Use of serology in reptile medicine

Use of Serology in Reptile Medicine Elliott R. Jacobson, MS, DVM, PhD, and Francesco Origgi, DVM

Serologic assays are emerging as powerful tools for both diagnosing and screening collections of reptiles for exposure to and infection with certain pathogens. For the most part, these tests were developed in research laboratories with a specific interest in diseases of captive and free-ranging reptiles. Relatively few are commercially available, with most offered through individual research laboratories or universities. Tests have been developed for exposure to paramyxovirus of snakes, mycoplasma of tortoises and crocodilians, herpesvirus infection of marine turtles and tortoises, cryptosporidium of snakes, and spirorchid trematodes of sea turtles. Of the various serologic tests, enzymelinked immunosorbent assay (ELISA) is becoming the test with the most application. Although ELISA is relatively simple and has certain positive attributes such as high sensitivity and specificity, in the indirect format it does require specific anti-reptile immunoglobulins that recognize and bind to the antibody being assayed. In this report, w e review reptile humoral immunity for the major orders of reptiles, assays available for determining exposure of reptiles to specific pathogens, and factors affecting the immune response of reptiles. Copyright 9 2002 by W.B. Saunders Company. Key words: Serology, infectious diseases, chelonians, crocodilians, lizards, snakes.

erology is based on the principle that m o s t foreign molecules are capable of eliciting a distinct h u m o r a l i m m u n e response in vertebrates (except Agnatha) that can be assayed. Assays may either measure the presence of antibody itself directly by specific binding to antigen or indirectly by measuring or d e t e r m i n i n g changes in the antigen itself such as what occurs w h e n an antigen clumps or precipitates f r o m solution. Originally, antibody was m e a s u r e d in s e r u m of i m m u n i z e d humans; therefore, these assays were called serologic assays. Today, plasma, lymph, cerebrospinal fluid, and o t h e r fluids also are included within the scope o f serology. For purposes of this report, s e r u m and plasma are considered as the same type of sample. Serology is a powerful tool that allows animals or h u m a n s to be screened for exposure to an almost infinite array of foreign proteins. T h e information provided can be used to evaluate b o t h individual animals and their exposure status and, for free-ranging wildlife, the serologic

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status of a population. As such, this can be used along with o t h e r m e t h o d s for evaluating the health status of populations. Although single samples only provide i n f o r m a t i o n on exposure, a change in titer in a second sample collected 2 to 4 weeks after the first sample may indicate a recent infection. Thus, i n f o r m a t i o n on exposure and infection can be o b t a i n e d f r o m a s e r u m / plasma sample. In reptiles, the major classes of antibodies are i m m u n o g l o b u l i n (Ig) M and IgY (IgG-like). As in m a m m a l s and birds, after exposure to an antigen, IgM appears first, with IgY developing several weeks after exposure. Although the time sequence for this response has b e e n d e t e r m i n e d for certain antigens used in assessing the imm u n e response of reptiles, little information is available for the time sequence following pathogen exposure.

Reptile Humoral Immunity H u m o r a l immunity is the best studied aspect of the reptilian i m m u n e system 1 and is depend e n t on a n d influenced by a n u m b e r of factors including t e m p e r a t u r e , antigen concentration, route of inoculation, type of antigen, and type of adjuvant. 1,2 T h e r e are a wide variety of published studies on antibody synthesis in all m a j o r groups of reptiles. These studies used m a n y different antigens a n d adjuvants a n d were c o n d u c t e d over a wide range of temperatures. T h e rate and extent of antibody f o r m a t i o n in reptiles are depend e n t on body t e m p e r a t u r e of the host, and by manipulating environmental t e m p e r a t u r e the h u m o r a l response can be varied. In this report, we review the physiochemical properties of antibodies for the 4 m a j o r groups of reptiles. From the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, GainesviUe, FL. Address correspondence to Elliott R. Jacobson, MS, DVM, PhD, Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610 Copyright 9 2002 by W.B. Saunders Company. 1055-937X/02/1t01-0006535.00/0 doi:l O.1053/saep. 2002.28239

Seminars in Avian and Exotic Pet Medicine, Vol 11, No 1 (January), 2002: pp 33-45

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Jacobson and Origgi

Chelonians: Turtles and Tortoises Sea turtles have 3 major classes of immunoglobulins: a 17S IgM, a 7S IgY, and a 5.7S IgY. 3,4 As in mammals, IgM is considered to be the first immunoglobulin to respond when a reptile is challenged with a foreign protein. Subsequently, it declines and is replaced by production and elevation of 7S IgY, which is supposed to function similar to IgG in mammals. Although the exact role of 5.7S IgYis unknown, it is t h o u g h t to be involved in chronic inflammation. 5 Whereas a 7S IgY appeared within 5 weeks in i m m u n i z e d green turtles (Chelonia mydas), a 5.7SIgY was detected 3 to 4 months later. 4 In painted turtles (Chrysemys picta) maintained at 22~ to 25~ and immunized with dinitrophenylhydrazine (DNP)bovine serum albumin (BSA), 6 during the first m o n t h , a high molecular weight, 1 8.5S, antibody appeared. After 1 month, a low molecular weight, 7.0S, antibody appeared. In a n o t h e r study, 4 types of immunoglobulins were found: 19.0S and 7.0S 2-mercaptoethanol sensitive types a p p e a r e d early on and later, 2 7.0S 2-mercaptoethanol resistant types appeared. 7 H e r m a n ' s tortoises (Testudo hermanni), kept at 25~ and imm u n i z e d with DNP-BSA and DNP-horse serum albumin, showed a response, detected by passive hemagglutination, after day 10 and a maximal response a t d a y 50 (1:5120). s Precipitating antibodies were detected in the late stages during the secondary response. This indicated that more than one antibody might be present: a hemagglutinating antibody and an antibody that is precipitating and of low molecular weight. This also showed that tortoises recognize the DNP group attached to a carrier protein as an antigen and develops antibody against it. In a m o r e recent study immunizing green turtles with DNP-BSA in Ribi's adjuvant, an antibody response developed within 5 weeks and rem a i n e d high for 9 months. 4 T h e response was to the DNP hapten because the response was inhibited by soluble DNP or by rabbit anti-DNP-specific anti-sera.

