Development of a peptide-generated antibody to rabbit hemorrhagic disease virus 2 VP60 and its immunohistochemical application in natural cases.

Rabbit hemorrhagic disease virus 2 (RHDV2) has spread across the United States infecting and causing death in domestic and wild rabbits. Immunohistochemistry (IHC) would be a useful tool for the detection of RHDV2 antigen in tissues as it is inexpensive and readily achievable in most diagnostic laboratories. However, there is no readily available antibody for this purpose. To fill this void, we generated an RHDV2 capsid protein VP60-specific antibody in chicken eggs and validated the antibody using formalin-fixed tissues from 5 domestic rabbits naturally infected with RHDV2. Viral antigen was detected immunohistochemically in various tissues, most prominently in hepatocytes and macrophages in liver, and in macrophages in spleen and cecal lymphoid tissue. Intravascular mononuclear cells in lung and renal tubular and biliary epithelium also were immunolabeled. Both nuclear and cytoplasmic immunolabeling were observed. This peptide-generated antibody is a potentially useful tool as an adjunct to reverse-transcription PCR or in situ hybridization for detection of RHDV2 in tissues.

Plasma and interstitial fluid antibiotic levels of subcutaneously implanted compounded florfenicol calcium sulfate beads in New Zealand White rabbits (Oryctolagus cuniculus).

OBJECTIVE: To determine antibiotic levels in plasma and interstitial fluid (ISF) after SC placement of compounded florfenicol (FF) calcium sulfate beads (CSBs) in New Zealand White rabbits (Oryctolagus cuniculus). ANIMALS: 6 juvenile female rabbits (n = 5 treatment and 1 control). METHODS: An ultrafiltration probe and CSBs were placed SC in 6 rabbits (n = 5 for FF CSBs and 1 for control CSBs). Plasma (3, 6, 12, 24, and 48 hours and 7, 14, and 21 days) and ISF (daily for 21 days) samples were collected, and FF was measured by HPLC for pharmacokinetic analysis. Hematology, biochemistry, and histopathology were assessed. RESULTS: Means ± SD for the area under the curve, maximum concentration, time of maximum concentration, terminal half-life, and mean residence time to the last data point for plasma and ISF were 16.63 ± 8.16 and 17,902 ± 7,564 h·µg/mL, 0.79 ± 0.38 and 245 ± 223 µg/mL, 2.90 ± 0.3 and 59 ± 40 hours, 30.81 ± 16.9 and 27.3 ± 9.39 hours, 23.4 ± 10 and 73.7 ± 13 hours, respectively. Plasma FF was < 2 µg/mL at all time points. The ISF FF remained > 8 µg/mL for 109.98 to 231.58 hours. One rabbit death occurred during treatment, but the cause of death was undetermined. Local tissue inflammation was present, but no clinically significant systemic adverse effects were found on hematology, biochemistry, or histopathology in the remaining rabbits. CLINICAL RELEVANCE: Florfenicol CSBs maintained antibiotic concentrations in ISF at levels likely to be effective against bacteria sensitive to > 8 µg/mL for 5 to 10 days while maintaining low (< 2 µg/mL) plasma levels. Florfenicol CSBs may be effective for local antibiotic treatment in rabbit abscesses.

Causes of morbidity and mortality in wild cottontail rabbits in the eastern United States, 2013-2022.

