Diseases of the respiratory tract of chelonians.

Diseases of the respiratory tract commonly occur in captive chelonians, and several diseases also have occurred in wild chelonians. Infectious causes include viruses, bacteria, fungi, and parasites. Herpesviruses have surfaced as important pathogens of the oral cavity and respiratory tract in Hermann's tortoise (Testudo hermanii), spur-thighed tortoise (Testudo graeca), and other tortoises in Europe and the United States. Herpesvirus-associated respiratory diseases also have been reported in the green turtle, Chelonia mydas, in mariculture in the Cayman Islands. Of diseases caused by bacteria, an upper respiratory tract disease caused by Mycoplasma sp has been reported in free-hanging and captive gopher tortoises in the southeastern United States and in desert tortoises in the Mojave Desert of the southwestern United States. Mycotic pulmonary disease is commonly reported in captive chelonians, especially in those maintained at suboptimal temperatures. An intranuclear coccidia has been seen in several species of captive tortoises in the United States, and, in one case, a severe proliferative pneumonia was associated with organisms in the lung. The most common noninfectious cause of respiratory disease in chelonians results from trauma to the carapace. Although pulmonary fibromas commonly occur in green turtles with fibropapillomatosis, for the most part, tumors of the respiratory tract are uncommon in chelonians.     

Intranuclear coccidiosis in tortoises: nine cases

Chelonian intranuclear coccidiosis has been reported once, in two radiated tortoises (Geochelone radiata), and is apparently rare. We describe intranuclear coccidiosis diagnosed histologically in two radiated tortoises, three Travancore tortoises (Indotestudo forstenii), two leopard tortoises (Geochelone pardalis), one bowsprit tortoise (Chersina angulata), and one impressed tortoise (Manouria impressa). Infection was systemic and involved alimentary, urogenital, respiratory, lymphoid, endocrine, and integumentary systems. Trophozoites, meronts, merozoites, macrogametocytes, microgametocytes, and nonsporulated oocysts were seen histologically or by electron microscopy. Intracytoplasmic and extracellular stages of parasite development also were identified histologically. Sequencing of a coccidial 18S rRNA consensus polymerase chain reaction (PCR) product revealed a novel sequence that provided phylogenetic information and may be useful for further diagnostic test design. Intranuclear coccidiosis was associated with variable degrees of inflammation in all cases, was considered the cause of death in six tortoises, and was a substantial contributing factor to the cause of death in two tortoises.     

Cytobrushing of the oral mucosa as a possible tool for early detection of testudinid herpesvirus in Horsfield’s tortoises with nonspecific clinical signs

Forty-five Horsfield’s tortoises (Testudo horsfieldii; syn. Agrionemys horfieldii, Russian tortoise) belonging to different owners had decreased appetite and respiratory issues. Twenty-nine tortoises had epiphora, dyspnea, and white necrotic diphtheroid oral plaques (group G1). Ten of the remaining 16 tortoises had serious dehydration, appetite disorder, and depression (G2). The last 6 tortoises had only decreased appetite and moderate conjunctival discharge (G3). During the physical examination of all 45 tortoises, a cytologic sample and an oral swab for herpesvirus and Mycoplasma agassizii PCR testing were taken. In 20 of 29 specimens from G1, in 8 of 16 from G2, and 0 of 6 from G3, the cytologic exam revealed intranuclear acidophilic inclusion bodies, multinucleate cellular syncytia, and further abnormalities caused by herpesviral infection. Moreover, all 45 tested subjects were found to be positive for testudinid herpesvirus 1; 2 were positive for M. agassizii. This prospective study suggests that Horsfield’s tortoises with such signs would benefit from this screening procedure, given that it was effective in a significant proportion of infected and symptomatic animals, and no negative effects were seen.     

