A novel group of negative-sense RNA viruses associated with epizootics in managed and free-ranging freshwater turtles in Florida, USA.

Few aquatic animal negative-sense RNA viruses have been characterized, and their role in disease is poorly understood. Here, we describe a virus isolated from diseased freshwater turtles from a Florida farm in 2007 and from an ongoing epizootic among free-ranging populations of Florida softshell turtles (Apalone ferox), Florida red-bellied cooters (Pseudemys nelsoni), and peninsula cooters (Pseudemys peninsularis). Affected turtles presented with similar neurological signs, oral and genital ulceration, and secondary microbial infections. Microscopic lesions were most severe in the softshell turtles and included heterophilic/histiocytic meningoencephalitis, multi-organ vasculitis, and cytologic observation of leukocytic intracytoplasmic inclusions. The virus was isolated using Terrapene heart (TH-1) cells. Ultrastructurally, viral particles were round to pleomorphic and acquired an envelope with prominent surface projections by budding from the cell membrane. Viral genomes were sequenced from cDNA libraries of two nearly identical isolates and determined to be bi-segmented, with an ambisense coding arrangement. The larger segment encodes a predicted RNA-directed RNA polymerase (RdRP) and a putative zinc-binding matrix protein. The smaller segment encodes a putative nucleoprotein and an envelope glycoprotein precursor (GPC). Thus, the genome organization of this turtle virus resembles that of arenaviruses. Phylogenetic analysis shows that the RdRP of the turtle virus is highly diverged from the RdRPs of all known negative-sense RNA viruses and forms a deep branch within the phylum Negarnaviricota, that is not affiliated with any known group of viruses, even at the class level. In contrast, the GPC protein of the turtle virus is confidently affiliated with homologs from a distinct group of fish hantaviruses. Thus, the turtle virus is expected to become the founder of a new taxon of negative-sense RNA viruses, at least with a family rank, but likely, an order or even a class. These viruses probably evolved either by reassortment or by intrasegment recombination between a virus from a distinct branch of negarnaviruses distant from all known groups and a hanta-like aquatic virus. We suggest the provisional name Tosoviridae for the putative new family, with Turtle fraservirus 1 (TFV1) as the type species within the genus Fraservirus. A conventional RT-PCR assay, targeting the TFV1 RdRP, confirmed the presence of viral RNA in multiple tissues and exudates from diseased turtles. The systemic nature of the TFV1 infection was further supported by labeling of cells within lesions using in situ hybridization targeting the RNA of the TFV1 RdRP.

Multisystemic Enterococcosis in Brown Anoles (Anolis sagrei) from Florida, USA.

Beginning in July 2019, numerous free-ranging brown anoles (Anolis sagrei), an invasive lizard species in Florida, USA, were reported with large, soft, subcutaneous masses and disfiguring facial swellings. Postmortem evaluations of six affected animals, including cytology, histology, and electron microscopy, identified the presence of myriad chain-forming coccoid bacteria surrounded by a prominent clear capsule and abundant lightly basophilic matrix material with minimal associated granulomatous inflammation and effacement of normal tissue. Standard PCR and sequencing of the lesions revealed 100% nucleotide identity to Enterococcus lacertideformus. This bacterium was first observed in 2014 as the cause of a severe, multisystemic infection in several species of lizards (geckos and skinks) on Christmas Island, an Australian external territory in the Indian Ocean. Previously, analysis of E. lacertideformus had been hindered by an inability to grow the bacterium in standard culture conditions. We successfully cultured the organism on primary anole kidney cells. Given the growing recognition of host species diversity and geographic distribution noted for this organism, there is potential concern for spread to native North American lizards, especially the green anole (Anolis carolinensis), whose population numbers have apparently decreased due to introduced brown anoles.

Heavy Rainfall, Sewer Overflows, and Salmonellosis in Black Skimmers (Rynchops niger).

