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ZEBRA FOUNDATION GRANT REPORT

First published in the BVZS ZooMed Bulletin, September 2004

INVESTIGATION OF PARAMYXOVIRUS HAEMAGGLUTINATION INHIBITION TITRES FROM

PREHENSILE TAILED SKINKS (Corucia zebrata) WITHIN UK COLLECTIONS

Georgina Yates BVetMed MRCVS

Abstract

Paramyxovirus is considered the most important pathogen affecting captive snakes especially the viperids

(Jacobson et al, 1999). The purpose of this study was to look for antibodies in prehensile tailed skinks (Corucia

zebrata) from a range of UK reptile collections to determine whether exposure to ophidian paramyxoviruses

(OPMV) is confined to previously known positive specimens or is more widespread. Comparison of the reaction

of skink antibodies to a range of viruses was also undertaken. Serum was collected from 34 C. zebrata specimens

from 9 collections across the UK. Haemagglutination inhibition assays were performed using this serum against

avian paramyxovirus (APMV) serotypes -1 to -9, a Sendai virus isolate and an OPMV isolate serologically similar

to APMV 7. Six specimens had mono-specific reactions to OPMV 7, nine had negative reactions to all viruses and

the remaining specimens had weak non-specific reactions to two or more viruses.

Introduction

Respiratory diseases account for considerable disease and death in collections of reptiles and whilst bacteria,

fungi and parasites play a part in this, ophidian paramyxovirus (OPMV) is the most important pathogen affecting captive

snakes especially Viperids (Jacobson et al, 1999). PMV has been isolated from a number of healthy lizard species, but

there has only been one report of disease due to OPMV in lizards (Ahne et al, 1999, Jacobson et al. 2001, Lloyd 2002,

Marschang et al. 2002). In a screening of 54 healthy reptiles at London Zoo, 3 Rhinoceros Iguanas (Cyclura cornuta

cornuta), 2 Blotched Blue Tongued Skinks (Tiliqua nigrolutea) and 3 Prehensile Tailed Skinks (Corucia zebrata) were

found to have positive antibody titres to an OPMV 7 (Lloyd 2002).

The aims of this study were to:

1) Look for antibodies in C. zebrata from a range of UK reptile collections to determine whether exposure to

paramyxoviruses is confined to previously known specimens or is more widespread.

2) Examine which viruses of avian PMV (APMV) -1 to -9, Sendai virus and OPMV 7 skink antibodies react to in a

serotype specific manner.

To achieve these aims serum was collected from 33 specimens in nine reptile collections across the UK.

Haemagglutination inhibition (HI) assays were used to achieve antibody titres to the named viruses. HI was chosen as

Corucia zebrata from Birmingham Nature Centre

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it is frequently used in serological analysis of OPMV outbreaks and is a quick and relatively easy test that would be

possible to complete within the three weeks available for this project (Jacobson et al, 1999).

Literature review

Background to Paramyxovirus infection of snakes

In 1972 a new reptilian virus was isolated from a Fer-de-Lance viper (Bothrops atrox) housed in a private

collection in Switzerland (Clark et al, 1979). The collection had suffered an outbreak of a fatal respiratory disease. The

paramyxo-like virus was isolated from lung tissue of one of the dead animals and named Fer-deLance virus (FDLV)

(Clark et al, 1979).

In 1980 the death of Rock Rattlesnakes (Crotalus lepidus) from a breeding colony in Florida was reported and

a PMV was isolated (Jacobson et al. 1980). The affected vipers had suffered from head tremors and loss of equilibrium

with delayed irregular tongue flickering and exudate was seen from the respiratory tract ( Jacobson et al, 1980).

In 1981 another PMV outbreak was reported by Jacobson et al in Louisiana and again only vipers were affected

(Jacobson et al, 1981). The clinical signs in this case involved mouth gaping followed the next day by convulsions then

death. Some snakes had been regurgitating food for the past 1 - 3 weeks; some died with no previous clinical signs

(Jacobson et al, 1981). On post-mortem grossly the only abnormality was a mucoid or caseous exudate within the lung.