Crocodilians: Alligators, Caiman, Crocodiles T h e r e is limited information on physiochemical characteristics of crocodilian immunoglobulins, with most involving the American alligator,

Alligator mississippiensis. Two distinct light chains, 19S and 7S Igs, were f o u n d in American alligators after immunization with Salmonella2 Two distinct H chains are also present. 1~Early studies showed t e m p e r a t u r e - d e p e n d e n t effects in alligators vaccinated with tetanus toxin. 11 No antitoxin f o r m e d at 20~ whereas a maximum a m o u n t developed in alligators kept at 32~ to 37~

Lizards Light (7S) and heavy (19S) weight immunoglobulins have been demonstrated in several species of lizards in response to various antigens (normal pig serum, bovine gamma globulin) including the desert iguana, Dipsosaurus dorsalis, 12 and the anguid, Ophisaurus apodus. 13 The shingle-back skink, Tiliqua rugosus, in response to rat erythrocytes and BSA, developed an early 19S immunoglobulin followed by a light weight 7S immunoglobulin. 14 Two distinct immunoglobulins have been purified from the agamid lizard, Calotes versicolor: a high molecular weight immunoglobulin similar to h u m a n IgM and a low molecular weight immunoglobulin designated as IgY. 15 Both can be dissociated into distinct light and heavy chains. A n u m b e r of studies in lizards have shown temperature effects on antibody production. T h e lizard SteUio caucasica, following injection of the tick-borne encephalitis virus, had high antibody titers to the virus when maintained at 37~ but n o n e when kept at 4~ 16 Similar temperature effects were shown in the chuckawalla (Sauromalus obesus) and desert iguana (D. dorsalis) when injected with the H-antigen of Salmonella. 1237,1s A good antibody response was measured when lizards were kept at 35~ whereas in those kept at 25~ and 40~ the response was low. A delayed antibody response also has been observed when lizards are kept at suboptimal temperatures. 19 Lizards may or may n o t show an anamnestic response after a second injection of an antigen. In desert iguanas receiving a second injection of keyhole limpet hemocyanin, antibody titers increased at 16 days after the second injection, z~ In contrast, desert iguanas and chuckawallas showed no m e m o r y after a second injection with H-antigen of SalmoneUa. is

Serology in Reptile Medicine

Snakes T h e i m m u n e response of black rat snakes, fox snakes, and black racers to BSA given subcutaneously or intraperitonealy in c o m p l e t e F r e u n d ' s adjuvant was studied at 20~ a n d 28~ 21 A 19.6 to 19.9S antibody a n d a 7S antibody were produced. T h e primary response was first detectable after 4 weeks and the titer rem a i n e d constant for 3 months. M e m o r y was shown by a secondary response yielding higher titers, a maximal response after just 2 weeks of imnmnization, a n d a response that lasted 4 months. Garter snakes i m m u n i z e d with h e n egg albumin, h u m a n g a m m a globulin, a n d keyhole limpet h e m o c y a n i n p r o d u c e d 3 types of i m m u noglobulins: a high molecular weight 20S antibody, which a p p e a r e d first; a low molecular weight 9S antibody detectable after 31 days; and a low molecular weight 8.5S antibodyY 2 F r o m the above studies, it is clear that variability exists in physiochemical properties o f antibodies between the m a j o r groups of reptiles. W h e t h e r this is a reflection of major differences in the c o m p o n e n t s of the h u m o r a l system of different groups of reptiles or based o n different m e t h o d s used in characterizing reptile antibodies is unknown. Much of the work is 20 to 30 years old and relatively few reports have b e e n published recently on this topic. But f r o m what can be discerned, only IgM and IgY exist in reptiles.

Types of Serologic Assays O f the various serologic tests available for d e t e r m i n i n g exposure of animals to pathogens, only a few have b e e n developed for use in reptile medicine. Serum neutralization (SN), hemagglutination inhibition (HI), and enzyme-linked i m m u n o s o r b e n t assay (ELISA) are the m o s t widely used semiquantitative serologic assays. O t h e r less c o m m o n l y used methods, including immunofluorescence, immunoperoxidase, and Western blot, can be used to show p r e s e n c e of antibody against specific proteins of pathogens. In SN assays, serial dilutions of a heat-inactivated s e r u m sample are mixed with a standardized a m o u n t of virus, which is then i n c u b a t e d for periods and at temperatures that vary according to the specific properties of the virus a n d o f the host. T h e v i r u s / s e r u m mixture is t h e n tested for

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"residual infectivity" o n a biological substrate that could be cells in culture, e m b r y o n a t e d eggs, or e x p e r i m e n t a l animals. T h e SN titer corresponds to the highest dilution at which no residual infectivity is detected. This test is considered the gold standard for serologic diagnosis of viral infection, 23 and it offers a strong confirmation of viral exposure. However, the test is laborious and requires the p r o d u c t i o n o f neutralizing antibodies by the host. Nonspecific neutralization activity in some plasma samples can sometimes cause misleading results. T h e H I test operates on a similar principle by detecting the presence of antihemagglutinating antibodies directed against the target hemagglutinating antigen. T h e ELISA is one o f the m o s t p o p u l a r and widely used of the various serodiagnostic tests available a n d is used in b o t h direct a n d indirect formats. In the simplest format, the direct ELISA, the antigen is generally adsorbed on the surface of the wells of a microtiter plate and the antibody specific for the antigen is conjugated with an enzyme that reacts with a c h r o m o g e n substrate, which is a d d e d after the antibody and antigen have h a d time to react. A m e a s u r a b l e colorimetric reaction develops. T h e indirect form a t is most c o m m o n l y used to d e t e r m i n e presence of specific antibody. In indirect ELISA, the primary antibody that binds to the antigen becomes the target of the secondary antibody conj u g a t e d to the enzyme. T h e secondary antibody is either a polyclonaI or m o n o c l o n a l antibody that recognizes and binds to the tested animal's i m m u n o g l o b u l i n . For reptiles, no secondary antibodies are commercially available and are generally p r o d u c e d by individual investigators. Competitive and capture "sandwich" ELISA techniques also exist.

