Comparative evaluation of diagnostic methods for detection of Theileria spp. in cows.

Theileriosis is a hemoprotozoan illness of cattle in tropical regions that poses a severe economic loss to dairy farmers in the form of production loss and mortality. We designed and optimized a multiplex real-time PCR by using Taq-Man® probe for detection and quantification of Theileria orientalis and Theileria annulata simultaneously by targeting 18 s rRna and MPSP (surface merozoite protein) genes, respectively. Fifty-five EDTA blood samples from clinically Theileria-suspected cows of three Theileria-endemic districts of Odisha were processed using acridine dye based fluorescent microscopy, Giemsa staining, and PCR. PCR revealed T. annulata and T. orientalis in 11/42 (26.11%) and 24/42 (57.14%) cases, respectively. Mixed infection due to both the Theileria spp. was recorded in 7/42 (16.66%). On comparison with gold standard test (PCR), the accuracy, sensitivity, and specificity were 92.72, 95.12, and 85.71% for Giemsa staining and 96.36, 97.56, and 92.85% for acridine orange dye. Multiplex real time PCR using Taq-Man probe detected two species of T. annulata and T. orientalis simultaneously. Acridine dye based fluorescent microscopy is comparatively easy and rapid method in detection of Thelieria spp.

Characterization and Whole Genome Sequencing of Chromobacterium violaceum OUAT_2017: A Zoonotic Pathogen Found Fatal to a Wild Asiatic Elephant.

This study reports a rare fatal case of Chromobacterium violeceum OUAT_2017 strain infection in an Asiatic elephant calf in India. Necropsy revealed pus-filled nodules in liver, spleen, and lungs. Nutrient broth cultures of nodule content showed sediment of violet pigment whereas smooth, non-diffusible, violet-pigmented, homogeneous colonies appeared on nutrient agar. The organism was found to be non-haemolytic and resistant to 8 of the 24 antibiotics tested in vitro. Partial 16S rRNA gene sequence measuring 1410 bp revealed 97% homology with C. violeceum. The bacterial genome composed of 64.87% of G + C content with total size of 4,681,202 bp. The genome annotation has 42 genes responsible for multidrug antibiotic resistance with the presence of Aminoglycoside-modifying enzymes (AAC (6')) that targets streptomycin and spectinomycin. Our findings corroborated the lethal effect of C. violeceum in a new host (elephant) that enriched scientific information on epidemiological picture and whole genome sequencing as well. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01047-4.

Comparative Molecular Characterization of Novel and Known Piscine Toti-Like Viruses.

Totiviridae is a virus family well known to infect uni-cellular organisms like fungi and protozoa. In more recent years, viruses characterized as toti-like viruses, have been found in primarily arthropods, but also a couple in planarians and piscine species. These toti-like viruses share phylogenetic similarities to totiviruses; however, their genomes also includes additional coding sequences in either 5' or 3' ends expected to relate to more advanced infection mechanisms in more advanced hosts. Here, we applied next generation sequencing (NGS) technologies and discovered three new toti-like viruses, one in wild common carp and one in bluegill from the USA and one in farmed lumpsucker from Norway. These are named common carp toti-like virus 1 (CCTLV-1), bluegill toti-like virus 1 (BGTLV-1), and Cyclopterus lumpus toti-like virus (CLuTLV), respectively. The genomes of these viruses have been characterized and compared to the three previously known piscine toti-like viruses, piscine myocarditis virus (PMCV) found in Atlantic salmon and the two from golden shiner, now named golden shiner toti-like virus 1 and 2 (GSTLV-1 and -2), and also to totiviruses and other toti-like viruses. We found that four piscine toti-like viruses had additional gene(s) in the 3' end of the genome, and also clustered phylogenetically based on both capsid and RdRp-genes. This cluster constituted a distant branch in the Totiviridae, and we suggest this should be defined as a separate genus named Pistolvirus, to reflect this major cluster of piscine toti-like viruses. The remaining two piscine toti-like viruses differentiated from these by lacking any additional 3' end genes and also by phylogenetical relation, but were both clustering with arthropod viruses in two different clusters.