Histologically an interstitial pneumonia was seen and a paramyxo-like virus was isolated from the lung tissue (Jacobson

et al, 1981). HI assays reacting with this isolate and with serum samples from 22 of the surviving snakes at the collection

gave antibody titres of 1/20 to 1/2560 (Jacobson et al, 1981). Positive titres were achieved from vipers and non-vipers

for at least five months post infection (Jacobson et al, 1981). The HI assays confirmed that this virus isolate was

antigenically similar to the existing Rock Rattlesnake isolate (Jacobson et al, 1981).

In subsequent outbreaks in USA, Germany, UK, and Canary Islands snakes from a number of families have

been infected including Viperidae, Crotalidae, Colubridae, Boidae (Jacobson et al, 1981, Manvell et al, 2000, Oros

et al, 2001). Only infections of adult animals have been reported and no sex predilection has been seen.

OPMV particles range in size from 126 - 500 nm and are pleomorphic spheroidal or filamentous and enveloped

with a peripheral fringe of spines (Clarke et al, 1979, Homer et al, 1995, Kania et al, 2000). Classification as a

paramyxovirus was based on electron microscopic appearance, ability to haemagglutinate chick erythrocytes and to

grow in chicken eggs, sensitivity to ether and possession of an RNA nucleocapsid core (Clark et al, 1979, Homer et al,

1995, Kania et al, 2000, Lunger and Clark 1979a). The virus has been found to replicate in cells of mammalian, avian

and reptilian origin but is most efficient in reptilian cell cultures especially rattlesnake cell lines (Ahne W., Adrian J.,

and Mayr A. 1999, Clarke et al, 1979, Homer et al, 1995, Lunger and Clark 1979a and 1979b). Syncytium and plaque

formation is induced by the virus in cell culture (Clark et al, 1979, Homer et al, 1995, Kania et al, 2000). Disease was

not produced in mice up to one year after

inoculation in a number of ways with the FDLV isolate (Clark et al, 1979). Among paramvxoviruses OPMV has a

uniquely low optimum temperature range of 25-30°C and replication is severely impeded at 37°C suggesting that it is

specifically adapted to ectothermic hosts (Ahne W. Adrian J. and Mayr A. 1999, Clark et al, 1979, Homer et al, 1995,

Lunger and Clark 1979a and I979b).

In 1997 Jacobson et a1 fulfilled Koch's postulates using an OPMV originally isolated from an Aruba Island

Rattlesnake and propagated in Vera cells. Virus was inoculated into the tracheas of Aruba Island Rattlesnakes to produce

characteristic respiratory disease and lesions and the inoculated virus was subsequently isolated from the trial animals

(Jacobson et al, 1997). None of the animals produced antibody to the inoculated virus but as they all died less than 23

days post inoculation they may not have lived long enough to do so (Jacobson et al, 1997).

OPMV in lizards

In 1998, wild iguanas (Iguana iguana rhinolopha, Ctenosaura bakeri, C. similis) from Honduras were found to

have weak positive antibody titres (1/8 – 1/32) to an OPMV 7 isolate from a healthy monitor lizard (Varanus prasinus) housed with snakes in a PMV epidemic (Gravendyck et al, 1998). HI and virus neutralisation test (VN) were used. VN

generally gave higher titres than HI (Gravendyek et al, 1998). Antibodies were not detectable, in these samples, to an

APMV 1 or OPMV l isolate by HI or VN and no virus was isolated (Gravendyck et al, 1998).

Three epidemics of infection in Caiman lizards (Dracaena guianensis) imported from Peru to USA were

reported in 2001 (Jacobson et al, 2001). The lizards died rapidly, some displaying clinical signs of depression, anorexia,

muscle wastage and possibly respiratory distress (Jacobson et al, 2001). Proliferative interstitial pneumonia was

diagnosed at necropsy (Jacobson et a1, 2001). PMV was seen in lung tissue by electron microscopy and

immunoperoxidase staining using rabbit antibodies to an Aruba Island rattlesnake OPMV isolate (Jacobson et al, 2001).