Herpesvirus infection in tortoises (Malacochersus tornieri and Testudo horsfieldii)

Large numbers of pancake tortoises (Malacochersus tornieri) and Horsfield tortoises (Testudo horsfieldii) in three consignments imported into Japan died soon after arrival. Some tortoises in the first consignment were dead on arrival. Postmortem examination of two of the pancake tortoises and four of the Horsfield tortoises revealed necrotizing lesions of the oral mucosa in both species, primarily in the tongue. Eosinophilic to amphophilic inclusion bodies were visible in the nuclei of mucosal epithelial cells in the lesions. Similar inclusion bodies were observed in the liver, spleen, adrenal glands, stomach, lungs, kidneys, small and large intestines, pancreas, and cerebrum of the pancake tortoises and in the liver, spleen, and pancreas of the Horsfield tortoises. Electron microscopic examination of the cells containing inclusion bodies showed herpesvirus-like particles about 100 nm in diameter in the cytoplasm. Nested polymerase chain reaction analysis using a herpesvirus consensus primer method confirmed the presence of a characteristic herpesvirus base sequence in tissue from these lesions.     

Adenovirus group II-like infection in chukar partridges (Alectoris chukar)

Seven live 5-to-6-wk-old chukar partridges (Alectoris chukar) were examined because of increased lacrimation, swollen eyelids, and increased mortality. Gross lesions consisted of mildly enlarged and mottled white spleens, swollen eyelids with external scab formation, and watery intestinal contents. Microscopically, there were increased numbers of mononuclear phagocytic system cells in the spleen, some of which contained faintly staining basophilic intranuclear inclusion bodies, blepharoconjunctivitis, enteritis associated with coccidia and crop mycosis. Transmission electron microscopy of the spleen revealed icosahedral virus particles 65 to 75 nm in diameter, consistent with the morphology of adenovirus. Three out of seven chukars were positive for hemorrhagic enteritis virus by serology.     

Calcium and phosphorus values and their derivatives in captive tortoises (Testudo species)

Objective : To evaluate the relationships between total calcium and phosphorus and ionised calcium and phosphorus values in clinically healthy tortoises. Methods : Jugular blood samples were obtained from 25 tortoises, as part of a health screen of the population. These comprised Hermann’s tortoises, Testudo hermanni boettgeri, spur-thighed tortoises, Testudo graeca ibera, marginated tortoises, Testudo marginata, and horsfield tortoises, Testudo horsfieldi. Plasma from these samples were analysed for total calcium, ionised calcium and phosphorus levels. These samples were taken in the immediate posthibernation period, before the onset of reproductive activity. Results : Females exhibited statistically significantly higher levels of phosphorus. Ionised calcium and total calcium levels were elevated in females compared with males, but this was not statistically significant. Females did have statistically significantly higher calculated solubility indexes and statistically significantly lower ratios compared with males. Clinical Significance : This study has provided an insight into the ratios and solubility indexes in tortoises, and this information may lead to further understanding of the significance of these parameters in chelonians.     

Molecularbiological diagnosis of herpes virus infection of a juvenile Russian tortoise (Agrionemys horsfieldii) with skin and lung lesions

Herpesvirus infections are significant in the care of turtles and tortoises. Clinical signs range from unspecific symptoms, due to the variety of organ manifestations, to the "classical" picture of rhinitis-stomatitis. The presented case study showed the typical disease only with respect to clinical symptoms following hibernation, but lacks stomatitis, erosions or plaques in the oral mucosa. On the other hand, skin lesions on the extremeties, causative with herpesvirus infection, could be diagnosed. In this case study, various symptoms, sampling procedures and diagnostics using two different PCR methods are presented. Following hibernation, samples from a Russian tortoise (Agrionemys horsfieldii) were taken ante mortem and post mortem and screened with respect to virology, pathology, bacteriology and parasitology. DNA-fragments specific for tortoise herpesvirus were detected in various organs and body liquids. Furthermore basophilic intranuclear inclusion bodies were found. The bacteriological examination showed a high level of Pasteurella testunis in the lungs. By parasitological examination nematodes (Oxyridae) were diagnosed. A potential prophylaxis tortoise herpesvirus is discussed.     

