Pre‐Clinical Evaluation of a Real‐Time PCR Assay on a Portable Instrument as a Possible Field Diagnostic Tool: Experiences from the Testing of Clinical Samples for African and Classical Swine Fever Viruses

African swine fever (ASF) and classical swine fever (CSF) are two highly infectious transboundary animal diseases (TADs) that are serious threats to the pig industry worldwide, including in China, the world's largest pork producer. In this study, a duplex real‐time PCR assay was developed for the rapid detection and differentiation of African swine fever virus (ASFV) and classical swine fever virus (CSFV). The assay was performed on a portable, battery‐powered PCR thermocycler with a low sample throughput (termed as ‘T‐COR4 assay’). The feasibility and reliability of the T‐COR4 assay as a possible field method was investigated by testing clinical samples collected in China. When evaluated with reference materials or samples from experimental infections, the assay performed in a reliable manner, producing results comparable to those obtained from stationary PCR platforms. Of 59 clinical samples, 41 had results identical to a two‐step CSFV real‐time PCR assay. No ASFV was detected in these samples. The T‐COR4 assay was technically easy to perform and produced results within 3 h, including sample preparation. In combination with a simple sample preparation method, the T‐COR4 assay provides a new tool for the field diagnosis and differentiation of ASF and CSF, which could be of particular value in remote areas.     

Evidence of porcine circovirus‐like virus P1 in piglets with an unusual congenital tremor

Outbreaks of trembling and shaking were reported among pigs at two pig farms in Jiangsu Province, China. Serum and tissue samples tested positive for porcine circovirus‐like virus P1 and negative for classical swine fever virus, porcine circovirus type 2, astrovirus and porcine pestivirus using PCR/RT‐PCR and immunohistochemical techniques. High P1 viral genome loads were identified in sera, brain and lymph node tissue samples by qPCR. In addition, one of the most notable pathological changes was dissolution of the nucleus in Purkinje cells. The results of this study provide molecular evidence of an association between congenital tremor in pigs and P1 virus.     

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.     

Virus Load in Pigs Affected with Different Clinical Forms of Classical Swine Fever

Summary Classical swine fever (CSF) is an endemic disease in India, but the real magnitude of the problem is not known as only outbreaks of acute CSF are reported and many cases of chronic and clinically inapparent forms of the disease, which manifest a confusing clinical picture, remain undiagnosed. The real status of classical swine fever virus (CSFV) infection can only be known by testing pigs with highly specific and sensitive diagnostic assays. To obtain the baseline prevalence of CSFV infection among pigs in an endemic region where no vaccination was being performed, a real‐time PCR assay was used to detect viral genetic material in tissue samples collected from a slaughterhouse in the northern state of Uttar Pradesh in India. In total, 1120 slaughtered pigs were examined for the presence of CSF suggestive pathological lesions and tissues from suspected cases were tested for the presence of CSFV antigen and nucleic acids by indirect immuno‐peroxidase test and real‐time PCR, respectively. Based on the detection of viral genetic material in the tonsils, the prevalence of CSFV infection among slaughtered pigs was found to be 7.67%. Pigs detected positive for viral genome by quantitative real‐time PCR assay when categorized into different forms of CSF, depending upon the pathological lesions observed, the viral load in the tonsils of some of the pigs with chronic or clinically inapparent form of the disease was similar to that detected in pigs with acute CSF. The results of the study suggested that the risk posed by pigs with chronic disease or those infected but showing no clinical disease may be relatively higher as they can transmit the virus to new susceptible hosts over a longer period of time.     

