Disturbances of

the calcium, phosphorus and vitamin D3 metabolism may lead to insufficiently mineralized

bones, rickets (COOPER and GIMBI, 1994; SPEER 1996; MORROW et al. 1997).

In contrast to wing fractures, fractures of the legs occur commonly in ratites and are

associated with muscular degeneration, laceration, haemorrhages, local circulatory

disturbances and result occasionally in internal exsanguination (STEWART 1994). Additional

skeletal disorders include degenerative joint disease in ostriches and emus (SPEER 1996),

subchondral cysts, bone sequestration, and arthritis (BURBA et al. 1996, TULLY et al. 1996).

2.2.3 Diseases of the lymphatic and haematopoietic systems

Infectious bursal disease virus causes severe destruction of the lymphatic tissue of the

bursa of Fabricius (SPEER 1996). Severe depletion of lymphatic tissues is also observed in

adenovirus infection in ostriches (RAINES et al. 1997), Eastern Equine Encephalitis (EEE)


14

virus infection in emus (VEAZEY et al. 1994) and the fading ostrich syndrome (TERZICH

and VANHOOSER 1993). Lymphoid leukosis with multiple organ involvement is reported in

an ostrich (GARCÍA-FERNANDEZ et al. 2000).

Various protozoal parasites are reported in ratites affecting cells of the circulatory

system including Plasmodium sp. (FOX et al. 1996; COOPER 2005), Leukocytozoon sp.

(HUCHZERMEYER 1998), Babesia sp. (JEFFERIES et al. 2008) and Hepatozoon sp.

(BLACK and GLATZ 2011).

2.2.4 Diseases of the cardiovascular system

Endocardiosis of the mitral valves with congestive heart failure is documented in an

adult captive ostrich (KUBBA and AL-AZREG 2013). Myocardial degeneration due to

avocado (Persea americana var. guatemalensis) poisoning is described in ostriches

(BURGER et al. 1994). A Turlock-like bunyavirus may cause lympho-histiocytic and

plasmacytic myocarditis in ostriches (SHIVAPRASAD et al. 2002). Non-suppurative

myocarditis is seen after Western equine encephalitis (WEE) virus infection (SHANE 1998).

Dissecting aneurysms with spontaneous rupture are often located at the aortic arch

occurring especially in overweight yearling ostriches, but also in emus (MITCHINSON and

KEYMER 1977; STEWART 1994; VANHOOSER et al. 1994; BAPTISTE et al. 1997;

FERRERAS et al. 2001). Vasculitis occurs in emus infected with WEE and EEE viruses

(AYERS et al. 1994; TULLY et al. 1992). Increased vascular permeability with widespread

haemorrhages occurs after Senecio sceleratus poisoning (COOPER 2007a). An intrathoracic

haemangiosarcoma is recorded in an ostrich (HEADLEY 2005).

2.2.5 Diseases of the respiratory system

Various subtypes of avian influenza A virus (AIV) are described in ratites

(ALEXANDER 2000). AIV infection in ratites may cause respiratory (e. g. ocular and nasal

discharge) and gastro-intestinal (e. g. diarrhea) clinical signs. The pathogenicity depends

especially on the particular virus subtype. Morphological findings include mucoid to

pyogranulomatous sinusitis, purulent laryngitis, muco-purulent to necrotising tracheitis,

necrotising bronchitis and bronchiolitis, pyogranulomatous pneumonia, fibrinous air

sacculitis, necro-suppurative hepatitis, necrotising enteritis, splenomegaly and nephritis with

greenish coloured urine (STEWART 1994; SWAYNE et al. 1996; HUCHZERMEYER 1999;

WOOLCOCK et al. 2000). Paramyxovirus type 1 (Newcastle Disease [ND] virus) causes

pneumonia and air sacculitis in ostriches and emus (JØRGENSEN et al. 1998).


15

Bacterial infections of the respiratory tract occur predominantly under predisposing

conditions (hypothermia, stress, overcrowding, poor air quality) and may be caused by

Klebsiella sp., Escherischia (E.) coli, Pseudomonas sp., Corynebacterium sp., Pasteurella sp.,

Bordetella sp., Haemophilus sp. [infectious coryza], Staphylococcus sp., Chlamydophila

psittaci, Streptococcus sp. and Mycoplasma sp. (SAMSON 1997; BLACK and GLATZ

2011). Morphological findings are variable depending on the manifestation of the infection

and may include rhinitis, sinusitis, laryngo-tracheitis, pneumonia and air sacculitis

(MOMOTANI et al. 1995; SHANE and TULLY 1996).

Respiratory mycosis occurs in ratites kept in enclosed, dusty environments and is

mainly caused by Aspergillus (A.) fumigatus, but also A. niger and A. flavus. Infection results

in granulomatous air sacculitis and pneumonia (ASOK KUMAR et al. 2014; PERELMAN

and KUTTIN 1992; SHANE 1998; CHANG REISSIG et al. 2002; PÉREZ et al. 2003;

COPETTI et al. 2004; YOKATA et al. 2004; COOPER 2005; KHOSRAVI et al. 2008).

Parasitic infestations of the respiratory tract include Syngamus trachea (ostrich, emu),

Paronchocerca struthionus (ostrich), and Cyathostoma variegatum (emu) (STEWART 1994;

CRAIG and DIAMOND 1996; SHANE 1998; NEMEJC and LUKESOVA 2012).

2.2.6 Diseases of the alimentary system

Congenital malformations of the beak are seen in ostrich chicks (HUCHZERMEYER,

1998). Partial or complete impactions of the proventriculus, mainly occurring in animals less

than six months, may be acute (within a few days) or chronic (within weeks to months), and

comprise soft (plant fibers) or hard (sand, rocks) materials (STEWART 1994; SPEER 1996;

SAMSON 1997; HUCHZERMEYER 1999; KUMMROW 2015). Stasis of gizzard

contractions may secondary lead to proventricular food accumulation (HUCHZERMEYER

1999; OCAL et al. 2006). Ingestion of sharp foreign bodies is a common problem in ratites

possibly leading to perforations of the alimentary tract (SAMSON 1997). Displacements of

the intestinal tract comprise intussusception, intestinal torsion, and cloacal prolapse, mainly in

chicks; all disorders are mostly associated with other enteric disorders (STEWART 1994;

SHWALUK and FINLEY 1995; SPEER 1996; FRASCA and KHAN 1997; SAMSON 1997;

BERRIDGE et al. 1998).

Proventriculitis and ventriculitis may be caused by gastric impaction, Macrorrhabdus

ornithogaster (HUCHZERMEYER et al. 1993), or fungal infection such as zygomycetes or

Candida sp., the latter may also responsible for fibrinous pharyngitis and oesophagitis


16

(JEFFREY et al. 1994; STEWART 1994; SHANE and TULLY 1996; SHANE 1998;

HUCHZERMEYER 1999; GULBAHAR et al. 2000; COOPER 2005).

Enteritis is mainly observed in chicks and juvenile ratites caused by a multitude of

micro-organisms resulting in various forms of inflammation such as catarrhal, fibrinous,

ulcerative, necrotizing, granulomatous, haemorrhagic and proliferative. Avian influenza, EEE

virus, ND, adeno-, rota- and coronavirus represent viral causes for enteritis in ratites (HINES

et al. 1995; RAINES et al. 1997; JØRGENSEN et al. 1998; SHANE 1998). Pathogenic

bacterial species comprise E. fergusonii, E. coli, Salmonella sp., Klebsiella sp., Aeromonas

sp., Pseudomonas sp., Campylobacter jejuni, Clostridium (C.) perfringens, Serpulina-like

spirochetes now termed Brachyspira sp., Lawsonia intracellularis, Chlamydia sp.

(SAGARTZ et al. 1992; KOLB et al. 1993; BUCKLES et al. 1997; LEMARCHAND et al.

1997; SAMSON 1997; SHANE 1998; VERWOERD 2000; KWON et al. 2004; HERRÁEZ et

al. 2005; FRANҪA et al. 2009; MAPPLEY et al. 2014; KEOKILWE et al. 2015). Protozoal

organisms isolated from the gastro-intestinal (GI) tract of ratites include coccidia (e. g.

Cryptosporidium sp., Isosporum sp. and/or Eimeria sp.), amoebae (e. g. Entamoeba sp.),

ciliates (e. g. Balatidium struthionis), flagellates (e. g. Histomonas melagridis, Giardia sp.,

Trichomonas sp.), Blastocystis sp. and microsporidia (CRAIG and DIAMOND 1996; GRAY

et al. 1998; SHANE 1998; PONCE GORDO et al. 2002; COOPER 2005; NEMEJC and

LUKESOVA 2012; QI et al. 2014). Ratites may harbour also numerous GI metazoic parasites

comprising the trichostrongylid nematodes Libyostrongylus sp. found in the crypts of the

glandular portion of the proventricus and gizzard wall of ostriches, Odontospiruria cetiopenis

and Sicarius uncinipenis in rheas. Furthermore, Trichostrongylus tenuis causing haemorrhagic

typhlitis in emus, Codiostomum struthionis and Heterakis dispar found in ostriches,

Capillaria sp., Paradeletrocephalus and Deletrocephalus sp. in rheas have been documented.

The principal cestode of ostriches and rheas is Houttuynia struthionis (STEWART 1994;

SHANE 1998; HUCHZERMEYER 1998, 1999; CRAIG and DIAMOND 1996;

HUCHZERMEYER 2002; PONCE GORDO et al. 2002; COOPER 2005; ZETTERMANN et

al. 2005; SCHULZE et al. 2006; NEMEJC and LUKESOVA 2012)

2.2.7 Diseases of liver and pancreas

Necrotizing hepatitis is described following infections with adenovirus (RAINES et al.

1997), avian influenza virus (HUCHZERMEYER 2002; COOPER 2004), EEE virus

(VEAZEY et al. 1994), ND virus (JØRGENSEN et al. 1998), E. coli, C. sordellii, C. difficile,

C. perfringens, Campylobacter coli and Campylobacter jejuni subsp. jejuni (POST et al.


17

1992; POONACHA and DONAHUE 1997; STEPHENS et al. 1998; SHIVAPRASAD 2003;

COOPER 2005;). Fasciola hepatica causes chronic cholangitis in emus and rheas

(VAUGHAN et al. 1997; NEMEJC and LUKESOVA 2012). In rheas, cases of biliary

cystadenoma and carcinoma are described (DELL´ARMELINA ROCHA et al. 2013;

DELL´ARMELINA ROCHA et al. 2015).

Pancreatic atrophy, lympho-plasmacytic inflammation and ductal epithelial necrosis

associated with Cryptosporidium sp. are documented in poorly growth ostriches (JARDINE

and VERWOERD 1997).

2.2.8 Diseases of the urogenital system

Nephrocalcinosis may be associated with high dietary calcium intake in ostrich chicks

(MORROW et al. 1997).

Malformations of the phallus may result in infertility (HICKS-ALLDREDGE 1998).

Intersex in ostriches is common and due to the lack of oestrogen the male´s feather are black

coloured (STEWART 1994). Damage, infection and prolapse of the phallus may occur after

prolonged eversion, however, phallus prolapse is described in association with cloacal and

bursal infection with Cryptosporidium sp. (PENRITH et al. 1994). Testicular tumours occur

rarely in ratites (HICKS-ALLDREDGE 1998).

Conformational defects of the oviductal infundibulum may result in internal ovulation

with subsequent infertility. Egg binding may be caused by genetic factors, malnutrition, cold

weather or lack of exercise (HICKS-ALLDREDGE 1998). Ovarian torsion with necrosis and

aseptic peritonitis is reported in an ostrich (SUÁREZ-BONNET et al. 2012). Ratites may

suffer from oviduct infections (salpingitis), metritis, egg retention, vaginal and uterine

prolapse, and egg-related peritonitis. Peritoneal hernias located in the caudal abdominal cavity

allow the intestines and uterus to prolapse into the pericloacal region (STEWART 1994).

Common bacterial pathogens infecting the reproductive tract include E. coli, Pseudomonas

sp., Acinetobacter sp., and Salmonella sp. The latter may be vertically transmitted to eggs.

Cystic ovarian changes may occur when eggs are continuously removed from the hen

resulting in permanent gonadotropin stimulation (HICKS-ALLDREDGE 1998).

2.2.9 Diseases of the nervous system

A hereditary lysosomal storage disease is identified in emus as mucopolysaccharidosis

type IIIB resembling human Sanfilippo syndrome type B (BERMUDEZ et al. 1995; KIM et

al. 1996; BERMUDEZ et al. 1997; FREISCHÜTZ et al. 1997; GIGER et al. 1997;


18

ARONOVICH et al. 2001). A spongiform degeneration of brain tissue causes neurological

signs in three ostriches kept in German zoos (SCHOON et al. 1991 a; b). Encephalomalacia in

emus may be associated with hypovitaminosis E (AYE et al. 1998).