Extreme weather events, particularly heavy rainfall, are occurring at greater frequency with climate change. Although adverse human health effects from heavy rainfall are often publicized, impacts to free-ranging wildlife populations are less well known. We first summarize documented associations of heavy rainfall on wildlife health. We then report a novel investigation of a salmonellosis outbreak in a colony of black skimmers (Rynchops niger) in Florida, USA. During June-September 2016, heavy rainfall resulted in the discharge of millions of gallons of untreated wastewater into the Tampa Bay system, contaminating the water body, where adult skimmers foraged. At least 48 fledglings died, comprising 39% of the colony's nesting season's offspring. Of eight examined deceased birds from the colony, six had a systemic salmonellosis infection. Isolates were identified as Salmonella enterica serotype Typhimurium. Their pulsed-field gel electrophoresis patterns were identical to each other and matched those from several human Salmonella sp. infections. Differences among whole-genome sequences were negligible. These findings and the outbreak's epidemic curve suggest propagated transmission occurred within the colony. A multidisciplinary and One Health approach is recommended to mitigate any adverse effects of climate change-driven stochastic events, especially when they place already imperiled wildlife at further risk.

Notoedric mange (Notoedres centrifera) in two species of free-ranging rabbits from Florida, USA.

Mange is a contagious skin disease caused by different mite species affecting numerous domestic and wild animals, worldwide. This report details notoedric mange in an eastern cottontail (Sylvilagus floridanus) and in a marsh rabbit (Sylvilagus palustris) from Florida, USA. Clinical examination revealed similar gross lesions including poor nutritional condition, multifocal alopecia and hyperkeratosis. Skin scrapings from both rabbits revealed numerous subcutaneous mites identified as Notoedres centrifera, a species previously only associated with rodents, primarily squirrels. Mites from both rabbits were identified based on morphology and confirmed by sequencing the internal transcribed spacer-2 (ITS-2) region. These cases emphasize the need for continued surveillance and accurate diagnostic evaluation to determine the cause and characterization of the skin disease, while distinguishing it from other potential pathogens that may manifest similarly in rabbits, such as Notoedres cati, Sarcoptes scabiei or Psoroptes cuniculi.

Mortality in Common (Sterna hirundo) and Sandwich (Thalasseus sandvicensis) Terns Associated with Bisgaard Taxon 40 Infection on Marco Island, Florida, USA.

Widely distributed aquatic species such as terns are highly dependent on, and can serve as indicators of, the global health of marine and other aquatic environments. Documented mass mortality events in terns have been associated with anthropogenic, weather-related and, less commonly, infectious causes. This study describes a multispecies mortality event associated with brevetoxicosis and Bisgaard taxon 40-induced sepsis involving common (Sterna hirundo) and sandwich (Thalasseus sandvicensis) terns off the southwest coast of Florida, USA, in November and December 2018. During an approximately 6-8-week period, a large number of birds were found dead or displayed weakness, ataxia or other neurological signs. Many were admitted to a wildlife hospital for evaluation, but most died or were euthanized due to poor prognosis. Necropsy of 12 birds revealed minimal or non-specific gross lesions. Initial toxicology screening of tissues for brevetoxins revealed levels that could be consistent with brevetoxicosis. However, histology revealed multiorgan inflammation and necrosis associated with a gram-negative bacillus. A bacterium isolated on aerobic culture of liver and heart tissues was unidentifiable in the MALDI-TOF database. Subsequently, 16 S rRNA gene sequencing revealed that the isolate shared 99.33% homology with Bisgaard taxon 40 from the Pasteurellaceae family. While the source of the bacterium and potential association with brevetoxin exposure are unclear, histopathology suggests that the bacterium was the proximate cause of clinical signs and mortality in all birds examined as well as the scale of the mortality event. This report highlights the need to conduct detailed investigations into wildlife mortality events and expands on the current, limited knowledge of the effects of novel Pasteurellaceae bacteria on avian health.

Detection of Alphacoronavirus vRNA in the Feces of Brazilian Free-Tailed Bats (Tadarida brasiliensis) from a Colony in Florida, USA.

