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.     

Detection of Bovine Viral Diarrhoea Virus 2 as the Cause of Abortion Outbreaks on Commercial Sheep Flocks

Outbreaks of abortions and lambs born with nervous clinical signs and/or congenital malformations affected different sheep farms in Spain. Initial diagnosis of ‘border disease‐like’ was established, based on clinical signs, serology and/or RNA detection by a pan‐pestivirus RT‐PCR. However, further investigation using immunohistochemical and molecular techniques identified BVDV‐2b as the aetiological agent.     

Cell‐Mediated Immune Response During Experimental Acute Infection with Bovine Viral Diarrhoea Virus: Evaluation of Blood Parameters

Acute infections with bovine viral diarrhoea virus (BVDV), a major pathogen of cattle, are often asymptomatic or produce only mild clinical symptoms. However, they may play an important role in the bovine respiratory disease complex by exerting a marked immunosuppressive effect, as a result of the death of the immunocompetent cell populations involved in controlling innate and adaptive immune responses, together with a marked reduction of both cytokine expression and co‐stimulatory molecule synthesis. Although experimental research and field studies have shown that acute BVDV infection enhances susceptibility to secondary infection, the precise mechanism involved in BVDV‐induced immunosuppression remains unclear. The present study is aimed at measuring a range of blood parameters in a single group of fourteen calves infected with non‐cytopathic BVDV‐1. Focus has been put on those related to the cell‐mediated immune response just as leucocyte populations and lymphocyte subpopulations, serum concentrations of cytokines (IL‐1β, TNF‐α, IFN‐γ, IL‐12, IL‐4 and IL‐10) and acute phase proteins [haptoglobin, serum amyloid A (SAA), fibrinogen and albumin], as well as BVDV‐specific antibodies and viremia. After non‐cytopathic BVDV‐1 infection, clinical signs intensity was never more than moderate coinciding with the presence of viremia and leucocyte and lymphocyte depletion. An early increase in TNF‐α, IFN‐γ and IL‐12 levels in contrast to IL‐1β was observed in line with a raise in haptoglobin and SAA levels on the latest days of the study. As regards IL‐4 levels, no evidence was found of any changes. However, a slight increase in IL‐10 was observed, matching up the TNF‐α decline during the acute phase response. These findings would help to increase our knowledge of the immune mechanisms involved in acute infection with non‐cytopathic BVDV‐1 strains, suggesting the existence of a clear tendency towards a type 1 immune response, thereby enhancing resistance against viral infections.     

Phylogenetic Analysis of the Spike (S) Gene of the New Variants of Porcine Epidemic Diarrhoea Virus in Taiwan

New variants of porcine epidemic diarrhoea virus (PEDV), which emerged in Taiwan in late 2013, have caused a high morbidity and mortality in neonatal piglets. To investigate the molecular characteristics of the spike (S) gene of the emerging Taiwan PEDV strains for a better understanding of the genetic diversity and relationship among the Taiwan new variants and the global PEDVs, full‐length S genes of PEDVs from nine 1–7 day‐old piglets from three pig farms in the central and southern Taiwan were sequenced and analysed. The result of phylogenetic analysis of the S gene showed that all the Taiwan PEDV strains were closely related to the non‐S INDEL strains from US, Canada and China, suggesting a common ancestor for these strains. As compared with the historic PEDVs and CV777‐based vaccine strains, the nine Taiwan PEDV variants shared almost the same genetic signatures as the global non‐S INDEL strains, including a series of insertions, deletions and mutations in the amino terminal as well as identical mutations in the neutralizing epitopes of the S gene. The high similarity of the S protein among the Taiwan and the globally emerged non‐S INDEL PEDV strains suggests that the Taiwan new variants may share similar pathogenesis and immunogenicity as the global outbreak variants. The development of a novel vaccine based on the Taiwan or the global non‐S INDEL strains may be contributive to the control of the current global porcine epidemic diarrhoea outbreaks.     

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.     

A Report on Bovine Ephemeral Fever Virus in Turkey: Antigenic Variations of Different Strains of EFV in the 1985 and 2012 Outbreaks Using Partial Glycoprotein Gene Sequences

We described the aetiological agents of outbreaks of bovine ephemeral fever (BEF) that occurred in 1985 and 2012 in Turkey, and identify mutations in the viruses from both outbreaks. Outbreaks have emerged periodically every 4–5 years in the same regions in Turkey. Because these regions are located in a subtropical climatic zone, good conditions for vector populations exist. The results of this study show that the BEFVs from outbreaks in Turkey vary significantly. Effective prevention will require a vaccine that contains BEFVs from different genetic clusters.     