Identification of a novel herpesvirus from a California desert tortoise (Gopherus agassizii)

Herpesviruses are significant pathogens of tortoises, causing upper respiratory tract disease and necrotizing stomatitis, with infections often associated with high mortality rates. Herpesvirus infection in a captive California desert tortoise (Gopherus agassizii) was detected by light microscopic observation of intranuclear inclusion bodies in various tissues followed by transmission electron microscopic observation of herpesvirus-like particles, and amplification of herpesvirus nucleic acid sequences using polymerase chain reaction. Using an indirect enzyme linked immunosorbent assay, anti-tortoise herpesvirus antibodies were detected one month after initial onset of clinical signs. This novel herpesvirus is distinct from the previously described tortoise herpesvirus (tortoise herpesvirus-1, THV-1) sharing 83% sequence identity of 60 amino acids of a portion of the DNA polymerase gene and 79% sequence identity across 120 amino acids of a portion of the ribonucleotide reductase gene. Similar to THV-1, this novel herpesvirus, tortoise herpesvirus-2 (THV-2), also clusters with the alphaherpesviruses.     

Biochemical and hematologic values for 18 clinically healthy radiated tortoises (Geochelone radiata) on St Catherines Island, Georgia

BACKGROUND: The radiated tortoise (Geochelone radiata) is a critically endangered species in its native land, the southern portion of the island of Madagascar. Captive breeding programs have generated data on the breeding behavior and ecology of G radiata; however, hematologic and biochemical data also are critically important in managing populations. OBJECTIVE: The purpose of this study was to evaluate sex and seasonal effects on hematologic and biochemical data from captive radiated tortoises. METHODS: Whole blood was collected in January and August 2001 from 18 radiated tortoises (10 male, 8 female) housed at the Wildlife Survival Center on St Catherines Island, Georgia, as part of a routine health assessment. Routine hematologic and plasma biochemical analyses and electrophoresis were done using standard methods. Data from male and female tortoises were compared within and between seasons using 2-way ANOVA. RESULTS: RBC and HCT values were significantly higher in summer than in winter and were higher in males than in females. Total protein concentration did not differ significantly between males and females; however, female tortoises had significantly higher concentrations of alpha1- and beta-globulins in winter and summer compared to males. Male tortoises had significantly higher sodium and uric acid concentrations and LDH activity during winter, and higher urea concentration and LDH and CK activities in summer, compared with females. Female tortoises had significantly higher triglyceride and phosphorus concentrations in winter, and higher phosphorus, cholesterol, and triglyceride concentrations in summer, compared with males. CONCLUSION: Sex and seasonal differences in hematologic and biochemical values for radiated tortoises likely reflect vitellogenesis and egg production in females, and altered hydration status and activity in summer. Data from the tortoises in this study will be useful for the seasonal health assessment of this species.     

Concurrent experimentally induced infection with Eimeria bovis and coronavirus in unweaned dairy calves.

Over a period of 3 summers, 21 colostrum-fed Holstein bull calves, 1 to 3 days old, were assigned to 7 replicates, each consisting of 3 calves. Within each replicate of 3 calves, 2 were selected at random, to be given 100,000 to 146,000 sporulated coccidia oocysts (principally Eimeria bovis) orally 60 hours after arrival at the college research farm. On the thirteenth day after coccidia inoculation, 1 of the 2 calves that had been given coccidia and the third calf that had not been inoculated, were given coronavirus by intranasal and oral routes. Calves were observed daily, and consistency of feces was scored visually. Nasal swab specimens for indirect immunofluorescent antibody testing for coronavirus and fecal samples for oocyst determination were obtained approximately every third day. Of 7 calves that were given only coronavirus, 3 developed diarrhea of short duration. Of 7 calves that were given only coccidia oocysts, 6 developed diarrhea. All 7 calves inoculated initially with coccidia and subsequently with coronavirus developed diarrhea. For 5 of 7 replicates, calves that were given coccidia and coronavirus developed diarrhea first. When overall severity, measured by fecal score and by blood in the feces, was compared, calves inoculated with coccidia followed by coronavirus were more severely affected (P less than 0.05) than were calves that were given only coronavirus. Calves that were given only coccidia oocysts appeared more severely affected than calves that were given only coronavirus, but differences were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimicrobial resistance genes present in the faecal microbiota of free‐living Galapagos tortoises (Chelonoidis porteri)

