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.     

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.     

Evidence for Tunisian‐Like Pestiviruses Presence in Small Ruminants in Italy Since 2007

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV‐1, BVDV‐2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5′‐UTR and the whole N^pro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV‐1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian‐like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian‐like pestivirus from this species.     

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.     

HoBi‐Like Virus RNA Detected in Foetuses Following Challenge of Pregnant Cows that had Previously Given Birth to Calves Persistently Infected with Bovine Viral Diarrhoea Virus

The ability of ruminant pestivirus including bovine viral diarrhoea virus (BVDV) and the related emerging pestivirus, HoBi‐like virus, to establish persistent infection (PI) following foetal infection is central to keeping these viruses in circulation. Non‐PI dams carrying BVDV PI calves develop high levels of immunity due constantly viral exposure. A study to determine whether the immunity developed following the generation of a BVDV PI is enough to prevent HoBi‐like virus infection of a subsequent foetus was performed. This study consisted of nine pregnant cows, four had birthed BVDV‐1 PI calves in a previous pregnancy, three cows had birthed BVDV‐2 PIs and two had birthed pestivirus negative calves. From this, six pregnant cows were challenged with HoBi‐like virus about day 85 of gestation (four BVDV‐1 and two BVDV‐2 cows) and three non‐challenged cows (negative control). At the day of challenge, the serum neutralizing titres against the homologous BVDV strains of the first inoculation ranged from 1148 to 5793. At day 6 post‐challenge, HoBi‐like RNA was detected in the serum of all four BVDV‐1 cows but not in the two BVDV‐2 cows. The foetuses harvested from five of the exposed dams (three BVDV‐1 and two BVDV‐2 cows) at day 30 post‐challenge were positive for HoBi‐like virus RNA. The sixth cow, BVDV‐1 cow #541, while pregnant at the time of exposure, had no foetus 30 days after exposure. Foetuses from HoBi‐like virus exposed dams were significantly smaller and lighter than control foetuses. HoBi‐like RNA was detected in samples of all challenged foetuses. The identification of viral RNA in the serum of 4 cows at day 6 post‐challenge, as well viral RNA detection in all foetuses 30 days post‐inoculation, indicates that the foetuses of dams with high antibodies titres against BVDV‐1 or BVDV‐2 would not be protected from challenge with a HoBi‐like virus.     

HoBi‐Like Pestivirus and Its Impact on Cattle Productivity

The clinical features and economic impact of the infection caused by an emerging group of pestiviruses, namely HoBi‐like pestivirus, in a cattle herd of southern Italy are reported. In 2011, the virus was first associated with respiratory disease, causing an abortion storm after 1 year and apparently disappearing for the following 3 years after persistently infected calves were slaughtered. However, in 2014, reproductive failures and acute gastroenteritis were observed in the same herd, leading to a marked decrease of productivity. A HoBi‐like strain closely related to that responsible for previous outbreaks was detected in several animals. Application of an intensive eradication programme, based on the detection and slaughtering of HoBi‐like pestivirus persistently infected animals, resulted in a marked improvement of the productive performances.     

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.     

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.     

Epidemiological factors and mitigation measures influencing production losses in cattle due to bovine viral diarrhoea virus infection: A meta‐analysis

