MANAGEMENT OF ENTAMOEBA INVADENS IN THE HERPETOLOGICAL COLLECTION AT THE SINGAPORE ZOO.

Amebiasis caused by Entamoeba invadens is an important disease in reptile collections, causing severe morbidity and mortality. Surveillance of the parasite at the Singapore Zoo was carried out over a 4-yr period by PCR testing on reptiles that presented with lethargy and enteritis for disease investigation. Asymptomatic reptiles sharing the same enclosures as positive individuals were also tested as part of outbreak investigation. Animals in the collection that tested positive for the parasite were treated with metronidazole at various doses, with the addition of paromomycin for two cases, until a negative PCR test result was obtained at the end of the treatment course. A total of 97 samples from 49 individuals across 19 species of reptiles were obtained, of which 24 samples (24.7%) from 19 animals were positive for E. invadens. Of these positive samples, 11 samples were for disease investigation, eight samples for outbreak surveillance, and five samples for treatment monitoring. Treatment was initiated for 10 animals, four of which were showing clinical signs of disease. The parasite was cleared in nine of these 10 animals (90%), with eight animals receiving metronidazole as a sole therapeutic agent. A total of nine animals died of the disease, four of which (44.4%) presented dead or died within 24 h of presentation. Necrotizing enteritis was a consistent postmortem finding resulting in gastrointestinal perforation in two cases, and coelomic adhesions and hepatic trophozoites were each seen in five animals. The results suggest that the management of Entamoeba epizootics in the collection requires prompt outbreak investigation. Diagnosis of the disease with advanced diagnostic tools like PCR, endoscopy, and ultrasonography and treatment with metronidazole in both symptomatic and asymptomatic animals may reduce mortalities during an outbreak.

MANAGEMENT OF A FATAL OUTBREAK OF STRONGYLOIDIASIS IN A CAPTIVE POPULATION OF PANTHER CHAMELEONS (FURCIFER PARDALIS) WITH IVERMECTIN.

An outbreak of the nematode Strongyloides sp. occurred in a population of 18 male and 29 female panther chameleons (Furcifer pardalis) at the Singapore Zoo. The parasite was first detected in one individual during routine microscopic examination of feces using the direct examination and magnesium sulfate flotation methods. The parasite was later found to have a closest match (98.96%) with Strongyloides sp. Okayama by DNA sequencing. Over a period of 6 mon, 97.9% (46/47) of the panther chameleons tested positive for the parasite, and 25.5% (12/47) of the animals died due to the disease. All the animals that died were female. Of the positive tests, magnesium sulfate flotation identified the parasite 98.1% (105/107) of the time, compared to direct fecal microscopy, which identified the parasite only 43.9% (47/107) of the time. Parasite eggs were detected in 100% (105/105) of the positive magnesium sulfate flotation tests but only 66.0% (31/47) of the positive direct fecal microscopy tests. Parasite larvae were detected in 61.7% (29/47) of the positive direct fecal microscopy tests but only 9.5% (10/105) of the magnesium sulfate flotation tests. Treatments with fenbendazole and pyrantel pamoate at published doses were ineffective at eliminating the parasite. Ivermectin (0.2 mg/kg PO q2wk for two doses) was successful at treating the parasite, with all animals testing negative for the parasite at the end of the treatment course without any observed adverse reactions. However, complete eradication of the parasite could not be achieved, as Strongyloides sp. could still be detected in the population on routine coproscopy intermittently over 3 yr. There were no further mortalities due to the disease with prompt treatment with ivermectin. Strongyloidiasis may cause high morbidity in panther chameleons, but severe disease leading to mortality can be prevented with the use of ivermectin.

Population distribution and causes of mortality of smooth-coated otters, Lutrogale perspicillata, in Singapore.