An OPMV was isolated from lung tissue. Seven months after the first epidemic, surviving lizards housed with the fatally

infected animals were blood sampled for serology. Of 17 animals, 10 had HI titres >1/20 and <1/80 showing sustained

low antibody levels (Jacobson et al, 2001).

A PMV was isolated from a cloacal swab of a healthy wild caught Mexican lizard (Xenosaurus patyceps)

(Marschang et al, 2002). Serology testing of the virus isolate with polyclonal antisera to APMV -1 to -9 gave the highest

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titres to APMV 7 (Marschang et al, 2002). Four of the wild caught Mexican lizards in this study (X. grandis, X.

platyceps and Abronia graminea) were shown to have antibodies to the OPMV isolated with titres >1/32 (Marschang

et al, 2002). This isolate has been used in this study and by Lloyd 2002.

Investigation of PMV infections Suspicion of OMPV infection occurs on visualisation of suggestive lesions such as epizootic proliferative

pneumonia. To confirm infection, antemortem virus isolation and/or demonstration of a rising titre of OPMV specific

antibodies using HI are used. VN can also be used. Postmortem demonstration of replicating virus particles

by electron microscopy and/or immunohistochemistry detection can be used in addition to N17irus isolation (Homer et

a1,1995, Oros et al, 2001, Jacobson et al, 2001, Jacobson et al, 1999). An enzyme linked immunosorbent staining assay

(ELISA) has been investigated to assess antibody reactivity to a number of antigens rather than just one as HI does

(Kania et al, 2000). However reagents to detect Ig from all major families of reptiles are needed before an ELISA can

be of widespread use for detection of seropositive reptiles (Kania et al, 2000). A polymerase chain reaction (PCR ) using

oral and cloacal swabs is currently being investigated for viability (E Holdsworth pers. comm. 2003).

Complement fixation tests titrating the reference FDLV isolate against guinea pig sera produced against a

number of paramyxoviruse isolates including APWV -1 to -5 but not Sendai virus produced negative results as did

titrating sera to FDLV against the other paramyxovirus isolates (Clark et al, 1979).

HI assays using two OPMV isolates from a disease outbreak in Germany and polyclonal antisera to APMV

serotypes -1 to -9 have been attempted (Blahak et al, 1991 ). The highest positive titres were to APMV 7 with a high

cross-reaction to APMV 1 with one isolate and to APMV 1 with a high cross-reaction to APMV 7 with another isolate

(Blahak et al, 1991). This has led to OPMV isolates being known as either --1 or -7 serotypes (Blahak et al, 1991). A

low cross-reaction to all the other antisera was achieved by both isolates (Blahak et al, 1991). The first OPMV strain

failed to bind monoclonal antibodies to OPMV 7 or APMV 1 but the second did

bind monoclonal antibodies to APMV 1 (Blahak et al, 1991). Serum from the snakes involved in this outbreak showed

positive HI antibody titres to both OPMV isolates, and APMV -1 but not APMV -3 or -8 (Blahak et al, 1991). An

OPMV from an Ottoman viper (Vipera xanthina xanthina) was reported to have a positive HI titre to parainfluenza

2 antisera (Homer et al, 1995). Chelodians with rhinitis have been found to have high antibody titres against Sendai

virus but no virus was isolated and so whether Sendai-like viruses actually cause disease is currently unknown

(Jackson and Needham 1983).

Sequence analysis of OPMV

The predominant problem with serodiagnosis is that multiple strains of OPMV have now been recognised to

exist so their relatedness needs to be assessed before the accuracy of serology methods can be determined (Cranfield

and Graczyk 1996, Ahne et al, 1999).

Analyses of the large (L) and haemagglutinin-neuramininidase (HN) protein sequences of 16 reptilian

paramyxoviruses by Ahne et al showed that there are two well defined subgroups/species of OPMV and a set of

intermediate isolates which may represent additional distinct species (Ahne et al, 1999). Isolates of one species were

mainly collected from outbreaks within USA and isolates of the other were mainly from Germany (Ahne et al, 1999).