Titer, Titrations, Quantitations, and Pitfalls T h e primary goal of all serologic tests is to detect and quantify antibodies to a pathogen. T h e a m o u n t of antibodies detected is generally expressed as a titer. It is very i m p o r t a n t for the clinician to u n d e r s t a n d that the titer is not a quantitative measure of the antibodies present in the tested samples, but at the most, it represents an ordinal measure of specific antibodyY 4 What is m e a s u r e d is the intensity of a colorimetric reaction (ELISA a n d i m m u n o p e r o x i d a s e ) ,

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the efficacy of neutralizing the cytopathic effect of a virus (serum neutralization) or one of its biological properties (HA or HI). This result is n o t only d e p e n d e n t and influenced by the a m o u n t of a specific antibody present in the sample tested, but is also influenced by several o t h e r factors such as the antibody affinity for the antigen, the integrity of the antigens used in the binding of antibody, the availability o f the antib o d y present in the samples, and the p r o p e r preservation of t h e samples. An SN titer of 1:64 does n o t necessarily m e a n t h a t t h e sample contains m o r e antibody than a sample that h a d a titer equal to 1:8. Although the a m o u n t of antibody could be very similar in absolute terms, a h i g h e r affinity o f the antibodies for the antigen in 1 sample could result in quite different titers. A plasma sample that has n o t b e e n properly collected, preserved, or shipped may result in e r r o n e o u s antibody titers. T h e antigen needs to be standardized. Each serodiagnostic test provides somewhat different i n f o r m a t i o n a n d the test selected will be based on the availability a n d properties of the antigen used in the test a n d availability of specific reagents n e e d e d in the assay. It is strongly suggested that laboratories and p e r s o n n e l experienced in r u n n i n g a n d / o r developing the test of choice should be contacted. Positive a n d negative controls are essential, a n d the best plasma samples used as positive controls are those obtained f r o m animals specifically i m m u n i z e d with the antigen used in the assay.

Sensitivity, Specificity, Positive Predictive Values, and Negative Predictive Values All diagnostic tests are e x p e c t e d to give an answer to the specific question: "Hag the subject b e e n exposed to the pathogen?" Besides the positive or negative answer that we can obtain f r o m the test, it is crucial to know to which extent we can trust the test itself. An unreliable assay is likely to lead a clinician in the wrong direction in the diagnostic pathway. In the best o f possible worlds, an ideal serologic test would have a sensitivity and specificity equal to 100%, b u t it is very u n c o m m o n for any to be characteri z e d by this. O f t e n the result of a serologic test is used to m a k e / decisions that might have an i m p o r t a n t

impact either f r o m a clinical or conservational p o i n t of view. Two additional epidemiologic parameters that are used to evaluate the reliability of a diagnostic test are the positive (PPV) and the negative (NPV) predictive values. PPV is the probability that the subject has the disease given that the test result is positive, whereas NPV is the probability that the subject is disease-free given that the test result is negativeY 5 Similar to the sensitivity a n d specificity parameters, PPV and NPV should be as close as possible to 100% for the test to be Considered reliable.

Collection and Handling of Blood Samples T h e total a m o u n t o f b l o o d that can be safely withdrawn f r o m a reptile d e p e n d s on the reptile's size a n d health status. T h e total b l o o d volu m e of reptiles varies between species but as a generalization is approximately 5% to 8% of total body weight. 26,27 Thus, a 100 g snake has an estimated b l o o d volume of 5 to 8 mL. Because clinically healthy reptiles can acutely lose 10% of their blood volume without any detrimental consequences, f r o m a snake weighing 100 g, 0.7 m L of b l o o d can be withdrawn safely. Several sites can be used in obtaining b l o o d f r o m chelonians, including the heart, j u g u l a r vein, brachial vein, ventral coccygeal vein, orbital sinus, and t r i m m e d toenailsY 8-35 In crocodilians, blood samples can be obtained f r o m the supravertebral vessel located caudal to the occiput and immediately dorsal to the spinal cord. 36 O t h e r sites of blood collection that are c o m m o n l y used include the h e a r t (via cardiocentesis) and ventral coccygeal vein. 3v Blood samples can be obtained f r o m several sites. In large lizards, blood is easily obtained f r o m the ventral tail vein. 38 T o e nails can be clipped, a n d blood can be obtained in a microcapillary t u b e Y Microcapillary tubes also can be used to obtain b l o o d samples f r o m the orbital sinus, 4~ in a similar fashion for collecting b l o o d f r o m mice. In snakes, blood samples can be obtained f r o m a variety of sites, including the palatine veins and ventral tail vein, a n d via cardiocentesis, s
Serology in Reptile Medicine

T h e most i m p o r t a n t points to r e m e m b e r when submitting p l a s m a / s e r u m samples for serology are: (1) to use the same blood collection technique at all times; (2) to use a reliable anticoagulant (preferably heparin) if plasma is required; (3) to handle the b l o o d in a consistent fashion; (4) to centrifuge the b l o o d immediately after collection and remove the plasma immediately after centrifugation; (5) to freeze the sample after collection, preferably on dry ice, in liquid nitrogen, or at - 7 0 ~ and (6) to transp o r t the sample appropriately to the laboratory, preferably on dry ice.

Serology for Viral Exposure Tortoise Herpesvirus Herpesvirus-like agents have b e e n associated with disease in desert tortoises 42,4~ a n d with high mortalities in recently i m p o r t e d chaco tortoises (Geochelone chilensis). 44 In the latter case, redfooted tortoises (Ge0chel0ne carbonaria) i m p o r t e d a n d h o u s e d with the chaco tortoises r e m a i n e d clinically healthy. 44 O f 13 spur-thighed tortoises (Testudo graeca) f r o m 2 private colonies, herpeslike particles were detected by electron microscopy in 2 animals with stomatitis. 45 In a preliminary r e p o r t describing viral epidemics in pet trade, Mediterranean tortoises (T. hermanni a n d T. graeca) detailing 300 case histories were derived f r o m the Tortoise Trust, England, a n d it was concluded that a virus was the responsible agent. 46 T h e reservoir species was considered to be the pet trade collected Asia m i n o r spurthighed tortoise (T. g. ibera). In 16 H e r m a n n ' s tortoises and 8 spur-thighed tortoises with necrotizing glossitis/stomatitis, intranuclear inclusions containing herpesvirus particles were f o u n d in epithelial cells in the tongue, trachea, bronchi, alveolae, endothelial cells of capillaries of the glomeruli, a n d within n e u r o n s and glial cells in the medulla oblongata and diencephaI o n . 4~ T h e authors considered i m p o r t e d tortoises to be latent carriers of this virus. By electron microscopy, herpes-like particles have also b e e n seen in the intestinal contents of a Herm a n n ' s tortoise, several of which h a d caseous material in the u p p e r digestive tract, hepatomegaly, Jand enteritis. 48 The' first isolate of a tortoise herpesvirus was f r o m / a H e r m a n n ' s tortoise in a collection of

