Cell culture propagation of porcine rotavirus (reovirus-like agent).

Two isolates of porcine rotavirus (reovirus-like agent) were isolated and passaged in primary procine kidney cell cultures. Viral infectivity for cells was monitored by immunofluorescence because viral cytopathic effect was moderate. Successful passage of virus in cell culture required that viral suspensions obtained from infected cell cultures be treated with pancreatin prior to inoculation onto cell monolayers. Porcine rotavirus passage in cell culture also was accomplished, using trypsin treatments in lieu of pancreatin treatments. Porcine rotavirus passaged 10 times in cell culture infected gnotobiotic pigs and caused diarrhea. Gnotobiotic pigs that recovered from this infection were resistant to challenge exposure with porcine rotavirus but were susceptible to challenge exposure with transmissible gastroenteritis virus. As determined by immunofluorescent cross reactions, porcine rotavirus was found to be antigenically related to the human and bovine rotaviruses but not to reovirus type 3 or to transmissible gastroenteritis virus.     

Molecular and biological characterisation of Cryptosporidium in pigs

OBJECTIVE: Genetic and biological characterisation of 12 isolates of Cryptosporidium from pigs and comparing them with Cryptosporidium isolates from humans and cattle. DESIGN: Cryptosporidium isolates from pigs were compared with those obtained from human and cattle using rDNA sequence analysis. The infectivity of two of the porcine isolates was determined in neonatal mice and the clinical history of the infected pigs recorded. RESULTS: Pig-derived isolates of Cryptosporidium exhibited two distinct genotypes; a porcine genotype and a bovine genotype, which is common to cattle and other livestock. The porcine genotype did not produce any infection in neonatal mice whereas the bovine genotype did. CONCLUSION: Two distinct genetically and biologically differing strains of Cryptosporidium appeared to be associated with acute diarrhoea in pigs. Whether Cryptosporidium was a primary or secondary pathogen is unclear but warrants further investigation. As the bovine genotype is known to infect humans, the results suggest that pigs can act as reservoirs of cryptosporidial infections for humans and other live-stock. The zoonotic potential of the pig-adapted genotype is uncertain and requires further study.     

Postweaning multisystemic wasting syndrome produced in gnotobiotic pigs following exposure to various amounts of porcine circovirus type 2a or type 2b

In late 2005, a postweaning, high mortality syndrome spread rapidly through finishing barns in swine dense areas of the United States. Diagnostic investigations consistently detected porcine circovirus type 2 (PCV2) from diseased tissues. Subsequent genetic analysis revealed that the infectious agent was a PCV2 type termed "PCV2b". Prior to late 2004, only the PCV2a type, but not PCV2b, had been reported in North America. In this communication, we produce severe postweaning multisystemic wasting syndrome (PMWS) in gnotobiotic pigs using infectious PCV2a and PCV2b generated from DNA clones constructed from field isolates identified in the 2005 outbreak. Clinical signs exhibited by diseased pigs included anorexia, dyspnea and listlessness. Mortality was typically observed within 12h of onset of dyspnea. The most striking microscopic lesions in affected animals were severe hepatic necrosis and depletion of germinal centers in lymph nodes with associated abundant PCV2 viral antigen. Clinical signs and lesions observed in these studies were comparable to those reported in experiments with gnotobiotic pigs inoculated with a PCV2a isolate while concurrently receiving immune-stimulation or co-infection with porcine parvovirus or torque teno virus. The animals in these studies were confirmed to be free of detectable porcine parvovirus, porcine reproductive and respiratory syndrome virus, bovine viral diarrhea virus, swine hepatitis E virus, and aerobic and anaerobic bacteria. Seven out of 24 PCV2 inoculated pigs had a detectable congenital torque teno virus infection with no correlation to clinical disease. Thus, in these studies, both PCV2a and PCV2b isolates were singularly capable of inducing high mortality in the absence of any detectable infectious co-factor.     