Smooth-coated otters (Lutrogale perspicillata) were transient in Singapore before one resident family group was observed in 1998, presumably having recolonized from Peninsular Malaysia. A population survey in 2017 revealed a minimum of 11 groups and 79 individuals. Since then, movements of otter groups within urban areas have led to increasing numbers of human-otter encounters, including conflicts. We determined the current abundance, population structure, and distribution of smooth-coated otters in Singapore. We assessed seven sampling zones nationwide through verified sighting records and social media. Mortality records from 2019 to 2021 were sourced from the Otter Working Group and Wildlife Reserves Singapore. In early 2021, there were a minimum of 17 groups and 170 individuals. Groups ranged from 2 to 24 individuals. Smooth-coated otters occupy coastal areas, waterways, reservoirs, and sites within the city center in urban gardens and ponds. Following territorial conflicts at waterways, smooth-coated otter groups moved into the urban matrix. Vehicle collisions are the main cause of mortality and are frequent at dams separating freshwater and coastal habitats. While there is a clear increase in smooth-coated otter numbers since 2017, there remain multiple natural and human-caused threats to otter persistence.

Systemic and Cardiac Amyloidosis in Captive Celebes Crested Macaques (Macacanigra).

Systemic amyloidosis has been described in many animals and the most common form is reactive systemic AA amyloidosis. However, cardiac amyloidosis leading to heart failure is rare in animals. We now describe systemic and cardiac amyloidosis in two captive endangered Celebes crested macaques (Macaca nigra) at the Singapore Zoo. Both animals were geriatric and had chronic morbidities. Physical examination, radiography and ultrasonography revealed cardiac arrhythmia, pleural effusion, pulmonary oedema and ascites, consistent with cardiac failure. Amyloidosis was suspected and confirmed as type AA by immunohistochemistry.

Management of Entamoeba histolytica in the non-human primates at the Singapore Zoo.

Amebic dysentery caused by Entamoeba histolytica accounts for significant morbidity in the non-human primates (NHP) at the Singapore Zoo. This includes the animals in the collection as well as a sizeable free-roaming wild crab-eating macaque (Macaca fascicularis) population. The disease is of great concern because of its zoonotic potential. Passive surveillance, both ante and post-mortem, of NHP displaying clinical symptoms and active surveillance of NHP assessed to be at a higher risk of infection were carried out via fecal real-time polymerase chain reaction (PCR) testing for 4 years. Treatment of the disease with 25 mg/kg metronidazole BID for 10 days followed by 15 mg/kg paromomycin BID for 7 days achieved good clinical resolution in most cases that tested positive. Three diseased NHP with severe clinical signs of weight loss, lethargy, and diarrhea were anesthetized for veterinary diagnostic investigation. Mesenteric lymphadenopathy was consistently seen on ultrasound examination in these severe cases of entamoebiasis. Two animals eventually died of severe chronic enteritis due to the disease. The eradication of entamoebiasis in the NHP at the Singapore Zoo may be complicated by the maintenance of a disease reservoir in wildlife, but a combination of timely treatment and efforts at maintaining biosecurity can help manage the disease in the collection.

Fatal Septicemia and Mastitis in Wild Sunda Colugos (Sunda Flying Lemurs; Galeopterus variegates) in Singapore.

Two free-ranging female Sunda colugos (Sunda flying lemur; Galeopterus variegates) were found dead in Singapore in November 2018 and February 2019. Septicemia and mastitis were diagnosed in both animals on postmortem examination. Infectious diseases have not previously been established as a cause of death in wild Sunda colugos.

First Report of Co-invasion by the Reptile Nematode Ozolaimus megatyphlon (Nematoda: Pharyngodonidae) with Invasive Green Iguanas (Iguana iguana) in the Asia-Pacific.

