Serological relationship between a novel ovine pestivirus and classical swine fever virus

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV‐1) and type 2 (BVDV‐2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross‐neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.     

Evidence for Circulation of Bovine Viral Diarrhoea Virus Type 2c in Ruminants in Southern Italy

Recently, bovine viral diarrhoea virus type 2c (BVDV‐2c) was responsible for a severe outbreak in cattle in northern Europe. Here, we present the results of an epidemiological survey for pestiviruses in ruminants in southern Italy. Pooled serum samples were obtained from 997 bovine, 800 ovine, 431 caprine and eight bubaline farms, and pestiviral RNA was detected by molecular methods in 44 farms consisting of 16 cattle and one buffalo herds and of 21 sheep and six goat flocks. Twenty‐nine and 15 farms were infected by BVDV‐1 and BVDV‐2 strains, respectively. BVDV‐1 strains were recovered mainly from cattle and were heterogeneous, belonging to the subtypes 1b, 1u, 1e, 1g and 1h. In contrast, all BVDV‐2 viruses but two were detected in sheep or goats and were characterized as BVDV‐2c by sequence analysis of 5′UTR. These strains displayed high genetic identity to BVDV‐2c circulating in cattle in northern Europe and were more distantly related to a BVDV‐2c isolate recovered from a cattle herd in southern Italy more than 10 years before. The circulation of a BVDV‐2c in small ruminants suggests the need for a continuous surveillance for the emergence of pestivirus‐induced clinical signs in southern Italian farms.     

HoBi‐like is the most prevalent ruminant pestivirus in Northeastern Brazil

The ruminant pestiviral species BVDV ‐1, BVDV ‐2 and BDV , along with the putative species HoBi‐like, may cause substantial economic losses in cattle, sheep and goats. Brazil's large size, variable biomes and wide range of ruminant animal production within different geographic regions suggest that the presence and prevalence of ruminant pestivirus may differ by regions within Brazil. This study investigated the genetic diversity of ruminant pestiviruses and determined the frequency of active infections within two states of the Northeast Region of Brazil, Maranhão and Rio Grande do Norte. Serum samples from 16,621 cattle and 2,672 small ruminants from 569 different herds residing in this region were tested by RT ‐PCR followed by DNA sequencing. Seventeen positive cattle were detected (0.1%) from fifteen different herds (2.64%). All isolates were classified as HoBi‐like pestiviruses based on phylogenetic analysis. All small ruminant samples tested negative. The findings presented herein suggest that the Northeast Region of Brazil has a uniquely high prevalence of HoBi‐like viruses. The increasing reports of HoBi‐like viruses detected in cattle in the field suggest that natural infection with these viruses may be more widespread than previously thought. The identification of HoBi‐like viruses as the most prevalent type of ruminant pestivirus circulating in the Northeast Region of Brazil indicates the need for both continued monitoring and determination of the extent of economic losses associated with HoBi‐like virus infections. In addition, it must be taken into account in the choice of diagnostic tests and in vaccine formulations.     

Genetic Diversity of Brazilian Bovine Pestiviruses Detected Between 1995 and 2014

Pestivirus infections in ruminants result in significant economic losses worldwide. The aetiological agents are three species from the genus Pestivirus, family Flaviviridae, including bovine viral diarrhoea virus type 1 (BVDV‐1), BVDV‐2, border disease virus (BDV), and an atypical pestivirus named HoBi‐like pestivirus. In this study, eighty‐nine pestivirus isolates that were collected in Brazil between 1995 and 2014 and that originated from either cattle, fetal bovine serum (FBS) or as cell culture contaminants were genotyped based on a comparison of gene sequences from their 5′ untranslated regions (5′UTR), N‐terminal autoprotease (N^pro) and envelope glycoprotein 2 (E2). Of these isolates, 53.9% of the sequences were genotyped as BVDV‐1, 33.7% as BVDV‐2 and 12.4% as HoBi‐like pestivirus. The prevalence of subgenotypes within the species was as follows: BVDV‐1a (35.9%), BVDV‐2b (31.4%), BVDV‐1b (10.1%), BVDV‐1d (6.7%), BVDV‐2c (2.2%) and BVDV‐1e (1.1%). BVDV‐2c and BVDV‐1e were detected for the first time in Brazil. This study revealed extensive genetic diversity among Brazilian pestivirus isolates, and the combination of pestiviruses that was detected is unique to Brazil. This information may serve as a foundation for designing and evaluating diagnostic tools and in the development of more effective vaccines; therefore, it may potentially contribute to pestivirus control and eradication.     

