Companion animals are susceptible to a variety of coronaviruses, and recent studies show that felines are highly susceptible to SARS-CoV-2 infection. RT-PCR diagnostic is currently the method of choice to detect the presence of SARS-CoV-2-specific viral nucleic acids in animal samples during an active infection; however, serological assays are critical to determine whether animals were exposed to the virus and to determine the seroprevalence of SARS-CoV-2-specific antibodies in a defined population. In this study, we utilized recombinant nucleocapsid (N) protein and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 expressed in E. coli (N) and mammalian cells (N, RBD) to develop indirect ELISA (iELISA) tests using well-characterized SARS-CoV-2-positive and -negative cat serum panels from previous experimental cat challenge studies. The optimal conditions for the iELISA tests were established based on checkerboard dilutions of antigens and antibodies. The diagnostic sensitivity for the detection of feline antibodies specific for the N or RBD proteins of the iELISA tests was between 93.3 and 97.8%, respectively, and the diagnostic specificity 95.5%. The iELISAs developed here can be used for high-throughput screening of cat sera for both antigens. The presence of SARS-CoV-2-specific antibodies in a BSL-2 biocontainment environment, unlike virus neutralization tests with live virus which have to be performed in BSL-3 laboratories.
African swine fever (ASF) is an infectious viral disease caused by African swine fever virus (ASFV), that causes high mortality in domestic swine and wild boar (Sus scrofa). Currently, outbreaks are mitigated through strict quarantine measures and the culling of affected herds, resulting in massive economic losses to the global pork industry. In 2019, an ASFV outbreak was reported in Mongolia, describing a rapidly progressing clinical disease and gross lesions consistent with the acute form of ASF; the virus was identified as a genotype II virus. Due to the limited information on clinical disease and viral dynamics within hosts available from field observations of the Mongolian isolates, we conducted the present study to further evaluate the progression of clinical disease, virulence, and pathology of an ASFV Mongolia/2019 field isolate (ASFV-MNG19), by experimental infection of domestic pigs. Intramuscular inoculation of domestic pigs with ASFV-MNG19 resulted in clinical signs and viremia at 3 days post challenge (DPC). Clinical disease rapidly progressed, resulting in the humane euthanasia of all pigs by 7 DPC. ASFV-MNG19 infected pigs had viremic titers of 108 TCID50/mL by 5 DPC and shed virus in oral secretions late in disease, as determined from oropharyngeal swabs. Whole-genome sequencing confirmed that the ASFV-MNG19 strain used in this study was a genotype II strain highly similar to other regional strains. In conclusion, we demonstrate that ASFV-MNG19 is a virulent genotype II ASFV strain that causes acute ASF in domestic swine.
Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic disease that was first identified in humans in 2012 in Saudi Arabia. MERS-CoV causes acute and severe respiratory disease in humans. The mortality rate of MERS in humans is ∼35% and >800 deaths have been reported globally as of August 2020. Dromedary camels are a natural host of the virus and the source of zoonotic human infection. In experimental studies, Bactrian camels are susceptible to MERS-CoV infection similar to dromedary camels; however, neither the virus, viral RNA, nor virus-specific antibodies were detected in Bactrian camel field samples so far. The aim of our study was to survey Mongolian camels for MERS-CoV-specific antibodies. A total of 180 camel sera, collected in 2016 and 2017, were involved in this survey: 17 of 180 sera were seropositive with an initial enzyme-linked immunosorbent assay (ELISA) test performed at the State Central Veterinary Laboratory in Mongolia. These 17 positive sera plus 53 additional negative sera were sent to the Rocky Mountain Laboratories, NIAID/NIH, and tested for the presence of antibodies with a similar ELISA, an indirect immunofluorescence assay (IFA), and a virus neutralization test (VNT). In these additional tests, a total of 21 of 70 sera were positive with ELISA and 10 sera were positive with IFA; however, none was positive in the VNT. Based on these results, we hypothesize that the ELISA/IFA-positive antibodies are (1) non-neutralizing antibodies or (2) directed against a MERS-CoV-like virus circulating in Bactrian camels in Mongolia.
Nuclear hormone receptors (NHRs) are emerging target candidates against nematode infection and resistance. However, there is a lack of comprehensive information on NHR-coding genes in parasitic nematodes. In this study, we curated the nhr gene family for 60 major parasitic nematodes from humans and animals. Compared with the free-living model organism Caenorhabditis elegans, a remarkable contraction of the nhr family was revealed in parasitic species, with genetic diversification and conservation unveiled among nematode clades I (n ≈ 15), III (n ≈ 45), IV (n ≈ 35) and V (n ≈ 60). Using an in vitro biosystem, we demonstrated that 40 nhrgenes in a blood-feeding nematode Haemonchus contortus (clade V; barber’s pole worm) were responsive to host serum and one nhrgene (i.e., nhr-64) was consistently stimulated by anthelmintics (i.e., ivermectin, thiabendazole and levamisole); Using a high-throughput RNA interfering platform, we knocked down 43 nhr genes of H. contortus and identified at least two genes that are required for the viability (i.e., nhr-105) and development (i.e., nhr-17) of the infective larvae of this parasitic nematode in vitro. Harnessing this preliminary functional atlas of nhr genes for H. contortus will prime the biological studies of this gene family in nematode genetics, infection, and anthelmintic metabolism within host animals, as well as the promising discovery of novel intervention targets.