Vaccination of Pigs Against Hog Cholera (Classical Swine Fever) With a Detergent Split Vaccine

Four pigs were inoculated subcutaneously with a detergent (triton X 100) split hog cholera virus in Freund’s incomplete adjuvant. Four other pigs were in the same way inoculated with a detergent split bovine viral diarrhoea virus, also in Freund’s incomplete adjuvant. In the experiment were used 3 control pigs. The vaccinations were repeated after 3 weeks. All pigs were challenged with highly virulent hog cholera virus (Tübingen) 12 weeks after primary inoculations. Signs of hog cholera were only noted in the control pigs.This introductory experiment was succeeded by a larger experiment with subcutaneous inoculations of: 10 pigs with detergent split hog cholera virus in Freund’s incomplete adjuvant, 10 pigs with detergent split hog cholera virus in a saponin (Quil A) solution, 10 pigs with detergent split bovine viral diarrhoea virus in Freund’s incomplete adjuvant, 10 pigs with detergent split bovine viral diarrhoea virus in the Quil A solution plus 5 control pigs. The vaccinations were repeated after 3 weeks, and finally all pigs were challenged 9 weeks later with the highly virulent hog cholera virus strain.With the exception of 1 animal which died accidentally, all animals survived in the groups inoculated with the Quil A vaccines and in the group inoculated with the detergent split hog cholera virus/oil adjuvant vaccine. In the group inoculated with the detergent split bovine viral diarrhoea virus/oil adjuvant vaccine, some of the pigs died of hog cholera.     

Virus survival in slurry: analysis of the stability of foot-and-mouth disease, classical swine fever, bovine viral diarrhoea and swine influenza viruses

Farm slurry can be highly contaminated with viral pathogens. The survival of these pathogens within slurry is important since this material is often distributed onto farm land either directly or after heat treatment. There is clearly some risk of spreading pathogens in the early stages of an outbreak of disease before it has been recognized. The survival of foot-and-mouth disease virus, classical swine fever virus, bovine viral diarrhoea virus and swine influenza virus, which belong to three different RNA virus families plus porcine parvovirus (a DNA virus) was examined under controlled conditions. For each RNA virus, the virus survival in farm slurry under anaerobic conditions was short (generally ≤ 1 h) when heated (to 55°C) but each of these viruses could retain infectivity at cool temperatures (5°C) for many weeks. The porcine parvovirus survived considerably longer than each of the RNA viruses under all conditions tested. The implications for disease spread are discussed.     

Pathogenic and humoral immune responses to porcine reproductive and respiratory syndrome virus (PRRSV) are related to viral load in acute infection

Although much research has been performed on porcine reproductive and respiratory syndrome virus (PRRSV), little quantitative information is available on the relationships between virulence and in vivo virus replication, among isolates recovered at different times in the history of PRRS, or the relative levels of virulence associated with individual virus isolates. In this study, the in vivo growth properties of virulent field isolates and attenuated PRRSV isolates were compared. The results show that virulent PRRSV isolates exhibit longer and more elevated levels of viremia, induce faster and more intense humoral immune responses, negatively affect body weight gain, induce higher death rates, and cause more severe clinical signs in a respiratory disease model. We found that the more virulent field isolates grew to significantly higher levels in pigs than did cell-culture adapted isolates. We concluded that the pathogenic consequences and immunological responses of pigs to PRRSV are directly related to viral load in acute infection as reflected in viral titers in blood.     

Differential diagnosis of classical swine fever and border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine farm.]

During recent years neutralizing antibodies against Border Disease Virus (BDV) were found repeatedly in German pig herds. Consequently there was a demand for a differential diagnostic system. A permanent sheep cell line and BDV reference strain Moredun were chosen and were applied in a could be used case study. A pestivirus could be isolated from piglets on a mixed farm and was characterised as 'non-Classical Swine Fever' (CSF) by using monoclonal antibodies. Due to a CSF suspicion the pig herd was destroyed immediately. Serum samples of sheep from the same farm were used for further characterisation of the new virus isolate. A neutralization test of the sheep sera was performed against different pestiviruses and the new isolate. Neutralizing antibody titres against the new virus pig isolate were significantly higher than against all other pestiviruses. BDV strain Moredun recognised the antibodies clearly, whereas CSF viral strain Alfort 187 and several isolates of bovine viral diarrhoea virus (BVDV) strains scored the lowest cross reaction.     