Isolates within a single virus species were found in more than one snake species suggesting the virus does not have a

narrow host range (Ahne et al, 1999). It was also shown that OPMV forms a separate family of viruses more closely

related to each other than to the outgroup using Sendai virus (Ahne et al, 1999, Franke et al, 2001, Junqueira de Azevedo

et al, 2001).

Kindermann et al found similar results comparing the sequences of L and HN proteins from the same OPMV

isolates as Ahne et al. This latter study also related OPMV isolates to each other and to Sendai virus and human

parainfluenza -l, -2 and -3 isolates (Kindermann et al. 2001). The L protein and HN protein from these isolates were

found to be highly divergent from all the mammalian isolates but most closely related to the Sendai virus (62% and

47.1%) and least closely related to human parainfluenza virus 2 (40.4% and 26.1%) (Kindermann et al, 2001).

Franke et al also found similar results comparing the sequences of L protein and Fusion (F) protein in 18

OPMV isolates from Germany to those of a variety of mammalian PMV isolates (Franke et al, 2001).

Characterisation of the F protein from a PMV from the venom gland of a Fer-de-Lance viper (Bothrops jararaca)

was performed by Junqueira de Azevedo et al in 2001. The protein was found to be most related to parainfluenza

3, Sendai virus and human parainfluenza 1 when compared to sequences in the databanks that included those of

APMV -1 and -2 (Junqueira de Azevedo et al, 2001). The L gene was found to be more conserved than the HN

gene (Kindermann et al, 2001). The genetic relatedness of haemagglutinin from reptilian species to avian serotypes

has yet to be reported. However APMV and human parainfluenza 2 are classified within the Rubulavirus genus and

Sendai virus within the Respirovirus genus within the Paramyxoviridae family suggesting OPMV is not closely

related to APMV despite the serological evidence for this (van Regenmortel et al, 2000). Due to lack of sequence

data in 2000 OPMV is currently unassigned to a genus within the Paramyxoviridae family (van Regenmortel et al,

2000).

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Prehensile tailed skinks

The Corucia zebrata is commonly known as the Solomon Island or prehensile tailed skink and is a large green arboreal

skink with a strong prehensile tail that lacks the tail fractural planes possessed by other species of skink (Wright 1993).

The C. zebrata is a herbivorous skink originating from the Solomon Islands and Papua New Guinea (CITES 2003,

Wright 1993). The average snout to vent length is over 25cm and average adult weight is between 500 and 700g making

them large enough to blood sample safely, withdrawing enough blood for a number of tests (Wright 1993).

The animals are also relatively easy to handle, so blood sampling from

the ventral tail vein can be performed without anaesthesia.

The C. zebrata is a CITES appendix II animal classified as “threatened” but, it is felt that due to pressures on

wil As a result, a UK captive breeding programme was started involving 55 animals at the last count (Basford 2001).

Most of the animals in this study are involved in that programme making this the most widespread OPMV screening of

the UK breeding population of C. zebrata to be undertaken to date. Being able accurately to assess the PMV status of

these animals is important to the breeding programme so that appropriate decisions can be made regarding transferring

positive and negative titre specimens between collections.

The purpose of this project was to explore the use of HI assays to detect carrier animals in UK collections.

APMVs 1-9, Sendai virus and OPMV 7 were used as antigens.

Materials and Method

Samples

Blood samples were collected by the veterinary surgeon working for each collection. Blood was taken from

the ventral tail vein of conscious specimens and spun down to remove the serum. Serum was stored at -20 °C until

needed.

Preparation of day old chicken blood

The day old chicken blood (TCS Biosciences Ltd) was washed three times with Phosphate Buffered Saline

solution (Oxiod). For haemagglutination, the washed cells were diluted in Phosphate Buffered Saline (PBS) to produce

a 1% chick erythrocyte suspension. The blood and 1% suspension were refrigerated when not in use.