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tortoises with stomatitis a n d rhinitis. 49 Five tortoises were submitted for p o s t m o r t e m evaluation. By light microscopy, characteristic eosinophilic intranuclear inclusions were seen in the epithelial cells of the esophagus, trachea, and tongue of all tortoises. Samples of brain, lung, trachea, and intestine a n d liver f r o m different tortoises were p r e p a r e d and inoculated onto T e r r a p e n e heart cells (TH-1). Herpesvirus was isolated f r o m all the tissue samples. Subsequent reports included properties of the tortoise herpesvirus in cell cultures a n d serologic evaluation (serum neutralization) o f tortoises exposed to herpesvirus.50-52 T h e r e are conflicting reports of differential morbidity a n d mortality of M e d i t e r r a n e a n tortoises infected with herpesvirus. In the spring of 1995, a G r e e k tortoise was a d d e d to a collection of 21 H e r m a n n ' s tortoises a n d 2 G r e e k tortoises. 5~ At the e n d of the s u m m e r of the same year, several H e r m a n n ' s tortoises started showing clinical signs of stomatitis a n d rhinitis with nasal and ocular discharge, necrotic lesions of the tongue, swelling of the neck and the lower jaw, anorexia, a n d lethargy. As of the mid-aut u m n of the same year, 13 o f the original 21 H e r m a n n ' s tortoises h a d died. Several tortoises were submitted for necropsy a n d although no inclusion bodies were observed in any tissues using light microscopy, herpesvirus was isolated f r o m 10 of the 11 pharyngeal swabs collected f r o m live tortoises in the collection, f r o m the buffy coat of 1 live tortoise, a n d f r o m several tissue samples obtained f r o m necropsied tortoises. Although blood samples were collected f r o m tortoises in the collection, n o n e of the H e r m a n n ' s tortoises h a d titers to herpesvirus. In contrast with these observations, Muro et aP 3 r e p o r t e d a b o u t a higher sensitivity of Greek tortoises to herpesvirus infection w h e n c o m p a r e d with H e r m a n n ' s , and 4-toed tortoises (Agrionemys horsfieldi). In an epidemic of chronic rhinitis in 1990 in a private collection of Mediterranean tortoises in Spain, consisting o f 50 adult Greek tortoises, 12 H e r m a n n ' s tortoises, and 3 4-toed tortoises, only the Greek tortoises (n = 33) were affected. N o n e of the H e r m a n n ' s a n d 4-toed tortoises showed clinical signs. O f the ill Greek tortoises, 8 died a n d 12 were euthanized during the same year. By light microscopy, lesions were limited to the oral cavity and the respiratory system, varying in severity between different tor-

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toises. In the m o s t severe cases, eosinophilic intranuclear inclusions were detected in the epithelial cells of the u p p e r respiratory tract and m u c o s a of the tongue. Electron microscopy revealed particles of size, morphology, and structure consistent with those of herpesvirus. Recently, herpesvirus infection was docum e n t e d in pet tortoises in Japan. 54 An o u t b r e a k of herpesvirus was r e p o r t e d in recently i m p o r t e d p a n c a k e tortoises (Malachochersus tornieri) and 4-toed tortoises. Degenerate primers directed to anneal with a conserved portion of the nucleotide sequence of the herpesviral DNA polymerase g e n e c o n f i r m e d the presence of herpesvirus. A step further in the molecular diagnostics of tortoise herpesvirus was the d e v e l o p m e n t o f an in situ hybridization technique for the detection of tortoise herpesvirus nucleotide sequences in formalin-fixed p a r a f f i n - e m b e d d e d tissues. 55 In this report, the ability of the DNA p r o b e to hybridize with allegedly different herpesvirus strains f r o m distant geographic locations suggested the possibility of a high degree of conservation in the nucleotide sequence of the helicase gene a m o n g the tortoise herpesviruses. Phylogenetic analysis based on the partial s e q u e n c e of the helicase gene suggested the positioning of tortoise herpesvirus within the alpha subfamily. T h e first serologic test used to detect exposure to herpesvirus in tortoises was an SN assay. 5~ In this assay, 25 to 100 ~L of plasma is required, varying with the desired n u m b e r s of replicates to be p e r f o r m e d . SN, in general, is considered the gold standard for serodiagnosis of viral infection. 23 Overall, SN is an, excellent d i a g n o s t i c test that can be p e r f o r m e d in any laboratory once the necessary p a r a m e t e r s are well established and standardized. According to o u r experience, a seronegative tortoise should be retested after 5 to 9 weeks before b e i n g int e r p r e t e d as negative. It appears that the lowest SN titer m i g h t not be detected for several weeks after e x p o s u r e . O t h e r p r o b l e m s with this test include an abSence of defined sensitivity a n d specificity a n d an absence of a universally recognized reference strain. Despite the fact that the SN test is normally considered serotype-specific, in a transmission study with Greek tortoises at the University of Florida, we showed that the SN test was n o t able to distinguish between an isolate originating f r o m a tortoise in E u r o p e and f r o m a tortoise in the United States.

T h e limitations of the SN test were an incentive in the d e v e l o p m e n t of an ELISA for detecting herpesvirus antibody in tortoises. 57 This assay was d e t e r m i n e d to be statistically as reliable as the SN test, easier to p e r f o r m , and able to detect seroconversion of experimentally infected tortoises 2 to 5 weeks earlier than the SN test. Consistent with the SN test, the ELISA could not distinguish between different herpesvirus isolates. 58 T h e sensitivity a n d the specificity for this test were 97% a n d 98%, respectively. T h e positive and negative predictive values were 92% and 99%, respectively, d e m o n s t r a t i n g the reliability of this assay. A negative feature of the current assay is that the whole virus is n e e d e d as the antigen in this test. This makes the procedure somewhat costly and laborious because large amounts of virus have to be harvested and purified. We are currently working on the expression of different r e c o m b i n a n t viral proteins to be used eventually as a surrogate for the whole viral antigen. This would reduce the cost of the test a n d allow better standardization of the antigen. This ELISA has b e e n validated for G r e e k and H e r m a n n ' s tortoises a n d is currently b e i n g standardized for o t h e r species of tortoises. Along with the SN a n d the ELISA test, direct (DIP) and an indirect (IIP) i m m u n o p e r o x i d a s e based tests are currently available at the University of Florida. ~9 T h e IIP test allows detection of antibody to tortoise herpesvirus, whereas the DIP can detect the presence o f herpesvirus antigen in paraffin-embedded, formalin-fixed tissue. The DIP a n d IIP serve as c o m p l e m e n t a r y tests for SN and ELISA a n d conserve as a n o t h e r level of validation for exposure and infection with herpesvirus.