Clinical Presentation Resembling Mucosal Disease Associated with ‘HoBi’‐like Pestivirus in a Field Outbreak

The genus Pestivirus of the family Flaviviridae consists of four recognized species: Bovine viral diarrhoea virus 1 (BVDV‐1), Bovine viral diarrhoea virus 2 (BVDV‐2), Classical swine fever virus (CSFV) and Border disease virus (BDV). Recently, atypical pestiviruses (‘HoBi’‐like pestiviruses) were identified in batches of contaminated foetal calf serum and in naturally infected cattle with and without clinical symptoms. Here, we describe the first report of a mucosal disease‐like clinical presentation (MD) associated with a ‘HoBi’‐like pestivirus occurring in a cattle herd. The outbreak was investigated using immunohistochemistry, antibody detection, viral isolation and RT‐PCR. The sequence and phylogenetic analysis of 5′NCR, N^pro and E2 regions of the RT‐PCR positive samples showed that four different ‘HoBi’‐like strains were circulating in the herd. The main clinical signs and lesions were observed in the respiratory and digestive systems, but skin lesions and corneal opacity were also observed. MD characteristic lesions and a pestivirus with cytopathic biotype were detected in one calf. The present study is the first report of a MD like presentation associated with natural infection with ‘HoBi’‐like pestivirus. This report describes the clinical signs and provides a pathologic framework of an outbreak associated with at least two different ‘HoBi’‐like strains. Based on these observations, it appears that these atypical pestiviruses are most likely underdiagnosed in Brazilian cattle.     

Identification of Natural Infections in Sheep/Goats with HoBi‐like Pestiviruses in China

The natural infections of HoBi‐like pestiviruses in cattle have been reported in South America, Europe and Asia. In China, although the detections of HoBi‐like pestivirus have been reported, the epidemiological investigation was limited. From January 2014 to October 2015, several flocks of sheep/goats in Henan province in central China suffered respiratory diseases which were recovered slowly after antibiotics treatment. To test whether it is the HoBi‐like pestivirus caused this symptom, 49 serum samples and 22 nasal swabs were then collected for analysis by serology and RT‐PCR. Serological result revealed that prevalence of pestivirus in small ruminants was 12.2% (6/49) in central China. Sequence analysis of partial 5′‐UTR nucleotides of pestivirus‐positive samples suggested that HoBi‐like pestivirus might have circulated in sheep/goats of China for a period and have evolved into new genotype clusters. It is apparent that the study provides the molecular evidence of natural infections in goat/sheep species with HoBi‐like pestiviruses in China.     

Identification and Epidemiology of a Rare HoBi‐Like Pestivirus Strain in Bangladesh

The genus pestivirus of the family flaviviridae consists of four recognized species: bovine viral diarrhoea virus 1 (BVDV‐1), bovine viral diarrhoea virus 2 (BVDV‐2), classical swine fever virus and border disease virus. A new putative pestivirus species tentatively named as either ‘HoBi‐like pestivirus’ or BVDV‐3 has recently been identified in Brazil, Italy and Thailand. Despite reports of serological evidence of BVDV in Bangladesh, the types of the virus circulating in cattle have not been identified. We conducted surveillance in cattle from May 2009 to August 2010 in three government veterinary hospitals to characterize BVDV in cattle of Bangladesh. We tested serum for BVDV using an antigen‐capture ELISA. Of 638 cattle samples, 3% (16/638) tested positive for BVDV antigen. The ELISA‐positive samples were selected for further molecular detection and characterization of BVDV. Molecular analysis of the partial 5′ untranslated region (UTR) nucleotide sequences of BVDV‐positive samples identified the rare HoBi‐like pestivirus or BVDV‐3 virus circulating in cattle of Bangladesh. The identification of this rare HoBi‐like pestivirus or BVDV‐3 strain in Bangladesh warrants further surveillance to evaluate its impact on livestock production.     

