Identification, Isolation, and Molecular Characterization of Betacoronavirus in Oryx leucoryx.

Coronaviruses (CoVs) are enveloped viruses with a large RNA genome (26 to 32 kb) and are classified into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. CoV infections cause respiratory, enteric, and neurologic disorders in mammalian and avian species. In 2019, Oryx leucoryx animals suffered from severe hemorrhagic diarrhea and high morbidity rates. Upon initial diagnosis, we found that the infected animals were positive for coronavirus by pancoronavirus reverse transcriptase RT-PCR. Next, we detected the presence of CoV particles in these samples by electron microscopy and immunohistochemistry. CoV was isolated and propagated on the HRT-18G cell line, and its full genome was sequenced. Full-genome characterization and amino acid comparisons of this viral agent demonstrated that this virus is an evolutionarily distinct Betacoronavirus belonging to the subgenus Embecovirus and the Betacoronavirus 1 species. Furthermore, we found that it is most similar to the subspecies dromedary camel coronavirus HKU23 by phylogenetic analysis. Here, we present the first report of isolation and characterization of Betacoronavirus associated with enteric disease in Oryx leucoryx. IMPORTANCE CoVs cause enteric and respiratory infections in humans and animal hosts. The ability of CoVs to cross interspecies barriers is well recognized, as emphasized by the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of novel CoV strains and surveillance of CoVs in both humans and animals are relevant and important to global health. In this study, we isolated and characterized a newly identified Betacoronavirus that causes enteric disease in a wild animal, Oryx leucoryx (the Arabian oryx). This work is the first report describing CoV infection in Oryx leucoryx and provides insights into its origin.

Characterization of Winter Dysentery Bovine Coronavirus Isolated from Cattle in Israel.

Bovine coronavirus (BCoV) is the causative agent of winter dysentery (WD). In adult dairy cattle, WD is characterized by hemorrhagic diarrhea and a reduction in milk production. Therefore, WD leads to significant economic losses in dairy farms. In this study, we aimed to isolate and characterize local BCoV strains. BCoV positive samples, collected during 2017-2021, were used to amplify and sequence the S1 domain of S glycoprotein and the full hemagglutinin esterase gene. Based on our molecular analysis, local strains belong to different genetic variants circulating in dairy farms in Israel. Phylogenetic analysis revealed that all local strains clustered together and in proximity to other BCoV circulating in the area. Additionally, we found that local strains are genetically distant from the reference enteric strain Mebus. To our knowledge, this is the first report providing molecular data on BCoV circulating in Israel.

First isolation of foot and mouth disease virus from wild boars in the Middle East.

Domestic pigs are susceptible to foot and mouth disease virus (FMDV) infection and suffer from severe clinical disease. Our knowledge on the clinical manifestations of FMD in and its transmission by wild boars (Sus scrofa) is very limited. During an FMD outbreak in the Golan Heights in 2018, wild boars grazing in close proximity to cattle were observed showing lameness. Infectious FMDV was isolated from throat and heart tissues of two young wild boars with FMD clinical signs. This is the first report of wild boars clinically infected with FMDV in the Middle East.

The serological response against foot and mouth disease virus elicited by repeated vaccination of dairy cattle.

In Israel, cattle are annually vaccinated against foot and mouth disease (FMD). If infections with FMD virus occur in dairy farms it mainly involves heifers and calves, while older dairy cows seldom become infected. We hypothesized that this difference in susceptibility between adult cows and the young heifers and calves is due to stronger and more stable immune response elicited by multiple vaccinations. In order to test this hypothesis, 99 dairy cattle, divided into six groups according to number of prior vaccinations, were annually vaccinated with a trivalent vaccine (A, O and Asia-1) and followed during two consecutive years. In total 988 sera were sampled at 11 time points. Virus neutralization tests (VNT) were performed in order to determine the neutralizing antibody titers (NAT) against the vaccine homologous serotypes: O-4625, O-Manisa, Asia-1-Shamir and the heterologous serotype A-Turkey-20/2006. A similar NAT pattern was observed to all serotypes and therefore statistical analysis was restricted to O-4625 serotype. In the 'high vaccination' groups (cows that were vaccinated at least four times before the study), high NAT were found on the beginning of the trial and no or only a mild increase of NAT was observed following further vaccinations. Additionally, in the 'high vaccination' groups, the percentage of cows that had a NAT higher than 2.0 (log10) by the end of the 1st year was significantly higher than in the 'low vaccination' groups (cows vaccinated only three times or less before the study). We conclude that starting from the 5th vaccination, the NAT increase following vaccination is mild and NAT are persistent, suggesting reduction of the frequency of routine vaccination after multiple vaccinations is possible.