The First Identification and Complete Genome of Senecavirus A Affecting Pig with Idiopathic Vesicular Disease in China

Senecavirus A (SVA) infection was recently confirmed in pigs in Brazil. In March, 2015, an outbreak of vesicular disease occurred in Guangdong, China, characterized by vesicular lesions in sows and acute death of neonatal piglets. Cumulative incidence of porcine idiopathic vesicular disease in farm A was 258, which had a total number of 5500 sows. Sows in farm B displayed typical vesicular symptoms by May, 2015, which also had 5500 sows. A total of 278 and 142 of 5500 sows in farm B demonstrated lame and presented vesicles, respectively, associated with a total of 186 mortality in piglets. Routine differential diagnoses for swine vesicular disease were carried out to exclude infection with foot‐and‐mouth disease virus, swine vesicular disease virus, vesicular exanthema of swine virus and vesicular stomatitis virus. In this study, seven pairs of primer were designed to amplify the complete genome of SVA in RT‐PCR assays. Sequence alignment showed that this Chinese strain shares 94.4–97.1% sequence identity to other eight strains of SVA. This is the first report of SVA in China and provides information about the association between SVA infection and vesicular disease.     

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.     

Fully automated and integrated multiplex detection of high consequence livestock viral genomes on a microfluidic platform

Differential diagnosis of diseases that share common clinical signs typically requires the performance of multiple independent diagnostic tests to confirm diagnosis. Diagnostic tests that can detect and discriminate between multiple differential pathogens in a single reaction may expedite, reduce costs, and streamline the diagnostic testing workflow. Livestock haemorrhagic diseases like classical swine fever (CSF), African swine fever (ASF), and vesicular diseases, such as foot‐and‐mouth disease (FMD), vesicular stomatitis (VS), and swine vesicular disease (SVD) can have an enormous impact on the livestock industry and economy of countries that were previously free of the diseases. Thus, rapid diagnosis of these diseases is critical for disease control. Here, we describe the development and initial laboratory validation of a novel fully automated user‐developed assay for simultaneous detection and differentiation of multiple viruses of veterinary importance in a single reaction with minimal user‐intervention. The user only performs sample loading, placement of consumables and reagents, selection and initiation of assay while all other processes (i.e., nucleic acid extraction, multiplex RT‐PCR, reverse dot blot detection and result reporting) are performed fully automated. The current assay has a turn‐around time of approximately 6 hr and can simultaneously process up to 24 samples. The automated assay accurately and specifically detected 37 laboratory amplified strains of the five target viruses, including all seven serotypes of FMD virus, three genotypes of CSF virus, and two serotypes of VS virus. The assay also detected targeted viruses in a variety of clinical samples collected from infected animals, such as oral fluid, oral swab, nasal swab, whole blood, serum, as well as tonsil, spleen, kidney, and ileum. No cross‐reactivity was observed with 15 nontarget viruses that affect livestock and samples from clinically healthy animals. To our knowledge, this is the first fully automated and integrated assay for simultaneous detection of multiple high consequence veterinary pathogens.     

Foot‐and‐Mouth Disease in Feral Swine: Susceptibility and Transmission

Experimental studies of foot‐and‐mouth disease (FMD) in feral swine are limited, and data for clinical manifestations and disease transmissibility are lacking. In this report, feral and domestic swine were experimentally infected with FMDV (A24‐Cruzeiro), and susceptibility and virus transmission were studied. Feral swine were proved to be highly susceptible to A‐24 Cruzeiro FMD virus by intradermal inoculation and by contact with infected domestic and feral swine. Typical clinical signs in feral swine included transient fever, lameness and vesicular lesions in the coronary bands, heel bulbs, tip of the tongue and snout. Domestic swine exhibited clinical signs of the disease within 24 h after contact with feral swine, whereas feral swine did not show clinical signs of FMD until 48 h after contact with infected domestic and feral swine. Clinical scores of feral and domestic swine were comparable. However, feral swine exhibited a higher tolerance for the disease, and their thicker, darker skin made vesicular lesions difficult to detect. Virus titration of oral swabs showed that both feral and domestic swine shed similar amounts of virus, with levels peaking between 2 to 4 dpi/dpc (days post‐inoculation/days post‐contact). FMDV RNA was intermittently detectable in the oral swabs by real‐time RT‐PCR of both feral and domestic swine between 1 and 8 dpi/dpc and in some instances until 14 dpi/12 dpc. Both feral and domestic swine seroconverted 6–8 dpi/dpc as measured by 3ABC antibody ELISA and VIAA assays. FMDV RNA levels in animal room air filters were similar in feral and domestic swine animal rooms, and were last detected at 22 dpi, while none were detectable at 28 or 35 dpi. The FMDV RNA persisted in domestic and feral swine tonsils up to 33–36 dpi/dpc, whereas virus isolation was negative. Results from this study will help understand the role feral swine may play in sustaining an FMD outbreak, and may be utilized in guiding surveillance, epidemiologic and economic models.     