Viruses affecting the central nervous system of ratites causing non-suppurative

inflammation of meninges and/or neuroparenchyma include avian paramyxovirus (Newcastle

Disease), WEE virus, Borna disease virus and Turlock-like bunyavirus (RANDOLPH et al.

1994; ASHASH et al. 1996; SHANE and TULLY 1996; SHANE 1998; HUCHZERMEYER

1999; HUCHZERMEYER 2000; SHIVAPRASAD et al. 2002; COOPER 2004).

Botulism in ostriches comprises total paralysis, paresis and ataxia caused by an ostrich

carcass in the camp in which the birds were kept (ALLWRIGHT et al. 1994).

Verminous encephalitis in ratites is caused by Baylisascaris procyonis (KAZACOS et

al. 1982, KAZACOS et al. 1991) and Chandlerella quiscali (LAW et al. 1992).

2.2.10 Diseases of the eyes and ocular adnexae

Disorders of the eye may be caused by laceration, abrasion and ulceration due to direct

contact to objects (STEWART 1994), viral infections such as avian influenza and pox virus

(PERELMAN et al. 1988; ALLWRIGHT et al., 1993), bacterial infections including

Moraxella phenylpyruvica (SAROGLU et al. 2003; GÜREL et al. 2004), Mycobacterium

avium complex (POCKNELL et al. 1996; ŠEVČİKOVÁ et al. 1999; GARCÍA et al. 2001),

Staphylococcus hyicus subsp. hyicus (CHIRINO-TREJO and WHELER 1989),

Staphylococcus aureus and E. coli (SAHINDURAN 2004), Chlamydia psittaci (KÖSTERS et

al. 1996), as well as parasitic infestations in ostriches and rheas with the trematode

Philophthalmus gralli (GREVE and HARRISON 1980; MUKARATIRWA et al. 2005;

VEROCAI et al. 2009; NEMEJC and LUKESOVA 2012; CHURCH et al. 2013) and the

thribs Limothrips denticornis (COOPER 2007b). Ingestion of the toxic plant Lantana camara

caused severe conjunctivitis (COOPER 2007a). Cataracts commonly occur in older ostriches

(STEWART 1994).

2.2.11 Systemic diseases

High mortality in ostrich fetuses may be caused by malpositioning and severe

subcutaneous oedema during artificial incubation (STEWART 1994; BROWN et al. 1996;

SPEER 1996). The “fading ostrich syndrome” (FOS; Syn.: “chick fading syndrome”) that also

occurs in rheas (FERNÁNDEZ et al. 2013) is a multifactorial disease complex characterized

by growth retardation, weight loss, anasarca, ascites, delayed or inappropriate intake of food,


19

gastric stasis with accumulated ingesta, absence of faeces, yolk sac retention and increased

mortality in 1 to 3-week-old chicks (PHILBEY et al. 1991; STEWART 1994;

HUCHZERMEYER 2002). It is associated with improper management, stress and possibly

infectious pathogens such as circovirus (EISENBERG et al. 2003) or adenovirus (EL-

ATTRACHE et al. 2001).

The ostrich is the only avian host susceptible to Bacillus anthracis

(HUCHZERMEYER 1998). Infection results in septicaemia followed by sudden death,

splenomegaly, hepatomegaly, vascular congestion and tarry blood exuding from the body

orifices (SHANE 1998; THEILER 1912; VERWOERD 2000; COOPER 2005).

Infection with Erysipelothrix rhusiopathiae, described in emus (GRIFFITHS and

BULLER 1991; MORGAN et al. 1994) and ostriches (SHANE 1998), is characterized by

serosal petechiae, venous congestion, splenomegaly and hepatomegaly.

Mycobacteriosis is commonly caused by Mycobacterium (M.) avium, rarely by M.

bovis (KELLY et al. 2013), and may result either in localized lesions, e. g. granulomatous

inflammation of the cloaca, eye, phallus, or in a generalized infection with granulomas in the

GI tract, liver and spleen (SHANE et al. 1993; SHANE 1998; DONELEY et al. 1999;

HUCHZERMEYER 1998, 1999; GARCÍA et al. 2001; KELLY et al. 2013).

Paralysis with intestinal haemorrhages, multifocal hepatic necroses and splenomegaly

is described in ostriches associated with C. chauvoei infection (LUBLIN et al. 1993).

Septicaemia caused by Pasteurella multocida resulting in hepato-splenomegaly and vascular

congestion is rarely documented in ratites (OKOH 1980). Chlamydia psittaci infection may

cause generalized disease with high mortality rates in ostriches (HUCHZERMEYER 1999).

2.2.12 Diseases of the yolk sac

Omphalitis is seen in chicks less than 1 week of age, often as a result of incorrect

working conditions of the incubator and/or hatcher. Ascending bacterial infection may lead to

yolk sac infection. A persistent yolk sac occurs commonly in artificially raised ratites

associated with incorrect management conditions and poor hygiene leading to insufficient

uptake of nutrients and antibodies and, after decomposition of the yolk sac, to absorption of

bacterial toxins (SPEER 1996; HUCHZERMEYER 1998, 1999; STEWART 1994; BLACK

and GLATZ 2011). Occasionally exposure of small or larger parts of the yolk sac is

encountered which predisposes for suppurative bacterial infections (STEWART 1994; SPEER

1996; BLACK and GLATZ 2011). Transovarian or transoviductal infection with Salmonella

sp. may result in embryonic infection (SPEER 1996).

C h a p t e r 3 : M a t e r i a l a n d M e t h o d s

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Chapter 3: Materials and Methods

3.1 Study design

Data records and histological preparations of 71 captive ratites originating from

German zoological gardens and private farms in northwestern Germany were reviewed

retrospectively. Animals included 54 ostriches (Struthio camelus), 5 emus (Dromaius

novaehollandiae) and 12 rheas (Rhea americana) comprising 37 adults, 23 juveniles and 11

neonates and eggs. The animals were necropsied between 1968 and 2014 at the Department of

Pathology, University of Veterinary Medicine Hannover, and at the Chemisches und

Staatliches Veterinäruntersuchungsamt Arnsberg, North Rhine-Westphalia.

The body condition had been scored as good, moderate, poor and cachectic. Animals

in a good body condition revealed vast amounts of fat tissue within the furcular and

abdominal regions whereas animals with a moderate body condition showed reduced amounts

of corporal fat tissue. Animals in a poor body condition possessed only low amounts of fat

reserves frequently associated with mild pectoral muscle atrophy. In contrast cachectic

animals lacked fat reserves, displayed a serous atrophy of the coronal myocardial cardiac

coronary fat tissue and moderate to severe atrophy of the pectoral muscles. Detailed

information about the investigated animals including species, gender, age, nutritional status,

and pathologic findings is given in Annex Table 1.

This study was carried out in accordance to the German animal welfare act. All

animals were dead at the time of submission for necropsy and post-mortem examinations

were carried out by order of the operators of the zoological gardens and private farms to

investigate the causes of illness or death.

3.2 Histopathology

Samples for histopathology had been taken from the majority of animals and included

skin, musculoskeletal system, respiratory system, alimentary system, reproductive system,

urinary system, cardiovascular system, endocrine and lymphatic organs, nervous system and

eye. Samples had been fixed in 10% neutral buffered formalin, dehydrated and embedded in

paraffin wax according to standard laboratory procedures. Tissue sections of 3 μm thickness

had been mounted on glass slides (Engelbrecht Medizin- und Labortechnik GmbH,

Edermünde, Germany) and stained with haematoxylin and eosin (HE). Applied

histopathological stainings were performed according to standard procedures as described

(RIEDELSHEIMER and WELSCH 2010). In selected cases additional sections were stained

C h a p t e r 3 : M a t e r i a l a n d M e t h o d s

21

with HE, periodic acid-Schiff-(PAS) reaction, Gram, Congo red, von Kossa´s, Ziehl-

Neelsen´s, and Heidenhain's Azan trichrome stain, Grocott's methenamine silver, or Warthin-

Starry´s silver impregnation according to standard laboratory protocols (RIEDELSHEIMER

and WELSCH 2010).

3.3 Immunohistochemistry

For the detection of tick-borne encephalitis (TBE) virus, Borna disease virus (BDV),

equines herpesvirus (EHV), rabies virus, avian influenza virus (AIV), Lawsonia

intracellularis and Listeria monocytogenes pathogen-specific antibodies and the avidin–

biotin-peroxidase complex (ABC) method (Vectastain Elite ABC Kit, Vector Laboratories,

Burlingame, California) was performed as previously described (GUEDES and GEBHART

2003; SCHWAB et al. 2007; BRANDT et al. 2010; WOHLSEIN et al. 2011; NICHOLLS et

al. 2012).

C h a p t e r 4 : R e s u l t s

22

Chapter 4: Results

Spontaneous Diseases in Captive Ratites (Struthioniformes) in Northwestern

Germany: A Retrospective Study

Aimara Bello1¶, Samuel Frei1, 2¶, Martin Peters3, Anne Balkema-Buschmann4,

Wolfgang Baumgärtner1, Peter Wohlsein1*

1Department of Pathology, University of Veterinary Medicine Hannover Foundation,

Hannover, Lower Saxony, Germany.

2Zoo Wuppertal, Wuppertal, North Rhine-Westphalia, Germany

3Chemisches und Veterinäruntersuchungsamt Westfalen, Arnsberg, North Rhine-

Westphalia, Germany

4Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institute,

Greifswald, Insel Riems, Mecklenburg-Western Pomerania, Germany.

¶ These authors contributed equally to this work.

* [email protected]

Running head: Spontaneous Diseases in Captive Ratites


23

Abstract

A retrospective study was carried out to define the spectrum of spontaneous

diseases in captive ratites, order Struthioniformes, in northwestern Germany. The

investigation included 71 ratites necropsied between 1968 and 2014. They consisted

of 54 ostriches, 5 emus, and 12 rheas with 37 adults, 23 juveniles and 11 neonates

and eggs. Necropsy reports were reviewed, histologic preparations were re-

examined and additional histochemical and immunohistochemical stains were carried

out in selected cases. In many animals more than one morphologic diagnosis

attributable to different disease processes was found. In adult animals (n = 37), the

most commonly altered organ systems were the musculoskeletal system (49%), the

digestive system (46%), and the cardiovascular system (46%) affected by traumatic

lesions, inflammatory and degenerative changes, respectively. Lesions of the

nervous system (35%) included spongy degeneration; however,

immunohistochemistry and western blotting failed to detect accumulations of

pathological prion protein. In juvenile animals (n = 23), the musculoskeletal system

(44%) and the digestive system (43%) were predominantly affected by traumatic and

inflammatory lesions, respectively. In eggs and neonates (n = 11) the major cause of

death was the fading ostrich syndrome (64%). Summarized, most of the lesions

observed in adult and juvenile ratites living in northwestern Germany are related to

trauma, inflammatory and degenerative disorders, whereas in eggs and neonates the

fading ostrich syndrome was found predominantly. Surprisingly, a degenerative

encephalopathy with spongy degeneration of unknown cause and pathogenesis that

await further studies was found in adult animals.

Key words: emu, ostrich, pathology, ratites, retrospective investigation, rhea


24

Introduction

―Ratite‖ is not a strict taxonomic term, but it is used to refer to flightless birds that lack

a keel at their sternum and have a rather flat ―raft-like‖ breast. In general, ratites

belong to the order Struthioniformes, and common ratite species include ostriches (e.

g. North African ostrich [Struthio camelus camelus]), emus (Dromaius

novaehollandiae), rheas (e. g. the Common or Greater Rhea [Rhea Americana]),

cassowaries (e. g. Southern cassowary [Casuarius casuarius]), and kiwis (e. g.

Brown kiwi [Apteryx australis]) [1]. Interestingly, they are not a homogenous group

and show numerous anatomical and physiological differences [2, 3]. The natural

habitat of ratites is the southern hemisphere, i. e. ostriches in Africa, rheas in South

America, emus in Australia, kiwis in New Zeeland, and cassowaries in Australia and

New Guinea [3].

Historically, ratites have been very popular animals in zoological collections. In

addition, there has been a worldwide interest in farming ostriches, emus and rheas in

recent decades, because of the assumed healthy red meat and versatile use of the

skin in the leather industry, and recently they are appreciated as an alternative

livestock in some countries [3]. A concern was raised in the European Union (EU)

that ostriches, which are adapted to hot dry climates, would experience difficulties to

live under cold winter conditions [4]. The EU Council Directive 98/58 ―Concerning the

protection of animals kept for farming purposes‖ states that breeding of animals is

prohibited if it causes adverse effects on their health or welfare [4]. The EU also

recommended that the environmental conditions in areas where ostrich farms are

located should allow the birds to be kept outside most of the day in any season, to

satisfy their need for exercise and grazing [4].

