First line of defence: Skin microbiota may protect anurans from infective larval lungworms.

Functional roles of the rich microbiota of the skin are not fully understood, but include protection against microbial diseases and other environmental challenges. In experimental studies, we show that reducing the microbiota from cane toad (Rhinella marina) skin by gently wiping with absorptive gauze resulted in threefold higher rates of infection by lungworms (Rhabdias pseudosphaerocephala) following standardised exposure to infective skin-penetrating larvae. Higher concentrations of microbial DNA were associated with lower rates of lungworm entry. Our data suggest that microbial activity on the anuran skin comprises an important line of defence against attack by macroparasites as well as by fungi and other microbes.

Pathology Associated With an Outbreak of Entamoebiasis in Wild Cane Toads (Rhinella marina) in Tropical Australia.

Infection due to Entamoeba spp. is known to cause serious disease in primates (Entamoeba histolytica) and snakes (Entamoeba invadens), but there are no detailed descriptions of the pathology associated with Entamoeba spp. infection in amphibians. In 2014, an outbreak of entamoebiasis associated with a novel species of Entamoeba induced clinical illness and poor body condition in free-ranging cane toads in Australia's Northern Territory. Here, we describe the gross pathology, histology, and clinical pathology linked to the outbreak. The study compared 25 toads with invasive entamoebiasis, defined as histologically visible amoebas within tissue, and 12 toads without invasive entamoebiasis. Grossly, affected toads had mild to marked congestion of colonic serosal vasculature, with variable thickening of the intestinal wall and serosanguineous to hemorrhagic colonic content. Histologically, invasive entamoebiasis manifested primarily as moderate to severe, variably hyperplastic to ulcerative colitis. The small intestine was affected in 10 of 25 toads, and 5 of 25 toads also had gastric lesions. Amoebas consistent in morphology with Entamoeba sp. were commonly intermingled with mucosal epithelium, frequently along the basement membrane, with deeper invasion into the superficial lamina propria in only 5 toads. Toads with invasive entamoebiasis had neutrophilia, monocytosis, and lymphopenia, and thus elevated neutrophil to lymphocyte ratios, suggestive of an inflammatory and/or stress leukogram.

Dermatological conditions of farmed Crocodilians: A review of pathogenic agents and their proposed impact on skin quality.

The control of pathogens that target crocodilian skin is essential to the long-term success and sustainability of intensive farming operations worldwide. To understand the impact these pathogens may have on the skin, a brief overview of skin histology is given. A review of the known viral, bacterial, fungal and helminth taxa associated with skin conditions in commercially significant crocodilian species is presented. Best management practices are discussed, with an emphasis on addressing extrinsic factors that influence transmission and pathogenicity. It is argued that, in the past, reduced immune function arising from inadequate thermal regulation was the leading cause of skin disease in captive crocodilians. Consequently, innovations such as temperature control, coupled with the adoption of more stringent hygiene standards, have greatly reduced the prevalence of many infectious skin conditions in intensively farmed populations. However, despite improvements in animal husbandry and disease management, viral pathogens such as West Nile virus, herpesvirus and poxvirus continue to afflict crocodilians in modern captive production systems.

Divergent bornaviruses from Australian carpet pythons with neurological disease date the origin of extant Bornaviridae prior to the end-Cretaceous extinction.

Tissue samples from Australian carpet pythons (Morelia spilota) with neurological disease were screened for viruses using next-generation sequencing. Coding complete genomes of two bornaviruses were identified with the gene order 3'-N-X-P-G-M-L, representing a transposition of the G and M genes compared to other bornaviruses and most mononegaviruses. Use of these viruses to search available vertebrate genomes enabled recognition of further endogenous bornavirus-like elements (EBLs) in diverse placental mammals, including humans. Codivergence patterns and shared integration sites revealed an ancestral laurasiatherian EBLG integration (77 million years ago [MYA]) and a previously identified afrotherian EBLG integration (83 MYA). The novel python bornaviruses clustered more closely with these EBLs than with other exogenous bornaviruses, suggesting that these viruses diverged from previously known bornaviruses prior to the end-Cretaceous (K-Pg) extinction, 66 MYA. It is possible that EBLs protected mammals from ancient bornaviral disease, providing a selective advantage in the recovery from the K-Pg extinction. A degenerate PCR primer set was developed to detect a highly conserved region of the bornaviral polymerase gene. It was used to detect 15 more genetically distinct bornaviruses from Australian pythons that represent a group that is likely to contain a number of novel species.

Using experimental de-worming to measure the immunological and pathological impacts of lungworm infection in cane toads.

