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1.  Phylogenetic comparison of the S3 gene of United States prototype strains of bluetongue virus with that of field isolates from California. 
Journal of Virology  1996;70(8):5735-5739.
To better define the molecular epidemiology of bluetongue virus (BTV) infection, the genetic characteristics and phylogenetic relationships of the S3 genes of the five U.S. prototype strains of BTV, the commercially available serotype 10 modified live virus vaccine, and 18 field isolates of BTV serotypes 10, 11, 13, and 17 obtained in California during 1980, 1981, 1989, and 1990 were determined. With the exception of the S3 gene of the U.S. prototype strain of BTV serotype 2 (BTV 2), these viruses had an overall sequence homology of between 95 and 100%. Phylogenetic analyses segregated the prototype U.S. BTV 2 strain to a unique branch (100% bootstrap value), whereas the rest of the viruses clustered in two main monophyletic groups that were not correlated with their serotype, year of isolation, or geographical origin. The lack of consistent association between S3 gene sequence and virus serotype likely is a consequence of reassortment of BTV gene segments during natural mixed infections of vertebrate and invertebrate hosts. The prototype strain of BTV 13, which is considered an introduction to the U.S. like BTV 2, presents an S3 gene which is highly homologous to those of some isolates of BTV 10 and especially to that of the vaccine strain. This finding strongly suggests that the U.S. prototype strain of BTV 13 is a natural reassortant. The different topologies of the phylogenetic trees of the L2 and S3 genes of the various viruses indicate that these two genome segments evolve independently. We conclude that the S3 gene segment of populations of BTV in California is formed by different consensus sequences which cocirculate and which cannot be grouped by serotype.
PMCID: PMC190544  PMID: 8764098
2.  Genetic characterization of rabies field isolates from Venezuela. 
Journal of Clinical Microbiology  1996;34(6):1553-1558.
Twenty samples from cases of rabies in humans and domestic animals diagnosed in Venezuela between 1990 and 1994 and one sample from a vampire bat collected in 1976 were characterized by reactivity to monoclonal antibodies against the viral nucleoprotein and by patterns of nucleotide substitution in the nucleoprotein gene. Three antigenic variants were found: 1, 3, and 5. Antigenic variant 1 included all samples from dogs and humans infected by contact with rabid dogs. Unique substitutions permitted identification of two separate outbreaks of dog rabies in the Maracaibo Depression and Los Llanos region and in the Andean region of Venezuela. Samples from the vampire bat and two head of cattle were characterized as antigenic variant 3 and showed a nucleotide sequence homology of 96 to 98% to each other and to samples of vampire bat-associated rabies throughout Latin America. Ten of the remaining 12 samples were characterized as antigenic variant 5. Genetic studies indicated that 11 of these samples formed a highly homologous and distinctive group but were closely related to samples of vampire bat-associated rabies. The 12th sample of variant 5 (from a cat) showed only 78 to 80% genetic homology to samples of rabies associated with vampire bats. The application of antigenic and genetic typing to rabies surveillance in Latin America is essential to improve control programs. Recognition of the source of outbreaks of dog rabies and identification of wildlife species maintaining sylvatic cycles of rabies transmission permit better utilization of public health resources.
PMCID: PMC229062  PMID: 8735118
3.  Immune responses to Mycoplasma bovis vaccination and experimental infection in the bovine mammary gland. 
This study characterized the immune responses in four vaccinated and four control cows in response to vaccination and experimental intramammary inoculation with Mycoplasma bovis. Specific antibody responses occurred in serum and milk in response to vaccination and experimental infection. Lymphocytes from peripheral blood, but not from the mammary gland of vaccinated cows had increased responsiveness to mitogens. No lymphocytes tested were responsive to M. bovis antigen. Both vaccination and experimental infection resulted in skin test reactivity. These results imply that vaccination results in immune responses which may alter the course of experimental M. bovis mastitis, but may contribute to cellular inflammation.
PMCID: PMC1255462  PMID: 3167718
4.  Genome segment reassortment between two serotypes of bluetongue virus in a natural host. 
Journal of Virology  1987;61(9):2670-2674.
The occurrence of genome segment reassortment between two antigenically related orbiviruses was demonstrated in cattle. Individual virus clones were isolated by cell culture plaque assays directly from the blood of a calf infected with two serotypes of bluetongue virus. The majority (89%) of progeny viruses isolated from the calf represented reassortant viruses. A minimum of six genome segments participated in reassortment, with 16 unique reassortant constellations being identified. Such genome segment reassortment between unique, though antigenically related, orbiviruses has undoubtedly played a major role in generating the extensive phenotypic and genotypic diversity that is characteristic of this serogroup.
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PMCID: PMC255769  PMID: 3039160
5.  Resistance of C3H/HeJ mice to the effects of Haemophilus pleuropneumoniae. 
Infection and Immunity  1986;53(3):474-479.