Bats are natural reservoirs of coronaviruses and other viruses with zoonotic potential. Florida has indigenous non-migratory populations of Brazilian free-tailed bats (Tadarida brasiliensis) that mostly roost in colonies in artificial structures. Unlike their counterparts in Brazil and Mexico, the viruses harbored by the Florida bats have been underexplored. We report the detection of an alphacoronavirus RNA-dependent RNA polymerase (RdRp) gene sequence in the feces of two of 19 different T. brasiliensis that were capture/release bats that had been evaluated for overall health. The RdRp sequence is similar but not identical to previously detected sequences in the feces of two different species of bats (T. brasiliensis and Molossus molossus) in Brazil. In common with the experience of others doing similar work, attempts to isolate the virus in cell cultures were unsuccessful. We surmise that this and highly related alphacoronavirus are carried by Brazilian free-tailed bats living in a wide eco-spatial region. As various coronaviruses (CoVs) that affect humans emerged from bats, our study raises the question whether CoVs such as the one detected in our work are yet-to-be-detected pathogens of humans and animals other than bats.

Molecular Diversity of Trypanosoma cruzi Detected in the Vector Triatoma protracta from California, USA.

BACKGROUND: Trypanosoma cruzi, causative agent of Chagas disease in humans and dogs, is a vector-borne zoonotic protozoan parasite that can cause fatal cardiac disease. While recognized as the most economically important parasitic infection in Latin America, the incidence of Chagas disease in the United States of America (US) may be underreported and even increasing. The extensive genetic diversity of T. cruzi in Latin America is well-documented and likely influences disease progression, severity and treatment efficacy; however, little is known regarding T. cruzi strains endemic to the US. It is therefore important to expand our knowledge on US T. cruzi strains, to improve upon the recognition of and response to locally acquired infections. METHODOLOGY/PRINCIPLE FINDINGS: We conducted a study of T. cruzi molecular diversity in California, augmenting sparse genetic data from southern California and for the first time investigating genetic sequences from northern California. The vector Triatoma protracta was collected from southern (Escondido and Los Angeles) and northern (Vallecito) California regions. Samples were initially screened via sensitive nuclear repetitive DNA and kinetoplast minicircle DNA PCR assays, yielding an overall prevalence of approximately 28% and 55% for southern and northern California regions, respectively. Positive samples were further processed to identify discrete typing units (DTUs), revealing both TcI and TcIV lineages in southern California, but only TcI in northern California. Phylogenetic analyses (targeting COII-ND1, TR and RB19 genes) were performed on a subset of positive samples to compare Californian T. cruzi samples to strains from other US regions and Latin America. Results indicated that within the TcI DTU, California sequences were similar to those from the southeastern US, as well as to several isolates from Latin America responsible for causing Chagas disease in humans. CONCLUSIONS/SIGNIFICANCE: Triatoma protracta populations in California are frequently infected with T. cruzi. Our data extend the northern limits of the range of TcI and identify a novel genetic exchange event between TcI and TcIV. High similarity between sequences from California and specific Latin American strains indicates US strains may be equally capable of causing human disease. Additional genetic characterization of Californian and other US T. cruzi strains is recommended.

Analysis of serum and whole blood values in relation to helminth and ectoparasite infections of feral pigs in Texas.

In the summers of 1996 and 1997, 60 wild pigs (Sus scrofa) were necropsied from three sites in south Texas (USA) to test the hypothesis that serum and whole blood parameters vary significantly (P < or = 0.05) with the prevalence and intensity of parasites infecting wild pigs. We found ten parasite species: five nematodes (Metastrongylus salmi, Metastrongylus pudentotectus, Stephanurus dentatus, Oesophagostomum dentatum, and Physocephalus sexalatus); four ixodid ticks (Amblyomma cajennense, Amblyomma maculatum, Amblyomma americanum, and Dermacentor variabilis); and one trematode (Fascioloides magna). Among juvenile pigs, the intensity of the four species of ticks, collectively, was negatively correlated (P < or = 0.05) with whole blood principal component number one (PC-1); this factor was positively associated with lymphocytes and eosinophils. Lungworm intensity (Metastrongylus spp.) among adult pigs was negatively correlated (P < or = 0.05) with whole blood PC-2; this factor was negatively associated with segmented neutrophils and monocytes. There were no significant correlations found between parasite prevalences and either serum or whole blood principal component factors. The correlations observed between parasite intensities and serum and whole blood parameters generally were weak. Thus, we found no strong evidence that serum and whole blood parameters provided good predictive information on parasite infections in wild pigs for most practical management decisions.

