Electropherotypic analysis of rotaviruses isolated from turkeys

From 1980 to 1984, several flocks of turkeys in Minnesota exhibiting signs of clinical enteritis were examined for viruses. Electron microscopic (EM) examination of fecal specimens from 35 flocks revealed the presence of rotavirus particles. Rotaviruses were successfully isolated in cell cultures from only 24 of these positive fecal specimens. Double-stranded RNA (dsRNA) preparations made from these 24 cell-culture isolates and from the remaining 11 fecal samples that were rotavirus-positive on EM examination were analyzed by polyacrylamide gel electrophoresis for genetic differences in their genomes. The study revealed eight distinct electropherotypes among the rotavirus dsRNA preparations. Atypical dsRNA migration patterns were recognized only in preparations of dsRNA from fecal materials.     

Comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection

An indirect, double-antibody sandwich-type ELISA for detection of transmissible gastroenteritis virus (TGEV) was developed, using a solid phase of rabbit hyperimmune serum and a pool of 3 antipeplomer monoclonal antibodies to trap and to detect the virus, respectively. The technique was used to detect viral antigen in feces of pigs that had been infected with the virulent Miller strain, the attenuated Purdue strain, or the Erica strain (a Dutch field isolate) of TGEV. The results were compared with those of a solid-phase immunosorbent electron microscopy (SPIEM) technique for virus detection. Both techniques detected shedding of virulent virus in feces obtained from pigs on the first or second day after infection, and virus excretion continued for 6 to 8 consecutive days. Virus shedding started later in pigs infected with the attenuated Purdue strain of TGEV and lasted only 2 to 4 days. In comparison with the 2 virulent strains, infection with the attenuated strain appeared to be limited to a smaller portion of the small intestine. Of 242 fecal specimens that were tested by use of ELISA and SPIEM, 119 had positive results in both tests. Additionally, virus could be detected by ELISA in 21 and by SPIEM in 16 specimens. Fecal specimens obtained from pigs before infection always reacted negatively by ELISA for TGEV antigen; there was no cross-reactivity with fecal specimens containing porcine rotavirus or porcine epidemic diarrhea virus. The ELISA and SPIEM were found to be specific and sensitive for the detection of TGEV in feces.     

Detection of transmissible gastroenteritis virus in feces from pigs by reversed passive hemagglutination

A reversed passive hemagglutination (RPHA) method was developed for the detection of transmissible gastroenteritis (TGE) virus in the fecal specimens from pigs. Ovine erythrocytes fixed with glutaraldehyde and treated with tannic acid were coated with anti-TGE virus swine antibodies, which were purified by affinity chromatographic technique linked with purified TGE virus. The RPHA test was done by the Microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by TGE virus, but not by porcine rotavirus or porcine enterovirus. The reaction was specifically inhibited by antiserum against TGE virus, confirming the specificity of the reaction. A litter of seven 3-day-old pigs was orally inoculated with TGE virus, and fecal specimens were obtained once a day and serum was obtained every 4th day. With the RPHA test, TGE virus was detected in the diarrheal feces; all of the inoculated pigs developed virus-neutralization antibody for the TGE virus. The RPHA test detected TGE virus in feces from pigs with naturally occurring diarrhea. The RPHA test detected TGE virus in 5 of 6 fecal specimens (80%), whereas the positive rate was only 50% (3/6) for the immunofluorescent staining of primary cultures of porcine kidney cells inoculated with the specimens. The advantages of the RPHA method are simplicity, high sensitivity, and rapid to do.     

Isolation of rotavirus from calves, foals, dogs and cats in New Zealand

The incidence of rotaviruses in calves, foals, dogs and cats in the Dunedin urban and rural areas was investigated using electron microscopy and enzyme-linked immunosorbent assays. Of the 283 faecal specimens examined, 26% were positive for rotavirus. Comparison of the genetic electropherotypes was made by separating the viral dsRNA segments using polyacrylamide gel electrophoresis. It is possible that rotavirus infection is a zoonotic disease.     