Detection of border disease virus in Mexican cattle

The genus Pestivirus within Flaviviridae is comprised of four recognized species, namely, bovine viral diarrhoea virus 1 (BVDV ‐1), bovine viral diarrhoea virus 2 (BVDV ‐2), border disease virus (BDV ) and classical swine fever virus (CSFV ). BDV , while primarily infecting sheep and goats, has also been reported in cattle and wild animals. Infections of sheep and goats result in economic loss due to abortions and the birth of persistently infected animals that have poor production and reduced life expectancy. In this study, we report the detection of BDV in cattle serum collected as part of pestivirus surveillance programme from six regions of Mexico, where a 67.1% of BVDV seroprevalence was calculated previously. Phylogenetic analyses based on comparison of the 5′UTR region typed the Mexican strains as BDV ‐1. Border disease (BD ) is listed as an exotic disease in Mexico, and the origin of BDV found in these cattle is unclear. This is the first identification of BDV in Mexican cattle.     

Diagnosis of Peste des Petits Ruminants in Wild and Domestic Animals in Xinjiang,China, 2013–2016

Peste des petits ruminants viruses (PPRVs) re‐emerged in China at the end of 2013 and then spread rapidly into 22 provinces through movement of live goats and sheep. In this study, 96 samples of domestic animals and 13 samples of wildlife were analysed for the presence of PPRV infection by ELISA or RT‐PCR. Of 96 samples from sheep and goats, 91 were PPRV positive, whereas all of the 13 samples from three wild species, Capra ibex (Capra ibex sibirica), argali (Ovis ammon) and Goitered gazelle (Gazella subgutturosa), were found to be positive. Five wildlife‐origin isolates from the above samples were identified as the lineage IV by a multiple alignment of the partial sequences in N gene.     

Molecular Characteristics of Bovine Virus Diarrhoea Virus 1 Isolates from Turkey: Approaches for an Eradication Programme

Forty pestivirus isolates sampled from cattle in Turkey between 2002 and 2007 were characterized according to 5′ untranslated region (5′UTR) sequences and autoprotease (N^pro) gene sequences. The sampling of Bovine virus diarrhoea viruses (BVDVs) from 15 farms in five different regions indicated that BVDV 1‐l (18/40, 45%) was the predominant genotype in Turkey; the samples also contained the genotypes 1‐f (10/40, 25%), 1‐b (7/40, 17.5%), 1‐d (3/40, 7.5%), and 1‐a (2/40, 5%), respectively.     

Genetic Detection and Characterization of Atypical Bovine Pestiviruses in Foetal Bovine Sera Claimed to be of Australian Origin

Summary Two European laboratories independently detected atypical bovine pestiviral nucleic acids in three commercial batches of foetal bovine serum (FBS) that was claimed by the producers to be of Australian origin. Additional batches of FBS were obtained directly from Australia to exclude possible contamination of the Australian FBS with that of South American origin during manufacturing/packaging in European countries. RT‐PCR amplification of partial 5′untranslated region and the complete N^pro gene yielded a specific band with expected size, which was confirmed by DNA sequencing. Bayesian analysis of sequence data demonstrated a closer phylogenetic relation of the newly detected atypical bovine pestiviruses to those of South American origin, which were related to the recognized bovine pestivirus species, bovine viral diarrhoea virus. Taken together, the results indicated the presence of atypical bovine pestiviruses in the Australian FBS, and most likely in Australian Continent.     

