Concurrent infection of Bluetongue and Peste‐des‐petits‐ruminants virus in small ruminants in Haryana State of India

Bluetongue (BT ) and peste‐des‐petits‐ruminants (PPR ) are major transboundary diseases of small ruminant, which are endemic in India. Testing of bluetongue virus (BTV ) and peste‐des‐petits‐ruminants virus (PPRV ) from recent outbreaks (2015–2016) in different regions of Haryana State of India revealed that 27.5% of the samples showed the presence of dual infection of BTV and PPRV . Analysis of Seg‐2 of BTV (the serotype‐determining protein) showed the presence of BTV ‐12w in several isolates. However, analysis of N gene fragment amplicons showed that viruses belong to lineage IV were most closely related to a pathogenic strain of PPRV from Delhi. This is the first report of co‐circulation of PPRV lineage IV and bluetongue virus serotype 12 in the state.     

Bluetongue Virus Serotype 8‐Associated Congenital Hydranencephaly in Calves

Hydranencephaly, the almost complete absence of the cerebral parenchyma, induced by infection with modified live bluetongue virus (BTV) crossing the placenta has previously been reported in sheep and rarely in cattle in the USA and in South Africa. The current study describes 29 cases of hydranencephaly in bovine foetuses and ‘dummy’ calves up to 3 months of age in Belgium associated with natural BTV serotype 8 infection very early in gestation. Histological examination of the remaining cerebral parenchyma showed moderate to severe atrophy of the neural tissue. The lesions observed support the hypothesis of BTV‐induced destruction of precursor cells. However, in several calves a slight infiltration of the walls of venules and arterioles with T lymphocytes (vasculitis) was observed as well, which seems to be responsible for at least some of the lesions. Bluetongue viral RNA was detected in 15 animals using a BTV‐specific real‐time RT‐PCR with a much higher success rate in brain tissues compared with blood and spleen samples. Virus isolation in embryonated eggs was unsuccessful. In conclusion, hydranencephaly in calves can be associated with natural wild‐type BTV‐8 intra‐uterine infection.     

Detection and Phylogenetic Analysis of Peste des Petits Ruminants Virus Isolated from Outbreaks in Punjab,Pakistan

Peste des Petits Ruminants (PPR) is an important viral disease of small ruminants and is endemic in Pakistan. In the following study, samples from two outbreaks of PPR in goats have been subjected to laboratory investigations. The Peste des Petits Ruminants virus (PPRV) genome was detected using both conventional and real‐time PCR. Genetic characterization of the local PPRV field isolates was conducted by sequencing 322 bp of the fusion (F) gene and 255 bp of the nucleoprotein (N) gene. The phylogenetic tree based on the F gene clustered samples from both outbreaks into lineage 4 along with other Asian isolates, specifically into subcluster 1 along with isolates from Middle East. Analysis of N gene revealed a different pattern. In this case, the Pakistani samples clustered with Chinese, Tajikistani and Iranian isolates, which probably represents the true geographical pattern of virus circulation. This is the first report presenting the phylogenetic tree based on N gene as well as performing a parallel comparison of the trees of F and N gene together from Pakistani isolates. The results of this study shed light on the PPRV population in Pakistan and emphasize the importance of using molecular methods to understand the epidemiology. Such understanding is essential in any efforts to control the number and impact of outbreaks that are occurring in endemic countries such as Pakistan, especially in the current scenario where OIE and FAO are eager to control and subsequently eradicate PPR from the globe, as has been achieved for Rinderpest.     

Re‐Emergence of Bluetongue Virus Serotype 8 in France, 2015

At the end of August 2015, a ram located in central France (department of Allier) showed clinical signs suggestive of BTV (Bluetongue virus) infection. However, none of the other animals located in the herd showed any signs of the Bluetongue disease. Laboratory analyses identified the virus as BTV serotype 8. The viro and sero prevalence intraherd were 2.4% and 8.6% in sheep and 18.3% and 42.9% in cattle, respectively. Phylogenetic studies showed that the sequences of this strain are closely related to another BTV‐8 strain that has circulated in France in 2006–2008. The origin of the outbreak is unclear but it may be assumed that the BTV‐8 has probably circulated at very low prevalence (possibly in livestock or wildlife) since its first emergence in 2007–2008.     