The isolation of rotavirus from calves,foals, dogs and cats in New Zealand.

The incidence of rotaviruses in calves, foals, dogs and cats in the Dunedin urban and rural areas was investigated using electron microscopy and enzyme-linked immunosorbent assays. Of the 283 faecal specimens examined, 26% were positive for rotavirus.

Comparison of the genetic electropherotypes was made by separating the viral dsRNA segments using polyacrylamide gel electrophoresis. It is possible that rotavirus infection is a zoonotic disease.     

Differences in virulence between two strains of Streptococcus suis type II after experimentally induced infection of newborn germ-free pigs

Fifteen newborn germ-free pigs were inoculated with 2 strains, D-282 and T-15, of Streptococcus suis type II. Some pigs also were preinoculated with Bordetella bronchiseptica, which successfully predisposed them to S suis infection. The 2 streptococcal strains were differentiated by muramidase treatment, which released certain high molecular-weight proteins, termed muramidase-released proteins (MRP), from the cell wall of strain D-282, but not from the cell wall of strain T-15. Only strain D-282 (MRP-positive) induced clinical signs of disease and markedly increased neutrophil numbers in pigs. Streptococci were more frequently isolated from fecal swab specimens obtained from pigs inoculated with strain D-282 (MRP-positive) than from specimens obtained from pigs inoculated with strain T-15 (MRP-negative). Both strains were isolated from nasal swab specimens obtained from all infected pigs. Postmortem examination revealed fibrinopurulent meningitis, polyserositis, and polyarthritis in pigs inoculated with strain D-282; this strain was isolated from the CNS, serosae, visceral organs, heart, and joints. Whereas strains D-282 caused several pathologic changes, strain T-15, isolated from the lungs, caused only pneumonia. Both strains were isolated from the tonsils of all pigs. Virulence differed distinctly between the MRP-positive and the MRP-negative strains.     

Evaluation of killed and modified live porcine rotavirus vaccines in cesarean derived colostrum deprived pigs

Twenty-eight cesarean derived, colostrum deprived (CDCD) piglets were used to evaluate the efficacy of killed and modified live rotavirus (MLV) vaccines against challenge with virulent A-1 and A-2 rotaviruses. Two killed rotavirus vaccines were evaluated: an experimental vaccine and a commercially available vaccine. Efficacy parameters included: average daily weight gains, rotavirus shedding in feces, morbidity incidence and duration, and rotavirus serum antibody conversion post-vaccination and post-challenge. Piglets vaccinated orally/intramuscularly with the modified live vaccine were completely protected from A-1 and A-2 virulent rotavirus challenge. Nonvaccinated control piglets and piglets receiving killed rotavirus vaccines developed diarrhea, shed virus and exhibited reduced weight gains post-challenge. Only the MLV rotavirus vaccine was able to prevent virus shedding in feces after virulent challenge. Both controls and pigs which received killed vaccines intraperitoneally, orally or intramuscularly shed virus in the feces for 7 days post-challenge and virus peak titers approached 10(7) fluorescent antibody infectious dose (FAID)50/g feces. These studies clearly reflected the inability of killed rotavirus vaccines to induce active local immunity to rotaviral diarrhea in piglets.     

Experimental establishment of persistent infection in swine with a zoonotic strain of Salmonella newport