Frequent infectious and non-infectious spontaneous diseases in ostriches and

other ratites have been reviewed [1, 3, 5-13], describing diseases that occur mostly

in ratites farmed in countries in and outside of their areas of origin. It was noted that

commercialization of ratite production goes along with increased disease frequency

and parasitism[8]. This increment was related at least partially to improper

management of farmed ratites in most cases [4]. Surveys about spontaneous

diseases of ratites in Europe are scarce [14]. Due to the increase and development


25

of ostrich and other ratites farming in Germany since the late 1980s, this

retrospective analysis focuses on the characterization of frequently observed gross

and histopathological findings and will highlight rare salient lesions in different age

groups of ratites held in captivity in northwestern Germany.

Materials and Methods

Cases records

The study was conducted on 71 ratites of the order Struthioniformes including 54

ostriches (Struthio camelus), 5 emus (Dromaius novaehollandiae) and 12 rheas

(Rhea americana) kept in northwestern Germany that had been submitted for

necropsy between 1968 and 2014. The records on medical history, animal data

(gender, age at time of death), macroscopic and histological findings were analyzed.

Furthermore, available results of additional investigations, such as

immunohistological, parasitological, microbiological and virological investigations

were included. The body condition had been scored as good, moderate, poor and

cachectic. Animals in a good body condition revealed vast amounts of fat tissue

within the furcular and abdominal regions whereas animals with a moderate body

condition showed reduced amounts of corporal fat tissue. Animals in a poor body

condition possessed only low amounts of fat reserves frequently associated with mild

pectoral muscle atrophy. Cachectic animals lacked fat reserves, displayed serous

atrophy of the coronary fatty tissue and moderate to severe atrophy of the pectoral

muscles. Detailed information about the investigated animals including case number,

animal identification number, species, age group, sex, nutritional status, husbandry,

manner of death and major pathologic findings is given in table 1. This study was

carried out in accordance to the German animal welfare act. All animals were dead at

the time of submission and the authors confirm that no animal was sacrificed for the

purpose of this study. Postmortem examinations were carried out to investigate the

cause of illness or death.


26

Histopathology

Each case was reviewed retrospectively, except case Nos. 7, 8 and 12, because

paraffin blocks and sections were not available any longer. Histologic sections,

stained with hematoxylin and eosin, were re-evaluated. In selected cases additional

sections were stained with hematoxylin and eosin (HE) or special stains including

periodic acid-Schiff (PAS) reaction, Gram, Congo red, von Kossa´s, Heidenhain's

Azan, Ziehl-Neelsen´s stain, Grocott's methenamine and Warthin-Starry silver

impregnation according to standard laboratory protocols [15].

Immunohistochemistry

For the detection of tick-borne encephalitis virus, Borna disease virus, equine

herpesvirus, rabies virus, influenza A virus, Lawsonia intracellularis and Listeria

monocytogenes, pathogen-specific antibodies were applied as previously described

[16-20], and the avidin–biotin-peroxidase complex method was used according to the

manufacturers´ instructions (VECTASTAIN Elite ABC Kit, Vector Laboratories,

Burlingame, California). For the detection of Brachyspira sp., a direct

immunofluorescence test using a polyclonal rabbit antibody against Brachyspira

hyodysenteriae was applied (kindly provided by Prof. G. Amtsberg, Institute of

Microbiology, University of Veterinary Medicine Hannover, Germany).

For the visualization of accumulated pathologic prion protein (PrP), paraffin

sections were autoclaved at 121°C for 20 min in citrate buffer, pH 6.0 after dewaxing

and incubation for 30 min in 3% H2O2 in methanol. The PrP-specific primary

monoclonal antibodies (mabs) F99/97.6.1 (VMRD, Inc.; Pullman, WA, USA)[21] or

mab 6C2 (Central Veterinary Institute Wageningen, NL) [22] were applied in Tris-

buffered saline with 10% goat serum and 0.03% sodium azide, at a concentration of

0.2 µg/ml and 0.7 µg/ml and incubated for 2 hours at room temperature. As negative

control, sections were incubated with 0.2 µg/ml of a monoclonal antibody directed

against glycoprotein GP5 of the porcine reproductive and respiratory syndrome virus

(PRRSV) used as a null-mab. As a conjugate, the avidin-biotin-peroxidase complex

method was used as described above. For both methods 3, 3′-diaminobenzidine-


27

tetrahydrochloride (DAB, Sigma-Aldrich, Munich, Germany) was used as chromogen.

Sections were counterstained with Mayer`s hematoxylin, and mounted.

Polymerase chain reaction

For the detection of genome fragments of Brachyspira hyodysenteriae and Lawsonia

intracellularis polymerase chain reactions (PCRs) were performed as previously

described [23].

Western Blot

In order to test the binding of mammalian PrP-specific antibodies to ratite-associated

PrP, 10% cattle, ostrich and rhea brain homogenates were prepared in Sucrose-

DOC-NP40 (0.42 M Sucrose, 0.5% deoxycholic acid sodium, 0.5% Nonidet P40).

Samples were mixed with gel loading buffer containing 6% sodium dodecyl sulphate

(SDS) and analyzed in a SDS-PAGE as described previously [24]. Mabs F99/97.6.1

and 6C2 were applied as detection antibodies at a concentration of 0.05 µg/ml and

0.07 µg/ml respectively, which are well established for the diagnostic of transmissible

spongiform encephalopathy in ruminants. Both antibodies bind to conserved regions

with little variation in the prion protein sequence between ruminants and ratites.

Results

Between 1968 and 2014, a total of 71 ratite including 54 ostriches, 5 emus and 12

rheas were submitted for pathological examination. Spontaneous death was

recorded in 19 adults, 17 juveniles and 10 neonates and eggs, whereas 8 adults, 5

juveniles and one neonate had been euthanized for humane reasons. For the

remaining 11 cases the mode of death was not recorded. 83% of the animals (n = 59)

were kept in zoological institutions and 17% (n = 12) in private farms in northwestern

Germany. 52% (n = 37) of the ratites were adults (older than 1.5 years), 32% (n = 23)

were juvenile birds (older than 5 days and under 1.5 years of age), and 15% (n = 11)

were neonates (under 5 days of age) or eggs.

The body condition was regarded as good in 57% (n = 41), as moderate in 10%

(n = 7), and as poor in 17% (n = 15). In 6% (n = 4) of the ratites a cachectic body

condition was noted and in 10% (n = 7) the body condition was not mentioned.


28

In many animals more than one morphologic change attributing to a different

disease processes was found.

Musculoskeletal system

Adult ratites

Diseases of the musculoskeletal system were diagnosed in 18 (49%) animals (case

Nos. 3, 5, 6, 8, 13, 21, 23, 32, 47, 48, 52 - 55, 61, 62, 70, 71). Traumatic injuries to

the legs and trunk associated with muscular degeneration, lacerations, hemorrhage

and local circulatory disturbances characterized by edema and vascular thrombosis

were present in 11 cases (case Nos. 3, 5, 13, 21, 23, 32, 48, 52, 54, 70, 71). Two

animals (case Nos. 32, 53) had fractured legs. One of these (emu No. 53) suffered

from an intraosseous fibrosarcoma (Fig. 1). Grossly, the left limb showed a focal

oblique pathologic fracture of the distal tarsometatarsus with concurrent lacerations

and hemorrhage in the adjacent muscles and skin. In the marrow cavity, a

fibrosarcoma with a proximo-distal extension of 9 cm was found. The tumor was

composed of interlacing bundles of moderately pleomorphic spindle-shaped cells

embedded in a collagen fiber-rich extracellular matrix and rare mitotic figures (Fig. 2).

Tumor-associated osteolysis of trabecular bone was multifocally observed.

Other changes comprised mild focal fibrino-purulent tarsitis and hip arthrosis in

one ostrich (case No. 8), articular gout in a rhea (case No. 48), and mild hyaline

muscular degeneration (case Nos. 11, 61, 62).

Juvenile ratites

Ten (44%) out of 23 juvenile animals presented morphological lesions in the

musculoskeletal system. Various forms of trauma were observed in six animals (case

Nos. 4, 15, 16, 28, 45, 50). Two ratites (case Nos. 4, 16) presented a fractured

vertebral column. One ostrich (case No. 15) showed multiple rib fractures and

swellings in the costochondral areas suggestive of rickets. Furthermore, slipped

tendons (perosis) with lateral displacement of the gastrocnemius tendon and

deformation of the distal tibiotarsal and tarsometatarsal bones were observed in two

ostriches (case Nos. 12, 14). In addition, two animals exhibited hyaline muscular

degeneration (case Nos. 26, 28) of unknown cause. Ostrich No. 60 showed


29

widespread hyaline degenerations of the musculus gastrocnemius, musculus

extensor metacarpi radialis, musculus iliotibialis lateralis and musculus elevator

caudalis as well as a degenerative cardiomyopathy associated clinically with severe

depression after transportation. The findings were indicative of capture myopathy.

Eggs and neonates

One neonatal ostrich (case No. 37) showed a traumatic fracture of the femur.

Digestive system

Adult ratites

Disorders of the alimentary system were observed in 17 (46%) out of 37 animals

(case Nos. 1, 3, 6, 17, 18, 21, 22, 32, 41, 48, 51 - 53, 56, 59, 61, 69). In one adult

rhea (case No. 41), proliferative enteritis with marked thickening of the mucosa (Fig.

3, 4), liquid dark brown intestinal content, infiltration of heterophils, macrophages,

lymphocytes and plasmacells, as well as severe hyperplasia of intestinal crypts was

found (Fig. 5, 6). Although Warthin-Starry silver impregnation and

immunohistochemistry for Lawsonia intracellularis antigen failed to detect the

pathogen, PCR identified genome fragments of Lawsonia intracellularis in samples

taken from the small intestine. In addition, Clostridium (C.) perfringens, C. sordellii,

Bacillus lentus, and Enterococcus durans were isolated from the large intestine of the

same animal. Moreover, severe enteritis was diagnosed in ostrich No. 6. In case

Nos. 48, 52, 56 and 59 intraluminal nematodes (not further identified) without

inflammatory reactions were observed.

Hepatic lesions comprised moderate to severe randomly distributed lympho-

histiocytic and lympho-plasmacytic hepatitis (case Nos. 21, 51, 53) as well as mild to

severe diffuse hepatic lipidosis (case Nos. 3, 32, 59, 61, 69).

Juvenile ratites

Disorders of the alimentary system were observed in 10 (43%) out of 23 animals

(case Nos. 9, 11, 31, 38, 58, 64 - 68).

Non-purulent and purulent inflammation of the glandular stomach was observed

in two cases. One rhea (case No. 66) showed multifocal mineralization of the mucosa

with associated lympho-plasmacytic infiltrates and one ostrich (case No. 68) had a


30

mild to moderate purulent proventriculitis associated with numerous intralesional

nematodes (not further identified) in the lamina propria mucosae.

Inflammatory changes of the intestine were found in four animals. In two cases

catarrhal and fibrinous enteritis (case No. 65, 67) and catarrhal typhlocolitis (case No.

38) were diagnosed. One rhea (case No. 31) suffered from fibrino-necrotizing

typhlocolitis characterized by numerous erosions with accumulation of cellular debris

as well as infiltration of heterophils and macrophages (Fig. 7, 8). Warthin-Starry silver

impregnation applied on the tissue sections and a Brachyspira-immunofluorescence

assay applied on feces revealed countless tortuous bacteria of up to 25 µm in length

(Fig. 8). Genome fragments of Brachyspira (B.) sp. were detected by PCR.

An intestinal intussusception with associated necrotizing and ulcerative

inflammatory changes and intralesional bacterial colonies were observed in an

ostrich (case No. 64). One ostrich (case No. 58) showed severe large intestinal

impaction of unknown cause with subsequent fibrinous serositis.

Eggs and neonates

Perforation of the muscular stomach wall due to ingestion of a sharp foreign body

associated with severe purulent and necrotizing inflammation of the gastric wall and

serosa was detected in one neonatal rhea (case No. 24).

Cardiovascular system

Adult ratites

Diseases of the cardiovascular system were diagnosed in 17 animals (46%).

Myocardial lesions were found in 7 and vascular changes in 10 animals, respectively.

In two ostriches and one rhea (case Nos. 2, 47, 60) a degenerative cardiomyopathy

with extensive hyaline degeneration of cardiomyocytes and histiocytic inflammation

was observed. In one ostrich (case No. 51), a severe chronic valvular endocarditis

with thrombus formation and intralesional Gram-positive bacteria was detected.

Ostrich No. 61 showed a focal moderate purulent epi- and myocarditis.

A female ostrich (case No. 57), weighing 80 kg, suffered from a severe left

ventricular hypertrophic cardiomyopathy with an absolute heart weight of 1.3 kg.

Streak-like changes were seen in the aortic intima consisting of fibrous tissue that


31

was interpreted as arteriosclerotic change. Morphological findings indicating

decompensation such as left atrial hypertrophy and dilatation, chronic pulmonary

congestion or effusions were not recorded.

A 15-year-old female ostrich (case No. 56), weighing 146 kg, died of hypovolemic

shock after rupture of a dissecting abdominal aortic aneurysm. Adjacent to the

rupture rims, the aortic wall showed multifocal mural calcifications and necrosis.