Antimicrobial resistance (AMR), encoded by plasmid‐mediated AMR genes (ARGs), is an increasing global public health threat. Wildlife play a fundamental role as sentinels, reservoirs and potential vectors of ARGs. For the first time in Galapagos, we have identified and quantified the presence of ARGs in free‐living giant tortoises (Chelonoidis porteri). We performed ARG analyses by quantitative PCR of faeces collected from the cloaca of 30 tortoises widely distributed across Santa Cruz Island. Validated samples (n = 28) were analysed by a panel of up to 21 different ARGs and all 28 tortoise samples were positive to one or more genes encoding resistance. Thirteen of 21 tested ARGs were present in at least one sample, and 10 tortoises (35.7%) had a multi‐resistant pattern. We recommend additional research so we may more fully understand resistance patterns across taxa and geographical locations throughout the Galapagos archipelago, and the implications of ARGs for the health of wildlife, domestic animals, and humans. In this study, we found 100% of sampled giant tortoises had ARGs present in their faeces, suggesting a large‐scale distribution of these genes within the archipelago.     

Concurrent infections with reoviruses and coccidia in broilers

These experiments investigated the interaction among two species of coccidia (Eimeria acervulina and E. mitis) and three strains of reovirus (virus 2035, a weak to moderate pathogen; and viruses 2408 and 1733, severe pathogens). When reoviruses were not present, high inoculation dosages (10(6) sporulated oocysts/bird) of both E. acervulina and E. mitis depressed weight gain, plasma pigment, and plasma protein. Low doses of coccidia (10(4) oocysts) in the absence of virus had no such effect on weight gain. When high doses of coccidia were present at the same time as virus 2035 or 2408, they resulted in a significantly greater depression of weight gain than when either virus or coccidia were present alone. With virus 2035, this greater depression was seen even when low doses of coccidia were used. Lesion scores due to coccidiosis and the number of oocysts produced were not affected by previous exposure to reovirus. Both coccidiosis and reovirus infections increased the frequency of some leg problems and other abnormal conditions. The most obvious interaction between coccidia and reovirus was the marked increase in swollen hocks seen when coccidia and virus 2035 were present together (20-27%) compared with either the virus or coccidia alone (0-10%). Virus 2408 interfered slightly with the development of immunity to coccidia. There was some indication that early coccidiosis could increase the ability of some virus isolates to infect various tissues of the host.     

Sedative Effects of Intranasal Midazolam and Dexmedetomidine in 2 Species of Tortoises (Chelonoidis carbonaria and Geochelone platynota)

The sedative effects of intranasal midazolam and dexmedetomidine were evaluated in 2 tortoise species as a means to facilitate handling and performing diagnostic procedures. Six red-footed tortoises (Chelonoidis carbonaria) and 6 Indian star tortoises (Geochelone platynota) received the following treatments in a randomized order with 2-week washout periods between the treatments: midazolam (0.5 and 1.5 mg/kg), dexmedetomidine (0.05 and 0.15 mg/kg), and saline control. Tortoises were evaluated and scored for sedation (using a previously published subjective method) by blinded observers, and results were averaged for each species and treatment group. Low-dose dexmedetomidine (0.05 mg/kg) in red-footed tortoises resulted in a significantly higher median sedation score at 5 minutes compared with other treatments. Control saline resulted in a higher median sedation score 5 minutes after administration in Indian star tortoises compared with red-footed tortoises. No other significant differences were observed between treatments or between species. The results suggest that intranasal administration of midazolam or dexmedetomidine, at the dosages used in this study, do not provide effective sedation in red-footed tortoises or Indian star tortoises, based on a subjective scoring system.     