Marine Turtle Herpesviruses T h r e e herpesviruses have b e e n identified in marine turtles. A virus with the m o r p h o l o g i c a p p e a r a n c e of herpesvirus was shown to be the causative agent of epizootics of skin lesions t e r m e d grey-patch disease in y o u n g g r e e n turtles (C. mydas) between 56 a n d 90 days after hatching in aquaculture. 6~ Skin lesions c o m m e n c e d as small circular papular lesions, which coalesced into spreading patches containing e p i d e r m a l cells that h a d basophilic intranuclear inclusions that contained enveloped viral particles measuring 160 to 180 nm. T h e m o s t severe epizootics

Serology in Reptile Medicine

o c c u r r e d in the summer, u n d e r stressful envir o n m e n t a l conditions of high water t e m p e r a t u r e (>30~ crowding, and organic pollution. G r e e n sea turtles m o r e t h a n 1 year old at Cayman Turtle Farm, G r a n d Cayman, British West Indies, are susceptible to a disease characterized by p n e u m o n i a , tracheitis, a n d conjunctivitis. 61 T h e disease was called lung, eye, a n d trachea (LET) disease. T h e disease spreads quickly t h r o u g h a tank of turtles and generally runs its clinical course in 2 to 3 weeks. Histologically, focal areas of ballooning d e g e n e r a t i o n a n d necrosis of tracheal mucosal epithelial cells, with a m p h o p h i l i c intranuclear inclusions, were seen in several turtles early in the course of the disease. A herpesvirus was identified in tissue section a n d isolated f r o m affected turtles. Recently, an ELISA was developed to d e t e r m i n e e x p o s u r e of marine turtles to the LET-associated herpesvirus. 62 Plasma samples were collected f r o m juvenile g r e e n turtles f r o m 3 sites in Florida, and some samples f r o m each site h a d the anti-LET virus antibody. Fibropapillomas (FP) were first r e p o r t e d in the g r e e n turtle, C. mydas, over 60 years ago w h e n tumors were identified in g r e e n turtles f r o m the Florida Keys. 63,64 Based on visual observation alone or histologic evaluation o f lesions, FP seems to be present in several species of m a r i n e turtles including l o g g e r h e a d turtles (Caretta caretta), hawksbills (Eretmochelys imbricata), a n d olive ridley sea turtles (Lepidochelys olivacea). T u m o r s are seen as papillary, arborizing masses on the body surface, but they can also occur internally. 65 A high prevalence of FP has b e e n s h o w n in g r e e n turtles in Florida, 66,67 a n d in 1991 herpesvirus infection was associated with FP for the first time. 68 In experimental transmission studies of FP in g r e e n turtles using cell-free t u m o r extracts, b o t h filtered and unfiltered t u m o r extracts successfully induced t u m o r d e v e l o p m e n t 69 and herpesvirus-like particles were identified in tumors. O n e of the genes in the fibropapilloma-associated herpesvirus has b e e n sequenced, and phylogenetic relationships with o t h e r herpesviruses have b e e n constructed. 7~ An i m m u n o p e r o x i dase-based serologic test was developed to perf o r m correlative studies looking at the presence on fibropapillomas and presence of circulating antiherpesvirus antibody in affected g r e e n turtles. 71 In this test, sections of tumors containing

39

herpesvirus intranuclear inclusions were used as the source of antigen in the assay.

Paramyxovirus Viruses in the family Paramyxoviridae are known to infect a n d cause disease in a variety of snakes. T h e first r e p o r t in 1975 described a dieoff o f vipers (Bothrops moojeni) in a s e r p e n t a r i u m in Switzerland, 72,7~ and since that time multiple isolates have b e e n obtained f r o m captive snakes in G e r m a n y a n d the United States. 74-m This g r o u p of viruses has b e e n collectively called ophidian (snake) paramyxoviruses. Transmission studies in Aruba Island rattlesnakes (Cr0talus unicolor) showed a causal relationship between a proliferative p n e u m o n i a seen in snakes and an Aruba Island rattlesnake isolate of ophidian paramyxoviruses, s2 Recent comparative analyses of partial gene sequences for the large protein and hemagglutinin-neuraminidase protein of 16 reptilian paramyxoviruses recovered f r o m multiple species of snakes f r o m different families indicated that there were at least 2 distinct subgroups of isolates a n d several intermediate isolates, s3 In addition to snakes, paramyxoviruses also have b e e n isolated f r o m lizards, s4,s5 Based on this work, it seems that reptilian paramyxoviruses do not have a narrow reptilian host range. An HI assay has been developed to determine presence of antibody against ophidian paramyxoviruses. 7s,sl T h e H I assay has b e e n m o s t useful because of its relative simplicity and rapid turna r o u n d time. Briefly, s e r u m samples collected by h e a r t p u n c t u r e or tail v e n i p u n c t u r e are diluted 1:10 in sterile physiologic saline at 56~ for 30 minutes to inactivate c o m p l e m e n t , t h e n absorbed with washed a n d pelleted chicken erythrocytes to remove nonspecific agglutinins (12 hours at 5~ Using mierotiter methodology, serial doubling s e r u m dilutions are m a d e (1:10, 1:20, 1:40, 1:80, etc.) using 0.05-mL volumes of phosphate-buffered physiologic saline containing 0.1% bovine serum albumin. T h e latter minimizes autoagglutination of the erythrocyte suspension used later to indicate whether active virus is present or not. An ophidian paramyxovirus suspension diluted to contain 8 H A units/50 /xL is a d d e d to each serum dilution. This previously has b e e n d e t e r m i n e d by titration, taking advantage of the fact that the virus

Jacobson and Origg'i

40

causes chicken erythrocytes to agglutinate. Virus-serum mixtures are incubated for 1 h o u r at r o o m t e m p e r a t u r e , then 0.05 m L of a 0.5% suspension of washed chicken erythrocytes is added. T h e plates are placed at 4~ for 2 to 3 hours to p e r m i t settling of the erythrocytes. If antibodies are present in a particular s e r u m dilution, they will bind to viral particles a n d prevent t h e m f r o m hemagglutinating the erythrocytes. T h e s e r u m antibody titer is r e a d as the reciprocal of the highest s e r u m dilution that causes HI. An H I titer --<1:20 is considered negative, 1:40 to 1:80 suspect, a n d >1:80 positive, indicating exposure to the virus. Snakes that survive paramyxovirus infections m a y have H I antibody titers exceeding 1:10,240, b u t a single sample is only indicative of the exposure status at the time the sample was collected. T o d e m o n strate an active infection, samples should be collected at 2- to 4-week intervals to show a rising titer. T h e cross reactivity between the various isolates o f paramyxovirus has not b e e n d e t e r m i n e d . It is u n k n o w n whether one isolate is a d e q u a t e to d e t e r m i n e exposure to all known isolates. In a zoologic collection of snakes experiencing an o p h i d i a n paramyxovirus outbreak, snakes sampled at 5 m o n t h s after the death o f the first snake showed elevated antibody titers; 3 m o n t h s later, m a n y snakes sampled h a d antibody titers <1:80. 79 Although H I antibody does n o t r e m a i n elevated for p r o l o n g e d periods in m a n y snakes, in others it may remain elevated for long periods o f time. It is u n k n o w n if snakes with elevated antibody have latent infections.