Perivascular hematomas, extensive granulation tissue and thrombosis of periaortic

blood vessels were additionally observed. Moreover, a moderate focal

granulomatous myocarditis was diagnosed in the same animal, but special stains

(Ziehl-Neelsen´s stain, PAS-reaction) failed to detect intralesional pathogens.

In eight cases (case Nos. 6 - 8, 19, 20, 22, 23, 33) a gradually variable

hypertrophy and hyperplasia of the tunica media and proliferation of smooth muscle

cells in the tunica intima of arterial vessel walls was diagnosed in various organs

(Fig. 9). In one ostrich disseminated intravascular coagulopathy associated with

multifocal intravascular fibrin thrombi of undetermined cause was seen (case No. 6).

Juvenile ratites

In one ostrich (case No. 9) disseminated intravascular coagulopathy associated with

meningoencephalitis and focal cortical encephalomalacia most likely caused by

hypoxia was found.

Nervous system

Adult ratites

In 13 (35%) out of 37 adult ratites lesions of the brain were detected. In 6 ostriches

(case Nos. 7, 8, 17, 19, 20, 34) and two emus (case Nos. 18 and 55) a degenerative

encephalopathy with spongy degeneration of various regions of the brain was found.

Reported clinical signs in ostriches Nos. 7, 8, 17 and 19 that were necropsied

between 1986 and 1993 comprised slowly progressive ataxia with disturbances of

balance, depression and anorexia. Ostrich No. 20 showed mild ataxia and possible

blindness. In addition, ostriches Nos. 17 and 19 exhibited unusual twisting of the

neck and increasing sternal recumbency. In animals Nos. 34 and 55 no nervous

signs were noticed. Histological findings in ostriches Nos. 17 and 19 consisted of


32

mild to moderate numbers of round to oval clear vacuoles in the perikaryon of

neurons of the substantia grisea centralis, nucleus nervi oculomotorii, and nucleus

reticularis pontis oralis and in the neuropil of the brain stem (Fig. 10). Spongy

changes were distributed bilaterally and symmetrically. The lesions of ostriches Nos.

7 and 8 have been described previously in detail [25, 26]. Briefly, severe vacuolation

was observed in the perikaryon of neurons of the nucleus ruber, nucleus vestibularis

and nucleus reticularis, and in the neuropil of the brain stem. In addition, a fine

granular, under UV light autofluorescent, PAS-positive and acid-fast pigment was

observed in the perikaryon of numerous neurons in the same animals interpreted as

lipofuscin.

In animals Nos. 34 and 55 single vacuoles admixed with a fine granular, golden-

brown, PAS-positive pigment interpreted as lipofuscin were present in the perikaryon

of some neurons of the brain stem (Fig. 11, 12).

The immunohistochemical evaluation of ostriches Nos. 17, 19, 34 and emu No.

55 failed to demonstrate specific PrP accumulations. Signals observed after

incubation of the sections with a null-mab directed against GP5 of PRRSV were

indistinguishable from those observed after incubation with both PrP-specific mabs.

Analysis of the binding affinity of the two PrP-specific mabs generally used for the

transmissible spongy encephalopathy diagnostics in ruminants (F99/97.6.1 and 6C2)

did not reveal PrP-specific bands to verify the binding of both antibodies to ratite PrP,

although especially in the case of mab 6C2, the binding epitope within the PrP

displays no mutation between ruminant and ratite PrP. In contrast, PrP-associated

bands were visible for the cattle brain sample loaded as a control (data not shown).

One emu (case No. 53) and one ostrich (case No. 54) with a clinical history of

acute trauma suffered from inflammatory lesions in the brain. In emu No. 53

moderate multifocal perivascularly accentuated granulomatous encephalitis was

detected predominantly in the telencephalon. However, Ziehl-Neelsen´s stain failed

to detect intralesional pathogens. In ostrich No. 54 moderate multifocal perivascular

lympho-histiocytic and plasmacytic encephalitis predominantly in the cerebellum and

in the brain stem was recorded. Immunohistochemical evaluations for tick-borne


33

encephalitis virus, Borna disease virus, equine herpesvirus, rabies virus, influenza A

virus and Listeria monocytogenes antigens remained negative in this case.

In ostriches Nos. 49, 52 and 59, mild to moderate golden-brown pigment

accumulations interpreted as lipofuscin were observed in the perikaryon of some

neurons of the brain stem.

Juvenile ratites

In 5 (22%) out of 23 juvenile ratites lesions of the brain were detected. Spongy

degeneration was found in three juvenile male ostriches (case Nos. 12, 29, 30).

According to a previous report [26], ostrich No. 12 that died in 1989 showed clinically

slowly progressive ataxia, including disturbances of balance and loss of appetite.

Lesions were characterized by severe vacuolation of the neuropil and few neurons of

the brain stem and medulla oblongata [26]. Ostriches no. 29 and 30 presented with

clinical signs of apathy, somnolence and opisthotonos. Mild intraneuronal

vacuolizations in Purkinje cells, the cortex cerebri and neuropil of the brain stem were

found. In addition, both animals were in a cachectic nutritional status.

Immunohistochemical evaluation failed to demonstrate specific PrP accumulations.

In ostrich No. 9 multifocal meningoencephalitis and focal cortical

encephalomalacia was found in association with disseminated intravascular

coagulopathy. One rhea (case No. 27) displayed severe diffuse granulomatous

inflammation of the choroid plexus without demonstrable pathogens using PAS-

reaction and Ziehl-Neelsen´s stain.

Respiratory system

Adult ratites

Seven (19%) out of 37 adult ratites showed significant changes of the respiratory

system. Findings included one case of mycotic rhinitis with numerous vascular

thrombi in an ostrich due to Aspergillus sp. (case No. 57) and one case of purulent to

necrotizing rhinitis in an ostrich (case No. 61). A rhea (case No. 62) with a history of

respiratory distress showed purulent laryngitis, multifocal to coalescing suppurative to

necrotizing laryngo-tracheitis (Fig. 13, 14) and lympho-plasmacytic parabronchitis.

Immunohistochemically intralesional influenza A virus nucleoprotein (Fig. 15) was


34

detected in trachea and lung. Molecular analysis performed at the Veterinary State

Reference Laboratory revealed subtype H7N7 of influenza A virus. Furthermore,

three ostriches (case Nos. 1, 34, 59) suffering from severe granulomatous

pneumonia with intralesional septated fungal hyphae indicative of Aspergillus sp.

were also recorded. One ostrich (case No. 17) showed a purulent aerosacculitis.

Juvenile ratites

In ostrich No. 68 nematodes (not further specified) were detected in the nasal cavity

associated with a severe diffuse ulcerative and purulent inflammation caused by a

secondary bacterial infection. Another ostrich (case No. 30) had a severe

granulomatous and necrotizing rhinitis with associated fungal hyphae, most likely

Aspergillus sp.

Eggs and neonates

In one neonatal ostriches (case Nos. 37) interstitial lympho-histiocytic pneumonia of

unknown etiology was observed.

Urogenital system

Adult ratites

Morphological changes of the urogenital system were present in 7 (19%) out of 37

adult ratites and included mild multifocal lympho-histiocytic interstitial nephritis (case

Nos. 21, 53, 55), multifocal tubulonephrosis and tubulonecrosis (case No. 2),

proliferative glomerulonephritis (case No. 62), and a renal adenoma in an adult rhea

(case No. 48). Cystic lesions in the testicle without other associated lesions were

detected in an ostrich (case No. 3).

Juvenile ratites

Mild multifocal tubulonephrosis was diagnosed in one ostrich (case No. 29.).

Systemic diseases

Adult ratites

In 3 (8%) adult animals (case Nos. 34, 49, 63) systemic diseases characterized by

granulomatous inflammations were observed. Using Ziehl-Neelsen´s stain

intralesional acid-fast bacterial rods were found in all cases. In ostrich No. 34,


35

granulomas were present in conjunctiva, lung, spleen, liver, ovaries and serosal

surface of the body cavity. In ostrich No. 49 granulomas were observed in spleen and

thymus. Microbiologic examination failed to isolate Mycobacterium (M.) sp. in both

cases. In emu No. 63, housed together with wallabies, M. avium subsp. avium was

identified in granulomatous lesions of trachea, lung, spleen and liver (Fig. 16, 17).

One of the wallabies was also reported to be infected with the same bacterium.

In two animals (no 34, 63) an additional granulomatous mycotic infection in lung, air

sacs and serosal surface of the body cavity characterized by intralesional septated

hyphae was observed.

Juvenile ratites

In ostrich No. 30, granulomatous inflammation with intralesional septated hyphae

most probably Aspergillus sp. was observed in nasal cavity, esophagus and body

cavity.

Eggs and neonates

Seven (64%; case Nos. 35, 36, 39, 40, 42 – 44) out of 11 ostriches with generalized

subcutaneous edema were diagnosed with the fading ostrich syndrome. In three

animals there was also a moderate to severe edema of the musculature present.

Additionally, purulent omphalitis (Fig. 18) most likely due to a bacterial infection was

found in two ostriches (case Nos. 35, 36). In ostriches Nos. 39 and 40, mild

mineralized concrements in renal tubuli were observed, and in ostriches Nos. 43 and

44 mild mineralizations were detected in the proventricular mucosa.

Miscellaneous disorders

Adult ratites

Thyroidal adenoma was found in an adult rhea (case No. 47).

Granulomatous dermatitis occurred in an ostrich (case No. 57), however, Ziehl-

Neelsen´s stain and PAS-reaction failed to detect intralesional pathogens. In two

ratites (case Nos. 34, 55) a granulomatous and proliferative conjunctivitis with

intralesional acid-fast bacterial rods and a lympho-plasmacytic conjunctivitis were

documented respectively. In one emu (case No. 18), a fibrino-purulent inflammation

of the body cavity was found and Haemophilus sp. was isolated from the lesion. One


36

emu (case No. 55) showed a bilateral retinal detachment associated with lympho-

plasmacytic uveitis of unknown etiology.

Discussion

In this retrospective study pathological findings and their frequency related to

different age groups of necropsied ratites in northwestern Germany are described.

The survey was conducted in order to elucidate the principal causes of disease in

various species including ostriches, emus and rheas kept in captivity.

Musculoskeletal system

Lesions of the legs and trunk with associated hemorrhage, muscular laceration and

degeneration most likely caused by traumatic impacts was the most prominent cause

of disease in adult ratites. The high number of traumatic injuries detected in adult

animals, supported by clinical evidence of trauma, may be caused by housing

situations on farms and in zoological institutions. Femoral and tibiotarsal fractures

frequently resulted in severe hemorrhages followed by hypovolemic shock and death.

The tibiotarsus is probably the most trauma-exposed bone of the ratite skeleton [27].

In addition, tarsometatarsal fractures are usually open with the risk of infection and

prolonged recumbency often resulting in muscular degeneration [7]. Injuries most

often occur when ratites are either chased or become frightened and run into fences

and other obstacles [6]. With the exception of one emu (case No. 53), evidence of

predisposing conditions for pathological fractures was not observed in adult ratites in

the current study. However other mechanisms or underlying causes due to metabolic

changes cannot be ruled out completely and would require additional investigations

including determination of calcium and phosphor values in the blood and bones. In

emu No. 53, a spontaneous pathological fracture due to an intraosseous

fibrosarcoma was found. Skeletal fibrosarcomas arise from connective tissue stroma

in either the medullary cavity, as in this case, or the periosteum. Fibrosarcomas of

different locations are among the most frequently observed neoplasm in pet birds

[28]. However, in ratites this variant has not been described before. Histologically,

this malignant tumor shares features with fibrosarcomas of other body sites and


37

species and is characterized by interlacing bundles of fusiform cells with vesicular

nuclei.

In ostrich No. 60, hyaline muscular degeneration (Zenker´s degeneration) of

various skeletal muscles was observed. With respect to the clinical history of

depression after transportation, this finding is suggestive of capture myopathy as

possible cause of disease and death. Capture myopathy, selenium or vitamin E

deficiency, furazolidone and ionophore intoxications are some of several etiologies

which reportedly cause degenerative myopathy in ratites [1, 7, 29, 30]. Clinical signs

in ratites include depression, reluctance to rise or move, and a rapid progression to

death. Transportation has been considered to be one of the most stressful

procedures in managing ratites [29, 31]. These bipedal animals with a height of up to

approximately 3 m in adult ostriches have their center of gravidity rather high above

the ground [29]. This causes difficulties in achieving a balance, particularly during

shipping on trucks. In addition, ratites are very nervous birds and highly susceptible

to capture myopathy, making their transportation on trucks more complex and

stressful than for food and companion animals like cattle and horses [29, 31].

However, in ostrich No. 60 an additional degenerative cardiomyopathy was present.