The long term effect of age and maternally derived antibodies against foot and mouth disease on the serological response following vaccination in young dairy calves.

In Israel, occurrence of foot and mouth disease (FMD) in dairy farms is rare. However, when FMD outbreaks occur, dairy calves are the most affected, despite routine vaccination. Contradictory findings exist regarding the effect of age and maternally derived antibodies (MDA) on the serological response following vaccinations against FMD in dairy calves. Furthermore, the long term effect of FMD vaccination regimen during early life was rarely assessed. This study was conducted in order to assess both the short and long term effects. In total 44 non-vaccinated calves were divided into four groups of different age. Calves were vaccinated up to four times and 484 serum samples were collected on 11 time points in a period of 70weeks. Virus neutralizing tests were performed in order to determine the neutralizing antibody titers (NAT) against the vaccine strains (homologous serotypes): O-4625, O-Manisa, ASIA-1-Shamir and the heterologous serotype A-Turkey-20/2006. A similar NAT pattern was observed to all serotypes and therefore statistical analysis was restricted to O-4625 serotype. The MDA titer was negatively associated with the age of the calves and the MDA half-life was 22days. We demonstrated that early vaccination of calves (younger than three months) resulted in low NAT, even after four repeated vaccinations, compared with vaccination of calves older than three months. The percentage of time in which these calves had a NAT above 2.0 (log10) between the age of six months and 1.5years was significantly lower compared to older calves (older than three months). Additionally, we found that by increasing the frequency of vaccination in calves older than three months, it is possible to reach high NAT by the age of one year. Adoption of such a vaccination regimen in Israel as well as other FMD endemic countries may allow better protection against FMD in dairy calves and reduction in FMD incidence.

Prevalence and risk factors for foot and mouth disease infection in cattle in Israel.

Foot and mouth disease (FMD) is a highly contagious viral disease with major economic consequences. In Israel, FMD epidemics recur almost every year and mostly affect cattle. The highest number of outbreaks occurs among beef cattle farms, followed by feedlot farms and dairy farms. We performed several cross-sectional serological studies in Israel during 2006-2014, aimed to reveal if the virus is endemic among cattle and to determine the sero-prevalence of antibodies directed against non-structural proteins (NSP) of FMD virus. Additionally we aimed to determine the risk factors for such sero-positivity. A risk based sampling was performed and the presence of anti-NSP antibodies was estimated using the PrioCHECK(®) ELISA kit. Beef cattle showed the highest sero-prevalence (13.2%, CI95%=10.8-15.8%). Higher FMD sero-prevalence in beef cattle sampled in 2014 was associated with previous FMD outbreaks in the farm and with age (adult cows versus calves (p<0.05)). Sero-prevalence in feedlot calves was significantly lower with only one sero-positive calf out of 256 (0.4%, CI95%=0-2.2%). Sero-prevalence among dairy cattle was 2.7% (CI95%=2-3.6%) with location of up to 3km from FMD outbreaks in multiple farms and location of up to 5km from the nearest border standing out as significant (p<0.05) risk factors for sero-positivity. The extremely low sero-prevalence of FMD in feedlot cattle and the significant association of infection in beef cattle with previous outbreaks suggest absence of virus circulation between these two populations during the study period, although previous data show that during outbreaks such transmission can occur. Low sero-prevalence in dairy cattle located in areas adjacent to previous FMD outbreaks may be attributed to intense routine vaccination and stringent control measures that were applied during outbreaks such as emergency vaccination and strict quarantine. Early detection of FMD outbreaks among grazing beef herds as well as the implementation of control measures among these farms are therefore the methods of choice to prevent future outbreaks in Israel.