Serology for Bacterial Exposure Chelonian Mycoplasmosis In most animals, respiratory mycoplasmosis is a slowly progressing, chronic, and seemingly clinically silent infection, which may be exacerb a t e d by environmental factors, stress, or o t h e r microbial agents. Most hosts have difficulty in eliminating the mycoplasma, even in the presence o f a strong i m m u n e response. In fact, the host i m m u n e response is critical for developm e n t o f lesions. Increased n u m b e r s of inflammatory/cells, particularly in loci, a n d lymphoid hyperplasia observed in the lesions are consistent,with respiratory mycoplasmosis in m o s t spe-

cies, including the desert and g o p h e r tortoise. Although overt clinical signs may be inapparent, lesions can range f r o m microscopic to gross, with eventual loss of the n o r m a l respiratory epithelium architecture. Thus for most mycoplasmas morbidity is quite high, b u t frank mortality is not c o m m o n until the end stages o f the disease. In 1988, a chronic u p p e r respiratory tract disease was recognized in desert tortoises (Gopherus agassizii) in the Desert Tortoise Natural Area, Kern County, California. a6 Partially because of this disease a n d because of r e p o r t e d population declines in the western Mojave Desert, desert tortoises n o r t h and west of the Colorado River were listed as t h r e a t e n e d by the United States D e p a r t m e n t o f the Interior in 1990. The disease was characterized clinically by serous, mucous, or p u r u l e n t nasal a n d ocular discharge, conjunctivitis, a n d palpebral edema. At a light microscopic level, there was infiltration of the nasal cavity m u c o s a and submucosa with inflammatory cells a c c o m p a n i e d by hyperplasia and d e g e n e r a t i o n of u p p e r respiratory tract epithelium. 8c~88Similar signs of disease were seen in free-ranging gopher tortoises (Gopherus polyphemus) in Florida. s9 A previously undescribed species of mycoplasma (Mycoplasma agassizii p r o p o s e d species novum) was subsequently cultured f r o m nasal lavages of affected tortoises a n d in transmission studies was identified as a causative agent of this disease, s7 An indirect ELISA was developed to detect the presence of anti-M, agassizii antibodies in tortoise plasma. 9~ Results of the ELISA are analyzed quantitatively but i n t e r p r e t e d categorically (negative, suspect, or positive). Two m o n o c l o n a l antibodies have b e e n d e v e l o p e d for use as the second antibody in this essay. O n e (HLl163) is a m o u s e antibody against tortoise IgM, the class of antibodies that a tortoise p r o d u c e s first in response to infection by M. agassizii. T h e second (HL673) is a m o u s e antibody against the light chain of b o t h IgM a n d IgY tortoise i m m u n o globulins. T h e advantages of diagnosing infection by ELISA include sensitivity a n d the ability to detect the stage of infection (early first time v chronic) by using different secondary antibodies. A disadvantage of ELISA testing is the c o n s u m p t i o n of plasma in the test. A positive test result proves past exposure but not c u r r e n t infection. T h e r e

Serology in Reptile Medicine

may be false positives caused by cross-reaction of tortoise antibodies to o t h e r bacteria with similar antigens. Consistent with well-documented imm u n e periodicity in other reptiles, there is some evidence of seasonal fluctuation in M. agassiziispecific antibody concentrations.

Crocodilian Mycoplasmosis T h e first r e p o r t of mycoplasmosis in a crocodilian involved y o u n g ranch-reared Nile crocodiles (Crocodylus niloticus) in Zimbabwe that h a d polyarthritis, m At necropsy, a mycoplasma was isolated f r o m infected joints a n d lungs and was Subsequently n a m e d M. crocodyli. 92 T h e disease was r e p r o d u c e d with e x p e r i m e n t a l inoculation of the m y c o p l a s m a in Nile crocodiles. In 1995, an epidemic of p n e u m o n i a with fibrinous polyserositis a n d multifocal arthritis was r e p o r t e d in a captive group o f American alligators (Alligator missippiensis) in Florida. 93 Mycoplasma aUigatoris sp. novum was cultured f r o m ill a n d d e a d alligators, and a subsequent transmission study showed a causal relationship. Alligator i m m u n o g l o b u l i n was purified f r o m alligator plasma and a polyclonal antibody was raised in rabbits against the purified i m m u n o g t o b u l i n . Polyclonal anti-alligator antibodies were purified a n d conjugated with biotin. This was used as the secondary antibody in an indirect ELISA that was developed for detecting exposure of alligators to M. alligatoris, g4 In a challenge study in A m e r i c a n alligators, broad-nosed caiman (Caiman latirostris), and Siamese crocodiles (Cr0codylus siamensis), the indirect ELISA detected seroconversion in all 3 species b e g i n n i n g 6 weeks after inoculation.

Serology for Parasite Exposure Cryptosporidiosis Cryptosporidium spp. (Apicomplex: Cryptosporiidae) are protozoal parasites that infect epithelial surfaces of the gastrointestinal, respiratory, biliary, and in some cases renal tissue of a wide variety of vertebrates. Multiple species are known. Since the first r e p o r t describing cryptosporidiosi s i n corn snakes (Elaphe guttata) in 2 zoologic ,collections, 95 there have b e e n n u m e r ous cases seen in o t h e r species o f snakes, lizards, a n d tortoises. Cryptosporidium serpentis is conside r e d the primary cryptosporidial p a t h o g e n in

41

reptiles. However, the t a x o n o m y of reptile crypt o s p o r i d u m has not b e e n studied very well, and it is expected that m o r e t h a n I species will be identified in reptiles. Both clinical and subclinical cryptospordiosis are known. In snakes, it is m o r e c o m m o n to see the disease in adult snakes rather than in neonates/juveniles. Major signs of disease include e n l a r g e m e n t o f the stomach wall and c o n c o m i t a n t regurgitation. Some snakes also may be seen with mucosal epithelial hyperplasia o f the d u o d e n u m . Extraintestinal sites of infection also have b e e n seen. Cryptosporidial sialoadenitis 96 a n d aural-pharyngeal polyps associated with cryptosporidum 97 were rep o r t e d in g r e e n iguanas. Renal cryptosporidiosis was seen in a g r e e n iguana a n d Parson's chameleon (Calumma parsonz) :96 Cryptosporidial infection of tortoises also has b e e n seen, but for the m o s t part is subclinical. An indirect ELISA was developed to determ i n e exposure of snakes to C. serpentis. 9s It is commercially available (AniLab, Baltimore, MD). However, the assay does not detect an antibody response in certain reptiles (such as leopard geckos) that are infected with cryptosporidium. This suggests that m o r e than 1 cryptosporidum exists in reptiles and that the C. serpentis antigen used in the ELISA does not cross-react with all reptile cryptosporidium.