Seroprevalence after Vaccination of Cattle and Sheep against Bluetongue Virus (BTV) Serotype 8 in Sweden

Sweden experienced its first outbreak of bluetongue virus (BTV) infection beginning in September 2008. Mandatory vaccination with an inactivated vaccine (BTVPUR Alsap8; Merial, Lyon, France) began 2 days after bluetongue was confirmed in the country. The aim of this study was to investigate whether the goal of 80% seroconversion by the susceptible population within the vaccination area was met during the initial phase of the Swedish vaccination campaign and whether there were discrepancies between subpopulations. Milk or blood samples were collected from 274 cattle randomly selected from the vaccinated population. Blood samples were also collected from ten ewes on each of 28 randomly selected vaccinated herds. The vaccination campaign in Sweden may be regarded as successful, as measured by apparent seroprevalence in the vaccinated population. The overall apparent seroprevalence was 77%, and in cattle, which constituted the majority of the susceptible population, the apparent seroprevalence was 82%. Factors that influenced the titres after vaccination were as follows: (i) the time span between vaccination and sampling and (ii) the age of the animals.     

蓝舌病毒湖北株对小鼠肾癌细胞体内外的杀伤效应

目的 观察蓝舌病毒湖北株(BTV-HBC3)对小鼠肾癌细胞株Renca体内外的杀伤效应.方法 接种1 MOI的BTV-HBC3于Renca细胞,观察Renca感染后的细胞病变效应(CPE),应用扫描电镜观察感染48 h后的超微结构,以噻唑蓝(MTT)比色法检测病毒分别以不同感染复数(MOI)感染Renca细胞后24、36、48 h的细胞存活率,并应用琼脂糖凝胶电泳分析感染后48 h的Renca细胞及其对照组病毒基因组.同时制备BALB/C小鼠Renca细胞荷瘤动物模型,观察应用BTV-HBC3处理后皮下荷瘤生长,并进行苏木素-伊红(HE)染色病理分析,应用免疫组织化学检测病毒蛋白的表达.结果 Renca细胞感染BTV-HBC348 h后,CPE程度大于90%,透射电镜下,胞质内可见增殖的病毒颗粒,呈晶格状排列.Renca细胞接种1 MOI的BTV-HBC3 24、36、48 h后细胞的存活率分别为(44.45±5.26)%、(30.47±4.77)%、(20.34±5.26)%,且该病毒对Renca细胞的杀伤作用呈现一定的量效关系;感染后细胞进行琼脂糖电泳可以检测到特异性长(L)、中(M)、短(S)3组分片段的病毒的dsRNA基因组;体内实验表明BTV-HBC3也明显抑制肿瘤生长(P<0.01);组织病理学观察中肿瘤组织未见坏死区,可见大片透明空泡样组织,肿瘤组织内可见腺体样结构,而小鼠其他各脏器组织切片中未见病毒感染,治疗组肿瘤组织可见病毒蛋白的阳性染色,其余组织器官经免疫组织化学分析未见病毒蛋白表达.结论 蓝舌病毒湖北株对小鼠肾癌细胞体内外具有显著的靶向杀伤效应.     

Bluetongue Virus in Lebanon

Since 2000, several incursions of bluetongue virus (BTV) occurred in the Mediterranean Basin involving European and surrounding Countries. The Middle East represents one of the most important gateways for the access of BTV in Europe. Limited data on the BTV situation in this area are available. In this perspective, an epidemiological survey on the presence of BTV in Lebanon was conducted. Of the 181 serum samples tested, 97 (mean = 53.6%; 95% CI: 46.3–60.7) resulted positive when tested for the presence of BTV antibodies by c‐ELISA, of these 42 (mean = 42%; 95% CI: 32.8–51.8) serum samples were from sheep and 55 (mean = 67.9%; 95% CI: 57.1–77.1) serum samples were from goats. Fourteen blood samples (14/110; mean = 12.7%; 95% CI: 7.8–20.3), 6 (6/66; mean = 9.1%; 95% CI: 4.4–18.5) from sheep and 8 (8/44; mean = 18.2%; 95% CI: 9.6–32.0) from goats, were positive by qRT‐PCR. The results with serum‐neutralization assay and typing performed by RT‐PCR confirmed that six BTV serotypes are currently circulating in Lebanon, and these serotypes are as follows: 1, 4, 6, 8, 16 and 24. This study is the first report that confirms the presence and circulation of BTV in Lebanon.     