As exertional myopathy caused by transportation is reported to be restricted to

skeletal muscles [6] differential diagnoses for degenerative skeletal and cardiac

muscular disorders have to be considered including selenium deficiency. Although a

variety of drugs and chemicals are potentially myotoxic and cardiotoxic to birds, there

are very few documented cases. Severe cardiomyopathy in ostriches due to avocado

(Persea americana var. guatemalensis) intoxication was reported in South Africa

[32]. Moreover, in many animal species, ionophore toxicity characterized by focal

degenerative cardiomyopathy and skeletal muscle degeneration was reported as an

important cause for cardiomyopathies [33], however, myocardial damage has not

been reported in ostriches [34]. Further laboratory investigations were not performed

in the described case.

In juvenile ratites, two cases of slipped tendon were encountered. Slipped tendon

occurs in all ratite species, but ostriches and emus are most frequently affected [8].

Slipped tendon is a lay term used to describe the lateral displacement of the


38

gastrocnemius tendon from the normal anatomical location between the condyles of

the distal tibiotarsal and proximal tarsometatarsal bones [8]. Causes include genetic

predisposition and malnutrition (manganese, biotin, pantothenic acid, folic acid and

choline deficiency) as possible predisposing factors [8, 35]. Elevated selenium levels

as well as a lack of copper and calcium were suggested to play a role in the

development of slipped tendon in ostrich chicks [36]. In the cases presented here, no

essential nutrients or mineral values were determined.

There are additional metabolic conditions that can affect the bone quality

especially in juvenile ratites. In the current study, one juvenile ostrich (case No. 15)

showed lesions suggestive of rickets and several rib fractures that were most likely

the result of trauma and inadequate mineralized bone. Similar lesions have been

described in rheas [37]. Fibrous osteodystrophy caused by secondary

hyperparathyroidism has been described in birds and can also lead to weakening of

bones with subsequent fractures [38-40].

Digestive system

In this study, gastro-intestinal disorders were the major cause of disease in juvenile

ratites. Common factors that can cause enteritis in chicks and juveniles are failure to

establish a balanced gut flora, the use of antibiotics, lack of fibers in the diet,

hypothermia, excessive coprophagy, and poor hygiene [1, 41]. Bacterial pathogens

causing enteritis include E. coli, Salmonella sp. Klebsiella sp., Aeromonas sp.,

Pseudomonas sp., certain strains of Campylobacter jejuni and Clostridium (C.)

perfringens may also be involved [1, 3, 8, 41-43]. In the current study, Lawsonia

intracellularis, C. perfringens, C. sordellii, Bacillus lentus and Enterococcus durans

(case No. 41), as well as Brachyspira sp. (case No. 31) were identified in diseased

ratites.

Lawsonia intracellularis is the sole species in the bacterial genus Lawsonia and

is often found in pigs, where it causes a proliferative enteropathy [44]. Other affected

species include hamsters, rabbits, ferrets, foxes, dogs, rats, sheep, deer, emus,

ostriches, nonhuman primates, and guinea pigs [12]. This disease is characterized

macroscopically by thickening of the intestinal mucosa due to enterocyte


39

proliferation. As a result, mild to severe diarrhea represents the major clinical sign

described in affected animals [12]. Histologically, thickened villi, crypt hyperplasia,

dilated glands filled with mucus and heterophils, and a dense infiltration of

heterophils, lymphocytes, macrophages and plasma cells in the lamina propria are

morphological findings reported in emus [12]. The intestinal changes in rhea No. 41

of this study correspond to the described lesions and PCR confirmed the presence of

Lawsonia intracellularis, which represents the first case in this ratite species.

In a juvenile rhea (case No. 31) a severe fibrino-necrotizing typhlocolitis with

intralesional spirochetes was found. Immunofluorescence and molecular analyses

confirmed the presence of Brachyspira sp. which is regarded as the causative agent

of disease in this animal. The fastidious, anaerobic spirochete Brachyspira is capable

of causing enteric disease in avian, porcine and human hosts, amongst others, with a

potential for zoonotic transmission [45]. Rheas naturally colonized with B.

hyodysenteriae commonly show severe typhlitis and mortality can be as high as 80%

[46, 47]. Severe disease is characterized by dilation of the caeca with thickened walls

and ulceration, severe mucosal necrosis, crypt elongation and goblet cell hyperplasia

[45]. Lesions were most common in rheas less than 3 months of age. Stress after

shipping was proposed as a contributing factor [46]. The rhea in this case was 3

months old which corresponds well with published findings [46].

Intussusception is often seen in ostrich chicks and juveniles following gastric

impaction. Impaction with foreign body material is associated with abnormal

compensatory picking (disorientation stress, desertion stress, or frustration caused

by finding food from change of location or diet) [1]. Other causes of intestinal

intussusception are chronic diarrhea, frequently associated with other intestinal

disorders such as gastro-intestinal displacement, torsion and volvulus, and abnormal

peristaltic contractions due to unknown reasons [8]. In the described ostrich (case

No. 64), no underlying cause could be found for the intussusception.

One case of poor body condition associated with mild to moderate purulent

inflammation of the glandular stomach associated with infestation of numerous

nematodes was diagnosed in one ostrich (case No. 68). A considerable number of

endoparasites can infect ratites and gastro-intestinal nematodes are of highest


40

importance as they cause economic losses in ratite farming [8, 48]. Nematodes such

as Libyostrongylus (L.) douglassii, L. dentatus and L. magnus are found in glands of

the proventriculus and under the coilin layer of the gizzard of ostriches [42, 48]. Only

the former two have been reported outside Africa [49] with L. douglassi also infecting

ostriches in Germany [50] and The Netherlands [51]. The resulting inflammation

inhibits gastric secretions and hinders digestion. The disease is called ―vrootmag‖ or

―rotten stomach‖ as food decays within the stomach and can lead to weight loss and

high mortality in ostrich chicks and juveniles [7, 8, 48]. Rheas can also be infected,

although the parasite seems to be rather host specific [1].

In one young rhea (case No. 24) perforation of the muscular stomach was

diagnosed, presumably due to ingestion of a foreign body. Ingestion of foreign bodies

is a common problem in ratites [8, 41], because these animals will swallow anything

that fits into their mouths, and their curiosity and enquiring eye ensure that they will

find many unusual items in their enclosure [8].

Cardiovascular system

Reports on cardiovascular disease in ratites are scarce and some of the vascular

changes described here were most likely incidental findings with no associated

clinical signs. One of the well described vascular disorders in ostriches is aortic

aneurysm. One adult ostrich in this study (case No. 56) was diagnosed with a

dissecting aortic aneurysm with degenerative changes of the aortic wall which

probably was a predisposing factor along with assumed hypertension. Several cases

of aortic aneurysm have been described in the literature so far in which no

atherosclerotic changes of the aorta were associated with the rupture site, however,

copper deficiency that predisposes to defective elastin was suggested as a possible

cause [52-55] which is also described in turkeys [56]. Other causes might be

systemic hypertension and a genetic predisposition [53]. In the presented case, no

copper values were determined.

Atherosclerosis is the most important vascular lesion in birds especially in parrots

and lesions can be found within the aorta, brachiocephalic trunks, and pectoral and

carotid arteries [57]. Atherosclerotic lesions in psittacines are characterized by


41

progressive accumulation of inflammatory cells, cholesterol, fat and cellular debris in

the intima and luminal side of the vascular media. These changes are classified into

seven lesion types of which the atheroma (type IV lesion) covered by a fibrous cap

(type V lesion) are both features of advanced lesions and are often associated with

clinical signs [58]. Complications of plaques include mineralization, ulceration,

superimposed thrombosis, intraplaque hemorrhages and aneurysmal dilation [33].

The vascular lesions identified in this study (case Nos. 6 - 8, 19, 20, 22, 23 and 33)

were characterized by gradually variable thickening of the tunica media of arteriolar

vessel walls of parenchymatous organs like kidney and spleen. These changes did

not resemble atherosclerosis and were regarded most likely as incidental findings.

In ostrich No. 57, a considerable hypertrophy of the myocardium was found with a

relative heart weight of 1.63%. The reference range of the absolute heart weight in

ostriches of about 122 kg body weight is reported to be 1.054 g +/- 172 g

corresponding to an absolute heart weight of 0.86% (+/- 0.14%). The cause of the

cardiac hypertrophy in the study case remains undetermined, however, evidence of

decompensation was not found. In the same animal streaks of fibrous tissue were

present in the aortic intima. Whether these vascular changes were pathogenetically

related to the cardiac disorder or represent a separate disease process remains

undetermined. Abnormal jets of blood flow or turbulences have to be considered as

possible causes.

Nervous system

The principal change encountered in the central nervous system was a spongy

degeneration in six adult ostriches (case Nos. 7, 8, 17, 19, 20, 34), two emus (case

Nos. 18, 55) and three juvenile ostriches (case nos. 12, 29, 30). The clinical and

morphological findings of three of these animals (case Nos. 7, 8, 12) from two

different zoological collections in Germany have already been described [25, 26]. In

case Nos. 17 and 19 the lesions were observed bilaterally in the brain stem,

specifically affecting the substantia grisea centralis, nucleus nervi oculomotorii, and

nucleus reticularis pontis oralis. The lesions are regarded most likely as cause of the

clinical nervous signs.


42

Spongy degeneration in domestic animals may affect either gray or white matter,

or both, in a bilateral and symmetrical fashion and is caused by cytotoxic edema

resulting in intracellular fluid accumulation due to disrupted osmoregulation [59].

Possible underlying mechanisms include toxins, gene defects and infectious as well

as metabolic processes. The cause of the spongy degeneration observed in the

present study remained undetermined. In case Nos. 7, 8 and 12 material was not

available for additional investigations on the role of pathologic prion proteins. The

temporal coincidence of case Nos. 7, 8, 12, 17 and 19 during the years 1986 to 1993

with the emergence of bovine spongiform encephalopathy in the United Kingdom and

the subsequent occurrence throughout many European countries since 1990 raised

the possibility of a causal link between both diseases. So far all investigations failed

to proof a link between transmissible spongy encephalopathy and the central nervous

system lesions in ratites [60]. Although the presented cases share clinical and

morphological features described for transmissible spongiform encephalopathies in

mammalian species, the application of two different PrP-specific antibodies that are

routinely used for the diagnostics of ruminant transmissible spongiform

encephalopathy revealed no accumulation of pathological PrP. However, the binding

affinity of the applied antibodies to ratite PrP could not be irrevocably demonstrated.

Spongy degeneration of unknown cause involving the gray matter has been

described in domestic animals [61]. However, there is little information concerning

similar changes in the central nervous system of birds. In addition, no indications for

inherited, toxin-related, dietary-related or metabolic disturbances that could result in

similar nervous lesions were found in the present cases.

In ostriches Nos. 34 and 55 the observation of marked lipofuscin deposits in

neurons and the absence of nervous signs suggest an age-related spongy

degeneration. Neuronal vacuolation in case No. 29 may possibly be related to the

poor nutritional condition that might be caused, at least in theory, by nutritional

deficiencies, metabolic disorders, and intoxications [60].

In one emu and an ostrich (case Nos. 53 and 54), inflammatory lesions of the

brain were found to be associated with multiple hemorrhages on the head and neck

most likely leading to circulatory failure. Presumably, acute neurologic disturbance


43

was the cause of the polytrauma in ostrich No. 54. The cause of the encephalitis

remained undetermined in both cases, but an infectious agent, most likely a virus,

has to be considered in these animals. Ratites are susceptible to a number of viruses

that affect the central nervous system such as Borna disease virus and avian

paramyxovirus [8]. Newcastle disease has been reported as an important contagious

notifiable viral infection of ostriches characterized by neurological clinical signs [6].

However, microscopic changes are non-specific and include hemorrhages of the

heart and an enlarged liver [5]. None of these morphological changes were observed

in the current case, and due to the lack of neurological clinical signs in this bird, an

infection with Newcastle disease virus was regarded as unlikely.

Respiratory system

Respiratory diseases were recorded in adult ostriches and rheas including two cases

(case Nos. 57, 61) with rhinitis, one case (case No. 62) with tracheitis, laryngitis and

parabronchitis due to influenza A virus infection, and three cases (case Nos. 1, 34,

59) with mycotic granulomatous pneumonia.

Fungal infections of the respiratory organs occur most frequently in neonates and

juveniles housed in enclosed facilities and exposed to dust or hay which is

alternatively wet or dry [42]. Aspergillus fumigatus and other Aspergillus sp. are

commonly isolated and can also affect older birds, commonly in conjunction with

immunosuppression or concurrent disease [1, 42]. Grossly, yellow to white

granulomas are observed most frequently in the lung and/or air sacs, rarely also in

viscera [42]. Fungal granulomatous rhinitis restricted to the nose as in case No. 57

has been described seldom [62].

Ostriches, emus and rheas are highly susceptible to avian influenza, and various

subtypes have been identified including H7N1, H5N9, H5N2, H9N2 and H10N7 [1].

Natural infection with the subtype H7N7 has not been documented in ratites before.