Preabsorption

Before testing, serum had to undergo preabsorption to remove any antigens to chicken red blood cells from

the samples (Cross 2002, R. Manvell pers. comm. 2003). 100μ1 samples of the serum were added to 251 packed

washed chicken erythrocytes. These were mixed and left to stand for one hour at room temperature. The suspension

was then centrifuged at 800g for 2 minutes and the antisera removed for use. These were stored in aliquots at -20 °C when not in use.

Haemagglutination assays

Haemagglutination assays (HA) were performed on each virus to calculate the titre of the virus and from this,

4 Haemagglutination Units (HAU), to be used in the HI Assays. The viruses used were APMV serotypes -1, -2, -3, -4,

Blood sampling a conscious prehensile tailed

skink from the ventral tail vein.

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-6, -7, -8, -9 supplied by R. Manvell (VLA, Weybridge). These stocks had been grown in chicken eggs and inactivated

with β-propiolactone before use (Russell 1989). Also used was a live Sendai virus (murine parainfluenza 1) grown in

chicken eggs, originally supplied by M-J. Gething (ICRF, London). A formalin inactivated OPMV isolate (VLA 186/00)

grown in chicken embryo fibroblasts, supplied by R. Manvell (VLA, Wevbridge), completed the panel. This OPMV

was isolated from a cloacal swab of a healthy Mexican Lizard (Xenosaurus platvseps) using Terrapene heart cells at

28˚C (Marschang et al,2002). HI titres were found of 1/8 and 1/128 to chicken antiserum to APMV -1 and -7 respectively

meaning the isolate cross matches with APMV 7 (R. Gough pers. comm. 2003, Marschang et al, 2002).

To perform the HA, 25μl PBS was placed in each well on a V-bottomed microtiter plate. An additional 20μ1

PBS and 5μl virus isolate were added to the first column well of each row. This was double diluted across the row

discarding the last 25μ1. 25μ1 PBS as a volume blank and 25μ1 1% washed chick erythrocyte suspension were then

added to each well. In the final row of each plate, 50μ1 PBS and 25μ1 1 % erythrocyte suspension were added to each

well as a negative control. The plate was left at room temperature for 45min. To read, the plate was stood at an angle to

allow streaming of non-haemagglutinated red blood cells.

Each virus was tested in triplicate and the HA titre assessed from the average result over all three rows. 1

Haemagglutinating Unit (HAU) was defined as the highest dilution to cause 100% agglutination of chicken red blood

cells. The virus was then diluted with PBS to give a 4 HAU solution. The viruses were stored at -70°C in their neat

state until needed.

Haemagglutination inhibition assays Once 4HAU had been determined for each virus, haemagglutination inhibition assays (HI) could be

performed using the preabsorbed sera. 25μ1 PBS were added to each well on a V-bottomed microtiter plate. 20μ1

PBS and 5μ1 of serum were added to the first column well of each row and double diluted across the row to produce

l/10 to 1/1280 dilutions. The last 25μ1 from each row was discarded. 25μ1 of 4HAU virus solution was added to

every row. In an additional row, an antigen titration of 4HAU virus was double diluted across five wells to produce

dilutions from 4HAU to 1/4HAU in 50μl volumes. 25μ1 PBS were added to each of these wells as a volume blank.

The plate was left at room temperature for 30min. 25μ1 1% chicken erythrocyte suspension was added to every well

on the plate. In the final row, as a chicken erythrocyte control, 50μl PBS and 25μl 1% chicken erythrocyte suspension

were added to every well. The plate was then tapped to mix the reagents and left at room temperature for 45min.

To read, the plate was stood at an angle to allow streaming and the HI titre was defined as the highest dilution

to prevent agglutination of erythrocytes. On each plate 4 to 5 sera were tested against one virus serotype. Each sera

was tested in duplicate and the HI titre was taken as the average of the results from both in the event that they provide

different titres.

