A blocking ELISA for detection of antibody to a subgroup-reactive epitope of African horsesickness viral protein 7 (VP7) using a novel gamma-irradiated antigen.

A novel gamma irradiated inactivated cell culture derived African horsesickness viral (AHSV) antigen was used in a blocking ELISA (B-ELISA) for detecting antibody to a subgroup-reactive epitope of AHSV. A monoclonal antibody (MAB), class IgM, against an epitope on African horsesickness (AHS) viral protein 7 (VP7) was developed in BALBc mice and used in the B-ELISA. The MAB, designated F9H, was blocked by 69 serums from equidae with antibody to AHS, but its binding activity was not appreciably affected by 301 serums that did not contain antibodies to AHS virus. An ELISA protocol using a blocking format is described.

Future international management of African horse sickness vaccines.

Three types of African horse sickness (AHS) vaccine, namely adult mouse brain, modified live vaccine and inactivated viral vaccine (IVV) are reviewed. The results of efficacy trials carried out with each vaccine type highlight the advantages of the IVV. Vaccination with African horse sickness virus serotype 4 IVV, given as 2 separate doses, provided full protection against subsequent, homologous challenge. The absence of any detectable viraemia after challenge would also prevent infection of insect vectors. The advantages of establishing international vaccine banks for AHS are discussed.

Rapid and sensitive detection of foot-and-mouth disease virus in tissues by enzymatic RNA amplification of the polymerase gene.

Direct detection of foot-and-mouth disease (FMD) virus from infected bovine and porcine tissue was investigated using a modified polymerase chain reaction (PCR) technique. A high degree of conservation was found in the genomic region coding for the viral RNA polymerase among the seven FMD viral (FMDV) serotypes. An oligomeric primer pair and probe were constructed from consensus sequence data within this area. First strand cDNA was synthesized using random hexamers and Moloney MuLV reverse transcriptase. The oligomeric primers used for PCR of the random primed cDNA yielded a 454-base-pair target amplification product. The PCR product was sized by agarose gel electrophoresis and hybridized strongly with the consensus sequence oligomeric probe. The PCR product was further examined by digestion with NcoI, confirming the predicted internal restriction enzyme site. All seven serotypes of FMDV RNA were amplified in a few hours and the PCR product tested positive. The sensitivity of the enzymatic amplification for detection of FMDV was 10 TCID50 by gel electrophoresis and less than 1 TCID50 when combined with hybridization to a labeled probe. The technique was specific, as determined by examination of at least 12 other viruses, including enteroviruses and other agents of vesicular disease. In vitro enzymatic amplification of cDNA from FMDV RNA using the modified PCR technique is highly specific, rapid and at least as sensitive as presently used procedures for FMDV laboratory diagnosis.

Evaluation of techniques to demonstrate foot-and-mouth disease virus in bovine tongue epithelium: comparison of the sensitivity of cattle, mice, primary cell cultures, cryopreserved cell cultures and established cell lines.

Tongue epithelia infected with each of the 7 serotypes of foot-and-mouth disease virus (FMDV) were used to evaluate in vivo and in vitro systems for the detection of FMDV. Cattle inoculated by the intradermal route in the tongue (IDL) and suckling mice inoculated intraperitoneally were compared for susceptibility to FMDV with freshly prepared bovine thyroid cell cultures; cultures from cryopreserved bovine thyroid, bone marrow, mammary gland, myocardium, tongue, ovary and kidney cells; cultures from cryopreserved embryonic ovine kidney, newborn ovine kidney, ovine testicle, bone marrow, and chloroid plexus cells; and the continuous porcine kidney cell lines MVPK-1 and S6. The mean titers determined for each serotype in each system were statistically compared. The FMDV titers obtained in freshly prepared bovine thyroid cell cultures and by cattle IDL inoculation were the highest and were statistically indistinguishable. The titers obtained by suckling mouse inoculation were significantly lower than the titers obtained in thyroid cultures for serotypes A, C, Asia 1, and SAT 3. The cattle IDL assay was significantly more sensitive than the mouse assay for serotype A. The cell cultures from the cryopreserved newborn ovine kidney and embryonic ovine kidney were significantly less susceptible to serotype Asia 1 when compared with the fresh bovine thyroid cultures, but not significantly different when compared with the cattle assay for all serotypes. Cryopreservation of bovine thyroid cells directly after trypsinization resulted in the loss of susceptibility to FMDV serotype SAT 2. The other cryopreserved cell culture systems exhibited no or minimal susceptibility to all 7 serotypes, or exhibited considerable inconsistency. The established cell lines MVPK-1 and S6 were not susceptible to serotype A, and were less sensitive to serotype C than other culture systems. Quality control of cell cultures used to evaluate field specimens for FMDV was critical. The cell cultures of cryopreserved ovine kidney cells provided the most practical diagnostic system.