Avian influenza A virus causes respiratory (e. g. ocular and nasal discharge) and

gastro-intestinal (e. g. diarrhea) clinical disease [63]. Other signs are ruffled feathers,

and complications arising from secondary infections [63]. However, there are no

specific clinical signs linked with avian influenza infection in ratite species.


44

Characteristic postmortem findings include fibrinous aerosacculitis, mucoid sinusitis,

multifocal hepatic necrosis, splenomegaly and nephritis [3]. Rhea No. 62 showed

suppurative to necrotizing tracheitis, laryngitis, parabronchitis, and purulent hepatitis

most likely caused by secondary bacterial infection. The first influenza A virus

isolated from ratites was the highly pathogenic avian influenza A strain H7N1. It had

caused an epizootic in ostriches in South Africa in 1991 in which high mortality was

seen among young birds [63]. Young ostriches infected with this strain showed

multifocal hepatic necrosis, splenomegaly and, in advanced cases, nephrosis and

fibrinous aerosacculitis [8]. The same strain of avian influenza A virus was also

isolated from ostriches in The Netherlands, Denmark, Italy, and from emus and

cassowaries in The Netherlands [8, 63, 64]. The appearance of viruses of the same

strain in various European countries and in South Africa may be caused by

movement of infected domestic poultry and/or products as well as by wild and

migratory birds. Furthermore, the increase in international trade in ostriches and

other ratites may have contributed to the spread of influenza A virus [63]. For these

reasons, the observation of avian influenza A virus infection in a rhea from

Northwestern Germany is not surprising.

Ocular disorders

In ostrich No. 34, conjunctival manifestation as part of a systemic avian

tuberculosis was found which has been observed in ratites [65] [66]. In case of an

isolated mycobacterial lesion of the conjunctiva an entry of the organism through an

eyelid defect has to be considered. Systemic spread after primary conjunctival

infection cannot be excluded.

Urogenital system and miscellaneous disorders in other organs

Alterations observed in the urogenital system, endocrine glands and skin in animals

of this study were diagnosed as secondary or incidental findings without great clinical

importance.


45

Systemic disease

Three adult ratites showed granulomatous lesions in multiple organs with

intralesional acid-fast positive bacteria indicative of mycobacteriosis, presumably an

infection with M. avium subsp. avium. The susceptibility of ratites to avian

tuberculosis remains unknown. It has been rarely reported in ratites as compared to

other avian species [67]. The disease has been described mainly in emus and rheas

and occasionally in ostriches [67]. Tuberculosis has great potential economic

significance in emus, first due to loss of valuable birds and second due to the risk of

transmission to other avian and mammalian species [65]. Infections with other

species of mycobacteria are rarely reported in ratites. In one case, M. bovis was

isolated from granulomatous lesions in an ostrich [67].

In ratites, the disease occurs more commonly in multispecies exhibition places like

zoos than on single species farms [6]. One emu (case No. 63) was affected by

granulomatous lesions of the trachea, spleen and liver. The distribution of

granulomatous lesions suggests an oronasal infection as the possible portal of entry.

This animal was housed in a zoo together with wallabies, one of which was

diagnosed with mycobacteriosis caused by M. avium subsp. avium. Based on this

finding a spill-over infection of mycobacteria is likely.

In eggs and neonates, the most common cause of disease and death was the

fading ostrich syndrome (FOS). FOS occurs in animals of less than 3 weeks of age

and is a multifactorial disease complex that relates to improper incubation and

rearing conditions, and possibly viral infections. Affected chicks often hatch with

increased generalized edema, are weak, reluctant or unable to exercise, and show

delayed or inappropriate intake of food [68]. FOS is also characterized by depression,

anorexia, and death 3 to 5 days after onset of clinical signs in ostriches of less than 3

weeks old [69]. Except for the interstitial edema in the musculature the animals in this

study showed no morphological changes of the musculature which is in contrast to

mild, degenerative myopathy reported in another study [70]. Additionally, depletion of

fat reserves is also reported [71]. However, it is possible that this syndrome may refer

to more than one clinical condition or cause [68]. The causes of FOS are not known.

Inadequate management practices and husbandry may act as contributing factors of


46

these conditions. Vertically transmitted viral agents, such as adeno- or circoviruses,

which cause a similar syndrome in turkeys, may act as primary pathogens [72, 73].

Two ostrich adenovirus isolates (isolate 1781 and B492) have been found to be

noninfectious in inoculated ostrich chicks less than 8 weeks of age [71]. In addition,

E. coli and/or Klebsiella pneumoniae were isolated from various organs of affected

neonatal ostriches [68]. Chicks from the same study also showed a severe

lymphocytic depletion of the bursa of Fabricius [68], which was not a feature in this

study. Circovirus that possibly is transmitted vertically, has been suspected to be

involved in FOS on a farm in the Netherlands and a vertical transmission was

suspected [72].

As accidental findings, mineralized tubular concrements in the kidney were

observed in two neonate ostriches (case Nos. 39, 40). Mineralized tubular

depositions are frequently observed in embryos and unhatched eggs [8]. Mineral or

crystalline deposits comprise either calcium or uric acid. In low amounts these

deposits are regarded as clinically irrelevant [8]. The commonly known causes in

chicks include hypervitaminosis D3, and in embryos incubation-related dehydration

with subsequent urate accumulation in the tubules [8].

Multifocal mineralization of the proventriculus was detected in two other ostriches

(case Nos. 43 and 44). Similar deposits may occur in cases with hypercalcemia and

renal failure. However, widespread calcium deposits, e. g. pulmonary elastic fibers or

renal tubular basement membranes were not present in both cases and the cause of

the gastric mucosal mineralization remains unclear.

Conclusion

The high frequencies of trauma including fractures of the long bones, as well as

traumatic injuries of the neck of juvenile and adult captive ratites living in

northwestern Germany underline the susceptibility of ratites to mechanical injuries.

The cause of spongy changes of the central nervous system observed in adults and

juvenile animals in association with central nervous signs remains obscure. A similar

disorder is not known in wild ratites, and hereditary or exogenous toxic causes have

to be considered.


47

In juvenile animals, bacterial infections of the alimentary tract were an important

cause of disease in the present study. Whereas these disorders occur only very

rarely in wild ratites, they are often reported in ratites of this age group held in

captivity, even in their natural habitat.

Fading ostrich syndrome is a worldwide disease occurring in farm husbandry and

in zoological institutions. Among various factors particularly inadequate management

practices such as improper incubation conditions for eggs contribute to this disorder.

In addition, some salient lesions like intraosseous fibrosarcoma, isolated mycotic

rhinitis, infection with influenza A H7N7, that have not or only rarely been described

in ratites extend the spectrum of disorders in these species.

Acknowledgements

We thank Mrs. Buck, Mrs. Grünig, Mrs. Herrmann and Mrs. Schütz for the excellent

technical assistance. A. Bello received a grant from ―Fundación Gran Mariscal de

Ayacucho‖, Venezuela and from ―Deutscher Akademischer Austauschdienst‖

(DAAD), Germany. This study was in part supported by the Niedersachsen-Research

Network on Neuroinfectiology of the Ministry of Science and Culture of Lower

Saxony.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research,

authorship, and/or publication of this article.

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55

Figure legends

Figure 1-2. Intraosseous fibrosarcoma, left tarso-metatarsal bone, emu, case No. 53.

Figure 1. Adjacent to the gray-white neoplastic tissue that occupies partially the

marrow cavity (asterisk) the oblique fracture line of the bone is visible (arrow). Figure

2. The fibrosarcoma is composed of spindle-shaped neoplastic cells embedded in

abundant amounts of collagen fibers. Hematoxylin and eosin (HE).

Figure 3-6. Proliferative enteritis caused by Lawsonia intracellularis, intestine, rhea,

case No. 41. Figure 3. Red-brown discoloration of the intestinal mucosa with fleshy

appearance of the duodenal wall. P = pancreas. Figure 4. Marked thickening of the

intestinal mucosa (asterisks). Figure 5. Subgross magnification reveals severe

elongation of the intestinal crypts. HE. Figure 6. Severe crypt hyperplasia (asterisks)

and moderate inflammatory infiltration of the adjacent lamina propria. HE.

Figure 7-8. Fibrino-necrotizing typhlocolitis caused by Brachyspira sp., intestine,

rhea, case No. 31. Figure 7. Brown-greenish tinged fibrin attached to the cecal

mucosa (arrows). Figure 8. Multifocal fibrino-necrotic mucosal lesions (asterisks).

HE. Inset: Immunolabelling of spirochetes using a polyclonal antiserum against B.

hyodysenteriae. Immunofluorescence.

Figure 9. Hypertrophy and hyperplasia of the tunica media, cardiac artery, rhea,

case No. 22. Marked increase in thickness of the tunica media (asterisks). HE.

Figure 10. Spongy degeneration, brain, ostrich, case No. 19. Oval to spherical

vacuoles of variable diameter (arrows) are present in the perikaryon of several

neurons of the red nucleus. HE.

Figure 11-12. Spongy degeneration and lipofuscin deposition, brain, ostrich, case

No. 34. Figure 11. Oval vacuole (arrowhead) and deposition of a coarse granular

golden-brown pigment (arrow) in the perikaryon of some brain stem neurons. HE.


56

Figure 12. PAS-positive coarse granular pigment deposition suggestive of lipofuscin

(arrow) in a brain stem neuron. PAS-reaction.

Figure 13-15. Avian influenza, larynx and trachea, rhea, case No. 62. Figure 13.

Severe necro-suppurative laryngo-tracheitis with diffuse hyperemia and numerous

white, necrotic foci. Figure 14. Severe, diffuse, necro-suppurative tracheitis. HE.

Figure 15. Intralesional immunolabelling of influenza A virus nucleoprotein (arrows).

Immunohistochemistry.

Figure 16-17. Avian mycobacteriosis and aspergillosis, trachea and lung, emu, case

No. 63. Figure 16. Multifocal yellow granulomas (arrows) in the trachea caused by

M. avium subsp. avium and focal white granuloma caused by Aspergillus sp.

(arrowhead). Figure 17. Necro-granulomatous pneumonia. HE.

Figure 18. Fading ostrich syndrome, subcutis, ostrich, case No. 35. Generalized

subcutaneous edema (anasarca) and purulent omphalitis (arrow).


57

Figures


58


59


60


61

C h a p t e r 5 : D i s c u s s i o n

62

Chapter 5: Discussion

In this retrospective study about necropsied ratites kept in northwestern Germany the

pathological findings and the results of additional etiological investigations are described. The

frequency of lesions is related to different age groups. In many animals more than one

morphological diagnosis attributable to different disease processes was found.

5.1 General aspects

Ostriches represent with more than three-quarters the predominant ratite species of the

retrospectively evaluated necropsy cases indicating that they are probably the most prevalent

species in northwestern Germany. More than 80% of the cases originated from zoological

collections. Approximately 65% of the reviewed cases died spontaneously and about half of

the cases were adult individuals. The body condition was regarded as good in almost half of

all investigated animals which might indicate that fatal diseases or disorders requiring

euthanasia are of acute courses instead of chronic debilitating illnesses.

5.2 Musculoskeletal system

In adult ratites, alterations of leg, neck and trunk bones associated with soft tissue

injuries were most likely caused by traumatic injuries and represent the most frequent cause

of disease. This high incidence of trauma-related lesions in adult ratites has also been

observed in other studies (KÖSTERS et al. 1996). When ostriches and other ratites are chased

or frightened they run into fences or obstacles possibly favored by unsuitable housing

situations on farms and in zoological institutions (HUCHZERMEYER 1998). Fractures of

femur, tibiotarsus or tarsometatarsus may result in severe hemorrhage leading to fatal

hypovolemic shock. Surgical setting of leg fractures is extremely difficult and these cases

often end up in euthanasia (HUCHZERMEYER 1998).

A predisposing lesion for a fracture was found only in one individual, i. e. emu No. 53.

It suffered from an intraosseous fibrosarcoma with subsequent pathological fracture. In ratites

this tumor variant has generally not been described before. Avian fibrosarcomas are most

frequently seen in pet birds (REAVILL 2004) and may occur anywhere on the body,


63

particularly in the oral cavity, associated with long bones, or in the abdominal cavity

(LIGHTFOOT 2006).