Phylogenetic and evolutionary analysis of HoBi‐like pestivirus: Insights into origin and dispersal

The HoBi‐like pestivirus (HoBiPeV), currently classified as Pestivirus H species, is a pathogen associated with a broad spectrum of clinical manifestations in ruminants, particularly in cattle. Since HoBiPeV complete genome sequencing data is scarce, in the present study we described five nearly complete new Brazilian HoBiPeV genomes and further perform a more complete genetic and evolutionary characterization with all additional genome sequences available in the GenBank database. Entropy and selection pressure analysis showed the E2 gene, a surface glycoprotein, is the most variable gene, which also displays the greatest number of sites under positive selection. Phylogenetic and Bayesian inference based on complete genome and N^pro gene, respectively, from all HoBiPeV sequences available so far, confirms the existence of three main clades (a, b, and c). The abovementioned analysis suggests that this pestivirus species probably emerged in Asia and spread to different regions including Brazil, where only strains belonging to specific genetic group ‘a’ have been found. The hypothesis of the HoBiPeV introduction in Brazil (between 1,890 and 1,962), formulated based on Bayesian inference, coincides with a period of intensive importation of water buffalo (Bubalus arnee) and indicine cattle (Bos taurus indicus) from Asia to Brazil, suggesting that this could be the origin of the current Brazilian HoBiPeV genetic group ‘a’.     

First detection and molecular identification of Sarcocystis spp. in small ruminants in North‐West Tunisia

Sarcocystosis is a parasitic disease caused by varying Sarcocystis species infecting humans and animals. It is commonly found in small ruminants causing pathogenic effects. This contributes to detrimental economic loss for local farmers and the local economy due this disease. Although the distribution of Sarcocystis can be found all over the world, the species infecting small ruminants in Tunisia is still unknown. Through this study, we aim to estimate the molecular prevalence of natural infection with Sarcocystis spp. in sheep and goats using molecular identification. Also, phylogenetic analyses were used to identify the different species of this parasite infecting small ruminants in northern Tunisia for the first time. DNA was extracted from 198 and 121, sheep and goats meat samples, respectively. The molecular prevalence of Sarcocystis spp. in sheep and goats was 58.6% (116/198) and 50.4% (61/121), respectively. Compared to the Noire de Thibar and cross‐breeds, the Barbarine sheep had the highest infection prevalence (63.4%) (p  =  .004). Five of the 116 positive samples were sequenced identifying Sarcocystis tenella from sheep. For goats, the sequencing results showed that five positive PCR products belonged to Sarcocystis capracanis species.     

Serosurvey of Schmallenberg Virus Infections in Sheep and Goat Flocks in Lower Saxony,Germany

Schmallenberg virus (SBV) infections can cause congenital musculoskeletal and vertebral malformations as well as neurological failures in foetuses of several ruminant species if susceptible mother animals were infected during early gestation. Blood samples gained from 17 goat and 64 sheep flocks in Lower Saxony (LS), Germany (January–May 2012), which is located in the core region of the 2011/2012 epidemic were tested for antibodies against SBV by ELISA to detect past exposure to SBV. A SBV‐specific questionnaire was raised in all flocks. The calculated median within‐herd prevalence was 43.8% (min–max: 5.6–93.3%) for goats and 58.7% (min–max: 6.5–100%) for sheep, showing that small ruminants in LS, especially goats, are still at risk of novel SBV infections in the following lambing seasons as not all animals have seroconverted yet. Statistical analysis revealed that goats have a significantly lower risk of SBV infections than sheep which might be explained by different host preferences of Culicoides ssp. as main vectors for SBV and different housing conditions.     