Recent studies reported the detection of DNA from tick-borne pathogens (TBPs) of veterinary relevance such as Anaplasma marginale, Babesia bigemina, Babesia bovis and Theileria orientalis in bovine blood samples from Mongolia. These findings were unexpected, as the known tick vectors of these pathogens are not known to occur in Mongolia. We therefore conducted a study in May and June 2013 in six districts of Khentii province where DNA of the said TBPs was previously found. Ticks collected from the vegetation and rodents, as well as blood samples from cattle, were screened for the presence of TBPs by reverse line blot (RLB) hybridization. Tick larvae collected from rodents were pooled. A total of 310 adult ticks were collected from the vegetation, and 249 tick larvae were collected from 24 rodents. Adult ticks (n = 2,318) and blood samples were collected from 481 heads of cattle. All adult ticks were identified as Dermacentor nuttalli. DNA from Rickettsia raoultii (252/310; 81.3%), an uncharacterized Anaplasma species preliminary named Anaplasma sp. Mongolia (26/310; 8.4%), Candidatus Midichloria sp. (18/310; 5.8%), Theileria equi (16/310; 5.2%), Babesia caballi (5/310; 1.6%), T. orientalis (1/310; 0.3%), Borrelia afzelii (1/310; 0.3%) and Candidatus Neoehrlichia mikurensis (1/310; 0.3%) was detected in ticks collected from the vegetation. DNA of R. raoultii (27/28; 96.4%) and Midichloria sp. (2/28; 7.1%) was detected in the pooled tick larvae. Anaplasma sp. Mongolia, a species related to Anaplasma ovis based on a multi-locus analysis, was also detected in 153/481 (31.8%) of the bovine blood samples. DNA of B. bovis, B. bigemina and A. marginale was not detected in the ticks or bovine blood samples from Khentii district.
A total 22 (30.5%) camels were infected with 34 echinococcal cysts out of 72 slaughtered camels in Khurmen soum of Southgobi province. The prevalence of infection in camels between 5-7 years (14/22) was 18.2-22.7% and 8 years camels (6/22) were 27.3%. The fertile cyst rate was 40.9% and sterile cyst rate was 22.7%. Camel cystic echinococcosis cyst status was fertile, sterile, abscessed and calcified. Most of the cysts were located in the lungs 54.5%, liver 27.3% and lung-liver 18.2% and were spherical in shape, unilocular and 1-3 cysts located in lung and liver of one camel, cyst diameter was 2-10 cm and with cyst fluid ranging from 1 to 200 ml. Camel echinococcal cysts status and appearance were revealed as age dependent, as older camels echinococcal cysts were revealed as calcified statistically significant (p=0.0458). Histologically, leucocyte infiltration and mild hepatocellular degeneration and infiltration in the liver were noticed. In lungs, there was proliferation of fibrous connective tissue and infiltration of mononuclear cells.
African swine fever virus (ASFV), classical swine fever virus (CSFV), and foot-and-mouth disease virus (FMDV) cause important transboundary animal diseases (TADs) that have a significant economic impact. The rapid and unequivocal identification of these pathogens and distinction from other animal diseases based on clinical symptoms in the field is difficult. Nevertheless, early pathogen detection is critical in limiting their spread and impact as is the availability of a reliable, rapid, and cost-effective diagnostic test. The purpose of this study was to evaluate the feasibility to identify ASFV, CSFV, and FMDV in field samples using next generation sequencing of short PCR products as a point-of-care diagnostic. We isolated nucleic acids from tissue samples of animals in Mongolia that were infected with ASFV (2019), CSFV (2015), or FMDV (2018), and performed conventional (RT-) PCR using primers recommended by the Terrestrial Animal Health Code of the World Organization for Animal Health (WOAH). The (RT-) PCR products were then sequenced in Mongolia using the MinION nanopore portable sequencer. The resulting sequencing reads successfully identified the respective pathogens that exhibited 91–100% nucleic acid similarity to the reference strains. Phylogenetic analyses suggest that the Mongolian virus isolates are closely related to other isolates circulating in the same geographic region. Based on our results, sequencing short fragments derived by conventional (RT-) PCR is a reliable approach for rapid point-of-care diagnostics for ASFV, CSFV, and FMDV even in low-resource countries.
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