The immunological and pathological consequences of parasite infection can be more rigorously assessed from experimental manipulation than from correlational studies of natural infections. We used anthelmintic treatment to experimentally decrease intensities of lungworm infection in captive and free-ranging wild cane toads to assess parasite impacts on host immune responses. First, we administered the anthelmintic drug Ivermectin to both infected and uninfected toads, to distinguish drug effects per se from the impacts of killing lungworms. Worms began dying and decomposing <48 h after injection. The only immunological variables that were affected by anthelmintic treatment were bactericidal capacity of the blood which increased in parasitized toads (presumably triggered by decomposing worms in the lungs), and the phagocytic capacity of blood (which increased in both infected and uninfected toads); the latter effect presumably was caused by the injection of Ivermectin per se rather than removal of parasites. Second, we looked at correlates of variation in the infection intensity induced by de-worming (in both captive and free-ranging toads) over an eight-week period. Heavier lungworm infection was associated with increased phagocytic ability of the host's blood, and a reduction in the host's liver mass (and hence, energy stores). Experimental de-worming thus revealed pathological and immunological costs of the presence of lungworms, and of their removal by anthelmintic injection.

Diagnostic investigation of new disease syndromes in farmed Australian saltwater crocodiles (Crocodylus porosus) reveals associations with herpesviral infection.

Since 2006, 3 new disease syndromes have emerged in farmed saltwater crocodiles (Crocodylus porosus) in the Northern Territory of Australia. We describe the syndromes through a retrospective study of laboratory findings from 187 diagnostic cases submitted to Berrimah Veterinary Laboratories between 2005 and 2014. The first syndrome was characterized by conjunctivitis and/or pharyngitis (CP), primarily in hatchlings. Herpesviruses were isolated in primary crocodile cell culture, or were detected by PCR directly from conjunctiva or pharyngeal tissue, in 21 of 39 cases of CP (54%), compared with 9 of 64 crocodiles without the syndrome (14%, p < 0.0001). Chlamydiaceae were detected by PCR in conjunctiva or pharyngeal tissue of 55% of 29 CP cases tested, and of these, 81% also contained herpesvirus. The second syndrome occurred in juveniles and growers exhibiting poor growth, and was characterized histologically by systemic lymphoid proliferation and nonsuppurative encephalitis (SLPE). Herpesviruses were isolated or detected by PCR from at least 1 internal organ in 31 of 33 SLPE cases (94%) compared with 5 of 95 crocodiles without the syndrome (5%, p < 0.0001). The third syndrome, characterized by multifocal lymphohistiocytic infiltration of the dermis (LNS), occurred in 6 harvest-sized crocodiles. Herpesviruses were isolated from at least 1 skin lesion in 4 of these 6 cases. Although our study revealed strong associations between herpesvirus and the CP and SLPE syndromes, the precise nature of the role of herpesvirus, along with the pathogenesis and epidemiology of the syndromes, requires further investigation.

Molecular identification of three novel herpesviruses found in Australian farmed saltwater crocodiles (Crocodylus porosus) and Australian captive freshwater crocodiles (Crocodylus johnstoni).

As part of a larger investigation into three emerging disease syndromes highlighted by conjunctivitis and pharyngitis, systemic lymphoid proliferation and encephalitis, and lymphonodular skin infiltrates in farmed saltwater crocodiles (Crocodylus porosus) and one emerging syndrome of systemic lymphoid proliferation in captive freshwater crocodiles (Crocodylus johnstoni), cytopathic effects (CPE), including syncytial cell formation, were observed in primary crocodile cell lines exposed to clarified tissue homogenates from affected crocodiles. Ten cell cultures with CPE were then screened for herpesviruses using two broadly-reactive herpesvirus PCRs. Amplicons were obtained from 9 of 10 cell cultures and were sequenced. Three novel herpesviruses were discovered and the phylogenetic analysis of these viruses showed there was a 63% Bayesian posterior probability value supporting these viruses clustering with the subfamily Alphaherpesvirinae, and 100% posterior probability of clustering with a clade containing the Alphaherpesvirinae and other unassigned reptile herpesviruses. It is proposed that they are named Crocodyline herpesvirus (CrHV) 1, 2 and 3. CrHV1 and 2 were only isolated from saltwater crocodiles and CrHV3 was only isolated from freshwater crocodiles. A duplex PCR was designed that was able to detect these herpesviruses in formalin-fixed paraffin-embedded tissues, a sample type that neither of the broadly-reactive PCRs was able to detect these herpesviruses in. This work describes the isolation, molecular detection and phylogeny of these novel herpesviruses but the association that they have with the emerging disease syndromes requires further investigation.

Infection dynamics of the lungworm Rhabdias pseudosphaerocephala in its natural host, the cane toad (Bufo marinus), and in novel hosts (native Australian frogs).