An experiment was conducted to determine whether a persistent Salmonella newport infection could be established in swine, to determine duration of shedding and distribution of the organism in internal organs, and to determine whether changes occurred in antimicrobial susceptibility or plasmid profile of the organism during the course of long-term infection. Naturally farrowed Salmonella-free pigs (n = 22) were orally exposed to a multiply antimicrobial-resistant zoonotic strain of S newport when they were 7 weeks old. Tonsillar and rectal swab specimens were examined bacteriologically for S newport during the first week after exposure, then weekly for 7 weeks. Fecal samples were likewise examined weekly or every 2 weeks for 28 weeks after exposure. Necropsies of 2 or 3 randomly selected pigs were conducted at 2, 4, 8, 12, 16, 20, 24, and 28 weeks after exposure. A total of 45 specimens/pig representing the following internal organs or tissues were examined bacteriologically for S newport: liver, spleen, kidney, gallbladder, heart, heart blood, lung, stomach, and tonsils; segments of the intestinal tract with corresponding lymph nodes; and lymph nodes from lymphocenters of the head and neck, thoracic cavity, thoracic limbs, abdominal viscera, and abdominal wall. Exposure to S newport induced a mild and transient clinical response. The organism was recovered from 97% of tonsillar swab specimens and 89% of rectal swab specimens collected during 7 weeks after exposure and from 98% of fecal samples collected during 28 weeks after exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune electron microscopy of transmissible gastroenteritis virus and rotavirus (reovirus-like agent) of swine.

Immune electron microscopy (IEM) was developed as a diagnostic aid for detecting and identifying transmissible gastroenteritis virus and rotavirus (reovirus-like agent) in fecal and intestinal contents from cases of gastroenteritis in young pigs. Variables involved in use of direct IEM and its sensitivity were determined. Aggregates of virus coated with specific antibody were seen in virus samples mixed with homologous convalescent antiserum, but not in control samples containing preexposure serum or antibody directed against a heterologous virus. At least a ten fold enhancement of the sensitivity of direct IEM for virus detection was accomplished using indirect IEM employing rabbit anti-porcine IgG to further aggregate virus-antibody complexes. The technique was used to investigate the size and morphology of the porcine rotavirus. Particles ranged from 55 to 70 nm in diameter and had capsomere structures. Morphologically, the porcine rotavirus resembled the calf and human rotaviruses. By IEM, employing specific antiserums for each virus, porcine rotavirus was found to be antigenically related to these 2 viruses, but not to the reovirus type 3.     

The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination

This study investigated the efficacy of a bivalent swine influenza virus (SIV) vaccine in piglets challenged with a heterologous H1N1 SIV isolate. The ability of maternally derived antibodies (MDA) to provide protection against a heterologous challenge and the impact MDA have on vaccine efficacy were also evaluated. Forty-eight MDA(+) pigs and 48 MDA(-) pigs were assigned to 8 different groups. Vaccinated pigs received two doses of a bivalent SIV vaccine at 3 and 5 weeks of age. The infected pigs were challenged at 7 weeks of age with an H1N1 SIV strain heterologous to the H1N1 vaccine strain. Clinical signs, rectal temperature, macroscopic and microscopic lesions, virus excretion, serum and local antibody responses, and influenza-specific T-cell responses were measured. The bivalent SIV vaccine induced a high serum hemagglutination-inhibition (HI) antibody titer against the vaccine virus, but antibodies cross-reacted at a lower level to the challenge virus. This study determined that low serum HI antibodies to a challenge virus induced by vaccination with a heterologous virus provided protection demonstrated by clinical protection and reduced pneumonia and viral excretion. The vaccine was able to prime the local SIV-specific antibody response in the lower respiratory tract as well as inducing a systemic SIV-specific memory T-cell response. MDA alone were capable of suppressing fever subsequent to infection, but other parameters showed reduced protection against infection compared to vaccination. The presence of MDA at vaccination negatively impacted vaccine efficacy as fever and clinical signs were prolonged, and unexpectedly, SIV-induced pneumonia was increased compared to pigs vaccinated in the absence of MDA. MDA also suppressed the serum antibody response and the induction of SIV-specific memory T-cells following vaccination. The results of this study question the effectiveness of the current practice of generating increased MDA levels through sow vaccination in protecting piglets against disease.     

Resistance to fecal shedding of salmonellae in pigs and chickens vaccinated with an aromatic-dependent mutant of Salmonella typhimurium.

