Hemagglutinins HA) of H1N1 swine influenza viruses isolated in the United States have remained antigenically and genetically conserved for many years. In contrast to such conservation, the RA of A/Swine/Nebraska/1/92 (Sw/Neb) could readily be distinguished from those of contemporary porcine viruses. Twenty-eight amino acid mutations differentiated the HA of Sw/Neb and A/Swine/Indiana/1726/88, the most recent H1N1 swine influenza virus for which HA sequence data were available. Among these differences were mutations at potential asparagine-linked glycosylation sites and charge changes at many residues. The Sw/Neb virus also could be differentiated from other swine influenza viruses in hemagglutination-inhibition assays with monoclonal antibodies to recent H1 swine HA. Nonetheless, overall sequence analysis of the HA and the nucleoprotein genes of Sw/Neb indicated that this virus was more closely related genetically to classic H1N1 swine influenza viruses than to H1N1 avian or human viruses. Infection of swine with Sw/Neb under experimental conditions induced clinical signs and lesions typical of swine influenza. However, affected swine in the field had high, persistent fevers, but relatively mild signs of respiratory tract disease. This study indicated that an antigenically and genetically novel variant of swine influenza virus was detected in the United States.

Each of 5 US-origin serotypes of bluetongue virus (BTV) was inoculated into a separate pair of sheep. The duration of each animal's ensuing viremia was monitored, using a BTV serogroup-specific nested polymerase chain reaction (PCR) method and an embryonating chicken egg (ECE) inoculation procedure. Mean duration of viremia was 100 and 38 days for the PCR and ECE methods, respectively. This difference was significant (P < 0.001) and documents a more prolonged viremia in virus-exposed sheep than has been reported. A dual internal oligonucleotide solution hybridization procedure was developed for the rapid (2 hours) colorimetric detection and identification of BTV-specific PCR products. This enzyme-linked oligonucleotide sorbent assay (ELOSA) relied on annealing of separate biotinylated and fluoresceinated probes to the amplified BTV nucleic acid; these complexes were captured on streptavidin-coated microtitration wells and were detected, using a horseradish peroxidase-labeled antifluorescein antibody conjugate. End-point dilution analyses of PCR products indicated that the ELOSA was more sensitive than gel electrophoretic or comparable colorimetric slot-blot hybridization techniques. The BTV PCR-ELOSA system represents a more sensitive and expeditious means of diagnosing BTV-induced viremia than does the ECE procedure currently used. The combination of ELOSA with PCR should facilitate practical application of nucleic acid technology to diagnostic veterinary medicine.

In vitro transferability of penicillin, streptomycin, tetracycline, and erythromycin resistance from coagulase-negative staphylococci to Staphylococcus aureus and among the former species of bovine mammary gland origin was examined by bacterial mating on filters and by mixed-culture matings in broth and in skim milk. One hundred twenty-six (42 each on filter, in broth, and in skim milk) matings were performed among 37 isolates of different Staphylococcus species. Transfer of resistance to penicillin, tetracycline, or erythromycin was not detected. Of 51 matings performed to determine streptomycin-resistance transfer, 9 (3 each on filters, in broth, and skim milk) were successful. Nine strains representing 3 species of coagulase-negative staphylococci were tested as prospective donors of streptomycin resistance. Of these, 2 strains could transfer streptomycin resistance. A double-resistant donor, S hominis, not only transferred its streptomycin resistance to an S chromogenes strain lacking resistance, but also to an S aureus strain already carrying penicillin and tetracycline resistance. The transfer of streptomycin resistance from the donor S hominis, harboring 2 plasmids, to a plasmidless S chromogenes recipient strain was associated with apparent acquisition of the smaller plasmid of the donor by the recipient. The single-resistant donor, S epidermidis 681A, transferred streptomycin resistance to a tetracycline-resistant S aureus recipient. This strain however, failed to transfer its streptomycin resistance to another S aureus, 2 S hyicus, and 1 S xylosus recipient. Frequency of transfer of streptomycin resistance ranged from 1.1 X 10(-5) to 1 X 10(-4). When transfer of resistance was successful, attempts were made to characterize the transfer process. Conjugation appeared to be the mode of streptomycin-resistance transfer. Transfer of resistance between staphylococci of bovine mammary gland origin appears to be fairly uncommon. However, in view of the limitations of the procedures used, additional in vitro and in vivo work is needed to further assess the role of coagulase-negative staphylococci in dissemination of antibiotic resistance.

The ability of viral glycoproteins (VP) VP4/VP7 reassortant swine rotaviruses (RV) to induce cross-neutralizing antibody against parental serotypes was investigated in guinea pigs. Using selective culture conditions, we produced 10 reassortant viruses that contained gene segment 4 of the OSU RV strain and gene segment 9 of the Gottfried RV strain. These reassortant RV grew to high titer in cell culture and were neutralized by monospecific antisera against both parental RV strains. The reassortant RV were chemically inactivated with binary ethylenimine, adjuvanted with aluminum hydroxide, and used to produce antisera in guinea pigs. The hyperimmune antisera had high neutralization titer against both parent RV strains. These results indicate that several of the reassortant RV may be capable of inducing neutralizing antibodies to VP4 and VP7 and may have future use as bivalent vaccine strains.