Western Bluetongue virus serotype 3 in Sardinia,diagnosis and characterization

Over the last 20 years, Italy has experienced multiple incursions of different serotypes of Bluetongue virus (BTV), a Culicoides‐borne arbovirus, the causative agent of bluetongue (BT), a major disease of ruminants. The majority of these incursions originated from Northern Africa, likely because of wind‐blown dissemination of infected midges. Here, we report the first identification of BTV‐3 in Sardinia, Italy. BTV‐3 circulation was evidenced in sentinel animals located in the province of Sud Sardegna on September 19, 2018. Prototype strain BTV‐3 SAR2018 was isolated on cell culture. BTV‐3 SAR2018 sequence and partial sequences obtained by next‐generation sequencing from nucleic acids purified from the isolate and blood samples, respectively, were demonstrated to be almost identical (99–100% of nucleotide identity) to BTV‐3 TUN2016 identified in Tunisia in 2016 and 2017, a scenario already observed in past incursions of other BTV serotypes originating from Northern Africa.     

Bluetongue Serotype 2 and 9 Modified Live Vaccine Viruses as Causative Agents of Abortion in Livestock: A Retrospective Analysis in Italy

The recent outbreak caused by Schmallenberg virus, which affected sheep, goats and cattle in Europe, highlighted the importance of having a robust surveillance plan capable of monitoring abortions and malformations in the livestock offspring. In this context, bluetongue viruses (BTVs) represented and represent one of the major threats to the European livestock industry. Aiming to improve the understanding on BTV cross placental transmission and serotype involvement, in this retrospective study foetal spleens and/or brains of 663 ovines, 429 bovines, 155 goats and 17 buffaloes were tested for the presence of BTV by virus isolation. BTV vaccine strains were isolated from 31 foetuses (2.4%; 95% CI: 1.7–3.4%): 24 (3.6%; 95% CI: 2.4–5.3%) from ovine foetal tissues; 6 (1.4%; 95% CI: 0.6–3.0%) from bovine foetal tissues and 1 (0.6%; 95% CI: 0.2–3.5%) from the spleen of a caprine foetus. All foetuses were from animals vaccinated with either BTV‐2 or BTV‐2, and BTV‐9 modified live vaccines (MLVs) produced by Onderstepoort Biological Products (OBP), South Africa. Among the 31 isolated vaccine strains, serotype 9 (n = 28) was more frequently isolated (P < 0.05) than serotype 2 (n = 3). In two cases infectious vaccine strains were found in the foetal tissues 2 months after the vaccine administration. Other pathogens known to be causative agents of abortion in ruminants were not detected nor isolated. This study demonstrates, for the first time, that BTV‐2 and BTV‐9 vaccine strains are able to cross the placental barrier of sheep, cattle and goats. BTV‐2 and BTV‐9 vaccine strains are able to infect foetuses and cause abortions or malformations depending on the period of pregnancy at the time of vaccination.     