The purpose of this study was to evaluate the effectiveness of an aromatic-dependent mutant of Salmonella typhimurium as a parenteral vaccine for prevention of fecal shedding of Salmonella spp. Pigs and chickens were vaccinated IM, with 1 x 10(9) and 1 x 10(8) organisms, respectively, followed by a second identical vaccination 2 weeks later. Salmonella organisms were not detected by analysis of fecal or cloacal swab specimens from any animal after vaccination. Deleterious side effects were not noticed after vaccination. Pigs were challenge-inoculated PO with 1 x 10(12) virulent S typhimurium 1 week after the second vaccination. Chickens were challenge-inoculated PO with 3 x 10(8) organisms of either S enteritidis or the virulent parent strain of S typhimurium 3 weeks after the second vaccination. Vaccinated pigs shed Salmonella spp significantly less frequently than did nonvaccinated pigs. Vaccinated chickens challenge-inoculated with either S enteritidis or S typhimurium also shed Salmonella less frequently than the corresponding nonvaccinated control birds; however, the difference was not significant.     

Bovine viral diarrhoea virus infections in piglets born to sows vaccinated against swine fever with contaminated vaccine

On eight farms a congenital pestivirus infection in piglets was detected which could be traced to vaccination of the dams against swine fever (SF). Viruses isolated from the piglets were not recognised by monoclonal antibodies (MCAs) against swine fever virus (SFV) and were shown to be bovine viral diarrhoea virus (BVDV) or Border disease virus. The expected 'Chinese' strain of SFV could not be demonstrated in the batch of vaccine that had been used on these farms. Instead, a contaminating pestivirus was recovered which was not recognised by the MCAs against SFV. The contaminant had an unexpectedly high virulence for lambs and induced antibodies against BVDV in lambs and pigs. It could, therefore, be characterised as BVDV or Border disease virus.     

Vaccine genotype and route of administration affect pseudorabies field virus latency load after challenge

The influence of vaccine genotype and route of administration on the efficacy of pseudorabies virus (PRV) vaccines against virulent PRV challenge was evaluated in a controlled experiment using five genotypically distinct modified live vaccines (MLVs) for PRV. Several of these MLVs share deletions in specific genes, however, each has its deletion in a different locus within that gene. Pigs were vaccinated with each vaccine, either via the intramuscular or intranasal route, and subsequently challenged with a highly virulent PRV field strain. During a 2-week period following challenge with virulent PRV, each of the vaccine strains used in this study was evaluated for its effectiveness in the reduction of clinical signs, prevention of growth retardation and virulent virus shedding. One month after challenge, tissues were collected and analyzed for virulent PRV latency load by a recently developed method for the electrochemiluminescent quantitation of latent herpesvirus DNA in animal tissues after PCR amplification. It was determined that all vaccination protocols provided protection against clinical signs resulting from field virus challenge and reduced both field virus shedding and latency load after field virus challenge. Our results indicated that vaccine efficacy was significantly influenced by the modified live vaccine strain and route of administration. Compared to unvaccinated pigs, vaccination reduced field virus latency load in trigeminal ganglia, but significant differences were found between vaccines and routes of administration. We conclude that vaccine genotype plays a role in the effectiveness of PRV MLVs.     

Identification and clinical assessment of suspected vaccine-related field strains of porcine reproductive and respiratory syndrome virus

OBJECTIVE: To determine the origin and clinical relevance of selected strains of porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV). ANIMALS: 38 pigs without antibodies for PRRSV. PROCEDURE: A seemingly uncommon restriction endonuclease digestion site in a commercially available vaccine strain of attenuated PRRSV was tested for its stability and prevalence under defined conditions. Selected field strains of PRRSV, with or without the restriction-site marker, were subsequently tested in pigs for virulence and for their ability to replicate competitively in pigs simultaneously given the vaccine. RESULTS: Under experimental conditions, the restriction-site marker was stable during long-term infection of pigs. It was not detected in any of the 25 field strains of PRRSV that were isolated before use of the vaccine or 21 of 25 field strains that were isolated after use of the vaccine but that, on the basis of previous testing, were believed unrelated to the vaccine strain. Conversely, it was detected in 24 of 25 field strains that were isolated after use of the vaccine and that, on the basis of previous testing, were believed to be direct-line descendants of the vaccine strain. Putative vaccine-related strains caused more pronounced pathologic changes than did the vaccine strain alone, and they predominated during replication in pigs also given the vaccine strain. CONCLUSIONS: In some swine herds, the vaccine strain may have persisted and mutated to a less attenuated form. CLINICAL RELEVANCE: The potential for persistence and mutation of specific strains of virus should be an important consideration when designing vaccination programs involving attenuated PRRSV.     