Interest in causes of mortality of free-ranging, native North American lagomorphs has grown with the emergence of rabbit hemorrhagic disease virus 2 (RHDV2). Over the years 2013-2022, the Southeastern Cooperative Wildlife Disease Study received 119 Sylvilagus spp. case submissions from the central and eastern United States, comprising 147 rabbits. Most (86%) of these submissions occurred after detecting RHDV2 in the United States in 2020. Laboratory data from these rabbits were retrospectively evaluated for major causes, contributors to mortality, and pathogen detections. Gross and histologic examination was performed for 112 rabbits. Common primary causes of death included trauma (n = 49), bacterial disease (n = 31), emaciation (n = 6), and parasitism (n = 6). Among the 32 rabbits with bacterial disease, 12 were diagnosed with tularemia and 7 with pasteurellosis. Rabbits with pasteurellosis had disseminated abscessation, septicemia, and/or polyserositis. Less commonly, cutaneous fibroma (n = 2), notoedric mange (n = 2), encephalitozoonosis (n = 2), neoplasia (round-cell sarcoma; n = 1), and congenital abnormalities (n = 1) were diagnosed. RHDV2 was not detected in 123 rabbits tested. Although RHDV2 has not been detected in wild lagomorphs in the eastern United States, detections in domestic rabbits from the region emphasize the need for continued surveillance. Furthermore, continued surveillance for Francisella tularensis informs public health risk. Overall, increased knowledge of Sylvilagus spp. health furthers our understanding of diseases affecting these important prey and game species.

Trichomonad Disease in Wild Turkeys (Meleagris gallopavo): Pathology and Molecular Characterization of Histomonas, Tetratrichomonas, Tritrichomonas, and Simplicimonas spp.

The Wild Turkey (Meleagris gallopavo) is a geographically widespread, popular game bird and conservation icon in North America. Following successful reestablishment in parts of the US and Canada, regional declines have fueled population health concerns; therefore, understanding mortality causes and spatiotemporal patterns of disease is important to uncover potential ongoing and future health risks. Histomonosis, caused by the trichomonad Histomonas meleagridis, is a well-established and potentially fatal disease in Wild Turkeys; however, its prevalence and potential population health impacts remain poorly understood. Moreover, molecular tools recently have allowed for the detection of additional trichomonads that similarly can cause fatal disease in Wild Turkeys. We describe and compare disease due to H. meleagridis with that of Tetratrichomonas gallinarum, Tritrichomonas sp., and Simplicimonas sp. in Wild Turkeys in the southeastern US. Among 857 Wild Turkeys evaluated postmortem from 2002 to 2023, 34 (4.0%) were diagnosed with trichomonad disease, often assumed to be histomonosis prior to molecular testing. However, among 25 debilitating to fatal trichomonad disease cases for which etiologies were confirmed by PCR from 2015 to 2023, H. meleagridis was detected in 16/25 (64.0%), T. gallinarum in 6/25 (24.0%), Tritrichomonas sp. in 2/25 (8.0%) and Simplicimonas sp. in 1/25 (4.0%). These turkeys had similar clinical manifestations, and although lesion patterns varied to some extent, liver and/or intestinal tract was most commonly affected. Coinfections were common among all turkeys with trichomonad disease from 2015 to 2023 (21/25, 84.0%) and included viruses (lymphoproliferative disease virus, avian poxvirus), bacteria (Streptococcus gallolyticus, Listeria monocytogenes, Escherichia coli), and other protozoa (Sarcocystis sp., Haemoproteus sp.). Our results highlight the importance of molecular diagnostic testing in determining etiologies of trichomonad disease in Wild Turkeys. Further evaluation of the epidemiology and pathogenesis of trichomonad disease and its varied etiologies in Wild Turkeys and other birds is warranted to better understand risk factors and potential health impacts.

High Prevalence and Broad Distribution of Trichomonas gypaetinii in Bald Eagles (Haliaeetus leucocephalus) in the USA.

Trichomonas gypaetinii was detected in 117 (88%) of 133 Bald Eagles (Haliaeetus leucocephalus) and in 0/7 Golden Eagles (Aquila chrysaetos) in the USA, with no sex or age prevalence difference. All eagles lacked associated lesions. This study indicated that T. gypaetinii is common and widespread in Bald Eagles, but rarely associated with disease.

West Nile Virus Infections in an Urban Colony of American White Ibises (Eudocimus albus) in South Florida, USA.

