T cell function in tuatara (Sphenodon punctatus)

Tuatara are the sole survivors of an entire order of reptiles that thrived during the age of the dinosaurs. Therefore, knowledge of their physiology is critical to understanding the phylogeny of reptiles. Previous studies of the immune system of the tuatara did not assess T cell function. We analyzed T cell function among six captive tuatara by assessing concanavalin A (Con A), phytohemagglutinin (PHA) and mixed lymphocyte reaction (MLR) induced T cell proliferation. Peripheral blood mononuclear cells from six out of six and four out of four tuatara tested exhibited significant proliferative responses to Con A and PHA, respectively, as measured by an MTT reduction assay. A lower level of proliferation was detected in an MLR. However, Con A activated lymphocytes were not cytotoxic for a xenogeneic murine mastocytoma cell line (P815).     

Squamous cell carcinoma in a tuatara (Sphenodon punctatus)

CASE HISTORY: A mature male tuatara was presented with a history of recurrent cloacal prolapse. CLINICAL AND PATHOLOGICAL FINDINGS: The prolapsed tissue included a 12 mm diameter mass, which histologically showed cords and nodules of neoplastic epithelial cells. DIAGNOSIS: The mass was diagnosed as a squamous cell carcinoma with ulceration of the overlying stratified squamous epithelium and diffuse inflammation of the surrounding dermis. CLINICAL RELEVANCE: This case presented a rare opportunity to investigate squamous cell carcinoma in a long-lived lower vertebrate species. Future recurrence or metastasis of the mass may add useful information to the current base of knowledge of the behaviour of malignant neoplasms in reptiles.     

两种柞蚕微孢子虫病病原致病力的比较

近年柞蚕微孢子虫病的发生有了新的变化,病原种类中寄生率较低的Nosemasp.(一种未命名的小孢子)发生比例在逐年上升。比较原来的寄生优势种柞蚕微粒子虫与Nosemasp.对蚕的致病力,结果表明:Nosemasp.的致病力与柞蚕微粒子虫相当。发生比例上升的原因多数是镜检遗漏所致。     

Squamous cell carcinoma in a tuatara (Sphenodon punctatus)

CASE HISTORY: A mature male tuatara was presented with a history of recurrent cloacal prolapse.

CLINICAL AND PATHOLOGICAL FINDINGS: The prolapsed tissue included a 12 mm diameter mass, which histologically showed cords and nodules of neoplastic epithelial cells.

DIAGNOSIS: The mass was diagnosed as a squamous cell carcinoma with ulceration of the overlying stratified squamous epithelium and diffuse inflammation of the surrounding dermis.

CLINICAL RELEVANCE: This case presented a rare opportunity to investigate squamous cell carcinoma in a long-lived lower vertebrate species. Future recurrence or metastasis of the mass may add useful information to the current base of knowledge of the behaviour of malignant neoplasms in reptiles.     

Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals

Microsporidia are a ubiquitous group of obligate intracellular parasites that infect all major animal groups. Enterocytozoon bieneusi is the most commonly identified Microsporidia in humans and has also been reported worldwide in animals with importance in veterinary medicine (e.g. cats, dogs, horses, cattle and pigs). The identification of E. bieneusi in animals has raised the question of the importance of animal reservoirs in the epidemiology of this pathogen, and the implications of the infection with this pathogen in infected animals. Considerable genetic diversity within E. bieneusi has been found with over 90 genotypes identified based on the ITS nucleotide sequence of E. bieneusi spores recovered from the feces of infected humans and animals. Both host-adapted E. bieneusi genotypes with narrow host ranges and potentially zoonotic genotypes with wide host specificity have been identified. The information presented in this review should be useful in understanding the taxonomy, epidemiology, zoonotic potential, and importance in public health of E. bieneusi.     

Microsporidiosis in a Gouldian finch (Erythrura [Chloebia] gouldiae)

A 12-day-old nestling Gouldian finch (Erythrura [Chloebia] gouldiae) was presented for investigation of a mortality problem in nestling finches raised by Bengalese finch foster parents. On histological examination, large numbers of spores consistent with a microsporidian organism were present within the small intestinal mucosa. Electron microscopy and molecular studies (sequencing the 5' end of the ssu rRNA gene) further defined the organism as Encephalitozoon hellem. Sequence homology with other eukaryotes was determined using a BLASTN search from the NCBI GenBank database. The finch isolate sequences showed greater than 99% homology with those of previously reported human and avian isolates.     