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.     

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.     

Endemic outbreaks due to the re‐emergence of classical swine fever after accidental introduction of modified live LOM vaccine on Jeju Island,South Korea

After the unintentional vaccination of the LOM vaccine strain in 2014, classical swine fever virus (CSFV) reemerged in naïve pig herds on Jeju Island, South Korea, which had been a CSF‐free region with a non‐vaccination policy for a decade. Since the re‐emergence, endemic outbreaks of CSFV have occurred in the island, causing enormous damage to provincial pig farms. The present study reports the complete genome sequences and molecular characterization of the LOM‐derived field CSFV strains responsible for the current outbreaks on Jeju Island. The emergent Jeju LOM‐derived isolates shared 98.9%–99.7% and 98.7%–99.0% nucleotide sequence identity at the E‐gene and whole‐genome levels compared to the LOM vaccine strain respectively. Genetic and phylogenetic analyses indicated that the CSFV field isolates were closest to the LOM strains, but appeared to have undergone substantial evolution. The total number of nucleotide and amino acid differences between the LOM vaccine strain and LOM‐derived field isolates ranged from 111 and 28 to 148 and 42. These variations were found to be widely distributed throughout the genome and particularly accumulated in non‐structural proteins, which might be associated with the potential for LOM to revert to its original low pathogenic form and subsequent horizontal transmission in Jeju swine herds. These data improve our knowledge regarding safety of the LOM vaccine and inherent risk of reversion to natural virulence in host animals.     

Detection of a novel porcine circovirus‐like agent in aborted pig foetuses

A large outbreak of swine abortion, which began in January 2017, is ongoing in Jiangsu and Anhui provinces, China. We identified a new porcine circovirus‐like agent, designated P4, in the aborted foetuses from several of these cases of swine reproductive failure. PCR and qRT ‐PCR analyses confirmed that other common abortogenic agents were not present in these animals. P4 contains a 710‐nucleotide circular viral genome, and all strains examined in this study were closely related to the dominant genotype of porcine circovirus type 2. Further studies, such as in vivo analyses, are needed to confirm P4 as the aetiological agent.     

Detection of a novel sapelovirus in central nervous tissue of pigs with polioencephalomyelitis in the USA

An approximately 3,000 finishing swine operation in the United States experienced an outbreak of an atypical neurologic disease in 11‐weeks‐old pigs with an overall morbidity of 20% and case fatality rate of 30%. The clinical onset and progression of signs in affected pigs varied but included inappetence, compromised ambulation, ataxia, incoordination, mental dullness, paresis, paralysis and decreased response to environmental stimuli. Tissues from affected pigs were submitted for diagnostic investigation. Histopathologic examination of the cerebrum, cerebellum and spinal cord revealed severe lymphoplasmacytic and necrotizing polioencephalomyelitis with multifocal areas of gliosis and neuron satellitosis, suggestive of a neurotropic viral infection. Bacterial pathogens were not isolated by culture of neurologic tissue from affected pigs. Samples tested by polymerase chain reaction (PCR) were negative for pseudorabies virus and atypical porcine pestivirus. Immunohistochemistry for porcine reproductive and respiratory syndrome virus, porcine circovirus and Listeria was negative. Porcine sapelovirus (PSV) was identified in spinal cord by a nested PCR used to detect porcine enterovirus, porcine teschovirus and PSV. Next‐generation sequencing of brainstem and spinal cord samples identified PSV and the absence of other or novel pathogens. In addition, Sapelovirus A mRNA was detected in neurons and nerve roots of the spinal cord by in situ hybridization. The PSV is genetically novel with an overall 94% amino acid identity and 86% nucleotide identity to a recently reported sapelovirus from Korea. This is the first case report in the United States associating sapelovirus with severe polioencephalomyelitis in pigs.