The plates were read by two methods:

1) Taking streaming only as a true haemagglutination inhibition. Streaming was defined as where the

erythrocytes pooled to the bottom of the well leaving clear fluid until the plate was tipped when the cells stream in a

line towards the bottom edge of fluid.

2) Taking both streaming and buttoning as true haemagglutination inhibition. Clear buttoning was defined as

where the erythrocytes had pooled but no streaming was seen when the plate was raised to an angle.

Haemagglutination inhibition assay plate showing

buttoning and haemagglutination of chicken

erythrocytes

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Results

Samples were collected from:

1) Battersea Park Children's Zoo

2) West Midland Safari & Leisure Park Ltd, Bewdley

3) Birmingham Nature Centre

4) Bristol Zoo Gardens

5) Chessington World of Adventures

6) North of England Zoological Society, Chester

7) Cotswold Wildlife Park

8) Durrell Wildlife Conservation Trust Jersey

9) Zoological Society of London

A total of 33 samples were collected, 25 of these are included in the stud book for this species, 6 have been

moved into collections since the stud book was last updated and 2 are from a collection which does not participate in

the breeding programme. All of the animals were reported to be healthy apart from one at Chessington that has been

severely underweight since being rescued by the RSCPA in November 2001.

There were large variations in colour and consistency of the serum samples, and the appearance of each is noted in

table 1 along with specimen details. Many of the samples had coagulated into a gel with variable amounts of fluid

surrounding the gel. As only the fluid was usable for HI, some samples with only small volumes of fluid remaining

could not be tested against all the virus isolates.

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8

9

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Discussion

Analysis of results

When the results from the two methods of reading the HI plates are compared, the second method of counting

buttons as positive results leads to a high number of sera showing variable titres to different viruses. The first method

of just counting streaming samples as positives, gives much fewer positive and generally lower titres. This result agrees

with information from R. Manvell that only streaming wells should be regarded as true positives (R. Manvell pers.

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comm. 2003). Due to the highly variable results from the second method with no coherent pattern of reaction, these

results offer no convincingly valid conclusions. The results from the first method of reading plates are however specific

enough to draw conclusions from.

These results can be divided into weak specific positives, non-specific reactions and negative reactions. There

are serum samples that only produced a mono-specific positive result to one virus from the panel, those that non/poly-

specific positive titres to a number of isolates and those that produced no positive titres at all. A breakdown of these

results is given in Table 7 below. Six specimens out of the 33 sampled (18%) currently have low levels of antibodies to

OPMV 7 (186/00).

Of the six specimens with mono-specific positive titres of OPMV 7, two from London (nos.31, 32) also had

similar positive titres last year to the same OPMV 7 isolate as shown in Table 1 (VLA 186/00 (Lloyd 2002, Marschang

et al, 2002). Maintaining positive titres to OPMV 7, that are neither rising nor falling, could mean that the animals

have been exposed to OPMV infection in the past and have a long lived antibody response to it. Antibody levels, in

reptiles, can be mantained over long periods. Caiman lizards have been shown to have antibody levels of up to 1/80

to OPMV seven months after exposure (Jacobson et al, 2001). Antibody levels in snakes after an outbreak in

Louisiana, USA were high, declining to low values from 3 to 7 months after the initial outbreak (Jacobson et al,

1992). Alternatively, the two skinks could be carrying the virus, so antibody levels are maintained, or multiple

exposures could have occurred giving coincidentally similar titres. No other London specimens have become

positive over the year so there is no evidence for seroconversion by the positive specimens but it is unknown how

much contact occurs between animals.

Analysis of haemagglutination inhibition assays

Whilst low HI titres suggest low levels of antibody in the serum, a titre is not a quantitative measure but a

measure of the efficiency of neutralisation of haemagglutination (Jacobson 2002). The result is dependent on the

antibody affinity for antigen, the availability of the antibodies and proper preservation of the samples (Jacobson 2002).