Comparison of the effect of various chemical stabilizers and lyophilization cycles on the thermostability of a Vero cell-adapted rinderpest vaccine.

The thermostability of a rinderpest vaccine produced on Vero cells was evaluated using a variety of chemical stabilizers and lyophilization protocols. Three stabilizer preparations and three lyophilization schedules were examined using accelerated stability testing at 37 degrees C. The vaccine preparation exhibiting the greatest stability at 37 degrees C was tested at three additional temperatures, 42, 45 and 56 degrees C, and an Arrhenius plot was constructed from the data. The stability of the reconstituted vaccine produced with the two most efficacious stabilizers was examined using three different diluent preparations. The stabilization method and high Vero cell virus batch titers resulted in a lyophilized vaccine which maintained the minimum required dose of log10 2.5 TCID50 tissue culture infectious dose for more than 20 weeks at 37 degrees C.

The serological response to a thermostable Vero cell-adapted rinderpest vaccine under field conditions in Niger.

A lyophilized thermostable Vero cell-adapted ringerpest vaccine, stabilized with lactalbumin hydrolysate and sucrose, was tested for safety, serological response and suitability for use with an abbreviated cold chain under field conditions in Niger. A total of 480 cattle, 90 goats and 55 sheep of unknown serological status were vaccinated on government ranches and observed for at least 22 days. No untoward effects of the vaccine were detected. The serological response to the vaccine stored at environmental temperatures for 30 to 34 days was determined in 144 previously unvaccinated yearling calves. Seroconversion was demonstrated in 98% of the yearling calves using seroneutralization. The un-refrigerated vaccine retained a titer of 3.69 log10 TCID50 per dose through day 34.

Experimental equine herpesvirus-1 infection in llamas (Lama glama).

Three llamas (Lama glama) were experimentally infected intranasally with an isolate of equine herpesvirus-1 (EHV-1) from the brain of an alpaca that had experienced severe neurologic signs. Two of the 3 llamas developed severe neurologic disorders following inoculation; 1 died, and 1 was euthanized in a moribund state. The third llama showed only mild neurologic signs. The euthanized llama had preexisting antibodies to EHV-1, and the remaining 2 llamas were seronegative (virus neutralization titer less than 6) at the time of inoculation. One of the seronegative llamas died acutely without production of detectable antibodies, and the other developed antibodies typical of a primary immune response. The EHV-1 virus was recovered only from a sample of the thalamus of the llama that died acutely. Histopathologic lesions were observed in the brain and retina of the dead and euthanized animals. This study verifies the pathogenic potential of EHV-1 for llamas.

Analysis of the serologic relationship among San Miguel sea lion virus and vesicular exanthema of swine virus isolates. Application of the western blot assay for detection of antibodies in swine sera to these virus types.

Caliciviruses are positive-sense single-stranded RNA viruses with a single capsid protein. The serotypes of the marine mammal calicivirus, San Miguel sea lion virus (SMSV), are antigenically related to vesicular exanthema of swine virus (VESV) and are potentially hazardous to swine. Western blot assays using purified SMSV serotypes 1 and 4 were used to further examine the serologic relationship among SMSV and VESV isolates. With the exception of SMSV 8 and SMSV 12, rabbit polyclonal antisera generated against all the available SMSV and VESV isolates reacted positively, as assessed by western blot, with purified capsid protein from SMSV 1 and SMSV 4. Consequently, the SMSV 8 and SMSV 12 virus isolates may not be members of the SMSV/VESV calicivirus group. Using antisera from pigs experimentally inoculated with SMSV and VESV as positive controls, a western blot assay for these virus types was utilized to check for the presence of antibodies to calciviruses in swine sera. Sera from colostrum-deprived gnotobiotic pigs were used as a negative control in all experiments. Examination of sera from domestic and feral swine collected in Iowa, California, and Florida was completed using this technique. The presence of antibodies to these virus types was not detected in any of the porcine sera tested.

The isolation of lumpy skin disease virus and bovine herpesvirus-4 from cattle in Egypt.

Lumpy skin disease (LSD) virus (LSDV) was isolated for the first time from cattle in Egypt in 2 disease outbreaks. Bovine herpesvirus-4 (BHV-4) and LSDV were detected in a pooled sample from the first outbreak (Suez). Only LSDV was isolated from the second outbreak (Ismalia). The capripoxviruses were identified as LSDV by neutralization with specific antiserum and by their ability to produce generalized LSD in experimentally inoculated cattle.