湘黄鸡球虫病病原学调查与鉴定

鸡球虫病是危害养鸡业的主要疾病之一,为了解湘黄鸡球虫病病原学流行情况,选择湘黄鸡饲养较集中的衡东、衡山、衡南、衡阳县以及衡阳市郊区五个县区,以地面散养的湘黄鸡为病原学调查对象。通过粪样分肠段采集,采用琼脂薄板单卵囊的分离培养和球虫单卵囊感染试验。经鉴定,结果发现湘黄鸡球虫病的病原体有堆型艾美耳球虫、早熟艾美耳球虫、柔嫩艾美耳球虫、毒害艾美耳球虫、巨型艾美耳球虫等五种球虫。鉴定中,采用4%琼脂糖块将单卵囊分离最合适,此法与其他分离方法比较,简单易行,不需要专门的分离仪器。     

Occurrence of coccidiosis in calves at one to six months of age, which are housed in a large capacity barn]

One- to six-month-old calves, housed in the large-capacity calf house of the Nová Bystrice state farm in the district of Jindrich?v Hradec were studied for the occurrence of coccidia in the period from January 1974 to April 1975. The coccidia found in these animals included nine species of the genus Eimeria: E. zuernii, E. bovis, E. ellipsoidalis, E. subspherica, E. auburnensis. The species E. alabamensis, E. cylindrica, E. wyomingensis and E. bukidnonensis occurred just sporadically. Further, the findings included oocysts of Isopora spp. The oocysts of the coccidia E. wyomingensis and Isopora spp. were found for the first time in Czechoslovakia. The overall extensity of invasion (EI) and intensity of invasion (II) grow with age as follows: (chart: see text) The results suggest that even modern large-capacity calf houses are not free from invasion by coccidia; the calves in the house under study were invaded by ten species. Transition to vegetable feeding is accompanied by a marked rise of the EI of all the species of coccidia found in the animals.     

石榴皮提取物对离体培养兔球虫卵囊的杀灭作用

本试验旨在研究石榴皮提取物对离体培养兔球虫卵囊的杀灭作用。采集已感染球虫的18只家兔(体重2.0~2.5 kg)的新鲜粪便(24 h内,35 g/只),用饱和盐水漂浮法分离、提取球虫卵囊。球虫卵囊分别在蒸馏水、石榴皮水提物、石榴皮黄酮、石榴皮多酚、盐酸氯苯胍中培养24、48、72 h,光镜下观察不同浓度的各种药物对兔球虫卵囊数量的影响。结果显示,与蒸馏水组比较,5、10 mg/mL剂量组培养72 h,4种药物均使兔球虫卵囊数量明显减少。球虫卵囊的减少率石榴皮水提物组大于石榴皮黄酮组,石榴皮黄酮组大于石榴皮多酚组和盐酸氯苯胍组;起效时间约为48~72 h。1 mg/mL剂量组4种药物虽可使兔球虫卵囊数量减少,但无显著差异(P＞0.05)。结果提示,石榴皮提取物对体外培养的兔球虫卵囊有较强的杀灭作用,其中石榴皮水提物对兔球虫卵囊杀灭作用最强,石榴皮黄酮和石榴皮多酚次之,其杀灭作用呈一定的正相关量效关系。     

Eimeria tenella搭载科学卫星对虫体生物学特性影响的初探

为探讨微重力与太空辐射等因素对鸡球虫生物学特性的影响，我们首次将一株已孢化的柔嫩艾美耳球虫搭载于返回式科学试验卫星。卫星运行７天后返回地面。     

Tonometry in adult yellow-footed tortoises (Geochelone denticulata)