中英HEL项目暨国内“微孢子与微孢子虫病”专题研讨会在华南农业大学召开

为进一步加深国内外同行的合作与交流，2004年3月10日至13日在华南农业大学召开了“中英HEL项目暨国内微孢子虫与微孢子虫病”专题研讨会。     

Microsporidiosis in a Gouldian finch (Erythrura [Chloebia] gouldiae)

A 12-day-old nestling Gouldian finch ( Erythrura [Chloebia] gouldiae ) was presented for investigation of a mortality problem in nestling finches raised by Bengalese finch foster parents. On histological examination, large numbers of spores consistent with a microsporidian organism were present within the small intestinal mucosa. Electron microscopy and molecular studies (sequencing the 5' end of the ssu rRNA gene) further defined the organism as Encephalitozoon hellem . Sequence homology with other eukaryotes was determined using a BLASTN search from the NCBI GenBank database. The finch isolate sequences showed greater than 99% homology with those of previously reported human and avian isolates.     

Presence of antibodies to Salmonella in tuatara (Sphenodon punctatus) sera

Colonisation of a host by pathogenic microorganisms is a near constant threat to the health of all vertebrates and most species have evolved an efficient adaptive immune response which produces antibodies following exposure to a specific antigen. The strength of this response can be influenced by many factors including sex and season. Tuatara are exposed to Salmonella through contact with infected skinks and soil; however, no gastrointestinal colonisation of tuatara with Salmonella has been found. Using Western blot and flow cytometry we have demonstrated that tuatara possess antibodies which recognise Salmonella antigens, but many of these antibodies are not specific and are cross-reactive with two closely related and ubiquitous bacteria, Escherichia coli and Citrobacter koseri. Our study describes the anti-Salmonella immune responses in tuatara and will help to inform decisions around maintaining wildlife health, as well as providing important insights into the role and development of adaptive immunity in reptilian species.     

Failure to detect Salmonella species in a population of wild tuatara (Sphenodon punctatus)

AIM: To assess the prevalence of faecal excretion of Salmonella serovars by wild tuatara (Sphenodon punctatus) on Stephens Island, New Zealand. METHODS: One hundred cloacal swabs obtained as part of health-screening for the translocation of adult tuatara from Stephens Island were subjected to general aerobic culture and enrichment, and cultured specifically for Salmonella spp. RESULTS: No Salmonella spp were cultured from any of the cloacal samples, which suggests that, at the 95% confidence interval, the maximum prevalence of tuatara in the island population that were shedding Salmonella spp not detected by our sample size was 1.5%. Mixed bacteria were grown from the 70 cloacal swabs cultured aerobically. A predominant organism was evident in 30 cultures, and these were identified as Hafnia alvei type 1 (n=16) and type 2 (n=7), Corynebacterium spp (n=4), Klebsiella oxytoca (n=2), and Moraxella spp (n=1). CONCLUSIONS: The absence of intestinal carriage of Salmonella spp by the tuatara sampled in this study may indicate either lack of exposure, or an innate resistance to intestinal colonisation in tuatara. The significance of the other bacteria cultured as potential pathogens to the tuatara and as zoonotic risks is also uncertain. Wildlife managers should screen translocated reptiles for Salmonella spp, and thereby avoid exposing wild and managed populations to infection.     

Health screening for a translocation of captive-reared tuatara (Sphenodon punctatus) to an island refuge

AIMS: To screen tuatara undergoing translocation from a captive crèche to an island refuge for evidence of health and known diseases, and apply basic epidemiological techniques to assess the significance of disease test results.

METHODS: Tuatara (n=353) were physically examined and samples were taken from a random selection (n=30) for estimated white cell counts, screening for haemoparasites, and culture for Salmonella, Yersinia, Aeromonas and Campylobacter spp. Direct faecal smears were carried out on-site, and faecal floats were later performed to assess levels of endoparasitism with helminths and protozoa (n=69). Modified Ziehl-Neelsen staining was used to screen faecal smears, and positive specimens were further screened using an immunofluorescence antibody (IFA) test for Cryptosporidium oocysts.

RESULTS: There was no evidence of external parasites on any of the animals examined and only one animal had a gross abnormality. All estimated white cell counts were in the range 2.8– 17.5 × 10⁹/L. No haemoparasites were observed. There were no enteric pathogens cultured, indicating the intestinal carriage of these bacteria in the tuatara was 9.4%. Of the 69 individual faecal samples examined, 12 (17%) had unidentified coccidial oocysts, 21 (30%) had nematode ova of various kinds, and 12 (17%) had intestinal carriage of motile protozoa consistent with Trichomonas spp and another unidentified organism. Nineteen (28%) tuatara had acid-fast oocysts present; however, IFA staining failed to detect any Cryptosporidium oocysts.

CONCLUSIONS: Our understanding of the diversity of gastrointestinal endoparasites affecting tuatara is inadequate as many of the parasite ova seen could not be identified. This is the first record of tuatara as a host for Trichomonas spp of protozoa in the gastrointestinal tract.     

Detection of Paranannizziopsis australasiensis in tuatara (Sphenodon punctatus) using fungal culture and a generic fungal PCR

AIMS: To describe the methods used at the Animal Health Laboratory (AHL, Ministry for Primary Industries) to identify Paranannizziopsis australasiensis.