PURPOSE: Co-invasion of naïve ecosystems by non-native parasites is a serious threat to global biodiversity, though such events are difficult to detect early in the invasion process. Green iguanas (Iguana iguana) are an emerging invasive species and have colonised several countries in the Asia-Pacific. A survey was undertaken to determine whether parasites of the green iguana had co-invaded naïve ecosystems with their introduced host. METHODS: Over a 10-month period, wild green iguanas were trapped and euthanised in Singapore. All animals were necropsied and sampled for parasites. Parasites were then identified morphologically and subsequently characterised molecularly at the cytochrome c oxidase I (COI) locus. RESULTS: The reptile nematode Ozolaimus megatyphlon was found in 38% of the sampled green iguanas, with burdens of 100 + worms in all infected animals. This represents the first recorded co-invasion of this species with wild green iguanas in the Asia-Pacific. Based on the molecular characterisation of the cytochrome c oxidase I (COI) locus, the first DNA barcode is provided for O. megatyphlon. CONCLUSION: For the first time, the reptile nematode Ozolaimus megatyphlon is shown to be invasive and to have colonised the Asia-Pacific region with its introduced host, the green iguana. The DNA barcode provided here will facilitate future monitoring programmes as O. megatyphlon invades new habitats and countries.

Intralesional methylprednisolone injection as an adjunct treatment for peri-cloacal pyogranuloma in an African penguin (Spheniscus demersus).

An African penguin (Spheniscus demersus) presented with a large peri-cloacal mass. The mass was diagnosed as a pyogranuloma histologically, with multifocal to coalescing inflammatory responses in the subcutis and keratinized simple stratified squamous epithelium overlying the surface. The patient was prescribed 125 mg/kg oral clavulanic acid/amoxycillin twice a day and 20 mg/kg oral terbinafine once a day for 14 days, but there was no change in the size of the mass 4 days after therapy, so a decision was made to administer 1 mg/kg of intralesional methylprednisolone. Fourteen days later, the mass resolved with no further recurrence. This case demonstrated that intralesional methylprednisolone used with antibiotic and antifungals was effective in resolving a peri-cloacal pyogranuloma and without any side effects.

The use of a 3D-printed prosthesis in a Great Hornbill (Buceros bicornis) with squamous cell carcinoma of the casque.

The advent of new technologies in medical imaging and 3D printing in recent years has made customization of surgical tools and implants more accessible, revolutionizing many surgical fields. In many human diseases, these implants have led to superior surgical outcomes and greatly improved patients' quality of life. Thus, it is of great interest to apply these technologies to the treatment of animal diseases. In this study, we report the use of computed tomography (CT) and 3D printing for the treatment of a Great Hornbill at Jurong Bird Park that was diagnosed with squamous cell carcinoma of the casque. A 3D printed prosthesis that perfectly fitted the subject was implanted to replace its resected casque. The subject exhibited natural eating behaviour with no post-operative complications. Using this case as an example, the positive outcomes suggest a great potential in applying these technologies to the treatment of other wildlife diseases.

Tick-crocodilian interactions: a review, with the first record of tick (Acari: Ixodidae) infestation in the saltwater crocodile (Crocodylus porosus), and a concise host-parasite index.

Interactions between ticks and crocodilians (crocodiles, alligators, caiman, and gharials) are poorly studied but may have significant bearing on the ecology and health of these reptiles. The first record of tick infestation of the saltwater crocodile (Crocodylus porosus) is reported along with the first case of infestation by Amblyomma cordiferum on Cuvier's dwarf caiman (Paleosuchus palpebrosus). A review is also provided of tick-crocodilian interactions with a concise host-parasite index.

Correction to: The first authenticated record of the pangolin tick Amblyomma javanense (Acari: Ixodidae) in Singapore, with notes on its biology and conservation.

Due to an unfortunate turn of events, the surname of the third author appeared incorrectly in the original publication as it should have read Douay. The original article has been corrected, and the proper representation of the authors' names and their affiliation is also listed here.

The first authenticated record of the pangolin tick Amblyomma javanense (Acari: Ixodidae) in Singapore, with notes on its biology and conservation.