Native rodent species are unlikely sources of infection for Leishmania (Viannia) braziliensis along the Transoceanic Highway in Madre de Dios, Peru.

An estimated 2.3 million disability-adjusted life years are lost globally from leishmaniasis. In Peru's Amazon region, the department of Madre de Dios (MDD) rises above the rest of the country in terms of the annual incidence rates of human leishmaniasis. Leishmania (Viannia) braziliensis is the species most frequently responsible for the form of disease that results in tissue destruction of the nose and mouth. However, essentially nothing is known regarding the reservoirs of this vector-borne, zoonotic parasite in MDD. Wild rodents have been suspected, or proven, to be reservoirs of several Leishmania spp. in various ecosystems and countries. Additionally, people who live or work in forested terrain, especially those who are not regionally local and whose immune systems are thus naïve to the parasite, are at most risk for contracting L. (V.) braziliensis. Hence, the objective of this study was to collect tissues from wild rodents captured at several study sites along the Amazonian segment of the newly constructed Transoceanic Highway and to use molecular laboratory techniques to analyze samples for the presence of Leishmania parasites. Liver tissues were tested via polymerase chain reaction from a total of 217 rodents; bone marrow and skin biopsies (ear and tail) were also tested from a subset of these same animals. The most numerous rodent species captured and tested were Oligoryzomys microtis (40.7%), Hylaeamys perenensis (15.7%), and Proechimys spp. (12%). All samples were negative for Leishmania, implying that although incidental infections may occur, these abundant rodent species are unlikely to serve as primary reservoirs of L. (V.) braziliensis along the Transoceanic Highway in MDD. Therefore, although these rodent species may persist and even thrive in moderately altered landscapes, we did not find any evidence to suggest they pose a risk for L. (V.) braziliensis transmission to human inhabitants in this highly prevalent region.

White-nosed coatis (Nasua narica) are a potential reservoir of Trypanosoma cruzi and other potentially zoonotic pathogens in Monteverde, Costa Rica.

We studied white-nosed coatis (Nasua narica) in Monteverde, Costa Rica to evaluate their potential as a reservoir for the vector-borne zoonotic parasite Trypanosoma cruzi and other selected microorganisms. We live-trapped 20 coatis in July and August 2011 and tested them for T. cruzi by blood smear, molecular analysis of blood, culture of blood and anal gland secretions, and serology. Seven coatis (35%) were polymerase-chain-reaction-positive for T. cruzi and one coati was also culture positive. We did not detect T. cruzi in anal gland secretions. All coatis were positive for Mycoplasma and Babesia, but were negative for Baylisascaris, Anaplasma, Candidatus Neoehrlichia lotoris, Ehrlichia, Bartonella, and several apicomplexan parasites. The possible pathogenicity of T. cruzi, Babesia, and Mycoplasma in coatis and their transmission potential to humans and domestic animals warrants further investigation.

Alternaria fungal dermatitis in a free-ranging javelina (Pecari tajacu).

We report a javelina from Pinal County, Arizona, USA, with severe fungal dermatitis and cellulitis. Extreme emaciation and rostral disfiguration, including left-lateral displacement of the nasal planum, justified euthanasia. A pus-filled tract within the rostrum was observed. Histopathology revealed granulomatous inflammation with hyphae morphologically consistent with Alternaria sp. isolated by culture.

Salmonellosis in a free-ranging population of javelinas (Pecari tajacu) in south central Arizona.

The javelina, or collared peccary (Pecari tajacu), is indigenous to Arizona, New Mexico, and Texas in the United States and ranges throughout Latin America. From June 2004 to April 2005, an estimated 105 javelinas died in a mortality event that occurred in Tucson, Arizona, and neighboring areas. Clinical signs observed in sick animals included emaciation, dehydration, lethargy, and diarrhea. In addition, some animals showed labored breathing and hind limb weakness. We necropsied 34 animals, and enteritis was the most frequent clinical sign, followed by colitis, pulmonary congestion, and pneumonia. The only consistent findings were isolations of Clostridium perfringens type A and multiple Salmonella serotypes. Although it is likely that these javelinas ultimately succumbed to salmonellosis, it is unclear whether other unidentified underlying factors were involved. This is the first reported case of widespread salmonellosis in free-ranging javelinas.