Comparative virulence of two porcine group-A rotavirus isolates in gnotobiotic pigs

The virulence of 2 porcine group-A rotavirus isolates was compared. Forty hysterotomy-derived 3-day-old gnotobiotic pigs were inoculated orally with 2 ml of intestinal homogenate containing either the Ohio State University (OSU) or the South Dakota State University (SDSU) strain of porcine rotavirus or were inoculated with medium only. Clinical signs of disease, body weight, distribution of viral antigen, fecal excretion of virus, and histologic lesions (observed by light and scanning electron microscopy) were determined. Morphometric measurements of villi and crypts were made. In pigs inoculated with OSU or SDSU strains, diarrhea began at postinoculation hours (PIH) 19 to 48 and PIH 24 to 54, respectively. None of the virus-infected pigs died as a consequence of infection and all had similar clinical signs of disease, body weight changes, and virus-shedding patterns, regardless of the strain of rotavirus with which they were infected. Microscopic findings in the small intestine of virus-infected pigs were similar, except that the SDSU strain caused more severe villus atrophy and villus fusion in the duodenum at PIH 72 and 168 than was associated with the OSU strain. Viral antigen in the small intestine of pigs infected with either virus was observed by use of immunofluorescence at PIH 24 and 72, but was seldom seen at PIH 168.     

Assessment of various treatments to reduce carriage of Salmonella in swine.

In this study, different strategies to reduce carriage of Salmonella spp. in pigs were evaluated. Probiotics, prebiotics, vaccination, and acidification of drinking water were assessed as means of reducing Salmonella. Acidification of water, use of egg yolk-specific immunoglobulins, and vaccination with an endotoxin vaccine did not reduce Salmonella excretion in experimentally infected pigs. A reduction of Salmonella in the colonization of mesenteric lymph nodes was observed with the use of bambermycins and a live attenuated vaccine. A reduction in the shedding of S. Typhimurium was also observed after supplementation with fructooligosaccharides in drinking water. The use of probiotics and prebiotics appeared to change the pig fecal bacterial flora as indicated by Gram staining of smears from rectal swabs.     

Effects of a single intranasal dose of modified-live bovine respiratory syncytial virus vaccine on resistance to subsequent viral challenge in calves

OBJECTIVE: To determine whether a single intranasal dose of modified-live bovine respiratory syncytial virus (BRSV) vaccine protects calves from BRSV challenge and characterize cell-mediated immune response in calves following BRSV challenge. ANIMALS: 13 conventionally reared 4- to 6-week-old Holstein calves. PROCEDURES: Calves received intranasal vaccination with modified live BRSV vaccine (VC-group calves; n = 4) or mock vaccine (MC-group calves; 6) 1 month before BRSV challenge; unvaccinated control-group calves (n = 3) underwent mock challenge. Serum virus neutralizing (VN) antibodies were measured on days -30, -14, 0, and 7 relative to BRSV challenge nasal swab specimens were collected for virus isolation on days 0 to 7. At necropsy examination on day 7, tissue specimens were collected for measurement of BRSV-specific interferon gamma (IFN-gamma) production. Tissue distribution of CD3+ T and BLA.36+ B cells was evaluated by use of immunohistochemistry. RESULTS: The MC-group calves had significantly higher rectal temperatures, respiratory rates, and clinical scores on days 5 to 7 after BRSV challenge than VC-group calves. No difference was seen between distributions of BRSV in lung tissue of VC- and MC-group calves. Production of BRSV-specific IFN-gamma was increased in tissue specimens from VC-group calves, compared with MC- and control-group calves. Virus-specific IFN-gamma production was highest in the mediastinal lymph node of VC-group calves. Increased numbers of T cells were found in expanded bronchial-associated lymphoid tissue and airway epithelium of VC-group calves. CONCLUSIONS AND CLINICAL RELEVANCE: An intranasal dose of modified-live BRSV vaccine can protect calves against virulent BRSV challenge 1 month later.     