West Nile virus (WNV) is pathogenic in a wide range of avian hosts and is endemic in much of North America. This virus is responsible for population declines of some Passeriformes. We describe a WNV-associated mortality event in American White Ibis (Eudocimus albus) nestlings. This is a species, inherent to the Everglades ecosystem, which has recently begun nesting in urban areas. An urban colony in south Florida was monitored from March-July in 2020 as part of an ongoing study. Nestling carcasses were collected opportunistically and sent to the Southeastern Cooperative Wildlife Disease Study, University of Georgia within 24 h for diagnosis. Three ibis nestling deaths were confirmed to be caused by WNV infection based on histopathology, immunohistochemistry, and reverse transcription PCR. Serial plasma samples collected weekly from 36 healthy chicks of the same urban rookery were tested for WNV-neutralizing antibodies via plaque reduction neutralization test; four chicks were seropositive. Antibody titers in three seropositive chicks from which serial samples were collected waned over time, suggesting maternal antibody transfer. Ibis mortalities were consistent with a spike of WNV activity in this region of Florida. West Nile virus infection may be an important seasonal cause of mortality for wading bird nestlings.

Distinctive Gross Presentation in Free-Ranging White-Tailed Deer (Odocoileus virginianus) with Rabies.

The white-tailed deer (Odocoileus virginianus) is a popular game species in North America and often lives in close proximity to humans and domestic animals. Deer with neurologic signs are of high interest to the general public and wildlife managers because of disease and safety concerns. Our aim was to describe diagnostic findings from free-ranging white-tailed deer diagnosed with rabies from across the eastern US from 2000 to 2021, with emphasis on gross lesions in the skin and soft tissue overlying the skull. We reviewed diagnostic reports of white-tailed deer cases submitted to the Southeastern Cooperative Wildlife Disease Study for those diagnosed with rabies from 2000 to 2021. Rabies virus infection was confirmed by immunohistochemistry or fluorescent antibody test of brain, or both. Nine adult deer from five states were diagnosed with rabies, including seven (78%) females and two (22%) males. Three (33%) deer were found dead, and six (67%) were humanely dispatched for abnormal behavior. Six deer heads were examined grossly and had lesions, including forehead or periorbital alopecia, cutaneous erythema, abrasions and ulcers, and subcutaneous edema. Histologic examination was performed for eight of nine cases, all of which had intraneuronal eosinophilic inclusion (Negri) bodies in cerebrum, cerebellum, or both. Most (6/8; 75%) had perivascular lymphoplasmacytic encephalitis. Rabies should be considered a differential diagnosis in deer with this pattern of head lesions, suggestive of head rubbing or head pressing.

Listeriosis with viral coinfections in 8 gray foxes, 8 wild turkeys, and 2 young cervids in the southeastern United States.

Listeria monocytogenes is a bacterium that can cause disease in many species, including humans, livestock, and wildlife. Increased interactions via shared habitats may promote pathogen transmission among these groups. Our objectives were to evaluate the Southeastern Cooperative Wildlife Disease Study diagnostic data to characterize and compare L. monocytogenes-induced lesions and comorbidities in gray foxes and wild turkeys, and to describe cases of listeriosis in 2 cervids. From 1991-2020, 8 gray foxes, 8 wild turkeys, a neonatal elk, and a white-tailed deer fawn from several eastern states in the United States were diagnosed with listeriosis. All 8 foxes had hepatitis and/or hepatic necrosis with intralesional gram-positive bacilli, and concurrent canine distemper virus (CDV) infection; 2 of the foxes had been vaccinated recently for CDV. L. monocytogenes was cultured from the liver (6 of 8) or lung (2 of 8) of foxes. Lesions in wild turkeys included hepatocellular necrosis (3 of 8), heterophilic hepatitis (1 of 8), heterophilic granulomas (1 of 8), intrasinusoidal gram-positive bacilli without hepatic lesions (1 of 8), granulomatous dermatitis (1 of 8), and/or granulomatous myocarditis (2 of 8). Lymphoproliferative disease viral DNA was detected in 5 of 6 turkeys tested; reticuloendotheliosis viral DNA was detected in 2 of 3 turkeys tested. Both cervids had systemic listeriosis, with L. monocytogenes isolated from liver. Immunohistochemistry for Listeria spp. on select cases revealed immunolabeling in affected organs. Listeriosis was thus established as a cause of morbidity and mortality in 3 wildlife species, which often suffered from concurrent infections and likely immunosuppression.