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Unfortunately, the samples sent from one collection were not refrigerated for five days due to being sent over a bank

holiday. This could have denatured antibodies present in the samples. However mono-specific positive titres were

produced to APMV 7 by three of these samples (nos. 9, 10, 12). No. 12 has a relatively high titre of 1/80, so denaturing

appears not to have occurred, but without re-testing these animals it is not possible to know whether these titres would

have been any different or specific to OPMV 7 had the samples been chilled throughout. As little is known about the

full nature of OPMV strains, their cross reactivity and reptile immune responses to them, the specificity and sensitivity

of HI assays for antibodies to the virus are as yet unknown.

There was a problem with some of the serum samples which coagulated into a gel form preventing use of a

large part of the collected sample. This could have been avoided by collecting plasma rather than serum (Jacobson

2002). Another potential problem is that false negatives caused by toxins or bacteria in the serum can lyse chicken

erythrocytes so that they cannot bind to the antigen or causing agglutination (Jacobson 2002). Antisera present in the

samples against antigens on the erythrocytes could also cause agglutination (Jacobson 2002). Preabsorption should have

prevented these occurring by removing agents reacting with the erythrocytes, however the clear buttons could be a sign

that some remained (Jacobson 2002). Repeating preabsoption twice is occasionally done if this is suspected but there

was insufficient time available for this to be done in this study (R. Manvell pers. comm. 2003).

Reptile immunity involves immunoglobulin (Ig)M and IgY (reptilian equivalent of IgG), but it is currently

unknown which of these are measured by HI assay for PMV (Jacobson 2002, Ridi 1992). Much higher levels of IgM

are present in the blood than IgY and IgM in mammals, fish and presumably reptiles is poly-specific (Avrameas 1992,

Ridi 1992). Therefore if predominately IgM reacts in HI assays to PMV this could account for the non-specific results

seen. More investigation of this is required to assess whether investigation of just IgY levels to OPMV would give more

specific results.

Investigation into the prevalence of OPMV is still in its infancy especially as there is no gold standard test. HI

is a useful test as it is quick and simple to perform, but with the recent developments in characterisation of the many

virus isolates it is increasingly obvious that the choice of isolates used is vital to its accuracy. Without full knowledge

of the different serotypes of OPMV, and the relatedness of their haemagglutinin, it is currently not possible to be certain

of our ability to screen animals for OPMV. Testing samples against more strains of OPMV especially an OPMV 1 would

have been a useful adjunct to this study but none were available. HI is still an invaluable tool for testing for a specific

strain of OPMV, for example, to determine potential carriers in the face of an outbreak.

Serum neutralisation assays involve incubating the virus with serial dilutions of serum, then assessing for

residual infectivity on cell culture or another biological substrate. Serum neutralisation is considered the gold standard

test for serological diagnosis of viral infection and is more sensitive but also more laborious than HI (Jacobson 2002).

Although both HI and serum neutralisation can show antibody titres to PMV strains considered suspicious or positive

for PMV, they cannot prove if virus is still present even with paired samples. Had it been possible, virus isolation or an

ELISA to show shedding would have been a useful adjunct to this study.

Comparison of APMV and OPMV

When examining the cross reactions seen in this experiment it is helpful to look at known cross reactions

between the APMV viruses themselves. A common cross reaction is between APMV 1 and APMV 3 but lesser cross

reactions are also known to occur within two groups with APMV -1, -3, -4 , -7 and -9 in one group and APMV -2, -6

and -8 in the other (Alexander 1980, Russell 1989). In this study however, the main cross reactions occurred between

OPMV 7 and APMV 2 that are in separate grouping so are not expected to cross react (Russell 1989). Also some of

these samples show a higher HI titre to APMV 2 than OPMV 7 which is the opposite of what could be expected. This

suggests that these results are non-specific reactions and therefore the positive titre to OPMV 7 cannot always be relied

upon to indicate that there are specific antibodies present in that specimen to OPMV. The positive results to just one

isolate are more likely to be specific, especially as two of them (nos. 31, 32) correlate with previous results (Lloyd

2002). The one specimen (no. 17) showing a cross reaction between just OPMV 7 and APMV 7 is also likely to be

correct as there is a known cross reaction between them. It would be expected for the APMV to give a lower titre than

the OPMV rather than being the same, but these titres are very low, making differences harder to distinguish.