Phylogenetic Characterization Genome Segment 2 of Bluetongue Virus Strains Belonging to Serotypes 5, 7 and 24 Isolated for the First Time in China During 2012 to 2014

Bluetongue is endemic in China, and Bluetongue virus (BTV) strains belonging to eight different serotypes (BTV‐1, BTV‐2, BTV‐3, BTV‐4, BTV‐9, BTV‐12, BTV‐15 and BTV‐16) had been isolated between 1996 and 1997. However, there has been a long pause in investigating the epidemiology of BTV infection since then. During 2012–2014, eight BTV strains belonging to serotypes 5, 7 and 24 were isolated for the first time in Yunnan and Guangdong provinces from the blood of sentinel animals. Phylogenetic analyses of genome segment 2 of these Chinese BTV strains grouped them into nucleotypes E, F and A, respectively, along with the reference strains of the same serotype. For each serotype, Chinese strains cluster together closely to form a China's sublineage. In addition, these strains were most closely related to strains from Africa, indicating that they may share a recent common ancestry with African strains. To our knowledge, this is the first time that BTV‐5, BTV‐7 and BTV‐24 strains have been isolated in South‐East Asia. These data will be beneficial for understanding the BTV epidemiology and improving diagnostic assays and control measures against bluetongue in China and its neighbouring countries in the Asia–Pacific region.     

Epidemiology of Bluetongue in India

Bluetongue (BT) is an insectborne endemic disease in India. Although infections are observed in domestic and wild ruminants, the clinical disease and mortality are observed only in sheep, especially in the southern states of the country. The difference in disease patterns in different parts of the country could be due to varied climatic conditions, sheep population density and susceptibility of the sheep breeds to BT. Over the five decades after the first report of BT in 1964, most of the known serotypes of bluetongue virus (BTV) have been reported from India either by virus isolation or by detection of serotype‐specific antibodies. There have been no structured longitudinal studies to identify the circulating serotypes throughout the country. At least ten serotypes were isolated between 1967 and 2000 (BTV‐1–4, 6, 9, 16–18, 23). Since 2001, the All‐India Network Programme on Bluetongue and other laboratories have isolated eight different serotypes (BTV‐1–3, 9, 10, 12, 16, 21). Genetic analysis of these viruses has revealed that some of them vary substantially from reference viruses, and some show high sequence identity with modified live virus vaccines used in different parts of the world. These observations have highlighted the need to develop diagnostic capabilities, especially as BT outbreaks are still declared based on clinical signs. Although virus isolation and serotyping are the gold standards, rapid methods based on the detection of viral nucleic acid may be more suitable for India. The epidemiological investigations also have implications for vaccine design. Although only a handful serotypes may be involved in causing outbreaks every year, the combination of serotypes may change from year to year. For effective control of BT in India, it may be pertinent to introduce sentinel and vector traps systems for identification of the circulating serotypes and to evaluate herd immunity against different serotypes, so that relevant strains can be included in vaccine formulations.     

Sero‐prevalence and epidemiology of peste des petits ruminants in Libya

We conducted a cross‐sectional study during 2013 to quantify the serological prevalence of peste des petits ruminants ( PPR) infection and to investigate host factors associated with PPR infection in small ruminants in Libya. A two‐stage sampling design was carried out. A total number of 148 flocks owning at least 100 heads each were randomly selected. Sixteen to forty‐eight samples were collected from each selected flock. A total number of 3,508 serum samples from unvaccinated animals were collected and analysed at IZSLER Brescia, Italy, by using competitive ELISA, IDvet innovative diagnostics (IDvet 310, France). The overall serological prevalence among SR was 33% (95% CI: 31.4–34.5). Significant differences between the prevalence in the geographical branches were observed. The lowest prevalence level was observed in Zawiyah branch (16.1%), whereas the highest value was obtained for the Sabha branch (56.8%). Considering the age, a serological prevalence of 24.7%, 31.5% and 42.1% was observed in SR <1 year, between 1 and 2 years and more than 2 years, respectively. Statistically significant differences (p  <  .001) in the sero‐prevalence levels were also observed between the age groups. Our findings suggest that the southern part of Libya could be more exposed to the infections coming from the neighbouring countries and this should be better investigated to correctly identify wherever specific entry points can be considered at higher risk than others. The results also confirmed the endemic status of PPR in Libya, with a constant exposure to the infection of the animals during their life. In the framework of the global strategy for control and eradication of PPR, our results, even if obtained by a preliminary study, can contribute to the assessment of the epidemiological situation of PPR in Libya as required by the Stage 1 of the plan.     