Spirorchidiasis T h e family Spirorchidae contains digenetic trematodes that use fresh water a n d sea turtles as their final or definitive host. Spirorchids are known to infect l o g g e r h e a d (Caretta caretta), g r e e n ( C. mydas), and hawksbill (Eretmochelys imbricata) sea turtles; at least 8 g e n e r a a n d 20 species have b e e n described. 99 T h e i r life cycle is d e p e n d e n t o n an intermediate host, but n o n e has so far b e e n identified for any of the sea turtle spirorchids. Spirorchids are considered the m o s t pathogenic of all the parasites known to infect sea turtles, 1~176 a n d they are considered vascular syst e m generalists, with a p r e f e r e n c e for the heart and arterial system. 1~ Adults m a y cause an endocarditis, arteritis, and thrombosis of vessels, ~~ b u t eggs released within the vascular system may travel to r e m o t e areas of the turtle's b o d y where they lodge in small vessels a n d cause mild to severe granulomatous inflammation. 1~ The

42

Jacobson and Origgi

eggs can also migrate through the walls of vessels, causing tissue damage and inflammation in adjacent tissues. Parasitism with spirorchids may also allow secondary gram-negative bacterial infections, a03 An ELISA was developed using the surface glycocalyx crude antigen of adult blood trematodes Carettacola hawaiiensis, Haplotrema dorsopora, and Laeredius learedi for detecting antibodies in blood of Hawaiian green turtles naturally infected with these parasites.1 ~ For antigen preparation, all 3 species of trematodes were pooled. A direct ELISA using anti-reptilian/amphibian phosphatase-labeled IgG recognized green turtle immunoglobulin at a dilution of 1:12,800. Next, an indirect ELISA was used with the anti-reptilian/amphibian phosphatase-labeled IgG serving as the secondary antibody. Antibody to blood flukes was detected up to a dilution of 1:3,200. O f 59 samples collected from turtles at 5 sites, 47 (80%) were seropositive. T o look at the relationship of spirorchid infections and green turtle fibropapilloma, an indirect ELISA was developed using adult antigens of either Laeredius sp. or Haplotrema sp. and biotinylated monoclonal antibody HL857, which is specific for green turtle 7S IgY heavy chain. 71 Two wild green turtles, one with Learedius and one with Hapalotrema infection, were serologically evaluated. Both turtles with spirorchid infections had antispirorchid titers. Because each turtle was infected with only 1 of these parasites, considerable antigenic cross-reactivity was f o u n d between these 2 species of spirorchids. T h e ELISA was sensitive e n o u g h to detect exposure of green turtles to spirorchids even t h o u g h adult parasites could not be f o u n d in seropositive turtles at necropsy. Either there were too few parasites to find at necropsy or there wa s clearance of infection with persistence of antibody. It is unknown how long antibodies continue to circulate once there is clearance of infection.

Factors Affecting the Immune Response in Reptiles Seroconversion of reptiles after experimental immunization or natural influence can be influe n c e d by a n u m b e r of factors. The nature of the antigen, route o f administration, environmental conditions, and biology of the specific reptile are all variables that can affect the i m m u n e re-

sponse. All foreign proteins may not result in an i m m u n e response. In such cases, adjuvants may be used to maximize the response. The timing of immunization is critical because the ability of a reptile to b e c o m e i m m u n o c o m p e t e n t after birth varies between species. 1~ Some reptiles have a c o m p e t e n t i m m u n e system at birth, whereas others n e e d several months for the i m m u n e response to mature. Maternal transfer of antibody needs to be considered when interpreting neonate serology. 106 T h e i m m u n e response seems to respond maximally when the reptile has a body temperature within its thermal o p t i m u m zone. Reduced responses occur when a reptile is maintained at temperatures above and below this zone. 19A~ Seasonal cycles of lymphoid follicular involution and recrudescence occur in many reptiles, particularly those living in temperate regions. These cycles may correlate better with mating periods rather than changes in ambient temperature. During these periods, antibody levels may be low and corticosteroids and sex steroids high. 1~176 Sex-related differences also have been r e p o r t e d with males having lower antibody production during periods of high testosterone levels compared with females. Age, body temperature, season, and h o r m o n e s (sex) have to be considered when interpreting serologic findings.

References 1. Cooper EL, Klempau AE, Zapata AG: Reptilian immunity, in Gans C (ed): Biology of the Reptilia. Volume 14: Chicago, University of Chicago Press, 1985, pp 599-678 2. Ambrosius H: Immnnoglobulins and antibody production in reptiles, in Marchalonis JJ (ed): Comparative Immunology. Oxford, Blackwell Scientific, 1976, pp 298-334 3. Benedict AA, Pollard LW: Three classes ofimunoglobufins found in the sea turtle, Chelonia mydas. Folia Microbiol 17:75-78, 1972 4. Herbst LH, Klein PA: Monoclonal antibodies for the measurement of class-specific antibody responses in the green turtle, Chelonia mydas. Vet Immunol Immunopathol 46:317-335, 1995 5. Benedict AA, Pollard LW: The ontogeny and structure of sea turtle immunoglobulins, in Solomonx JB, Horton JD (eds): Developmental Immunology. Amsterdam, Elsevier, i977, pp 315-323 6. Coe JE: Immune response in the turtle (Chrysemys picta). Immunology 23:45, 1972 7. Grey HM: Phylogeny of the immune response. Studies on some physical, chemical and serologic characteristics of antibody produced in the turtle. J Immunol 91:819, 1963