Detection of Bovine Leukaemia Virus Antibodies and Proviral DNA in Colostrum Replacers

Great Britain has been bovine leukaemia virus (BLV) disease free since 1999. We recently reported three separate incidents of BLV seropositivity on farms with home‐reared cattle due to the use of colostrum replacer rather than infection with BLV (Emerg. Infect. Dis., 19 , 2013, 1027). These cases were all linked via the use of the same brand of colostrum replacer. Here, we investigate further by examining multiple brands of colostrum replacer for proviral DNA and BLV antibodies. BLV antibodies were detected in 7 of the colostrum replacers tested, with PCR concurring in two cases. Thus, the use of these BLV antibody‐positive colostrum replacers may also lead to false‐positive serological diagnostics.     

Molecular Characterization and Phylogenetic Analysis of VP1 of Porcine Enteric Picornaviruses Isolates in Italy

Porcine enterovirus (PEV), Porcine Teschovirus and Porcine sapelovirus, belonging to the family Picornaviridae, are ubiquitous and mainly cause asymptomatic infections in pigs. In this study, a total of 40 Italian porcine picornavirus isolates were characterized by sequencing the capsid VP1‐encoding gene. This procedure turned out to be a useful diagnostic tool for the molecular identification of porcine enterovirus, teschovirus and sapelovirus strains and for the study of molecular epidemiology and evolution of these viruses confirming the possibility of correlating virus genotype to serotype.     

Phylogenetic Analysis of Classical Swine Fever Virus Isolates from Peru

Classical swine fever (CSF) is considered to be endemic in Peru with outbreaks reported to the World Organization for Animal Health as recently as 2008 and 2009. Nevertheless, little is known regarding the genetic subgroup(s) of CSF virus that are circulating in Peru or their relationship to recent CSF viruses that have been isolated from neighbouring South American countries or other parts of the world. In this study, we molecularly characterize CSF viruses that were isolated from domestic pigs from different regions of Peru from the middle of 2007 to early 2008. All virus isolates were found to belong to genetic subgroup 1.1, consistent with the subgroup of viruses that have been identified from other South American countries. Although the Peruvian isolates are most closely related to viruses from Colombia and Brazil, they form a monophyletic clade, which suggests they have a distinct evolutionary history.     

Genetic Typing of Classical Swine Fever Virus Isolates from China

The E2 genes of 73 classical swine fever virus (CSFV) originated from CSF suspected cases in different regions of China were genetically characterized and compared with reference CSF viruses. All Chinese viruses that characterized were segregated into two major groups and subdivided into four subgroups. Most of isolates (61.6%) belonged to group 2 and were further divided into three subgroups: subgroup 2.1, 2.2 and 2.3. Subgroup 2.1 was the largest subgroup which contained 46.6% of isolates, whiles subgroup 2.3 was the smallest subgroup which contained only one isolate (1.4%). The remaining 38.4% of isolates were classified into subgroup 1.1 within group 1. However, no group 3 and subgroups 1.2 and 1.3 viruses were found in this study. This study has provided epidemiological information useful for assessing the virus origin and establishing a national prevention and control strategy against the disease.     

Molecular Characterization of Foot‐and‐Mouth Disease Virus: Implications for Disease Control in Bangladesh

Foot‐and‐mouth disease (FMD) is endemic in Bangladesh, and to implement an effective FMD control programme, it is essential to understand the complex epidemiology of the disease. Here, we report on the characterization of FMD virus (FMDV) recovered from FMD outbreaks in Bangladesh in late 2009. All isolated viruses belonged to the FMDV serotype O. The phylogenetic reconstruction showed that all isolates belonged to the Middle East–South Asia (ME–SA) topotype, but fell into two distinct sublineages, one named Ind‐2001 (the other has not been named). Within both sublineages, the 2009 Bangladesh isolates were most closely related to viruses from Nepal collected during 2008 and 2009. Additionally, both sublineages contained older viruses from India collected in 2000 and 2001. In South Asia, there is extensive cross‐border cattle movement from Nepal and India to Bangladesh. Both these findings have implications for the control of FMD in Bangladesh. Because of the porous borders, a regional FMD control strategy should be developed. Further, animal identification and monitoring animal movements are necessary to identify the cross‐border movements and market chain interactions of ruminants, leading to improved border and movement controls. Additionally, a vaccination strategy should be developed with the initial objective of protecting small‐scale dairy herds from disease. For any successful FMD control programme, long‐term Government commitment and adequate resources are necessary. A sustainable programme will also need farmer education, commitment and financial contributions.     