Emergence of a Novel Bluetongue Virus Serotype,China 2014

One hundred and twenty‐six blood samples were collected from healthy sheep and goats in Xinjiang, China, during July 2014. Seventy‐three samples (57.93%) were bluetongue virus (BTV) serology‐positive, and 39 samples (30.95%) were BTV NS1 gene‐positive. BTV strain XJ1407 was isolated from the blood of BTV NS1 gene‐positive animals and sequenced. Analysis of its genome sequence suggests that XJ1407 is a novel BTV serotype.     

Molecular epidemiology of peste des petits ruminants in Niger: An update

Like many West African countries, outbreaks of peste des petits ruminants (PPR), an economically important disease of goats and sheep, are regularly reported in Niger. The causative virus, peste des petits ruminants virus (PPRV), can be differentiated into four genetically distinct lineages. A publication in 2018 identified three PPRV lineages circulating in the country in 2001 (lineages I and II) and 2013 (lineage IV), respectively. In this present study, more recent samples were collected from goats and sheep in locations throughout Niger between 2011 and 2017. Twelve PPRV‐positive samples were characterized by sequencing of a segment of the nucleocapsid protein (N) gene. Phylogenetic analysis of the sequences identified viruses from lineages II and IV only. The analysis also indicated a shared origin of the viruses from Niger with PPRVs from neighbouring countries suggesting transboundary movement.     

First report of peste des petits ruminants virus lineage II in Hydropotes inermis,China

In this study, we investigated an outbreak of peste des petits ruminants (PPR ) at a Hydropotes inermis (water deer) farm in Anhui Province, China. These results demonstrated that PPR viruses (PPRV s) can infect H. inermis and also revealed that virulent lineage II PPRV s exist in China, where they have been responsible for the deaths of wild animals. The government should pay close attention to the threat of PPRV epidemiology in China.     

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.     

Molecular Characterization of Peste des Petits Ruminants Viruses From Outbreaks Caused by Unrestricted Movements of Small Ruminants in Pakistan

Peste des petits ruminants (PPR) is an endemic disease of small ruminants, and vaccination has been the method of control but outbreaks are continuously occurring in Pakistan. The following study presents a detailed investigation of an outbreak, suspected to be PPR, probably introduced by PPRV‐infected sheep and goats from Sindh Province (north‐west) to Punjab Province (central) of Pakistan during the flood relief campaign in 2011. A total of 70 serum samples from 28 different flocks were tested with competitive ELISA (H antibodies), which detected 24 (34.2%) samples positive for PPRV antibodies. Nasal swabs and faeces were tested with immunocapture ELISA (N antigen), which detected 18 (25.7%) samples positive for PPRV antigen. The RNA detected positive (n = 28, 40%) using real‐time PCR was subjected to conventional PCR for the amplification of the fusion and nucleoprotein genes. Sequencing of both genes and subsequent phylogenetic analysis indicated the grouping of all the sequences to be in lineage IV along with other Asian isolates of PPRV. However, sequences of both genes were divided into two groups within lineage IV. One group of viruses clustered with previously characterized Pakistani isolates, whereas the other group was distinctly clustered with isolates from the Middle East or India. The sequence identity indicated the introduction of at least one population of PPRV from a different source and circulation in the local flocks of small ruminants, which emphasized the need to obtain health clearance certificate before movement of animals. The results of this study provide baseline data for the genetic characterization of different PPRV populations in Pakistan.     

A novel Bluetongue virus serotype 3 strain in Tunisia,November 2016

Since 1998, southern Europe has experienced multiple incursions of different serotypes and topotypes of Bluetongue virus, a vector‐borne transmitted virus, the causative agent of Bluetongue (BT), a major disease of ruminants. Some of these incursions originated from northern Africa, likely because of wind‐blown dissemination of infected midges. In this report, we describe the detection and whole genome characterization of a novel BTV‐3 strain identified in a symptomatic sheep in Tunisia. Sequences were immediately deposited with the GenBank Database under Accession Nos KY432369‐KY432378. Alert and preparedness are requested to face the next vector seasons in northern Africa and the potential incursion of this novel strain in southern Europe.     

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.