Susceptibility of Pimephales promelas and Carassius auratus to a strain of koi herpesvirus isolated from wild Cyprinus carpio in North America.

Cyprinid herpesvirus-3 (CyHV-3, syn. koi herpesvirus) is an important pathogen worldwide and a common cause of mass mortality events of wild common carp (Cyprinus carpio) in North America, however, reference strains and genomes obtained from wild carp are not available. Additionally, it is unclear if fishes in North America are susceptible to CyHV-3 infection due to incomplete susceptibility testing. Here we present the first North American type strain and whole-genome sequence of CyHV-3 isolated from wild carp collected from a lake with a history and recent incidence of carp mortality. Additionally, the strain was used in an in-vivo infection model to test the susceptibility of a common native minnow (Pimephales promelas) and goldfish (Carrasius auratus) which is invasive in North America. Detection of CyHV-3 DNA was confirmed in the tissues of a single fathead minnow but the same tissues were negative for CyHV-3 mRNA and samples from exposed fathead minnows were negative on cell culture. There was no detection of CyHV-3 DNA or mRNA in goldfish throughout the experiment. CyHV-3 DNA in carp tissues was reproducibly accompanied by the detection of CyHV-3 mRNA and isolation on cell culture. Additionally, environmental CyHV-3 DNA was detected on all tank filters during the study. These findings suggest that fathead minnows and goldfish are not susceptible to CyHV-3 infection and that detection of CyHV-3 DNA alone in host susceptibility trials should be interpreted with caution.

Influence of Environmental Stressors on the Microbiota of Zebra Mussels (Dreissena polymorpha).

Host-associated microbiota play a critical role in host fitness by providing nutrition, enhancing digestion capabilities, and by providing protection from pathogens. Here, we investigated the effects of two environmental stressors, temperature, and salinity, on the microbiota associated with zebra mussels (ZMs), a highly invasive bivalve in North America. To examine this in detail, lake-collected ZMs were acclimated to laboratory conditions, and subjected to temperature and salinity stress conditions. The impact of these stressors on the diversity, composition, and dynamics of ZM-associated microbiota were assessed by using amplicon- and shotgun-based sequencing, and qPCR-based approaches. Elevated temperature was found to be the primary driver of ZM mortality, although salinity alone also increased its likelihood. Stressor-induced ZM mortality, which ranged between 53 and 100%, was concomitant with significant increases in the relative abundance of several genera of putative opportunistic pathogens including Aeromonas. These genera were only present in low relative abundance in ZMs obtained from the control tank with 0% mortality. Shotgun sequencing and qPCR analyses indicated that the relative and absolute abundances of pathogenic Aeromonas species (particularly A. veronii) were significantly greater in temperature-induced dead ZMs. Taken together, our results show that environmental stress, especially elevated temperature (> 25 °C), is associated with the rapid mortality of ZMs as well as the proliferation of putative opportunistic bacterial pathogens.

Koi herpesvirus and carp oedema virus: Infections and coinfections during mortality events of wild common carp in the United States.

Koi herpesvirus (KHV; cyprinid herpesvirus-3) and carp oedema virus (CEV) are important viruses of common and koi carp (Cyprinus carpio); however, the distribution of these viruses in wild common carp in North America is largely unknown. During the summers of 2017 and 2018, 27 mass mortalities of common carp were reported from four states in the USA (Minnesota, Iowa, Pennsylvania and Wisconsin), the majority of which were distributed across eight major watersheds in southern Minnesota. Samples from 22 of these mortality events and from five clinically healthy nearby carp populations were screened for KHV, CEV and SVCV using real-time polymerase chain reaction (qPCR). KHV was confirmed in 13 mortality events, CEV in two mortality events and coinfections of KHV/CEV in four mortality events. Nucleotide sequence analysis revealed that the KHV and CEV detected here are closely related to European lineages of these viruses. While molecular detection alone cannot conclusively link either virus with disease, the cases described here expand the known range of two important viruses. This is also the first reported detection of KHV and CEV coinfections in wild carp populations.