Genomic Characterization of Pseudorabies Virus Strains Isolated in Italy

In this study, we undertook the genomic characterization of 54 pseudorabies virus (PRV) strains isolated in Italy during 1984–2010. The characterization was based on partial sequencing of the UL44 (gC) and US8 (gE) genes; 44 strains (38 for gene gE and 36 for gC) were isolated on pig farms; 9 originated from dogs and 1 from cattle. These porcine PRV strains, which were closely related to those isolated in Europe and America in the last 20 years, and the bovine strain bovine/It/2441/1992 belong to cluster B in both phylogenetic trees. Six porcine strains that do not belong to cluster B are related in both gE and gC phylogenetic trees to the ‘old’ porcine PRV strains isolated in the 1970s and 1980s. In the last two decades, the presence of these strains in domestic pig populations has been reduced drastically, whereas they are prevalent in wild boar. The two remaining strains have an interesting genomic profile, characterized by the gC gene being closely related to the old porcine PRV strains, and the gE gene being similar to that of recently isolated strains. Three strains originating from working dogs on pig farms are located in cluster B in both phylogenetic trees. Five strains isolated from hunting dogs have a high degree of correlation with PRV strains circulating in wild boar. The last isolate has a gC gene similar to that in the two porcine strains mentioned previously, and the gE gene is correlated with the strains isolated from hunting dogs. These results provide interesting insight into the genomic characterization of PRV strains and reveal a clear differentiation between the strains isolated from hunting dogs that are related to the wild boar strains and those originating from domestic pigs.     

Experimental Infection of Young Pigs with an Early European Strain of Porcine Epidemic Diarrhoea Virus and a Recent US Strain

Outbreaks of porcine epidemic diarrhoea (PED) were reported across Europe during the 1980s and 1990s, but only sporadic outbreaks occurred in recent years. PED virus (PEDV) spread for the first time into the USA in 2013 and has caused severe economic losses. Retrospectively, it was found that two different strains of PEDV have been introduced into the United States, both are closely related to strains circulating in China where a new wave of the disease occurred from 2010 onwards. Since autumn 2014, new outbreaks of PED have occurred in Europe. In this study, weaned piglets were inoculated with an early European isolate (Br1/87) or faecal/intestinal suspensions derived from pigs infected with a recent European strain of PEDV (from Germany) or a US strain of PEDV. No evidence for infection resulted from inoculation of pigs with the German sample that contained high levels of PEDV RNA; there were no clinical signs, excretion of viral RNA or anti‐PEDV antibody production. In contrast, all the pigs in the other two groups showed evidence of infection. Mild clinical signs of disease, mainly diarrhoea, occurred in piglets inoculated with the Br1/87 and US PEDV strains. PEDV RNA was detected throughout the intestine in euthanized animals at 4 days post‐inoculation. In addition, within these animals, low levels of viral RNA were detected in lungs and livers with higher levels in spleens. Seroconversion against PEDV occurred in all surviving infected animals within 10 days. PEDV RNA excretion occurred for at least 2 weeks. The US PEDV RNA was detected at low levels in serum samples on multiple days. It is apparent that current diagnostic systems can detect infection by the different virus strains.     

Bovine Tuberculosis: A Review of Current and Emerging Diagnostic Techniques in View of their Relevance for Disease Control and Eradication

Existing strategies for long‐term bovine tuberculosis (bTB) control/eradication campaigns are being reconsidered in many countries because of the development of new testing technologies, increased global trade, continued struggle with wildlife reservoirs of bTB, redistribution of international trading partners/agreements, and emerging financial and animal welfare constraints on herd depopulation. Changes under consideration or newly implemented include additional control measures to limit risks with imported animals, enhanced programs to mitigate wildlife reservoir risks, re‐evaluation of options to manage bTB‐affected herds/regions, modernization of regulatory framework(s) to re‐focus control efforts, and consideration of emerging testing technologies (i.e. improved or new tests) for use in bTB control/eradication programs. Traditional slaughter surveillance and test/removal strategies will likely be augmented by incorporation of new technologies and more targeted control efforts. The present review provides an overview of current and emerging bTB testing strategies/tools and a vision for incorporation of emerging technologies into the current control/eradication programs.     