METHODS: Skin biopsy samples from two adult male tuatara were submitted to the AHL in March 2014. Approximately half of each sample was processed for fungal culture and incubated on mycobiotic agar containing cycloheximide at 30°C. Following morphological examination of the culture products, DNA was extracted from suspect colonies. PCR was used to amplify the internal transcribed spacer (ITS) region of fungal rRNA using primers ITS1 and ITS4. Positive amplicons were subjected to DNA sequencing and the results were compared to published sequences. In addition, DNA was extracted from the remaining skin samples and the same PCR was carried out to compare the results.

RESULTS: After 7 days of incubation, colonies morphologically resembling P. australasiensis were observed. DNA extracted from these isolates tested positive for P. australasiensis by PCR and DNA sequencing. Samples of DNA extracted directly from the infected skin samples tested negative for P. australasiensis using the generic fungal PCR.

CONCLUSIONS AND CLINICAL RELEVANCE: Isolation and identification of P. australasiensis was carried out using a combination of fungal culture and molecular testing available at AHL. Results were available in significantly less time than in the past, when isolates had to be sent overseas. PCR and sequencing of fungal isolates is a valuable tool for identification of species that have few, if any, unique macroscopic or microscopic features to aid identification. Further sampling from captive and wild New Zealand reptiles will provide important information on the epidemiology of P. australasiensis, and the conservation and management implications for tuatara and other native reptile species.     

Health screening for a translocation of captive-reared tuatara (Sphenodon punctatus) to an island refuge

AIMS: To screen tuatara undergoing translocation from a captive crèche to an island refuge for evidence of health and known diseases, and apply basic epidemiological techniques to assess the significance of disease test results. METHODS: Tuatara (n=353) were physically examined and samples were taken from a random selection (n=30) for estimated white cell counts, screening for haemoparasites, and culture for Salmonella, Yersinia, Aeromonas and Campylobacter spp. Direct faecal smears were carried out on-site, and faecal floats were later performed to assess levels of endoparasitism with helminths and protozoa (n=69). Modified Ziehl-Neelsen staining was used to screen faecal smears, and positive specimens were further screened using an immunofluorescence antibody (IFA) test for Cryptosporidium oocysts. RESULTS: There was no evidence of external parasites on any of the animals examined and only one animal had a gross abnormality. All estimated white cell counts were in the range 2.8- 17.5 x 10(9)/L. No haemoparasites were observed. There were no enteric pathogens cultured, indicating the intestinal carriage of these bacteria in the tuatara was <9.4%. Of the 69 individual faecal samples examined, 12 (17%) had unidentified coccidial oocysts, 21 (30%) had nematode ova of various kinds, and 12 (17%) had intestinal carriage of motile protozoa consistent with Trichomonas spp and another unidentified organism. Nineteen (28%) tuatara had acid-fast oocysts present; however, IFA staining failed to detect any Cryptosporidium oocysts. CONCLUSIONS: Our understanding of the diversity of gastrointestinal endoparasites affecting tuatara is inadequate as many of the parasite ova seen could not be identified. This is the first record of tuatara as a host for Trichomonas spp of protozoa in the gastrointestinal tract.     

Dermatomycosis caused by Paranannizziopsis australasiensis in five tuatara (Sphenodon punctatus) and a coastal bearded dragon (Pogona barbata) in a zoological collection in New Zealand

CASE HISTORY: Health monitoring of tuatara (Sphenodon punctatus) at Auckland Zoo between 2001 and 2009 showed that 58/93 tuatara had been affected by dermatitis of unknown origin. From 2011 onwards, cases of suspected fungal dermatitis underwent extensive diagnostic investigations.

CLINCAL FINDINGS: Six cases of dermatomycosis were attributed to Paranannizziopsis australasiensis, five in tuatara and one in a coastal bearded dragon (Pogona barbata). Cases presented typically as raised, yellow to brown encrustations on the skin. Severe cases progressed to necrotising ulcerative dermatitis, and in the bearded dragon to fatal systemic mycosis. Following topical and systemic treatments, lesions resolved in all five tuatara.

LABORATORY FINDINGS: Histopathological examination of skin biopsy samples revealed dermatitis with intralesional septate branching hyphae. Fungal culture yielded isolates morphologically resembling Chrysosporium species, and isolates were submitted for molecular confirmation and sequencing of DNA.

DIAGNOSIS: All six cases were confirmed as dermatitis due to infection with P. australasiensis, on the basis of fungal culture and DNA sequencing of isolates.

CLINICAL RELEVANCE: These are the first reported cases of dermatomycosis associated with P. australasiensis infection in tuatara, and the first cases in which systemic therapeutic agents have been used in the treatment of such disease. Tuatara at the Auckland Zoo are now routinely examined every 3 months and tissue samples from any lesions sent for histopathology and fungal culture. Further work to elucidate the epidemiology and significance of P. australasiensis infections in reptiles in New Zealand is important for both welfare and conservation purposes.