Host-parasite systems have often evolved over time, such that infection dynamics may become greatly modified from the time of initial contact of the host with the parasite. Biological invasions may be useful to clarify processes in the initial contact of hosts with parasites, and allow us to compare parasite uptake between the ancestral (coevolved) host and novel (noncoevolved) hosts. Cane toads (Bufo marinus) are spreading rapidly through tropical Australia, carrying with them a nematode lungworm (Rhabdias pseudosphaerocephala) congeneric with those found in Australian frogs. We investigated the dynamics of infections of the toad parasite by conducting histologic examinations of cane toads and three native Australian frogs (Litoria dahlii, Litoria nasuta, and Opisthodon ornatus) at 2, 6, and 10 days after experimental exposure to the toad lungworm. More worms were found in toads than in frogs, especially at longer periods postexposure. In toads, the infective larvae entered the skin and muscles within 2 days postexposure, passed into the coelom in 6 days, and reached the lungs at 10 days. In frogs, larvae were found in many organs rather than migrating to consistent target tissues; a few larvae reached the lungs of L. dahlii. Migratory larvae caused increasing inflammation (primarily granulomatous admixed with granulocytes then lymphocytes) through time, especially in frogs. Evolution has resulted in an enhanced ability of the lungworm to locate the target organ (the lungs) of the toad, and an increase in rates of parasite survival within this host.

Primary ocular melioidosis due to a single genotype of Burkholderia pseudomallei in two cats from Arnhem Land in the Northern Territory of Australia.

Melioidosis was diagnosed in two domestic crossbred cats presented for unilateral ocular disease. One patient was born and bred in Nhulunbuy, Arnhem Land, while the other had moved there 6 months previously from Townsville, Queensland. Both patients were presented with sudden onset of a 'red eye' and blepharospasm, which progressed to an enlarged, painful, firm globe with loss of pupillary light reflexes and vision. An obvious primary focus of infection outside the eye was not detected in either cat. In both patients, the affected eye was surgically removed and vitreal culture revealed a pure growth of Burkholderia pseudomallei. In each instance, the infection had penetrated the sclera to produce retrobulbar cellulitis, and in one case frank retrobulbar abscessation. Histologically, there was a pyogranulomatous uveitis with extensive destruction of intraocular structures. The first case was still alive approximately 1 year following enucleation and limited antimicrobial therapy using amoxicillin clavulanate and doxycycline. The second was euthanased when a localised abscess developed on the same side of the face as the healed surgical incision, despite appropriate antimicrobial therapy. Both cases were caused by the same multilocus sequence type of B pseudomallei (ST 116), which had only been isolated previously from two human patients, both living in the same isolated geographical area as the cats of this report. Apart from the geographical clustering, no epidemiological links were evident between the two cats and/or the two people. The presumptive pathogenesis of these infections is discussed in relation to current knowledge about melioidosis in northern Australia.

Adenoviral infection in captive moose (Alces alces) in Canada.

Adenoviral infection was associated with hemorrhagic enteritis, serosal hemorrhages, and severe pulmonary edema in six captive moose (Alces alces) in Toronto, Ontario, Canada: an adult female moose and three calves in 1985 and two calves in 1998. Adenoviral disease was suspected based on histological findings of systemic vasculitis and widespread thrombosis associated with amphophilic intranuclear inclusions in endothelial cells. Diagnosis was confirmed by immunohistochemistry using antiserum to bovine adenovirus type 5, transmission electron microscopic identification of viral particles consistent in morphology with adenovirus within nuclei of pulmonary endothelial cells in an affected calf, and virus isolation. The restriction pattern of virus isolated from the lung of one of the calves indicated that the virus was identical to a recently characterized adenovirus in black-tailed deer (Odocoileus hemionus) in California. The moose adenovirus reported here may have been endemic in the captive moose herd, or infection may have resulted from either direct or indirect contact with other species of captive or wild cervids. This is the first report of adenoviral infection in moose and of the presence of adenoviral disease in a cervid in Canada.

The amphibian eye.

Knowledge of the anatomy of the amphibian eye is important to differentiate normal from abnormal. Special features of the amphibian eye mark the transition from aquatic to terrestrial life. The most often reported spontaneous ocular disease in the anurans (frogs and toads) is lipid keratopathy. An experimental study showed that high dietary cholesterol played a significant role in the development of corneal lipid infiltration. Other diseases, like panophthalmitis, occur with bacterial septicemia.

Nasopharyngeal myxosarcoma in a Bengal tiger (Panthera tigris).

An 18- to 25-yr-old intact female Bengal tiger (Panthera tigris) was diagnosed antemortem and postmortem with nasopharyngeal myxosarcoma metastatic to the lung, chronic lymphoplasmacytic otitis media, and lymphoplasmacytic and eosinophilic stomatitis. Myxosarcomas are rare in domestic animals and seldom metastasize; this tumor has not been previously reported in an exotic felid. Computed tomography of the skull was used during the diagnoses.