Epidemiological investigation and phylogenetic analysis of caprine parainfluenza virus type 3 in sheep of China

Caprine parainfluenza virus type 3 (CPIV3) is a new member of the Respirovirus genus in the Paramyxivirudae family, mainly causing respiratory disease. Up to now, accumulating evidence has focused on CPIV3 infection in goats, with little understood about its epidemiology in sheep. To that end, we collected 1,163 sheep sera samples from nine provinces/autonomous regions in 2012 and 1,863 samples from six provinces/autonomous regions during 2016–2017, with serological prevalence of 50.3% (95% CI: 47.5, 53.3) and 64.9% (95% CI: 62.9, 67.2), respectively. Pathogenic detection by qRT‐PCR was also performed on serum samples collected in 2016–2017, and the percentage of CPIV3 positive samples was 21.5% (95% CI: 19.7, 23.5). Sequence alignment and phylogenetic analyses revealed 11 novel CPIV3 strains based on the M gene sequences. The M gene and full‐length sequences of CPIV3 strains derived from sheep shared high nucleotide similarity with goat‐origin strains, indicating conserved genome characteristics between the viruses. Furthermore, sequence evolution and epidemiological analysis show that CPIV3 is widespread throughout China. This is the first report describing CPIV3 infection in sheep in China, showing a high sero‐prevalence and contributes to the assessment of the epidemiology of CPIV3 in China.     

Serosurvey of Schmallenberg Virus Infection in the Highest Goat‐Specialized Region of France

The monitoring of both the spread and clinical impact of Schmallenberg virus (SBV) infection within its full host range is important for the control of the epidemic and potential new outbreaks. In France, a national surveillance plan based on voluntary notifications of congenital malformations in newborn ruminants revealed that goats were the less affected host species. However, seroprevalence studies only targeted sheep and cattle, preventing accurate estimations of the real impact of SBV infection in goats. Here, a serological survey was conducted in the highest goat‐specialized region of France between June 2012 and January 2013. A total of 1490 goat sera from 50 herds were analysed by ELISA. The between‐herd and within‐herd prevalences were estimated at 62% and 13.1%, respectively. Seroprevalence was not uniformly distributed throughout the territory and markedly differed between intensive and extensive herds. The low within‐herd seroprevalence demonstrates that a large fraction of the French goat population remains susceptible to SBV infection.     

Mycobacterium avium subsp. paratuberculosis Sheep Strains Isolated from Cyprus Sheep and Goats

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic incurable infection of intestinal tract of animals. Molecular characterization of Map isolates classifies them into two major groups, ‘Cattle’ or Type II and ‘Sheep’ or Type I/III with a different phenotype, epidemiology, virulence and pathogenesis. The aim of this study was to examine 192 Map ELISA‐positive sheep and goats from Cyprus using faecal culture and genotype Map isolates using IS1311 PCR and restriction endonuclease analysis (IS1311 PCR‐REA) with HinfI restriction enzyme. Map was isolated from only four (4.6%) faecal samples out of 88 sheep and 15 (14.4%) faecal samples out of 104 goats. Genotyping of the isolates using IS1311 PCR‐REA revealed that sheep and goat populations on the island are infected primarily by ‘Sheep’ strains. Only three Map isolates from goats originated from one farm were characterized as ‘Cattle’ strains.     

A molecular and pathological study of peste des petits ruminants virus (PPRV) from field outbreaks in Palestine, 2017–2019

Peste des petits ruminants (PPR) is a severe, highly contagious and fatal viral disease of small ruminants causing high economic losses. Therefore, the disease was targeted for eradication by 2030. The aim of this study was to investigate for the first time the molecular and pathological characterization of the circulating PPR virus (PPRV) in sheep and goat in Palestine. Samples were collected from suspected necropsy cases of sheep and goats during recent outbreaks in two provinces in Palestine between 2017 and 2019. In this study, severe PPR outbreaks occurred in sheep and goats causing typical lesions which include erosive and ulcerative stomatitis, bronchointerstitial pneumonia and severe enteritis. For the molecular investigation of PPRV, suspected animals were examined for the presence of PPRV by RT‐PCR. PPRV genome was detected in all samples. Subsequently, two samples were used for N gene sequencing and phylogenetic analysis of PPRV isolates. The nucleotide sequence and phylogenetic analysis indicated that the Palestinian PPRV isolates were genetically clustered within the lineage IV isolates of the virus among populations of sheep and goats which most prevalent in Asia, the Middle East and recently Africa. Further analysis showed that the Palestinian isolates were closely related to those described in Turkey and Iraq, suggesting a common origin of PPRV isolates in the region. This information is critical to understand the molecular epidemiology of this disease in the region and helps to develop appropriate control measures for eradication of this disease.     