Infection with bovine viral diarrhoea virus (BVDV) is associated with a loss in productivity in cattle farms. Determining which factors influence monetary losses due to BVDV could facilitate the implementation of mitigation measures to reduce the burden of BVDV. Mixed‐effect meta‐analysis models were performed to estimate the extent to which the costs of mean annual BVDV production losses per animal may be influenced by epidemiological factors such as BVDV introduction risk, initial prevalence, viral circulation intensity and circulation duration (trial 1). Additionally, changes in mean annual BVDV production losses per animal due to specific mitigation measures (i.e., biosecurity, vaccination, testing and culling, cattle introduction or contact with neighbouring cattle herds) were analysed (trial 2). In total, 19 studies were included in the meta‐analysis to assess mean annual BVDV production losses. The mean annual direct losses were determined to be €42.14 per animal (trial 1). The multivariate meta‐regression showed that four of the previously mentioned epidemiological factors significantly influenced the mean annual BVDV production losses per animal. Indeed, the per animal costs increased to €67.19 when these four factors (trial 1) were considered as “high or moderate” compared to “low”. The meta‐regression analysis revealed that implementation of vaccination and biosecurity measures were associated with an 8%–12% and 28%–29% decrease in BVDV production losses on average, respectively, when simulated herds were compared with or without such mitigation measures (trial 2). This reduction of mean annual BVDV production losses per animal due to mitigation measures was partially counteracted when farmers brought new cattle on to farm or allowed contact with neighbouring cattle herds. The influencing mitigation factors presented here could help to guide farmers in their decision to implement mitigation strategies for the control of BVDV at farm level.     

Evaluation of bovine viral diarrhoea virus control strategies in dairy herds in Hokkaido,Japan, using stochastic modelling

Bovine viral diarrhoea virus (BVDV ) infection in cattle can result in growth retardation, reduced milk production, reproductive disorders and death. Persistently infected animals are the primary source of infection. In Hokkaido, Japan, all cattle entering shared pastures in summer are vaccinated before movement for disease control. Additionally, these cattle may be tested for BVDV and culled if positive. However, the effectiveness of this control strategy aiming to reduce the number of BVDV ‐infected animals has not been assessed. The aim of this study was to evaluate the effectiveness of various test‐and‐cull and/or vaccination strategies on BVDV control in dairy farms in two districts of Hokkaido, Nemuro and Hiyama. A stochastic model was developed to compare the different control strategies over a 10‐year period. The model was individual‐based and simulated disease dynamics both within and between herds. Parameters included in the model were obtained from the literature, the Hokkaido government and the Japanese Ministry of Agriculture, Forestry and Fisheries. Nine different scenarios were compared as follows: no control, test‐and‐cull strategies based on antigen testing of either calves or only cattle entering common pastures, vaccination of all adult cattle or only cattle entering shared pastures and combinations thereof. The results indicate that current strategies for BVDV control in Hokkaido slightly reduced the number of BVDV ‐infected animals; however, alternative strategies such as testing all calves and culling any positives or vaccinating all susceptible adult animals dramatically reduced those. To our knowledge, this is the first report regarding the comparison of the effectiveness between the current strategies in Hokkaido and the alternative strategies for BVDV control measures.     

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.     

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’.     

Presence of atypical porcine pestivirus (APPV) in Brazilian pigs

Recently, a putative new pestivirus species, provisionally named as Atypical Porcine Pestivirus (APPV ), was associated with the congenital tremor in piglets in North America and consequently in Europe and Asia. The present research aimed to describe the detection and characterization of APPV employing NS 5B gene partial sequencing, gross pathology and histologic examination of piglets displaying congenital tremor from two different farms of Southern Brazil. No gross lesions were observed, and the histological findings revealed moderate vacuolization of the white matter of the cerebellum. RT ‐PCR followed by DNA sequencing and a phylogenetic analysis confirmed the presence of APPV in samples from the two farms, which the samples were distinct in nature. Phylogenetic reconstruction reinforced the high genetic variability within the APPV s previously reported. This is the first report of APPV in South America suggesting that this new group of viruses may be widespread in swine herds in other countries as it is in Brazil.     

Molecular prevalence of Chlamydia and Chlamydia‐like bacteria in Tunisian domestic ruminant farms and their influencing risk factors