When comparing pathogenesis information regarding OPMV and APMV some distinct similarities appear.

Alexander describes AMPV 1 as having four distinguishable forms that vary in severity from causing greater than 90%

mortality in outbreaks to mild or inapparent disease (Alexander 1980). The severest forms can cause death without

previous clinical signs; slightly more mild forms give respiratory and nervous signs as seen with OPMV (Alexander

1980). However sequencing has shown no relatedness of OPMVs to the Rubulavirus family containing APMVs

(Kindermann et al, 2001). This raises questions as to the reliabilty of cross reactions between OPMV and APMV.

Hopefully further sequence analysis will improve understanding of how cross reactions are occurring and which, if any,

are specific.

Significance of OPMV

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Recent findings of OPMV antibodies in healthy reptiles from wild populations reduces the chance that this is

an emerging infectious disease that, if released into wild populations, could cause mass mortalities and even extinctions

(Daszak et al, 2001). However, if different strains do have differing severities, possibly depending on the species

infected, this is still a risk and should be a major consideration when planning programmes to breed reptiles for release

into the wild (Daszak et al, 2001). Jacobson recommends that reptiles bred in captivity for release into the wild be

isolated away from the main collection to reduce infiltration of pathogens to the release group, and that these breeding

programmes should be established within the geographical range of the species (Jacobson 1993). Isolation of breeding

groups may be possible in some collections, but many will not have the facilities to allow this. Whilst breeding animals

in the vicinity of where they will eventually be released should be the objective, this again will not be possible in many

situations. Therefore, collections need to do as much as possible to eliminate entrance of pathogens to the collection as

a whole.

While it is unlikely that animals with weak positive titres pose a risk to collections, there is a small chance they

are carrying the virus and could infect other specimens. Combining serology with virus isolation from cloacal swabs,

ELISAs for OPMV antigens or PCRs for OPMV related sequences (once developed and validated) would allow this

risk to be better assessed. It is still unknown how OPMV is spread. though other PMVs are generally spread by aerosol

or faeces; it is possible fomites or even parasites are involved (Jacobson et al, 1999, van Regenmortel et al, 2000). This

makes it difficult to recommend anything less than total isolation of carriers and infected animals (Jacobson et al, 1999).

All collections should already have a good quarantine protocol and should practice isolation of any sick specimens.

With more information on the natural history of this virus, collections will be able to make better decisions about

precautions regarding OPMV.

Summary

Six C. zebrata specimens were found to have weak mono-specific HI titres to OPMV 7. All of these specimens were

from collections where OPMV has been previously recognised (S. Thornton pers. comm.. 2003). Positive titres were

also produced to some APMV serotypes but not with any consistent pattern. Without being able to look for virus as well

as antibodies it is difficult accurately to assess the risk posed by these positive specimens to others in the collection.

More research is needed into the relatedness of OPMV strains to each other and to APMVs before the specificity of HI

assays for OPMV can be accurately assessed.

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Acknowledgements

I would like to express my thanks to Peter Russell my project supervisor and to Ruth Manvell from VLA, Weybridge for

advice and supply of viruses. Also to Sue Thornton for advice and assistance as well as collecting blood samples. In addition, I am

grateful to Andrew Mackie for technical instruction and advice on HI assays. Thank you to all the veterinary surgeons and other

staff at the reptile collections involved with sampling including: Stephanie Sanderson at Chester Zoo; Javier Lopez at Durrell

Wildlife Conservation Trust; Sharon Redrobe at Bristol Zoo Gardens; Chris Marshall at West Midlands Safari Park; Peter Aylmer

at Cotswold Wildlife Park; Tai Strike at Zoological Society of London; staff at Chessington World of Adventures and Battersea

Park Children's Zoo; and Les Basford at Birmingham Nature Centre.