The first authenticated record of the co-endangered pangolin tick (Amblyomma javanense) is reported from the Republic of Singapore, based on specimens of males and females collected from the Sunda pangolin (Manis javanica). The biology of the species is reviewed and steps for its conservation are outlined to ensure its continued survival across its range.

Identification of possible nutritional and stress risk factors in the development of marmoset wasting syndrome.

Marmoset wasting syndrome (MWS) describes a series of symptoms in callitrichids that lead to general weakness and a failure to thrive in captive conditions such as zoological institutions. Though the cause of MWS has not been identified, the majority of hypotheses are linked to deficiencies of specific nutrients and increased stress levels. Questionnaires were sent to zoos requesting information on diets and housing of currently living and dead callitrichids before their deaths, as well as their postmortem reports. Risk factors for development and occurrence of MWS include close proximity of predator enclosures and high levels of dietary magnesium and zinc. Variables with effects which may protect against the development of MWS included provision of a nest box, natural trees within enclosure, reduced visibility to visitors, as well as dietary factors such as higher concentrations of potassium and fiber fractions. The protective effects of limited concentrate feeds and increased total dietary fiber may help reduce the risks of developing MWS. The minerals may not have biological implications in MWS per se, however, they may be reflective of diets too high in concentrates and too low in plant matter. Habitat designs that are less naturalistic and those which provide insufficient privacy or hide areas may increase chronic stress for callitrichid species, possibly because of visitor-related stress. Other causes of chronic stress in captive zoo populations should be the topic of further research to reduce occurrence of MWS.

Severe visceral pentastomiasis in an oriental small-clawed otter with functional thyroid carcinoma.

In January 2016, a 20-year-old female oriental small-clawed otter (Aonyx cinereus) from Night Safari in Singapore was euthanized and diagnosed with a thyroid gland carcinoma. Postmortem examination and histology also revealed metastasis to the regional lymph nodes and severe visceral pentastomiasis. Grossly, the lymph nodes were infested, and encapsulation was observed on the visceral serosal surface. Histopathologically, the lymph nodes were encysted by a thick fibrous connective capsule with minimal inflammatory response. Pentastomiasis has been previously reported in the smooth-coated otter (Lutrogale perspicillata) in Malaysia. This report is the first case of severe visceral pentastomiasis in an oriental small-clawed otter with functional thyroid carcinoma.

A nutritional nonalcoholic steatohepatitis minipig model.

BACKGROUND AND AIMS: The objective of this study was to elucidate whether a Western diet was associated with nonalcoholic steatohepatitis (NASH), and the relationship between NASH, autophagy and endoplasmic reticulum (ER) stress. METHODS: Four-month-old Lee-Sung minipigs were randomly assigned to two groups: control diet (C) and Western diet (W), for a 5-month experimental period. RESULTS: Feeding a Western diet produced a body composition with more fat, less lean and a greater liver weight. Compared with C pigs, W pigs also exhibited an elevated level of plasma insulin and free fatty acid. The W pigs displayed glucose intolerance, lower circulation antioxidant capacity and greater hepatic oxidative stress. Furthermore, pig fed the W diets had increased collagen accumulation in the liver and elevated systemic inflammation [tumor necrosis factor α and interleukin (IL)-6]. Compared with C pigs, W pigs had higher hepatic ER stress-related protein expression of GRP94, CHOP and caspase-12. The W pigs also had greater hepatic autophagy-related protein expression of p62 and LC3II. In an obesity antibody array analysis, W pigs had higher type 2 diabetes mellitus- (insulin-like growth factor 1, osteoprotegerin and resistin), atherosclerosis- (vascular endothelial growth factor, platelet-derived growth factor-AA and plasminogen activator inhibitor-I) and inflammation- [IL-1, macrophage-stimulating protein alpha, X-linked ectodermal dysplasia receptor and serum amyloid A (SAA)] related protein expressions. In addition, W pigs had greater plasma SAA concentration than C pigs and plasma SAA level was highly associated with IL-6. CONCLUSIONS: We successfully established a NASH pig model, and our findings suggested an association of NASH with ER stress and autophagy. The SAA has potential as a novel plasma biomarker for nonalcoholic fatty liver disease pigs.