Chlamydia and Chlamydia ‐like bacteria are well known to infect several organisms and may cause a wide range of diseases, particularly in ruminants. To gain insight into the prevalence and diversity of these intracellular bacteria, we applied a pan‐Chlamydiales real‐time PCR to 1,134 veterinary samples taken from 130 Tunisian ruminant herds. The true adjusted animal population‐level prevalence was 12.9% in cattle, against 8.7% in sheep. In addition, the true adjusted herd‐level prevalence of Chlamydiae was 80% in cattle and 25.5% in sheep. Chlamydiales from three family‐level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant herds. Our results showed that Parachlamydia acanthamoebae could be responsible for bovine and ovine chlamydiosis in central‐eastern Tunisia. Multivariable logistic regression analysis at the animal population level indicated that strata and digestive disorders variables were the important risk factors of bovine and ovine chlamydiosis. However, origin and age variables were found to be associated with bovine and ovine chlamydiosis, respectively. At the herd level, risk factors for Chlamydia positivity were as follows: abortion and herd size for cattle against breeding system, cleaning frequency, quarantine, use of disinfectant and floor type for sheep. Paying attention to these risk factors will help improvement of control programs against this harmful zoonotic disease.     

Evaluation of an IGM‐specific ELISA for early detection of bluetongue virus infections in domestic ruminants sera

Competitive‐ELISA (c‐ELISA) is the most widely used serological test for the detection of Bluetongue virus (BTV) viral protein 7 (VP7) antibodies (Ab). However, these BTV c‐ELISAs cannot to distinguish between IgG and IgM. IgM Ab are generated shortly after the primary immune response against an infectious agent, indicating a recent infection or exposure to antigens, such as after vaccination. Because the BTV genome or anti‐VP7 Ab can be detected in ruminant blood months after infection, BTV diagnostic tools cannot discriminate between recent and old infections. In this study, we evaluated an IgM‐capture ELISA prototype to detect ruminant anti‐BTV VP7 IgM on 1,650 serum samples from cattle, sheep, or goats. Animals were BTV‐naive, infected, or/and vaccinated with BTV‐1, ‐2, ‐4, ‐8, ‐9, ‐16, or ‐27, and we also included 30 sera from cattle infected with the Epizootic haemorrhagic disease virus (EHDV) serotype 6. Results demonstrated that this ELISA kit is specific and can detect the presence of IgM with satisfactory diagnostic specificity and sensitivity from 1 to 5 weeks after BTV infection in domestic ruminants (for goats and cattle; for sheep, at least up to 24 days). The peak of anti‐VP7 IgM was reached when the level of infectious viruses and BTV RNA in blood were the highest. The possibility of detecting BTV‐RNA in IgM‐positive sera allows the amplification and sequencing of the partial RNA segment 2 (encoding the serotype specific to VP2) to determine the causative BTV serotype/strain. Therefore, BTV IgM ELISA can detect the introduction of BTV (or EHDV) in an area with BTV‐seropositive domestic animals regardless of their serological BTV status. This approach may also be of particular interest for retrospective epidemiological studies on frozen serum samples.     

Seroprevalence of canine neosporosis and bovine viral diarrhoea in dairy cattle in selected regions of Kenya

Neospora caninum is the causative agent for canine neosporosis (CN), a disease of potential zoonotic importance causing reproductive losses in cattle while causing neuromuscular disease in dogs. Bovine viral diarrhoea on the other hand is caused by the bovine viral diarrhoea virus (BVDV) and is one of the most important reproductive diseases of cattle worldwide. In Kenya, these infections are of economic importance due to the losses they cause in farms in which they are diagnosed or are subclinical. Such losses include reduced milk production, reduced conception, early embryonic deaths and abortions which lead to reproductive wastage. This study was conducted between April 2017 and July 2018 and determined the seroprevalence of neoporosis and BVD in select dairy herds in Kenya. Kakamega, Nandi and Makueni Counties from where dairy farms were purposively sampled were used. Serum samples were collected from randomly selected dairy animals aged at least 2 years in the selected farms and screened for BVDV and CN antibodies. Seroprevalence of N. caninum was 24.1% (n = 552) and BVD, 52.3% (n = 545) across all the counties. Co‐infection where antibodies against the two infective agents were present was in 14.6% (n = 541) animals. Chi‐square tests of association between prevalence and county were significant for BVD (p = .000) but not for neosporosis (p = .626). Further chi‐square tests of association between the two infections were not significant (p = .105) neither were the associations of BVD (p = .575) and neosporosis (p = .626) on pregnancy status. These two diseases are rarely investigated as causes of bovine infertility. Detection of antibodies in the studied dairy herds underpins the need for enhanced surveillance by laboratories and for further studies to understand associated risk factors to formulate effective control strategies in dairy cattle to forestall abortions and production and reproduction losses.     