Serology in Reptile Medicine

8. Ambrosius H, Frenzel EM: Anti-DNP antibodies in carp and tortoises. Immunochemistry 9:65-71, 1972 9. Saluk PH, Krauss J, Clem LW: The presence of two antigenically distinct light chains (kappa and lambda?) in alligator immunoglobulins. Proc Soc Exp Biol Med 133:365-369, 1970 10. Cutchens M, McLean E, Clem LW: Lymphocytic heterogeneity in fish and reptiles, in Wright RK, EL Coope EL (eds): Phylogeny of Thymus and Bone MarrowBursa Cells. Elsevier North-Holland Biomedical Press, Amsterdam, 1976, pp 205-213 11. Metchnikoff E: L'immunite dans les maladies infectieuses. Masson et Cie, Paris, 1901 12. Evans EE: Comparative immunology. Antibody response in Diopsosaurus dorsalis at different temperatures. Proc Soc Exp Biol Med 112:531-533, 1963 13. Ambrosius H, Hemmerling J, Richter R, et ah Immunoglobulins and the dynamics of antibody formation in poikilothermic vertebrates, in Sterzl, Riha I (eds): Developmental Aspects of Antibody Formation and Structure. Prague, Academia, 1969, pp 727-774 14. WetherallJD, Turner KJ: Immune response of the lizard Tiliqua rugosa. Aust J Exp Biol Med Sci 50:79-95, 1972 15. Natarajan K, Muthukkaruppan VR: Immunoglobulin classes in the garden lizard, Calotes versicolor. Dev Comp Immunol 8:845-854, 1984 16. Vorobjeva MS: Experimental study of humoral immunity in reptiles infected with tick-borne excephalitis virus. Vopr Virusol 10:36-41, 1965 17. Evans EE, CoMes RB: Effect of temperature on antibody synthesis in the reptile, Dipsosaurus dorsalis. Proc Soc Exp Bio! Med 101:482-483, 1959 18. Evans EE, Kent SP, Attleberger C, et al: Antibody synthesis in poikilothermic vertebrates. Ann N Y Acad Sci 126:629-646, 1965 19. Tait NN: The effect of temperature on the immune response in cold-blooded vertebrates. Physiol Zoo1 42: 29-35, 1969 20. Wright RK, Schapiro HC: Primary and secondary immune response of the desert iguana, Dipsosaurus dorsalis. Herpetologica 29:275-280, 1973 21. Salinitro SK, Minton S: Immune response of snakes. Copeia 3:504-514, 1973 22. Portis JL, Coe JE: IgM, the secretory immunoglobulin of reptiles and amphibians. Nature 258:54%548, 1975 23. Murphy FA, Gibbs EPJ, Horzinek MC, et al: Laboratory diagnosis of viral diseases. Chapter 12, in Veterinary Virology (ed 3). New York, Academic Press, 1999, pp 193-224 24. Tyler JW, Cullor JS: Titers, tests and truism: Rational interpretation of diagnostic serologic testing. J Am Vet Med Assoc 194:1550-1557, 1989 25. Baldock FC: Epidemiological evaluations of immunological tests, in Burgess GW (ed): ELISA Technology in Diagnosis and Research, Graduate School of Tropical Veterinary Science. Townsville, Australia, James Cook University of North Queensland, 1988, pp 90-95 26. Lillywhite HB, Smits AN: Lability of blood volume in snakes and its relationship to activity and hypertension. J Exp Biol 110:267-274, 1984 27. Smits AW, Kozubowski MM: Partitioning of body fluids

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and cardiovascular responses to circulatory hypovolemia in the turtle Pseudemys scripta elegans. J Exp Biol 116:237-250, 1985 28. Avery HW, Vitt LJ: How to get blood from a turtle. Copeia 209-210, 1984 29. Gandal CP: Cardiac punctures in anesthetized turtles. Zoologica 43:93-94, 1958 30. Jacobson ER: Reptiles, in Harkness J (ed): Veterinary Clinics of North America: Small Animal Practice. Philadelphia, Sannders, 1987, pp 1203-1225 31. MaxwellJH: Anesthesia and surgery, in Harless M, Morlock H (eds): Turtles: Perspectives and Research. New York, J o h n Wiley and Sons, 1979, pp 127-152 32. McDonald HS: Methods for the physiological study of reptiles, in Gans C, Dawson WR (eds): Biology of the Reptilia. Volume 5. Physiology A. New York, Academic Press, 1976, pp 19-125 33. Rosskopf WJ: Normal hemogram and blood chemistry values for California desert tortoises. Vet Med Small Anim Clin 77:85-87, 1982 34. Stephens GA, Creekmore JS: Blood collection by cardiac puncture in conscious turtles. Copeia 522-523, 1983 35. Taylor RW, Jacobson ER: Hematology and serum chemistry of the gopher tortoise, Gopherus polyphemus. Comp Biochem Physiol 72A:425-428, 1981 36. Olson GA, Hessler JR, Faith RE: Techniques for blood collection and intravascular infusions of reptiles. Lab Anim Sci 25:783-786, 1975 37. Jacobson ER: Immobilization, blood sampling, necropsy techniques and diseases of crocodilians a review. J Zoo Anita Med 15:38-45, 1984 38. Esra GN, Benirschke K, Griner LA: Blood collecting techniques in lizards. J Am Vet Med Assoc 167:555-556, 1975 39. Samour HJ, Risley D, March T, et al: Blood sampling techniques in reptiles. Vet Rec 114:472-478, 1984 40. LaPointeJL, Jacobson ER: Hyperglycemic effect ofneurohypophysial hormones in the lizard, Klauberina reversiana. Gen Comp Endocr 22:135-136, 1974 41. Jackson OF: Clinical aspects of diagnosis and treatment, in Cooper JE, Jackson OF (eds): Diseases of the Reptilia. Volume 2. London, Academic Press, 1981, pp 507-534 42. Harper PAW, Hammond DC, Heuschele WP: A herpesvirus-like agent associated with a pharyngeal abscess in a desert tortoise. J Wildl Dis 18:491-494, 1982 43. Pettan-Brewer KCB, Drew ML, Ramsay E, et al: Herpesvirus particles associated with oral and respiratory lesions in a California desert tortoise (G0phr agassizii). J Wildl Dis 32:521-526, 1996 44. Jacobson ER, Clubb S, Gaskin JM, et al: Herpesviruslike infection in Argentine tortoises. J Am Vet Med Assoc 187:1227-1229, 1985 45. CooperJE, Gschmeissner S, Bone RD: Herpes-like virus particles in necrotic stomatitis of tortoises. Vet Rec 123:554, 1988 46. Highfield AC: Viral epidemics in Mediterranean tortoises: Distribution of symptoms and mortality statistics by species and origin. London, England, The Tortoise Trust, 1990 47. Muller M, Sachsse W, Zangger N: Herpesvirus-Epi-

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