Betanodavirus meningoencephalitis in an Atlantic blue marlin.

Viral nervous necrosis (viral encephalopathy and retinopathy) is caused by piscine nodavirus (Nodaviridae, Betanodavirus). Since 1986, this highly infectious virus has caused mass mortalities of up to 100% in farmed saltwater and freshwater fish around the world (with the exception of South America and Antarctica), affecting >60 species across 10 orders. The Atlantic blue marlin (Makaira nigricans Lacépède, 1802) is a top-level predator found throughout the tropical waters of the Atlantic and Indo-Pacific oceans. Despite their popularity as a sportfish, relatively little is known about the Atlantic blue marlin and other billfish. We describe here chronic betanodavirus infection in a juvenile Atlantic blue marlin, which is, to our knowledge, the first report of disease in M. nigricans.

Histologic lesions of giant African millipedes (Archispirostreptus gigas) from a zoological institution.

Histopathologic data of millipedes are scarce. Little is known about health and disease of these invertebrates despite their exhibition at zoological institutions and use in ecotoxicological studies. In a retrospective study of 69 zoo-housed giant African millipedes (Archispirostreptus gigas) submitted between 2018 and 2021, most deaths occurred during midwinter and in 2021. The most common lesion was inflammation (n = 55; 80%). Necrosis was seen concurrently in 31 (45%) millipedes and of these, bacteria (20; 29%) and fungi (7; 10%) were detected in lesions. Inflammation was seen in the head/collum (20; 29%), hemocoel (16; 23%), and appendages (9; 13%), specifically in perivisceral fat body (42; 61%), gut (16; 23%), tracheae (26; 38%), skeletal muscle (24; 35%), and ventral nerve (17; 25%). Inflammatory cell types and patterns included agranular hemocytes (61; 88%), granular hemocytes (39; 57%), and nodulation/encapsulation (47; 68%) often accompanied by melanization. The oral cavity or gut (ingestion), spiracles (inhalation), or cuticular defects were considered plausible routes of bacterial entry. Metazoan parasites (adult nematodes: 2, 3%; trematode ova: 2, 3%; and arthropods: 1, 1%) were associated with gut necrosis and inflammation in 5 millipedes. In addition, adult nematodes were noted in the gut of 4 millipedes without lesions. Neoplasia was not detected in any millipedes. Speculatively, environmental factors may have predisposed to disease, as most deaths occurred during winter months. Disease surveillance of millipedes is critical to optimize husbandry practices in zoo populations and investigate potential impacts of environmental degradation and climate change on wild millipedes.

DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS 1A IN ARCHIVAL TISSUE USING RNASCOPE® IN SITU HYBRIDIZATION.

Hemorrhagic disease due to elephant endotheliotropic herpesvirus infection (EEHV-HD) is an important cause of calf mortality in managed and free-ranging Asian (Elephas maximus) and African elephant (Loxodonta spp.) populations. Consequently, infection has profound implications for elephant population growth and sustainability. The mechanisms of disease caused by EEHV (i.e., infection, dissemination, shedding, latency) are relatively undefined, in part because of a lack of robust validated assays for detecting viral gene products in relevant samples. To address this issue, we used RNAscope® in situ hybridization (ISH) based on EEHV1A DNA polymerase and terminase genes to detect EEHV1A RNA in archival formalin-fixed, paraffin-embedded Asian elephant heart and tongue from PCR-confirmed cases (n = 4) of EEHV-HD and Asian elephants (n = 2) that died from other causes. EEHV1A-positive cases had positive hybridization signal in endothelial cell nuclei of both tissues for both DNA polymerase and terminase. EEHV-negative cases lacked signal. In positive cases, the number of positive nuclei was manually assessed to provide an estimate of the viral load and compare sensitivity of the two probes. In all cases, heart had greater signal than tongue for both probes (Wilcoxon rank test; P ≤ 0.01). Overall, terminase hybridization signal was greater than DNA polymerase signal (Wilcoxon rank test; P ≤ 0.01). Results indicate RNAscope ISH is a valuable tool for detection of EEHV in archival samples and for confirming infection. Additionally, the terminase gene is the optimal target and heart is preferable to tongue for detection in cases of EEHV-HD. Results will inform future investigations of viral tropism in EEHV-HD cases due to EEHV1A.

Development and validation of a probe hybridization reverse-transcription quantitative PCR for detection of mamastrovirus 2 in domestic cats.

Astroviruses are viral pathogens that have been associated with enteric and neurologic disease in a variety of species. The domestic cat is a prominent host, with reports of astroviral infection being both highly prevalent and widely distributed in the feline population. Despite the potential for inducing significant disease, especially within shelter environments, there is currently only one reliable method of detection: standard reverse-transcription PCR using pan-astrovirus degenerate primers (consensus RT-PCR) with product sequencing. Unfortunately, this process is relatively slow and costly. Quantitative real-time PCR (qPCR) represents an efficient, economical alternative, with the added benefit of viral load quantification. We developed a RT-qPCR assay using probe hybridization technique to detect conserved regions of mamastrovirus 2 extracted from fecal samples of domestic cats. Known positive and negative samples were tested, and results were compared with gold standard consensus RT-PCR and sequencing. A standard curve was employed to determine limits of detection. In order to assess analytic specificity, we tested several additional samples that had been collected from non-felid species and were known to contain non-target astroviruses. Discrepant results between consensus RT-PCR and RT-qPCR testing were further analyzed with a validation RT-PCR assay, using mamastrovirus 2-specific primers. Our probe hybridization RT-qPCR assay is reliable and effective for the detection of mamastrovirus 2. This assay will allow rapid, affordable detection and facilitate further research on astroviral infection within domestic cats.

Determination of the diversity of astroviruses in feces from cats in Florida.

Astroviruses are small, nonenveloped RNA viruses that have been linked to numerous diseases in a variety of species, including enteric disease in humans and cheetahs. Species Mamastrovirus 2, previously known as feline astrovirus, has been isolated from the feces of domestic cats and cheetahs. A total of 122 cat fecal samples from Alachua County, FL Animal Services and the Veterinary Community Outreach Program at the University of Florida were analyzed, and 35 contained astroviral RNA that was amplified and identified using consensus RT-PCR and sequence analysis. Using phylogenetic analysis, 19 of the astroviral sequences were identified as Mamastrovirus 2, making it the most prevalent astrovirus in this population. Three samples were identified as an astrovirus similar to viruses previously identified in foxes in The Netherlands and a cat in California, and one was similar to a bat astrovirus. One astroviral sequence was identified as an Avastrovirus. Although a causative relationship between mamastroviruses and enteric disease in cats has yet to be established, it is clear that mamastroviruses are prevalent, and an understanding of prevalence of astroviral types may help direct future test development.