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.     

Modelling the variation in skin‐test tuberculin reactions,post‐mortem lesion counts and case pathology in tuberculosis‐exposed cattle: Effects of animal characteristics,histories and co‐infection

Correctly identifying bovine tuberculosis (bTB ) in cattle remains a significant problem in endemic countries. We hypothesized that animal characteristics (sex, age, breed), histories (herd effects, testing, movement) and potential exposure to other pathogens (co‐infection; BVDV , liver fluke and Mycobacterium avium reactors) could significantly impact the immune responsiveness detected at skin testing and the variation in post‐mortem pathology (confirmation) in bTB ‐exposed cattle. Three model suites were developed using a retrospective observational data set of 5,698 cattle culled during herd breakdowns in Northern Ireland. A linear regression model suggested that antemortem tuberculin reaction size (difference in purified protein derivative avium [PPD a ] and bovine [PPD b ] reactions) was significantly positively associated with post‐mortem maximum lesion size and the number of lesions found. This indicated that reaction size could be considered a predictor of both the extent (number of lesions/tissues) and the pathological progression of infection (maximum lesion size). Tuberculin reaction size was related to age class, and younger animals (<2.85 years) displayed larger reaction sizes than older animals. Tuberculin reaction size was also associated with breed and animal movement and increased with the time between the penultimate and disclosing tests. A negative binomial random‐effects model indicated a significant increase in lesion counts for animals with M. avium reactions (PPD b− PPD a <  0) relative to non‐reactors (PPD b− PPD a =  0). Lesion counts were significantly increased in animals with previous positive severe interpretation skin‐test results. Animals with increased movement histories, young animals and non‐dairy breed animals also had significantly increased lesion counts. Animals from herds that had BVDV ‐positive cattle had significantly lower lesion counts than animals from herds without evidence of BVDV infection. Restricting the data set to only animals with a bTB visible lesion at slaughter (n  = 2471), an ordinal regression model indicated that liver fluke‐ infected animals disclosed smaller lesions, relative to liver fluke‐negative animals, and larger lesions were disclosed in animals with increased movement histories.     

Experimental infection of sheep,goats and cattle with a bluetongue virus serotype 4 field strain from Bulgaria, 2014

In 2014, a new bluetongue virus serotype 4 (BTV ‐4) strain was detected in southern Greece and spread rapidly throughout the Balkan Peninsula and adjacent countries. Within half a year, more than 7,068 outbreaks were reported in ruminants, particularly in sheep. However, the reported morbidity and case fatality rates in ruminants varied. The pathogenesis of a Bulgarian BTV ‐4 strain isolated from sheep during the BTV ‐4 epizootic was studied in different species. Therefore, four sheep, three goats and three cattle were experimentally infected with the isolate BTV ‐4/BUL 2014/15 and monitored for clinical signs up to several weeks. Serum and whole‐blood samples were collected at regular intervals and subjected to serological and virological analyses. In this context, BTV ‐4‐specific real‐time RT ‐PCR assays were developed. The infection kinetics were similar to those known for other traditional BTV serotypes, and only mild BT ‐like clinical signs were observed in goats and sheep. In cattle, no obvious clinical signs were observed, except a transient increase in body temperature. The study results contrast with the severe clinical signs reported in sheep experimentally infected with an African BTV ‐4 strain and with the reports of BT ‐like clinical signs in a considerable proportion of different ruminant species infected with BTV ‐4 in the Balkan region and Italy. The discrepancies between the results of these animal trials and observations of BTV ‐4 infection in the field may be explained by the influence of various factors on the manifestation of BT disease, such as animal breed, fitness and virus strain, as described previously.