Outbreaks in Quebec pig farms of respiratory and reproductive problems associated with encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) was isolated from tissues of aborted fetuses and weaned and suckling piglets from 4 different pig farms in Quebec. The farms were experiencing reproductive failure in sows of different parities concomitant to respiratory problems in suckling and postweaning piglets. At necropsy, gross lesions were confined to the lung and consisted of pulmonary congestion and edema of various degrees. Lesions of multifocal interstitial to proliferative pneumonia were found in the lungs of these piglets. Bacteriologic examination of various tissues from necropsied pigs yielded no pathogens in most cases. No significant antibody titers against 3 swine viruses (transmissible gastroenteritis virus, porcine parvovirus, and swine influenza virus) and two bovine viruses (bovine viral diarrhea and infectious bovine rhinotracheitis viruses) were detected in the sera of convalescent pigs. The Quebec EMCV isolates were antigenically related to the reference ATCC-VR129 strain of EMCV, as demonstrated by indirect immunofluorescence, serum neutralization (SN), and Western immunoblotting. However, one of the Quebec isolates could be distinguish by SN. EMCV-specific SN antibody titers up to 1:12,800 were detected in thoracic and ascitis fluids of aborted fetuses and in sera of convalescent pigs. A possible pneumotropic EMCV variant in swine may exist.     

Natural infections of pigs with bovine viral diarrhoea virus associated with signs resembling swine fever

Between 1976 and 1985 while using immunofluorescence in the laboratory diagnosis of swine fever (SF), 13 incidents of bovine viral diarrhoea virus (BVDV) infection were detected in the Netherlands. Ultimate differentiation between swine fever virus (SFV) and BVDV was based on herd evidence supported by comparative antibody studies on sera from pigs inoculated with the isolate and from contact pigs in the herd of origin, using reference strains of SFV and BVDV. Recently differentiation of SFV and BVDV has been facilitated by typing the isolates with monoclonal antibodies. Signs suspicious of SF were observed in pigs two to 16 weeks old and were always confined to animals of one litter. In most cases the affected litter died out gradually although once an animal recovered. Stillbirth and neonatal death as well as the late onset of disease and its limitation to a single litter in a herd suggested a congenital route of infection. Although transplacental infection of BVDV in pigs has been reported before, these cases are believed to be the first in which natural BVDV infections could be associated with clinical signs and pathological lesions indistinguishable from those observed in chronic SF.     

Virus neutralising antibodies against 22 bovine viral diarrhoea virus isolates in vaccinated calves.

Seven of nine colostrum deprived calves, free from bovine viral diarrhoea virus (BVDV), were vaccinated with a commercially available vaccine containing two inactivated strains of BVDV, an inactivated strain of bovine herpesvirus-1 and modified-live strains of bovine respiratory syncytial virus and para-influenza-3 virus. The two other calves were kept as controls. The virus neutralising (VN) antibodies induced by vaccination were tested against 22 antigenically diverse BVDV isolates, including reference strains and field isolates, both cytopathic and non-cytopathic, as well as genotypes I and II. The strains were isolated in Belgium, France, Germany, the United Kingdom and the USA. While there were variations in the VN titres of the individual calves against all the strains, serum from the seven animals neutralised 20 or more of the strains tested. From the results, it can be concluded that the vaccine can stimulate the production of VN antibodies capable of neutralising a wide range of European and American isolates of BVDV, including genotypes I and II.     

The development of an international reference panel of monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses.

A panel of 30 monoclonal antibodies was defined and characterized with respect to the binding capacity in immunoperoxidase assay to different strains of pestivirus. Using the panel it was possible to identify specifically all strains and isolates of hog cholera virus, hog cholera vaccines derived from 'C' strains, and most strains of bovine viral diarrhoea/border disease (BVD/BD) viruses (including those isolated from pigs). A small proportion of BVD/BD isolates from pigs and ruminants reacted only with the monoclonals specific for pestivirus group antigen. It is recommended that monoclonal typing methods be introduced into official procedures for the diagnosis of hog cholera/classical swine fever.     