Objective To determine intraocular pressure (IOP) in adult yellow‐footed tortoises using applanation tonometry. Animals Fifteen healthy adult captive yellow‐footed tortoises (eight males and seven females). Procedures Intraocular pressures were estimated for tortoises by using an applanation tonometer after topical anesthesia. Body length, measured from nuchal to anal scutes, ranged from 27.5 to 57.2 cm. Five measurements from each eye were obtained by a single observer in an ambient temperature of approximately 30 °C. Results Mean ± SEM IOP of 30 eyes of 15 yellow‐footed tortoises was 14.2 ± 1.2 mmHg. Range of IOP was 6–30 mmHg for tortoises. Significant differences were detected neither between right and left eyes (P = 0.357) of individual tortoises, nor between males and females (P = 0.524). Observer's readability was good (intraclass coefficient = 0.65), and IOP did not change over the ordered five measurements. Conclusions There was no significant difference in IOP between males and females in this specie. Tonometry values for normal eyes may represent a useful diagnostic methodology for recognition and treatment of ocular diseases in reptiles.     

Soluble scute proteins of healthy and ill desert tortoises (Gopherus agassizii)

OBJECTIVES: To characterize protein composition of shell scute of desert tortoises and to determine whether detectable differences could be used to identify healthy tortoises from tortoises with certain illnesses. ANIMALS: 20 desert tortoises. PROCEDURES: Complete postmortem examinations were performed on all tortoises. Plastron scute proteins were solubilized, scute proteins were separated by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were analyzed, using densitometry. Two-dimensional immobilized pH gradient-PAGE (2D IPG-PAGE) and immunoblot analysis, using polyclonal antisera to chicken-feather beta keratin and to alligator-scale beta keratin, were conducted on representative samples. The 14-kd proteins were analyzed for amino acid composition. RESULTS: The SDS-PAGE and densitometry revealed 7 distinct bands, each with a mean relative protein concentration of > 1 %, ranging from 8 to 47 kd, and a major protein component of approximately 14 kd that constituted up to 75% of the scute protein. The 2D IPG-PAGE revealed additional distinct 62- and 68-kd protein bands. On immunoblot analysis, the 14-, 32-, and 45-kd proteins reacted with both antisera. The 14-kd proteins had an amino acid composition similar to that of chicken beta keratins. There was a substantial difference in the percentage of the major 14-kd proteins from scute of ill tortoises with normal appearing shells, compared with 14-kd proteins of healthy tortoises. CONCLUSIONS AND CLINICAL RELEVANCE: The major protein components of shell scute of desert tortoises have amino acid composition and antigenic features of beta keratins. Scute protein composition may be altered in tortoises with certain systemic illnesses.     

Resistance of leopard tortoises and helmeted guineafowl to Cowdria ruminantium infection (heartwater).

Experimental infection trials were conducted to investigate susceptibility of leopard tortoises (Geochelone pardalis) and helmeted guineafowl (Numida meleagris) to infection with Cowdria ruminantium, the causative agent of heartwater, a tickborne disease of domestic and wild ruminants. Ten guineafowl were inoculated intravenously with a virulent dose of C. ruminantium derived from bovine endothelial cell cultures, and four leopard tortoises were exposed to C. ruminantium infection by the feeding of infected Amblyomma hebraeum ticks. Uninfected A. hebraeum ticks (on both tortoises and guineafowl) and Amblyomma marmoreum ticks (on tortoises only) were fed on the animals during weeks 2 and 3 post-exposure in an attempt to detect infection. These ticks were analyzed for C. ruminantium infection by xenodiagnosis and with the C. ruminantium-specific pCS20 polymerase chain reaction (PCR) assay. Attempts to detect infection in ticks fed on either species were negative by both tests. These results suggest that leopard tortoises and helmeted guineafowl are refractory to C. ruminantium infection and, therefore, are unlikely to be capable of introducing heartwater directly into new areas. However, leopard tortoises are efficient hosts of A. marmoreum and A. hebraeum and are likely to be important epidemiologically in the transport and maintenance of these tick vector species.