The use of thermostable Vero cell-adapted rinderpest vaccine as a heterologous vaccine against peste des petits ruminants.

The thermostable Vero cell-adapted rinderpest vaccine was evaluated in terms of immunogenicity as a heterologous vaccine against peste des petits ruminants. A titration to establish the minimum immunising dose was performed in American mixed breed goats by vaccinating test subjects with dilutions of Vero cell-adapted rinderpest vaccine and then challenging 26 days later with virulent peste des petits ruminants virus. All animals were followed for virus neutralising antibodies against both rinderpest and peste des petits ruminants virus after vaccination and challenge. The antibody response to vaccination was primarily against rinderpest virus with very low levels of cross-reactivity to peste des petits ruminants virus. Following challenge, animals which possessed anti-rinderpest neutralising antibodies remained clinically normal but mounted strong anti-peste des petits ruminants virus neutralising antibody responses indicating that replication of challenge virus took place without the induction of illness. The 50 per cent minimum goat immunising dose was 3 tissue culture infectious doses 50 per cent (TCID50) as established by serological response and protection against challenge. The thermostable Vero cell-adapted rinderpest vaccine is a suitable immunogen for the protection of goats against peste des petits ruminants.

A canine parainfluenza viral vaccine: immunogenicity and safety.

A canine parainfluenza viral vaccine was developed and shown to be safe by absence of clinical disease in vaccinated dogs and by inability to isolate vaccine virus from blood or nasopharyngeal swabs. Backpassage in susceptible dogs, using blood of vaccinated dogs, could not be demonstrated. The vaccine produced neutralizing antibody when administered either intramuscularly or subcutaneously; however, a significantly higher immune response was obtained by intramuscular inoculation. Differences in the antibody response were not produced by tenfold dilutions of vaccine virus ranging from 10(2.9) to 10(5.9) median tissue culture infective doses. The presence of neutralizing antibody was associated significantly with decreased respiratory shedding period of challenge virus by vaccinated dogs compared to seronegative control dogs. Six days after aerosol exposure to virulent challenge virus, 100% of the controls (n = 5) but only 15% of the vaccinated dogs (n = 3) shed virus. Seven days after challenge exposure, virus could not be recovered from the vaccinated dogs, but 80% of the control dogs shed virus. An anamnestic response occurred in vaccinated dogs but not in the seronegative control dogs following challenge exposure. A mild clinical disease was produced in 3 of the 5 seronegative control dogs but not in the 20 vaccinated dogs.

Isolation of bluetongue virus serotype 2 from cattle in Florida: serotype of bluetongue virus hitherto unrecognized in the Western Hemisphere.

A serotype of bluetongue virus (BTV) hitherto unrecognized in the Western Hemisphere was isolated from cattle in the United States. Clinical disease was not seen in the cattle which were part of a sentinel herd system in Florida designed for studying the epizootiology of BTV. The isolation of serotype 2 was the first recovery of a different serotype of BTV in the United States since 1967. At least 21 serotypes of BTV have been reported worldwide; the 5 serotypes of BTV now recognized in the United States are 2, 10, 11, 13, and 17.

Neutralizing antibodies to phocine distemper virus in Atlantic walruses (Odobenus rosmarus rosmarus) from Arctic Canada.

The first evidence of phocine distemper virus (PDV) infection in Atlantic walruses (Odobenus rosmarus rosmarus) from Nottingham Island, Northwest Territories, Canada, is reported. Blood samples were collected from three male walruses killed by Inuit hunters in the fall of 1990. Differential virus neutralization test for each animal yielded higher titers against PDV than against other members of the Morbillivirus genus including canine distemper, peste des petits ruminants, rinderpest and measles viruses. Thus, PDV infection may be enzootic in walruses of the eastern Canadian Arctic.

Viral serologic survey of bowhead whales in Alaska.

Serum samples from 21 of 36 Eskimo harvested bowhead whales (Balaena mysticetus) were positive by virus neutralization (50% endpoint titer > or = 1:28 and/or 100% endpoint titer > or = 1:20) for antibodies to at least one virus serotype from the calicivirus family, vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV). Many animals were positive to more than one serotype when using the Spearman-Karber (S-K) method for calculating antibody titers. The most common serotype detected was VESV F55 with 6 of 36 (17%) by the Monto and Bryan (MB) titer calculation method, and 17 of 36 (47%) by the S-K titer calculation method. Vesicular exanthema of swine virus 1934B antibody was detected in 3 of 36 (8%) and 5 of 36 (14%) whales using the MB and S-K methods, respectively. Vesicular exanthema of swine virus J56 antibody was detected in 3 of 36 (8%) by the S-K method only. All whales < 8.5 m (estimated yearlings, n = 6) were seronegative for VESV J56 and 1934B while 10% and 17% of the whales > 8.5 m were positive, respectively. Whales assumed to be sexually mature (> 13 m) had a higher prevalence of antibody to VESV 1934B and SMSV 8 than those < 13 m. Gender had an effect on seroprevalence of antibody to VESV 1934B as titers > or = 1:28 (S-K method) occurred in 18% of the females and 7% of the males. Antibody to other serotypes (SMSV 8 and 12) occurred less frequently (< 6%) at an antibody titer > or = 1:28 by the S-K method. All 36 whale sera were negative for antibody to VESV-A48, B51, C52, D53, E54, G55, H54, I55, and K54; Tillamook calicivirus, and dolphin morbillivirus; and SMSV-1, 2, 4, 5, 6, 7, 9, 10, 11, and 13 by the S-K method.

