James A. House

Differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide ELISA

We have identified continuous antigenic determinants within the amino acid sequences of the conserved nonstructural region containing proteins 2C and 3ABC of foot-and-mouth disease virus which can distinguish between the sera from vaccinated and infected animals. An ELISA based on a 3B peptide gave a positive reaction with sera from cattle, pigs, sheep and guinea pigs infected with all seven serotypes of the virus, but not with sera from vaccinated animals. In experiments with cattle and pigs to determine the duration of the antibody response, positive reactions were obtained as late as one year after infection. The advantages of using peptides from the nonstructural viral proteins instead of recombinant proteins for differentiating vaccinees from infected animals include their exquisite specificity, nonreactivity with antibodies against host cell-derived proteins (e.g. E. coli and insect cell proteins), and their ease of preparation.

Rinderpest Eradication: Appropriate Technology and Social Innovations

Killer Eradication Rinderpest is the second disease to be eradicated globally after smallpox. Mariner et al. (p. 1309) review the technical and social challenges that were overcome during the course of eradication. Key achievements were the development of a thermostable vaccine, the recruitment of the pastoralists themselves for training and administration of vaccine, and complete vaccination coverage, despite occasionally hazardous environmental and political conditions. Although these achievements offer important lessons for future human and animal health programs, institutional memories are surprisingly short-lived, and it is important to document these lessons for the next eradication campaign. Rinderpest is only the second infectious disease to have been globally eradicated. In the final stages of eradication, the virus was entrenched in pastoral areas of the Greater Horn of Africa, a region with weak governance, poor security, and little infrastructure that presented profound challenges to conventional control methods. Although the eradication process was a development activity rather than scientific research, its success owed much to several seminal research efforts in vaccine development and epidemiology and showed what scientific decision-making and management could accomplish with limited resources. The keys to success were the development of a thermostable vaccine and the application of participatory epidemiological techniques that allowed veterinary personnel to interact at a grassroots level with cattle herders to more effectively target control measures.

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.

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.

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.

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.

The isolation of lumpy skin disease virus and bovine herpesvirus- 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.

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.

Experimental equine herpesvirus-l 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 < 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.

African horse sickness and African carnivores

African horse sickness (AHS) is a disease that affects equids, and is principally transmitted by Culicoides spp. that are biological vectors of AHS viruses (AHSV). The repeated spread of AHSV from sub-Saharan Africa to the Middle East, northern Africa and the Iberian peninsula indicate that a better understanding of AHS epizootiology is needed. African horse sickness has long been known to infect and cause mortality among domestic dogs that ingest virus contaminated meat, but it is uncertain what role carnivores play in transmission of the virus. We present evidence of widespread natural AHS infection among a diversity of African carnivore species. We hypothesize that such infection resulted from ingestion of meat and organs from AHS-infected prey species. The effect of AHS on the carnivores is unknown, as is their role in the maintenance cycle of the disease.