Characterization of porcine and some ruminant pestiviruses by cross-neutralization

Serologic relationships between 11 pestivirus strains that originated from pigs and five that originated from cattle or sheep were studied by cross-neutralization. Experiments were performed with pig and sheep sera raised against the strains. The results were analysed by a computerized taxonomic procedure. The 16 viruses were classified into four distinct serologic groups. All hog cholera virus (HCV) strains were classified in one group; the other three groups consisted of strains that can infect pigs, but that are identified as bovine viral diarrhoea virus (BVDV) or border disease virus (BDV), or showed a closer relationship to BVDV and BDV than to HCV.     

Descriptive summary of an outbreak of porcine post-weaning multisystemic wasting syndrome (PMWS ) in New Zealand

CASE HISTORY: Investigations were conducted to determine the cause of an acute, multi-farm outbreak of porcine respiratory disease that included diarrhoea and subsequent loss of body condition in affected pigs. A definition for post-weaning multisystemic wasting syndrome (PMWS) including both clinical and pathological features, previously developed for the pig industry in New Zealand, was applied to the current outbreak. In addition to self-reporting by owners of affected farms, local veterinarians, disease and epidemiology consultants, and animal health officials from the Ministry of Agriculture and Forestry (MAF) were involved in conducting farm visits and submission of diagnostic specimens. CLINICAL FINDINGS AND DIAGNOSIS: Pathogens known to be endemic in the pig industry in New Zealand as well as likely exotic diseases were excluded as causative agents of the outbreak. Clinical signs including dyspnoea, diarrhoea, and rapid loss of body condition were consistent with the New Zealand case definition for PMWS. Interstitial pneumonia, pulmonary oedema, generalised lymph-node enlargement, and presence of porcine circovirus type 2 (PCV2) inclusion bodies were consistently identified in affected pigs. Classical swine fever virus (CSFv), Porcine reproductive and respiratory syndrome virus (PRRSv), and Influenza virus were ruled out, using molecular and traditional virological techniques. Spread of the disease between farms was hypothesised to be facilitated by locally migrating flocks of black-backed seagulls. The original source of the disease incursion was not identified. DIAGNOSIS: Based on the consistent presence of circovirus-associated lesions in lymphoid tissues in combination with generalised enlargement of lymph nodes, histiocytic interstitial pneumonia, clinical wasting, and poor response to antibiotic therapy, a diagnosis of PMWS was made. CLINICAL RELEVANCE: PMWS should be considered in the differential diagnoses of sudden onset of respiratory dyspnoea, diarrhoea, and rapid loss of body condition in young pigs in New Zealand pig herds.     

Antimicrobial susceptibility of pathogenic Escherichia coli isolated from sick cattle and pigs in Japan

We examined the 12 antimicrobial susceptibilities of 175 E. coli isolates from sick cattle and pigs by an agar dilution method. Resistance was found in 78.3% of isolates for oxytetracycline, 70.3% of isolates for dihydrostreptomycin, and 49.1% of isolates for ampicillin. When compared with healthy animals reported by Kijima-Tanaka et al., resistance rates for 11 antimicrobial agents were higher in sick cattle than in healthy cattle, and resistance rates for all antimicrobial agents were higher in sick pigs than in healthy pigs. Comparing cattle and pigs, resistance rates to colistin was higher in porcine isolates than in bovine isolates, but was lower in porcine isolates than in bovine isolates for cefazolin. With regard to the association of virulence factors, higher resistance rates to colistin and enrofloxacin were observed in STEC (61 strains) than in non-STEC (57 strains) among porcine isolates, while there were no significant differences in bovine isolates. In conclusion, these results can be considered helpful for adequate selection and prudent use of antimicrobial agents for several types of colibacillosis.     