Epizootic vesicular disease in captive California sea lions.

An epizootic of vesicular disease occurred in a group of semi-domesticated California sea lions (Zalophus californianus) during the months of April and May 1997. Ten castrated mature male sea lions, ages 12 to 19 yr, were housed in three adjacent open-ocean net enclosures in San Diego Bay (California, USA). Four animals (40%) developed oral and extremity vesicles, anorexia, and were reluctant to perform learned behaviors. One animal developed vesicles but maintained a normal appetite and behavior. The remaining animals showed no clinical signs of infection. Virus (designated FADDL 7005) was isolated from four of the five animals that developed vesicles. Serum antibody titers to FADDL 7005, a previously untyped calicivirus, were demonstrated in animals that showed any combination of clinical signs and in two animals that did not show any clinical signs. No virus was isolated from five fecal samples collected from four of the group animals. Clinical signs lasted 4 to 20 days in affected animals. All affected animals recovered from infection. An experimental swine was inoculated with FADDL 7005 and developed vesicular disease, which was transmitted to another experimental swine upon contact. It is proposed that FADDL 7005 is a new San Miguel sea lion virus.

Epizootiology of morbillivirus infection in North American harbor seals (Phoca vitulina) and gray seals (Halichoerus grypus).

A longitudinal study of morbillivirus infection among harbor (Phoca vitulina) and gray (Halichoerus grypus) seals on the Atlantic coast of North America was carried out between 1980 and 1994. Serology also was carried out on harbor seals from the Pacific northwest coast collected in 1992 and 1993. The prevalence of morbillivirus neutralizing antibodies was significantly (P < 0.0001) higher in gray (73%, n = 296) than in harbor seals (37%, n = 387) from the Atlantic. Titers were significantly (P < 0.0001) higher against phocine distemper (PDV) compared to any other morbillivirus. Antibodies were not detected in serum from Pacific harbor seals. During the winter of 1991 to 1992 an epizootic occurred among harbor seals on the northeast coast of the United States. The event was characterized by an increase in strandings and by a significant (P = 0.001) increase in PDV antibody prevalence to 83% (n = 36) in seals stranded that winter. Morbillivirus lesions and antigen were observed in six animals found stranded from southern Maine to Long Island, New York (USA), between November 1991 and April 1992. In addition, morbillivirus encephalitis was detected in tissues from a harbor seal that stranded in 1988. Enzootic infection appeared to be present in both seal species, although with a different prevalence of disease. We propose that enzootic infection among gray seals is facilitated by population size, high annual recruitment and innate resistance to clinical disease. Infection may be maintained in the smaller harbor seal population through casual contact with gray seals.

Economic impact of rotavirus and other neonatal disease agents of animals.

Methods for estimating the economic impact of disease agents were developed and utilized to assess the relative economic importance of rotavirus and other disease agents in calves. Based on incidence data from 2 sources, Escherichia coli was responsible for the most devastating economic losses (50.9% and 74.6%). Coronaviral (17.5% and 29.7% loss) and rotaviral (3.2% and 9.1% loss) infections ranked 2nd and 3rd, respectively. In one study, cryptosporidial infections (6.5% loss) were estimated to be similar in economic impact to rotaviral infection. Salmonellosis, mycotic gastroenteritis, infectious bovine rhinotracheitis, and bovine viral diarrhea infections accounted for minor losses. The estimated average annual loss of calves for the 7-year period, 1970 through 1976, was $95,500,000/year. Based on data from 2 studies, the estimated average annual loss from E coli was $48.6 and 71.2 million; from coronaviral infection, $16.7 and 28.4 million; from rotaviral infection, $3.1 and $8.7 million; and from cryptosporidial infection, from 1 study, $6.2 million. Estimates of economic impact of disease agents on calves, and likely in other species, indicate that rotaviral infections have a relatively minor role with respect to E coli and coronaviral infections.