Interactions of host IL-6 and IFN-gamma and cancer-derived TGF-beta1 on MHC molecule expression during tumor spontaneous regression.

Many tumors down-regulate major histocompatibility complex (MHC) antigen expression to evade host immune surveillance. However, there are very few in vivo models to study MHC antigen expression during tumor spontaneous regression. In addition, the roles of transforming growth factor betal (TGF-beta1), interferon gamma (IFN-gamma), and interleukin (IL)-6 in modulating MHC antigen expression are ill understood. We previously reported that tumor infiltrating lymphocyte (TIL)-derived IL-6 inhibits TGF-beta1 and restores natural killing (NK) activity. Using an in vivo canine-transmissible venereal tumor (CTVT) tumor model, we presently assessed IL-6 and TGF-beta involvement associated with the MHC antigen expression that is commonly suppressed in cancers. IL-6, IFN-gamma, and TGF-beta1, closely interacted with each other and modulated MHC antigen expression. In the presence of tumor-derived TGF-beta1, host IFN-gamma from TIL was not active and, therefore, there was low expression of MHC antigen during tumor progression. TGF-beta1-neutralizing antibody restored IFN-gamma-activated MHC antigen expression on tumor cells. The addition of exogenous IL-6 that has potent anti-TGF-beta1 activity restored IFN-gamma activity and promoted MHC antigen expression. IFN-gamma and IL-6 in combination acted synergistically to enhance the expression of MHC antigen. Thus, the three cytokines, IL-6, TGF-beta1, and IFN-gamma, closely interacted to modulate the MHC antigen expression. Furthermore, transcription factors, including STAT-1, STAT-3, IRF-1, NF-kappaB, and CREB, were significantly elevated after IL-6 and IFN-gamma treatment. We conclude that the host IL-6 derived from TIL works in combination with host IFN-gamma to enhance MHC molecule expression formerly inhibited by TGF-beta1, driving the tumor toward regression. It is suggested that the treatment of cancer cells that constitutively secrete TGF-beta1 should incorporate anti-TGF-beta activity. The findings in this in vivo tumor regression model have potential applications in cancer immunotherapy.

Use of chemical ablation with trichloroacetic acid to treat eyelid apocrine hidrocystomas in a cat.

CASE DESCRIPTION: A 7-year-old Persian cat was evaluated for recurrence of multiple cystic periocular masses. A number of cyst-like lesions had been resected from the left eyelids 18 months earlier, with lesions recurring within 6 months after surgery. The cat had blepharospasm and signs of discomfort following rupture of the largest cyst the day prior to examination. Previous histologic examination of the cysts had revealed apocrine hidrocystomas. CLINICAL FINDINGS: Multiple pigmented nodules were seen around the skin of the upper and lower left eyelids. The nodules were brownish to black, round, soft, and fluid-filled. Signs of pain were not evident during palpation of the nodules. TREATMENT AND OUTCOME: The largest cyst on the upper eyelid was removed by means of a V-shaped full-thickness excision. Histologic and immunohistochemical examination of the excised tissue confirmed the diagnosis of apocrine hidrocystoma. The remaining periocular cysts were surgically debrided and then treated topically with 20% trichloroacetic acid. All lesions healed rapidly without any signs of discomfort. During a recheck examination 12 months later, the upper and lower left eyelids appeared morphologically normal, and there was no evidence of recurrence. CLINICAL RELEVANCE: Findings suggested that chemical ablation with trichloroacetic acid may be a useful treatment for apocrine hidrocystomas in cats.