Systemic muscular degeneration in ratites may be caused by muscular overexertion,

nutritional muscular dystrophy related to selenium or vitamin E deficiency, furazolidone and

ionophore intoxications (STEWART 1994; BAIRD et al. 1997; MUSHI et al. 1998;

WOTTON and HEWITT 1999; MINKA and AYO 2008; MENON et al. 2014; KUMMROW

2015). Pale discoloration of the skeletal musculature is present at gross examination and

swelling, vacuolation, fragmentation, interstitial edema and necrosis of muscle fibers are

observed histologically (MUSHI et al. 1998). Chronic lesions are characterized by firm white

streaks representing dystrophic calcifications. In an 11-month-old ostrich (case No. 60),

muscular degeneration and death was suggestive of capture myopathy because the animal was

submitted with a history of transportation. Transportation is considered to be one of the most

stressful procedures in managing ratites (WOTTON and HEWITT 1999; MINKA and AYO

2008). However, the ostrich No. 60 suffered additionally from a severe multifocal myocardial

degeneration. Similar to monensin intoxication (BAIRD et al. 1997), capture myopathy is

reported to be restricted to the skeletal muscles (HUCHZERMEYER, 1998). Therefore, in

this case a nutritional muscular and cardiac degeneration most likely related to selenium or

vitamin E deficiency has alternatively or additionally to be considered, because this disorder

involves skeletal musculature, myocardium and the gizzard muscle (HUCHZERMEIER

1998). Furthermore, it cannot be excluded that the acute manifestation is simply based on a

subclinical chronic vitamin E and selenium deficiency triggered by transportation because an

insufficient supplementation is more commonly observed in young ratites due to higher

demands required for growth (STEWART 1994). Vitamin E and selenium values were not

determined in that case.

Slipped tendon, a lay term used for the lateral displacement of the gastrocnemius

tendon, occurred in juvenile ratites. In domestic fowl a similar disorder is termed “perosis”,

however, in this disorder the displacement of the tendon is medial (HUCHZERMEIER 1998).

Slipped tendon is reported in all ratite species, but ostriches and emus are most frequently

affected. Genetic predisposition and malnutrition (manganese, biotin, pantothenic acid, folic

acid and choline deficiency associated with deformation of the hock joint) are regarded as

possible predisposing factors in relation with a triggering trauma (MACWHIRTER 1994;


64

SPEER 1996). However, essential nutrient values were not determined in the cases of this

study.

Inadequate mineralization of bone may be caused by rickets due to a lack of vitamin D

supplementation (HUCHZERMEIER 1998) or hypophosphatemia (GRÖNE et al. 1995;

MORROW et al. 1997). Animals most likely affected by rickets, like case No. 15, are

typically juvenile and display soft bone tissue with fractures. As differential diagnosis fibrous

osteodystrophy caused by secondary hyperparathyroidism has to be considered (SCHMIDT

2015).

5.3 Alimentary system

In this study, gastro-intestinal disorders were the major cause of disease in young

ratites. Perforation of the stomach as in the young rhea No. 24 was presumably caused by

ingestion of a sharp foreign body. Ratites consume or try to consume most items they can

reach resulting in gastro-intestinal impaction or perforation often resulting in death. Examples

of swallowed foreign bodies include large amounts of earth, stones, wood, straw, thorns,

sharp metal objects, nails, bullets, cartridge cases, handkerchiefs, gloves, pencils, and wire

pieces, in one case up to 2.5 m of barbed wire (DEGEN et al. 1989; STEWART 1994; SPEER

1996; SAMSON 1997; HUCHZERMEIER 1998). Causes of foreign body ingestion are

overgrazing of the pasture, insufficient grazing, insufficient energy intake, lack of fiber in the

diet, desertion and disorientation stress (HUCHZERMEIER 1998).

Gastric endoparasitosis was observed in a juvenile ostrich (case No. 68) associated

with severe purulent inflammation of the proventriculus and cachexia. The localization of the

parasites is typical for the hematophagous trichostrongylid nematode Libyostrongylus sp. that

already has been described in ostriches in Germany (SCHULZE et al. 2006). This wireworm

is considered as a primary pathogen in ostriches. The parasite lives in deep mucosal glands of

the glandular stomach and under the koilin layer of the muscular stomach. Disruption of the

gastric barrier results in secondary bacterial infections and suppurative inflammation

(HUCHZERMEYER 1998, 2002).

Intestinal intussusception was observed in one poorly nourished juvenile ostrich (case

No. 64). Invagination of the intestine occurs occasionally in ostrich chicks (KEFFEN 1984;

MUSHI et al. 1998) and has also been described in rheas (FRASCA and KHAN, 1997).


65

Predisposing causes include enteritis that may be frequently associated with other intestinal

disorders such as gastro-intestinal displacement, torsion or volvulus, as well as tumors,

parasites and abrupt diet change with higher fiber content (STEWART, 1994). However, in

some cases, as in ostrich No. 64, the cause of abnormal peristaltic contractions remains

undetermined (KEFFEN 1984; SHANE and TULLY 1996).

Bacterial pathogens associated with enteritis in the current study include Lawsonia

intracellularis, C. perfringens, C. sordellii, Bacillus lentus and Enterococcus durans (case

No. 41), and Brachyspira sp. (case No. 31).

Lawsonia intracellularis has been described as cause of proliferative enteritis among

many species including ostriches (COOPER et al., 1997) and emus (LEMARCHAND et al.

1997). In this study, a rhea, case No. 41, showed morphological findings similar to those

described in the literature for other ratite species. The demonstration of genome fragments of

Lawsonia intracellularis in the intestinal content suggests a causal association with the

morphological changes which represents the first documented case in this avian species.

Infections of various ratite species with this not-host-specific pathogen may have

epidemiological implications, particularly in zoological collections with multi-species

exhibitions in the same enclosure.

Brachyspira sp. is considered as the causative pathogen of a severe fibrino-necrotizing

typhlocolitis with intralesional spirochetes in a juvenile rhea (case No. 31). The manifestation

in juvenile individuals as well as the morphological findings are similar to those already

described in rheas (SAGARTZ et al. 1992; BUCKLES et al. 1997; KUTZER et al. 2007), that

generally are termed avian intestinal spirochetosis (MAPPLEY et al. 2014). Spirochete-

associated enteritis causes high mortality rates up to 80% in rhea flocks (SAGARTZ et al.

1992). B. hyodysenteriae and B. pilosicoli have been identified in affected rheas (KUTZER et

al. 2007; MAPPLEY et al. 2014). However, in the case of this study detailed molecular

analysis of the causative agent was not performed. Transmission may occur by contact

between pigs and ratites. However, in cases without evidence of contact to pigs or pig manure

(KUTZER et al. 2007), feral birds, rodents and domestic animals that may harbour

Brachyspira sp. or contaminated water have to be considered as possible source of infection

(MAPPLEY et al. 2014).


66

The aetiology of the lympho-histiocytic and lympho-plasmacytic hepatitis observed in

three animals (case Nos. 21, 51, 53) remains undetermined. Infectious and non-infectious

causes have to be considered including intoxications, viral or bacterial infections.

5.4 Cardiovascular system

A degenerative cardiomyopathy with extensive hyaline degeneration of

cardiomyocytes and histiocytic inflammation was found in two ostriches and one rhea (case

Nos. 2, 47, 60). Vitamin E and/or selenium deficiency has to be considered as primary cause

in cases with skeletal muscle involvement (case No. 60), because ionophore intoxication and

capture myopathy in ratites typically lack cardiac degeneration (BAIRD et al. 1997;

HUCHZERMEIER 1998). The cause of the degenerative cardiomyopathy in the other two

cases remains undetermined. Possible aetiologies include local perfusion disturbances,

chronic myocarditis or intoxications, e. g. avocado (Persea americana var. guatemalensis)

poisoning (BURGER et al. 1994).

Valvular endocarditis with intralesional Gram-positive bacteria was detected in an

adult ostrich (case No. 51) indicative for a systemic bacterial infection. However,

involvement of other organs and tissues was not recorded and a portal of entry was not

evident in this case.

An adult ostrich in this study (case No. 56) died due to rupture of a dissecting aortic

aneurysm. As predisposing factor degenerative changes of the aortic wall were found in this

case. Aortic rupture occurs in growing and adult ostriches and may be associated with

overfeeding and lack of exercise (HUCHZERMEIER 1998). Furthermore, copper deficiency

has been incriminated to play a role in the pathogenesis (VANHOOSER et al. 1994;

BAPTISTE et al. 1997; FERRERAS et al. 2001). Hormonal influences, diet, lathyrism, zinc

deficiency, pharmaceuticals, and parasites have to be considered as additional factors (Van

VEEN 1999).

One ostrich (case No. 57; 80 kg body weight) suffered from a left ventricular

hypertrophic cardiomyopathy with an absolute heart weight of 1.3 kg corresponding to a

relative heart weight of 1.63%. The reference range for the absolute heart weight in adult

ostriches (122.1 ± 3.9 kg body weight) is reported to be 1.054 kg (± 172.34 g) with a relative

heart weight of 0.86% (+/- 0.14%) (TADJALLI et al. 2009). Hypertrophic cardiomyopathy


67

has been associated with degenerative valvular changes described as endocardiosis in adult

ostriches (KUBBA and AL-AZAREG 2013). However, semilunar and atrio-ventricular valves

in the case of this study displayed no morphological alterations and the cause of the

hypertrophic cardiomyopathy remained undetermined. Furthermore, morphological findings

indicating decompensation such as atrial hypertrophy and dilatation, chronic pulmonary

and/or hepatic congestion or effusions were not recorded. In addition, linear intimal elevations

in the aorta consisting of plaque-like fibrous tissue were observed in ostrich no. 57 and are

interpreted as plaque-like intima sclerosis or arteriosclerotic plaques (ROBINSON and

ROBINSON 2016). Similarly, the pathogenesis of this vascular change remained

undetermined.

In several animals of this study hypertrophy and hyperplasia of vascular smooth

muscle cells with variable extent were found in different organs. Increased blood pressure and

platelet- or monocyte/macrophage-derived growth factors are important triggering factors for

muscular hypertrophy and proliferation (ROBINSON and ROBINSON 2016). However, due

to the lack of other cardiovascular-related lesions these changes were regarded most likely as

incidental findings.

Disseminated intravascular coagulopathy (DIC) represents a pathological activation of

the coagulation system resulting in systemic consumption of clotting factors. Numerous

bacteria, helminths, protozoa, viruses, toxins, as well as particular conditions like neoplasms,

neoplasia, gastric dilation and volvulus, are known to induce DIC. Morphological findings in

animals that die of DIC include hyaline thrombi, granular thrombi consisting of platelets and

hyaline globules/”shock bodies” derived from fibrin degradation products (ROBINSON and

ROBINSON 2016). In the presented case (case No. 9), E. coli and Salmonella typhimurium

were cultured from the liver and lung suggesting septicaemia with concurrent DIC as the

possible cause of hypoxia and encephalomalacia observed in this juvenile ostrich. Gram-

negative bacterial endotoxins can cause endothelial damage, which exposes subendothelial

collagen and leads to activation of platelets and Hageman factor (ROBINSON and

ROBINSON 2016).


68

5.5 Nervous system

The principal change encountered in the central nervous system (CNS) was a spongy

degeneration in six adult ostriches (case Nos. 7, 8, 17, 19, 20, 34) two adult emus (case Nos.

18, 55) and three juvenile ostriches (case nos. 12, 29, 30). Clinical signs together with the

morphological findings in case Nos. 7, 8, 12, 17 to 20 during the years 1986 to 1994 and the

simultaneous emergence of bovine spongiform encephalopathy in the United Kingdom with

subsequent spread in Europe raised the possibility of a causal link between both diseases.

However, so far no causal relationship between bovine transmissible spongiform

encephalopathy and the central nervous system lesions in ratites was detected (WELLS et al.

2006). In addition, in this study different PrP-specific antibodies that are routinely used for

the diagnostics of ruminant transmissible spongiform encephalopathy did not show an affinity

to ratite PrP. Therefore, the aetiology of the spongy degeneration in ratites remains

unresolved. As alternate causes for spongy CNS changes toxins, gene defects and infections

as well as metabolic processes have to be considered.

In two cachectic juvenile ostriches (case Nos. 29, 30) apathy, somnolence and

opisthotonus occurred prior to death. Histologically, a spongy degeneration of the brain was

observed in the cerebral cortex, brain stem and cerebellum. Whether the clinical signs are

related to the CNS changes remains undetermined. In addition, the aetiopathogenesis of the

CNS lesions is unclear. A causal relationship to the poor nutritional status cannot be excluded

completely. In case Nos. 34 and 55, no nervous signs were reported and the spongy changes

were interpreted as possible age-related findings similar to neuronal lipofuscin accumulations.

In few other cases (case Nos. 49, 52 and 59) lipofuscin depositions were present without

spongy lesions in the CNS.

In one adult emu (case Nos. 53) and one adult ostrich (case No. 54), inflammatory

lesions of the brain were found associated with multiple haemorrhages on the head and neck.

Presumably, acute neurologic disturbance may have led to polytrauma in case No. 54. The

cause of the encephalitis remained undetermined in both cases, but an infectious agent, most

likely a virus, has to be considered.


69

5.6 Respiratory system

Respiratory diseases were recorded in ostriches and rheas including four cases of

rhinitis (case Nos. 30, 57, 61, 68), one case of tracheitis, laryngitis and parabronchitis (case

No. 62) due to influenza A virus infection, and three cases of granulomatous pneumonia (case

Nos. 1, 34, 59).