Seroprevalence of Foot-and-Mouth Disease in Susceptible Wildlife in Israel.

Foot-and-mouth disease (FMD) epidemics recur in Israel almost every year. Wild even-toed ungulates are seldom affected during these epidemics. The seroprevalence of FMD in wild ungulates during 2000 and 2005-2013 was estimated using anti-non-structural proteins ELISA. Overall, 209 samples were tested, comprising sera of 120 wild boar (Sus scrofa lybicus), 64 mountain gazelles (Gazella gazella gazella), 6 water buffaloes (Bubalus bubalis), and 19 Persian fallow deer (Dama dama mesopotamica). None of the tested animals presented clinical signs of FMD during blood collection. Sixteen samples [7.7% (95% confidence interval (CI95%) = 4.4-12.1%)] were found to be seropositive. Fifteen out of 120 samples (12.5%) from wild boar were seropositive, compared with only 1 out of 89 samples (1.1%) from all other species combined (Fisher's exact test: p = 0.003). Most of the positive samples obtained from wild boar [13/15 (86.7%)] were collected during 2007, and analysis was restricted to that year and species only. The seroprevalence of FMD in this species during 2007 was estimated at 54.2% (CI95% = 32.8-74.5%; n = 24). A significant infection cluster, comprising nine seropositive samples collected in three different locations, was identified in the north-eastern part of Israel. These findings indicate that wild boar was affected during the 2007 FMD epidemic, even though wild boar presenting FMD typical clinical signs were not observed during that year. The actual role of wild boar in the spread of FMD virus in this epidemic, however, could not be determined. The negligible seroprevalence of FMD found for all other surveillance years indicates that ongoing circulation of FMD among wildlife in Israel is unlikely. It is concluded that while the role of wildlife species in the dynamics of FMD in Israel is usually limited, there might be occasions, in which wildlife plays a part in the spread of the virus.

Prevalence and risk factors for foot and mouth disease infection in small ruminants in Israel.

During the last decade, 27% of the foot and mouth disease (FMD) outbreaks in Israel affected small ruminant (SR) farms. FMD outbreaks reoccur in Israel despite vaccination of all livestock and application of control measures. We performed a cross-sectional serological study, aimed at estimating the prevalence of FMD infection in SR in Israel and the possible risk factors for infection. Overall, 2305 samples of adult sheep (n=1948) and goats (n=357) were collected during 2011-14 in two separate surveys. One survey was based on random sampling of intensive management system farms and the other was originally aimed at the detection of Brucella melitensis at extensive and semi-intensive management system farms. Sera were tested by NS blocking ELISA (PrioCHECK(®)). The serological prevalence of antibodies against non structural proteins (NSP) of FMD virus was estimated at 3.7% (95% confidence interval (CI95%)=3.0% -4.5%). Additionally, a significantly lower infection prevalence (p value=0.049) of 1.0% (CI95%=0.1%-3.6%) was found in a small sample (197 sera) of young SR, collected during 2012. The positive samples from adult SR were scattered all over Israel, though two significant infection clusters were found by the spatial scan statistic. Occurrence of an outbreak on a non-SR farm within 5km distance was associated with a fifteen times increase in the risk of FMD infection of SR in the univariable analysis. Yet, this variable was not included in the multivariable analysis due to collinearities with the other independent variables. Multivariable logistic regression modeling found significantly negative associations (P value<0.05) of grazing and being in a herd larger than 500 animals with risk of infection. Grazing herds and herds larger than 500 animals, both represent farms that are intensively or semi-intensively managed. Higher maintenance of bio-safety, fewer introductions of new animals and higher vaccination compliance in these farms may explain their lower risk of infection by FMD virus. We conclude that despite the wide distribution of infection among SR farms, low farm level prevalence indicates that in Israel SR pose only limited role in the transmission and dissemination of FMD. This conclusion may be applicable for other endemic countries in which, similar to Israel, all livestock are vaccinated against FMD.