Experimental inoculation of pregnant swine with type 1 bovine viral diarrhoea virus

The ability of bovine viral diarrhoea virus type 1 (BVDV-1) to induce transplacental infection in pigs was evaluated. Control pigs (n = 4) were sham-inoculated while infected pigs (n = 4) were intranasally inoculated with BVDV-1 on day 65 of gestation. Blood samples were tested throughout the study for BVDV and antibody to BVDV. On day 110 of gestation, a Caesarean section was performed. Serum was obtained for virus isolation and antibody determination from all piglets, and all experimental animals were killed. Tissues were collected for virus isolation and histopathology. Bovine viral diarrhoea virus was isolated on days 5 and 7 after infection and seroconversion was demonstrated in all infected gilts; however, BVDV was only isolated from one fetus from an infected pig. Viraemia and seroconversion were demonstrated in the pregnant gilts; however, transplacental infection at day 65 of gestation in the pig was not consistently demonstrated.     

Identification of hog cholera viral isolates by use of monoclonal antibodies to pestiviruses

A collection of 90 field isolates of hog cholera virus (HCV) was used to test the specificity of four hybridoma cell lines secreting monoclonal antibodies against pestiviruses. Reaction of virus isolates and monoclonal antibodies was controlled by an indirect immunofluorescence assay (IFA). Two monoclonal antibodies which had been generated against HC virus strain "Alfort 187" were reactive only with HCV field isolates and an HCV reference strain but not with bovine viral diarrhoea virus (BVDV) reference strains. Two other monoclonal antibodies (generated against BVDV, strain NADL) reacted only with BVDV reference strains but not with HCV field isolates, although with 3 of these strains focal reactions involving only a few cells were detected. The ability to discriminate between both viruses is a diagnostic need which may be fulfilled by these monoclonal antibodies.     

Infection with porcine respiratory coronavirus does not fully protect pigs against intestinal transmissible gastroenteritis virus

Eight nine-week-old specific-pathogen-free pigs which had been infected with the transmissible gastroenteritis virus (TGEV)-related porcine respiratory coronavirus (PRCV) and four uninfected littermates were challenged with TGEV. The previous PRCV infection failed to protect them against the enteric TGEV infection. Virus excretion in faeces was detected by an ELISA in all the pigs for three to six consecutive days after inoculation. Although little diarrhoea was observed, the infection extended through much of the small intestine of one of the previously infected pigs four days after inoculation. Challenge with TGEV caused a secondary neutralising antibody response. By using a peroxidase conjugate of a monoclonal antibody which recognises a specific antigenic site on TGEV, antibodies against TGEV could be distinguished from antibodies against PRCV in an ELISA blocking test.     

Bovine Viral Diarrhea Virus in Swine: Neutralizing Antibody in Naturally and Experimentally Infected Swine

Neutralizing antibodies against bovine viral diarrhea virus (BVDV) were detected in sera of Iowa farm sows, their piglets derived by hysterectomy and in specific-pathogen-free (SPF) pigs in a closed herd. No antibodies against BVDV were detected in sera of 134 fetal pigs from 67 sows in an Iowa slaughter-house. Neutralizing antibodies against BVDV or hog cholera virus, produced experimentally in SPF pigs, were demonstrated to be due to different antigens rather than to a common antigen, although slight cross-neutralization reactions sometimes occur.     

Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies

Nineteen monoclonal antibodies (MAbs) with specificity for hog cholera virus (HCV) were prepared. They were used in an immune binding (peroxidase linked) assay to determine the reaction patterns of HCV isolates from Europe, Brazil, USA, Japan and Malaysia, as well as laboratory reference strains of the virus. A further panel of 17 MAbs raised against bovine virus diarrhoea virus (BVDV) was included in the study, together with 5 MAbs raised against a non-HCV pestivirus of porcine origin. All the MAbs were also tested against representative strains of BVDV and border disease virus. Six MAbs were HCV-specific, reacting with all isolates of HCV and none of the ruminant viruses. Among the other HCV MAbs geographical variation in reaction patterns was observed. There was evidence of antigenic distinction between recent European isolates, and archive material originally isolated more than 10 years ago.