Isolation and Characterization of a Moderately Virulent Classical Swine Fever Virus Emerging in China

Classical swine fever (CSF) is a devastating infectious disease of pigs caused by classical swine fever virus (CSFV). In China, CSF has been under control owing to extensive vaccination with the lapinized attenuated vaccine (C‐strain) since 1950s, despite sporadic or endemic in many regions. However, recently, CSF outbreaks occurred in a large number of swine herds in China. Here, we isolated 15 CSFV strains from diverse C‐strain‐vaccinated pig farms in China and characterized the genetic variations and antigenicity of the new isolates. The new strains showed unique variations in the E2 protein and were clustered to the subgenotype 2.1d of CSFV recently emerging in China in the phylogenetic tree. Cross‐neutralization test showed that the neutralizing titres of porcine anti‐C‐strain sera against the new isolates were substantially lower than those against both the highly virulent Shimen strain and the subgenotype 2.1b strains that were isolated in China in 2006 and 2009, respectively. In addition, experimental animal infection showed that the HLJZZ2014 strain‐infected pigs displayed lower mortality and less severe clinical signs and pathological changes compared with the Shimen strain‐infected pigs. The HLJZZ2014 strain was defined to be moderately virulent based on a previously established assessment system for CSFV virulence evaluation, and the virus shedding and the viral load in various tissues of the CSFV HLJZZ2014 strain‐infected pigs were significantly lower than those of the Shimen strain‐infected pigs. Taken together, the subgenotype 2.1d isolate of CSFV is a moderately virulent strain with molecular variations and antigenic alterations.     

Pathology and Virus Distribution in the Lung and Lymphoid Tissues of Pigs Experimentally Inoculated with Three Distinct Type 1 PRRS Virus Isolates of Varying Pathogenicity

Porcine reproductive and respiratory syndrome (PRRS) continues to be the most economically important disease of swine worldwide. The appearance of highly pathogenic PRRS virus (PRRSV) strains in Europe and Asia has raised concerns about this disease and initiated increased efforts to understand the pathogenesis. In this study, we have compared the pathology and the virus distribution in tissues of pigs experimentally inoculated with three different genotype 1 PRRSV isolates. Sixty 5‐week‐old pigs were inoculated intranasally with a) the Lelystad virus (LV), b) a field strain from the UK causing respiratory clinical signs (UK) or c) a highly pathogenic strain from Belarus (BE). Sixteen animals were mock‐infected and used as controls. The animals were euthanized at 3, 7 and 35 days post‐infection (dpi), and lung and lymphoid tissues collected for histopathological examination and PRRSV detection by immunohistochemistry (IHC). Histopathological lesions consisted of interstitial pneumonia with mononuclear cell infiltrates in the lungs, lymphoid depletion, apoptosis and follicular hyperplasia in the spleen, lymph nodes and tonsil and lymphoid depletion in the thymus. Porcine reproductive and respiratory syndrome virus was detected mainly in monocytes–macrophages. BE‐infected animals showed the highest pathological scores and the highest presence of virus at 3 and 7 dpi, followed by the UK field strain and then LV. Moderate lesions were observed at 35 dpi with lesser detection of PRRSV by IHC in each infected group. The highly pathogenic BE strain induced more severe pathology in both lungs and lymphoid organs of pigs compared with the classic field isolate and the prototype LV. The increased severity of pathology was in correlation with the presence of a higher number of PRRSV‐infected cells in the tissues.     

New Introduction of Clade 2.3.2.1 Avian Influenza Virus (H5N1) into Bangladesh

Since the first outbreak of highly pathogenic H5N1 avian inafluenza (HPAI) in Bangladesh in February 2007, a total of 519 disease events have been reported till 22 October 2011. Partial HA gene sequences of 11 selected H5N1 HPAI isolates of 2007 to 2011 were determined and subjected to phylogenetic analysis. The study revealed a recent introduction of clade 2.3.2 and 2.3.4 viruses into Bangladesh in 2011 in addition to clade 2.2 viruses that had been in circulation since 2007. Clade 2.3.2 virus isolates from Bangladesh are phylogenetically related to the newly designated clade 2.3.2.1 viruses, reported recently from Asia and Eastern Europe.