Development of the rickettsia, Anaplasma marginale, salivary glands of male Dermacentor andersoni exposed as nymphs or adult ticks, was studied indirectly by inoculation of susceptible calves with homogenates and directly by examination, using light microscopy and a DNA probe; some unfed ticks were incubated before tissues were collected. Salivary gland homogenates made from ticks in every treatment group caused anaplasmosis when injected into susceptible calves; prepatent periods decreased as the time that ticks had fed increased. Colonies of A marginale were seen only in salivary glands of ticks exposed as adults and not in those exposed as nymphs; the percentage of salivary gland acini infected in these ticks increased linearly with feeding time. However, the probe detected A marginale DNA in salivary glands of ticks from both groups; the amount of DNA detected increased as feeding time was extended. The amount of A marginale DNA appeared to remain constant in gut tissues, but to increase in salivary glands. Salivary glands of adult-infected male ticks that were incubated, but did not feed a second time, became infected with A marginale, and the pattern of infection of acini varied with incubation temperature. Development of A marginale in salivary glands appears to be coordinated with the tick feeding cycle; highest infection rate was observed in ticks exposed as adults.

The genetic basis of drug-resistant strains of Actinobacillus pleuropneumoniae in Japan was studied. The A pleuropneumoniae strains AV277 and AV281 that belong to serotype 2 were resistant to streptomycin (SM) and sulfonamide (SA). Both strains had an 8.1-kilobase (kb) SM-SA plasmid that was previously classified in the H1 group. The AV177 (serotype 1) strain was resistant to SM, SA, ampicillin, and kanamycin (Km), but did not have any plasmids. The AV319 and AV324 (serotype 1) strains were resistant to Sm, SA, tetracycline (TC), and chloramphenicol (CP). The AV318 (serotype 12) strain was resistant to SM, SA, TC, minocycline, and CP. These 3 strains (AV319, AV324, and AV318) had a 4.3-kb SM-SA plasmid and a 5.2-kb CP plasmid. The 4.3-kb plasmid was classified in the H2 group. The AV263 (serotype 1) strain was resistant to SM, SA, KM, TC, and CP. It had a 5.2-kb CP plasmid and a 6.6-kb SM-SA-KM plasmid. Both plasmids did not replicate stably in Escherichia coli strains. The former 5.2-kb plasmid was mobilized in E coli strains by plasmid RP4, which belonged to incompatibility P with broad host range, but the latter 6.6-kb plasmid was not so mobilized. Three 5.2-kb CP plasmids isolated from strains AV319, AV324, and AV318, had the same restriction endonuclease pattern after digestion with Ava I and EcoRI. They coexisted with H1 group plasmids in the incompatibility test, and coexisted also with H2 group plasmids of the original A pleuropneumoniae strains. Results indicated that the 5.2-kb CP plasmids could be classified in a new incompatibility group, H3. In this study, 4 types of plasmids were isolated, but no plasmids encoded TC and minocycline resistance.

A specific polymerase chain reaction (PCR) assay was devised, allowing detection of 1 bovine leukemia virus (BLV)-infected cell in 10(4) bovine lymphocytes. The efficacy of field application of the developed method was verified by evaluating the rate of viral transmission to calves from infected cows, whether they have persistent lymphocytosis. With this objective, 43 calves were simultaneously tested at birth and at 6 months of age for viral antibodies in serum and for proviral DNA in lymphocytes. At birth, 36 calves were BLV-negative and 3 were BLV-positive by results of serologic and DNA-based assays. Conversely, results for 4 calves had lack of correlation between the diagnostic methods. In particular, 2 calves were DNA-positive and antibody-negative for BLV and 2 other calves had the opposite test results. At 6 months of age, when the immunologic pattern more closely reflects the status of calves' immune response, independent of maternal antibodies, all calves DNA-negative for BLV at birth (n = 38), were consistently PCR- and antibody-negative for BLV. On the contrary, the cattle DNA-positive for BLV at birth (n = 5), whether seropositive or not, were PCR- and antibody-positive for BLV, at the time of the second screening. Thus, these results indicate reliability of the PCR to diagnose perinatal BLV infection. Furthermore, the observation that all calves found to be infected at birth were born to BLV-positive cows with persistent lymphocytosis, indicates that the persistent lymphocytosis status of the cow may represent a factor associated with BLV infection in utero.

Tissue homogenates were obtained from swine co-infected with 2 vaccine strains of pseudorabies virus (PRV). Viral isolates derived by serial plaque purification directly from tissue homogenates, without an intervening step of isolation and amplification on cell cultures, were characterized as recombinant and parental PRV genotypes on the basis of thymidine kinase and glycoprotein X gene combinations. Use of limiting dilutions and recovery of virus isolates as individual plaques minimized the likelihood of in vitro recombination serving as a confounding source of recombinant PRV. The thymidine kinase and glycoprotein X gene sequences were classified as wildtype or deleted, using a battery of polymerase chain reaction assays. Results substantiate the observation that PRV vaccine strains can form genetic recombinants in vivo after experimentally induced co-infection.

Fifteen isolates of Escherichia coli obtained from the blood and tissues of septic foals had plasmid DNA of size ranging from 2.5 to 93 megadaltons. These isolates grew in normal equine serum (serum resistant), a trait previously documented to be expressed by isolates obtained from blood and tissues of septic foals, but not by isolates obtained from the feces of clinically normal horses. Of these isolates, 3 contained conjugal plasmids that encoded resistance to multiple antimicrobial agents linked to serum resistance and, in 1 isolate, to production of aerobactin as well. Serum resistance and production of aerobactin are related to virulence of septicemic E coli from non-equine sources.

A method of extracting bacterial DNA from swine feces was developed and used in a molecular assay for the presence of ileal symbiont (IS) intracellularis, formerly known as the Campylobacter-like organism associated with swine with proliferative enteritis. Hybridization with a digoxigenin-labeled, IS intracellularis-specific probe detected the presence of IS intracellularis at a concentration of 10(7) organisms/g of feces. This method was sufficient to detect is intracellularis in the feces of swine with experimentally induced and naturally acquired infection. Results of the hybridization were in agreement with those from histologic postmortem examination.