Experimental infection of fattening pigs with pseudorabies (Aujeszky's disease) virus: efficacy of attenuated live- and inactivated-virus vaccines in pigs with or without passive immunity

The efficacies of attenuated live- and inactivated-virus vaccines against pseudorabies (PR) in fattening pigs were compared. Pigs born from vaccinated or nonvaccinated sows were vaccinated with one or the other vaccine and were challenge exposed at the end of the fattening period. The particular form of PR observed in fattening units in the field could be reproduced. A marked difference was seen between the control lot and the lots of the pigs vaccinated with the attenuated live- and inactivated-virus vaccines. The protection was real, but not absolute, in the vaccinated pigs. The inactivated-virus vaccine conferred a strong passive immunity to the young pigs of vaccinated dams which interfered with the development of an active immunity. The titer of the colostral antibodies in the sera of pigs born from the sows vaccinated with the attenuated-live virus vaccine was low and decreased rapidly. The active protection obtained with this vaccine was similar to that observed with the inactivated-virus vaccine. Thermal curves, weight losses, and time necessary for recovering the weight of pigs at the time of challenge exposure seemed to be good criteria for measuring the protection of fattening pigs against this particular form of PR. The conditions of the outcome of respiratory tract disorders in these pigs are discussed.     

Conjunctivitis associated with a Mycoplasma-like organism in swine

A midwestern producer reported high incidence of conjunctivitis and keratoconjunctivitis in a herd of crossbred finishing swine. Complete necropsy was performed on 3 pigs with bilateral mucopurulent conjunctivitis and chemosis; other gross lesions were not seen. Mycoplasma sp was isolated from conjunctival swab specimens obtained from 1 pig; small numbers of streptococci and coagulase-negative staphylococci were isolated from conjunctival swab specimens from all 3 pigs. Neither swine influenza virus nor pseudorabies virus was isolated from conjunctival swab specimens. Histologically, the 3 pigs had chronic lymphoplasmacytic conjunctivitis with lymphofollicular hyperplasia and foci of epithelial and goblet cell hyperplasia. Ultrastructural examination of conjunctival specimens from the 3 pigs revealed large numbers of Mycoplasma-like organisms adhered to superficial conjunctival cells. Mycoplasma-like organisms also were seen in membrane-bound vacuoles in superficial conjunctival cells. Bacteria (including chlamydiae) or viruses were not seen ultrastructurally. The lymphoproliferative nature of the conjunctival lesion and the evidence of adhered and intracellular organisms suggested an etiologic role for a Mycoplasma-like organism in the disease in these pigs.     

Evaluation of different adjuvants for foot-and-mouth disease vaccine containing all the SAT serotypes

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals that is primarily controlled by vaccination of susceptible animals and movement restrictions for animals and animal-derived products in South Africa. Vaccination using aluminium hydroxide gel-saponin (AS) adjuvanted vaccines containing the South African Territories (SAT) serotypes has been shown to be effective both in ensuring that disease does not spread from the endemic to the free zone and in controlling outbreaks in the free zone. Various vaccine formulations containing antigens derived from the SAT serotypes were tested in cattle that were challenged 1 year later. Both the AS and ISA 206B vaccines adjuvanted with saponin protected cattle against virulent virus challenge. The oil-based ISA 206B-adjuvanted vaccine with and without stimulators was evaluated in a field trial and both elicited antibody responses that lasted for 1 year. Furthermore, the ISA 206 adjuvanted FMD vaccine protected groups of cattle against homologous virus challenge at very low payloads, while pigs vaccinated with an emergency ISA 206B-based FMD vaccine containing the SAT 1 vaccine strains were protected against the heterologous SAT 1 outbreak strain.