In two cases (case Nos. 30, 57) mycotic rhinitis characterized by ulcerative,

granulomatous and necrotizing inflammations was found. As causative pathogen most likely

Aspergillus sp. has to be considered. Infection is usually contracted by inhalation of spores

from moulds growing in the environment, especially on rotten feed (HUCHZERMEIER 1998,

1999). In ostrich No. 57, the infection was restricted to the nasal cavity, which is unusual and

has been described in ratites only rarely (FITZGERALD and MOISAN 1995).

In three adult ostriches (case Nos. 1, 34, 59) and one adult emu (case No. 63)

granulomatous pneumonia with intralesional septated fungal hyphae indicative of Aspergillus

sp. was recorded. In case Nos. 34 and 63 mycotic pneumonia represents a concurrent

infection to a mycobacterial infection. Respiratory mycosis occurs preferentially in young

ostriches (COOPER 2005) and other ratites, but can also affect older birds under

immunosuppression or suboptimal management conditions (HUCHZERMEYER 1998;

KUMMROW 2015).

In ostrich No. 68 an ulcerative and purulent rhinitis was associated with intralesional

nematodes (not further specified). A nematode that specifically inhabits the nasal cavity of

ratites is not known. Therefore, an aberrant localization of a ratite parasite or an infection with

a facultative parasite similar to Halicephalobus deletrix in horses and humans (ONDREJKA

et al. 2010) has to be considered.

In rhea No. 62 an infection with low pathogenic influenza A virus H7N7 had caused a

severe laryngo-tracheitis and parabronchitis. Ratites are highly susceptible to avian influenza,

and various subtypes have already been isolated including H1N2, H3N2, H4N2, H4N6,

H5N2, H5N9, H6N2, H7N1, H7N3, H9N2, H10N4 and H10N7 (ALEXANDER 2000;

ABOLNIK et al. 2012), however, the low pathogenic subtype H7N7 has not been reported in

ratites so far. Morphological lesions and distribution of viral antigen share similarities with

findings reported in ostriches infected with other low pathogenic subtypes like H10N7

(WOOLCOCK et al. 2000). Wild and migratory birds may play an important role in


70

transmitting the virus by co-mingling with ratite flocks (SHANE and TULLY 1996). In

addition, the direct or indirect contact to domestic poultry and/or products may be implicated

in the spread of the disease (VERWOERD 2000).

5.7 Urogenital system

In the urogenital system, degenerative and inflammatory changes of mainly mild

extent were found and interpreted as secondary or incidental findings without great clinical

importance.

5.8 Eyes and ocular adnexae

Granulomatous conjunctivitis caused by Mycobacterium sp. infection in ostrich No. 34

was part of a disseminated mycobacteriosis. Conjunctival manifestation of systemic avian

tuberculosis in ratites is not unusual (POCKNELL et al. 1996; GARCÍA et al. 2001).

However, as differential aetiologies of conjunctival granulomas foreign bodies

(ŠEVČİKOVÁ et al. 1999), infections with fungi (HUCHZERMEIER 1998), or Morexella

phenylpyruvica (GÜREL et al. 2004) and granulomatous dacryoadenitis (SAROGLU et al.

2003) have to be considered.

Bilateral retinal detachment was recorded in an adult emu (case No. 55). The cause of

this disorder remained undetermined. Retinal detachment may be trauma-associated

(WILLIAMS 1994), but it has also been described as idiopathic bilateral ocular disorder in

pheasants (RANDALL et al. 1986).

5.9 Systemic diseases

Three adult ratites animals (case Nos. 34, 49, 63) showed granulomatous inflammation

of multiple organs with intralesional acid-fast positive bacteria indicative for mycobacteriosis,

presumably an infection with Mycobacterium avium subsp. avium. However, bacteriological

investigation failed to isolate the pathogen. In the emu (case No. 63) a spill-over from or to

wallabies housed together with the ratites is likely, because one wallaby was also infected

with the same bacterium. Typically, ratites are infected by repeated ingestion of the pathogen,

which is present in soil, water and feed. Avian mycobacteriosis may result in an atypical form

with diffuse enlargement of affected organs, a lepromatous form with circumscribed


71

subcutaneous lesions, and a tubercular form characterized by discrete granulomas

(DONELEY et al. 1999). The latter type was present in the cases of this study.

Fading ostrich syndrome (FOS), also termed “chick fading syndrome”, represented the

most common cause of disease and death observed in this study among eggs and neonates.

This syndrome occurs exclusively in animals held in captivity and is characterized by

generalized oedema (anasarca), depression, weakness, anorexia, reluctance or unability to

exercise, and high mortality rates 3 to 5 days after onset of clinical signs in ostriches of less

than 3 weeks (PHILBEY et al. 1991; KUMMROW 2015). There are several most likely

concurrent factors causing FOS including inadequate management practices and husbandry

like improper incubation and rearing conditions, and possibly viral infections such as

circovirus infection or immunosuppressive mycotoxins (SPEER 1996; EISENBERG et al.

2003; KUMMROW 2015). In addition, isolation of E. coli and/or Klebsiella pneumoniae

from various organs including umbilicus, yolk sac, liver and air sacs, has been regarded as

criterion for FOS (TERZICH and VANHOOSER 1993). Subsequently, chicks often develop

enteritis, yolk sac retention and infection, gastric stasis and impaction, bacterial hepatitis and

enteritis, fungal proventriculitis, pneumonia and septicemia (SPEER 1996; KUMMROW

2015).

5.10 Conclusions

In summary, the diseases in captive ratites that originated from zoos and farms in

northwestern Germany presented a broad spectrum of various disorders. The high frequencies

of musculo-skeletal and alimentary alterations as well as numerous cases of FOS are most

likely related to housing and management conditions. However, the retrospective casuistic

work-up revealed also disorders that have been not or only rarely described in ratites

including mycotic rhinitis, intraosseous fibrosarcoma, Lawsonia intracellularis infection in

rheas, spongy CNS degeneration, or infection with subtype H7N7 of avian influenza A virus.

Knowledge about ratites diseases is gaining particular importance in future because these

flightless birds are expected to be used more and more in commercial farming in Germany.

C h a p t e r 6 : S u m m a r y

72

Casuistic evaluation of necropsied ratites (Struthioniformes) in

northwestern Germany

- a retrospective study - By: Aimara Bello

Chapter 6: Summary

The spectrum of spontaneous diseases was retrospectively analysed in 71 captive

ratites including ostriches, emus, and rheas kept in zoos or farms in northwestern Germany.

Reports and histological slides of the animals necropsied between 1968 and 2014 were

reviewed, additional histo- and immunohistochemical stainings were performed and findings

were interpreted with respect to virological, microbiological and parasitological results.

In many animals more than one morphologic diagnosis attributable to different disease

processes was found. In adult ratites, the most commonly altered organ systems were the

musculoskeletal, the digestive, and the cardiovascular systems affected by traumatic lesions,

inflammation and degenerative changes, respectively. The high frequency of traumatic lesions

underlines the susceptibility of ratites to mechanical injuries. In one ostrich an intraosseus

fibrosarcoma, that has not been described in ratites so far, represented a predisposing lesion

for a pathological leg fracture. Lesions observed in the nervous system included spongy

degeneration; however, immunohistochemistry and western blotting failed to detect

pathologic prion protein. A similar disorder is not known in wild ratites, and hereditary or

toxic causes have to be considered. Lawsonia intracellularis was recognized as cause of

proliferative enteritis for the first time in a rhea. Severe necrotizing inflammation of the

respiratory tract in a rhea was caused by the low pathogenic avian influenza virus A H7N7, a

subtype that has not been reported in ratites so far.

In juvenile animals, the digestive system and the musculoskeletal system were

predominantly affected by inflammatory and traumatic lesions. Bacterial infections of the

alimentary tract were an important cause of disease in the present study. Whereas alimentary

disorders are very rare in wild ratites, they are often reported in captive birds, even in their

natural habitat. In eggs and neonates the major cause of death was the fading ostrich

syndrome that is a worldwide disease occurring exclusively in farm husbandry. Among

C h a p t e r 6 : S u m m a r y

73

various factors particularly inadequate management practices such as improper incubation

conditions for eggs contribute to this disorder.

The retrospective analysis of diseases in various ratite species revealed many disorders

that were associated with housing or management conditions in farms and zoos. Some

disorders were found that were never or rarely described in these flightless birds.

C h a p t e r 7 : Z u s a m m e n f a s s u n g

74

Retrospektive Untersuchung der Erkrankungs- und Todesursachen von

Ratiten (Struthioniformes) in Nordwest-Deutschland

Von: Aimara Bello

Chapter 7: Zusammenfassung

Das Spektrum der spontanen Erkrankungen von 71 in menschlicher Obhut gehaltenen

Laufvögeln (Strauße, Nandus, Emus) aus Zoos und Farmen in Nordwest-Deutschland wurde

retrospektiv analysiert. Die Sektionsberichte und histologischen Präparate der zwischen 1968

und 2014 obduzierten Tiere wurden geprüft, zusätzliche histochemische und

immunhistologische Färbungen durchgeführt. Alle Befunde wurden unter Berücksichtigung

der virologischen, mikrobiologischen und parasitologischen Befunde interpretiert.

Bei vielen Tieren waren mehrere, voneinander unabhängige, morphologische

Diagnosen zu stellen. Die am häufigsten veränderten Organsysteme adulter Ratiten waren der

Bewegungsapparat, der Verdauungstrakt und das Herz-Kreislauf-System, die traumatisch

bedingte Verletzungen, Entzündungen und degenerative Veränderungen aufwiesen. Die große

Zahl traumatischer Alterationen unterstreicht die Anfälligkeit von Laufvögeln für

mechanische Schädigungen. Bei einem Strauß bestand ein intraossäres Fibrosarkom, das

bislang bei Laufvögeln noch nicht beschrieben wurde. Zu den Veränderungen im zentralen

Nervensystem zählte eine spongiöse Degeneration. Allerdings gelang weder durch

Immunohistochemie noch durch Western-Blotting ein spezifischer Nachweis von

pathologischem Prion-Protein. Eine vergleichbare Erkrankung ist bei wildlebenden

Laufvögeln nicht bekannt. Eine erbliche oder toxische Ursache ist in Erwägung zu ziehen.

Lawsonia intracellularis wurde erstmals bei einem Nandu als Ursache einer proliferativen

Enteritis festgestellt. Eine ausgeprägte nekrotisierende Entzündung des Respirationstraktes

wurde durch das niedrig pathogene aviäre Influenza A-Virus H7N7 verursacht, ein Subtyp,

der bislang noch nicht bei Ratiten nachgewiesen wurde.

Bei Jungtieren waren vor allem der Verdauungs- und Bewegungsapparat von

entzündlichen und traumatischen Veränderungen betroffen. Bakterielle Infektionen des

Verdauungstrakts stellten in der vorliegenden Studie eine wichtige Krankheitsursache dar.

C h a p t e r 7 : Z u s a m m e n f a s s u n g

75

Während diese Erkrankungen bei wild lebenden Laufvögeln sehr selten sind, werden sie bei

in Gefangenschaft gehaltenen Laufvögeln häufig beobachtet, auch wenn sie in ihrem

natürlichen Lebensraum leben. Bei Eiern und Neugeborenen stellte das „Fading Ostrich

Syndrome“ die wesentliche Todesursache dar. Diese weltweit verbreitete Erkrankung tritt

ausschließlich bei in menschlicher Obhut gehaltenen Laufvögeln auf. Unter anderem tragen

insbesondere unzulängliche Management-Praktiken, beispielsweise ungeeignete

Inkubationsbedingungen für die Eier, zur Pathogenese bei.

Die retrospektive Analyse von Erkrankungen verschiedener Laufvogelarten ergab,

dass zahlreiche Alterationen mit den Haltungsbedingungen in Farmen und Zoos assoziiert

sind. Aber es wurden auch einige Erkrankungen festgestellt, die bislang noch nicht oder nur

selten bei diesen flugunfähigen Vögeln beschrieben wurden.

C h a p t e r 8 : R e f e r e n c e s

76

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C h a p t e r 9 : A c k n o w l e d g e m e n t s

103

Chapter 9: Acknowledgements

I would like to express my sincere gratitude to my supervisor Prof. Dr. Wolfgang

Baumgärtner, for his constant support and constructive criticism.

I would also like to express my thanks to Dr. Peter Wohlsein, for his helpful discussions,

great support and time.

It gives me immense pleasure to thank my Family and Mu, for understanding and supporting

me in their own ways, and also to Jessica Freundt, for all the great and not so great moments

we shared until now!!! You were always there for me and I think just saying thanks is not

enough.

My sincere thanks also goes to Daniel Kupka, meine Liebe!! You changed my live!!

I would also like to thank Felipe and Alejandro, life was boring before you guys.

I also would like to express thanks to Vanessa and Annika, my dear colleagues!! For your

friendship and supporting me all the time, to Annika and Dr. M. Peters for providing the great

pictures used in this presentation, and to all my colleagues and personnel from Pathology, for

your great support, understanding and the wonderful time I had in Hannover.

University of Veterinary Medicine Hannover Department of ...€¦ · University of Veterinary...

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