Coxiella burnetii Infections in Small Ruminants and Humans in Switzerland

The recent Q fever epidemic in the Netherlands raised concerns about the potential risk of outbreaks in other European countries. In Switzerland, the prevalence of Q fever in animals and humans has not been studied in recent years. In this study, we describe the current situation with respect to Coxiella (C.) burnetii infections in small ruminants and humans in Switzerland, as a basis for future epidemiological investigations and public health risk assessments. Specific objectives of this cross‐sectional study were to (i) estimate the seroprevalence of C. burnetii in sheep and goats, (ii) quantify the amount of bacteria shed during abortion and (iii) analyse temporal trends in human C. burnetii infections. The seroprevalence of C. burnetii in small ruminants was determined by commercial ELISA from a representative sample of 100 sheep flocks and 72 goat herds. Herd‐level seroprevalence was 5.0% (95% CI: 1.6–11.3) for sheep and 11.1% (95% CI: 4.9–20.7) for goats. Animal‐level seroprevalence was 1.8% (95% CI: 0.8–3.4) for sheep and 3.4% (95% CI: 1.7–6) for goats. The quantification of C. burnetii in 97 ovine and caprine abortion samples by real‐time PCR indicated shedding of >10⁴ bacteria/g in 13.4% of all samples tested. To our knowledge, this is the first study reporting C. burnetii quantities in a large number of small ruminant abortion samples. Annual human Q fever serology data were provided by five major Swiss laboratories. Overall, seroprevalence in humans ranged between 1.7% and 3.5% from 2007 to 2011, and no temporal trends were observed. Interestingly, the two laboratories with significantly higher seroprevalences are located in the regions with the largest goat populations as well as, for one laboratory, with the highest livestock density in Switzerland. However, a direct link between animal and human infection data could not be established in this study.     

The Distribution of Escherichia coli Serovars,Virulence Genes,Gene Association and Combinations and Virulence Genes Encoding Serotypes in Pathogenic E. coli Recovered from Diarrhoeic Calves,Sheep and Goat

Ruminants, especially cattle, have been implicated as a principal reservoir of one of the enterovirulent Escherichia coli pathotypes. The detection of the virulence genes in diarrhoeic calves and small ruminants has not been studied in Egypt. To determine the occurrence, serotypes and the virulence gene markers, stx1, stx2, hylA, Flic_h7, stb, F41, K99, sta, F17, LT‐I, LT‐II and eae, rectal swabs were taken from diarrhoeic calves, sheep and goats and subjected to bacterial culture and PCR. The E. coli prevalence rate in the diarrhoeic animals was 63.6% in calves, 27.3% in goat and 9.1% in sheep. The 102 E. coli strains isolated from the calves, goat and sheep were 100% haemolytic non‐verotoxic and fitted into the Eagg group. The isolates belonged to seven O serogroups (O25, O78, O86, O119, O158, O164 and O157). The eae gene was detected in six of the strains isolated from the calves. The 102 bovine, ovine and caprine E. coli strains isolated in this study were negative for stx1, stx2, F41, LT‐I and Flic_h7 genes. The highest gene combinations were found to occur in the form of 24/102 isolates (23.5%) that carried the F17 gene predominantly associated with eaeA, hylA, K99 and Stb genes in the calves, while the hylA, K99 and Sta were the only genes found to be in conjunction in both calves and goats (6/102; 5.9% each). Our data show that in Egypt, large and small ruminants could be a potential source of infection in humans.