Comparisons were made in the mortality associated with an inhaled dose of viable Haemophilus pleuropneumoniae type 5, strain J45, between adult C3H/HeN and C3H/HeJ mice. Mice of both strains were also challenged with Escherichia coli strains O111:B4 and J5. The 50% lethal dose (LD50) of H. pleuropneumoniae in C3H/HeN mice was calculated to be 10(6.5) CFU. At a mean dose of 10(6.7) CFU a 46% mortality rate occurred in C3H/HeN mice, whereas only 10% of the C3H/HeJ mice died (P less than 0.01). Deaths occurred significantly earlier in C3H/HeN mice (P less than 0.01). No deaths occurred later than 12 h postinfection in either group. Pulmonary lesions in the mice that died were similar to those in pigs that die during the acute phase of H. pleuropneumoniae infection. In surviving mice of both strains, a mild resolving interstitial and bronchopneumonia was present which was not typical of subacute H. pleuropneumoniae infections in swine. Quantitative bacterial isolations from the lungs, liver, and spleen indicate that H. pleuropneumoniae did not multiply in the lungs, was rapidly cleared, and did not become systemic. No deaths occurred in the mice inoculated with E. coli J5 or O111:B4 at mean doses of 10(6.3), 10(7.2), and 10(8.5) CFU, and 10(6.4), 10(7.5), and 10(8.2) CFU, respectively. The difference in the mortality rate between the C3H/HeN and C3H/HeJ mice suggests that endotoxin may be involved in acute deaths in pigs infected with H. pleuropneumonia. As indicated by the E. coli challenge, however, other factors are also likely to be involved. Because of the differences in the pathology and microbiology following H. pleuropneumoniae pulmonary infections in mice and pigs, mice do not appear to be an accurate model of the overall disease in swine.
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PMCID: PMC260813  PMID: 3527983
6.  Cytokine production by blue tongue virus-infected fetal sheep cells. 
Infection and Immunity  1979;25(1):403-407.
The migration inhibition of guinea pig peritoneal macrophages by a factor(s) from media obtained from blue tongue virus-infected monolayer cultures was studied. Medium from blue tongue virus-infected sheep fetal cell cultures inhibited migration of guinea pig macrophages from agarose droplets. Medium from control cultures and stock virus did not inhibit macrophage migration. Medium containing migration inhibiting factor(s) in vitro induced an inflammatory reaction in the skin of a newborn sheep. The inflammatory reaction was observed 20 h after intradermal inoculation. The skin reaction consisted of infiltrates of mononuclear leukocytes in the superficial dermis. Control medium and stock virus caused no skin reaction.
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PMCID: PMC414465  PMID: 225275
9.  Association of bluetongue virus gene segment 5 with neuroinvasiveness. 
Journal of Virology  1994;68(2):1255-1257.
Two strains (UC-2 and UC-8) of bluetongue virus were used to determine genetic factors influencing neuroinvasiveness. Reassortants were produced in vitro, and the parental origins of their genes were determined by polyacrylamide gel electrophoresis profiles and restriction endonuclease digestion. Gene segment 5 of UC-8 correlated with neuroinvasiveness of reassortants when inoculated subcutaneously into newborn mice.
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PMCID: PMC236572  PMID: 8289361
10.  Immune responses to the lipopolysaccharides and capsular polysaccharides of Haemophilus pleuropneumoniae in convalescent and immunized pigs. 
Infection and Immunity  1986;54(2):575-582.
The immunologic responses to a smooth-type lipopolysaccharide (LPS) (HpS-LPS), a rough-type LPS (HpR-LPS), and a capsular-enriched polysaccharide preparation (HpC-PS) purified from Haemophilus pleuropneumoniae were determined in pigs immunized with a commercial H. pleuropneumoniae cellular vaccine, in pigs experimentally infected with H. pleuropneumoniae, in control pigs, and in immunized rabbits. The ability of the preparations to induce lymphocyte blastogenesis and B-cell activation was determined in the pigs and compared with the responses induced by the LPS of Escherichia coli O111:B4 and the LPS of Salmonella minnesota Re595. All the LPS preparations acted to induce proliferation of peripheral blood lymphocytes (PBL) from all pigs. The blastogenic response of PBL from H. pleuropneumoniae-infected pigs to HpS-LPS and HpR-LPS was significantly (P less than 0.05) greater than that of PBL from immunized and control pigs. HpC-PS did not induce a blastogenic response in the PBL of control pigs but did in PBL from H. pleuropneumoniae-infected pigs and to a greater degree in immunized pigs. An increase in the response of PBL to the S. minnesota LPS occurred only in the H. pleuropneumoniae-infected pigs. Significantly more (P less than 0.05) immunoglobulin-secreting cells (ISC) were induced in a reverse hemolytic plaque assay by stimulation with HpS-LPS and HpC-PS of PBL isolated from pigs infected with H. pleuropneumoniae than of PBL from immunized pigs. Increasing the number of T cells increased the number of ISC induced by HpS-LPS in control and immunized pigs, but not in convalescent pigs. The presence of macrophages reduced activation of ISC by HpS-LPS in control pigs and to a lesser degree in immunized pigs, whereas in H. pleuropneumoniae-infected pigs macrophages enhanced the induction of ISC by HpS-LPS. In immunized pigs, macrophages acted to inhibit the ability of HpC-PS to induce ISC. Serologic studies indicate that HpC-PS contains strain- and serotype-specific antigens; that HpS-LPS has both serotype-specific and cross-reacting species-specific antigens; and that HpR-LPS does not contain detectable serotype-specific antigens but does have both non species- and species-specific antigens. These studies show that the serotype-specific protection provided by immunization of pigs with an H. pleuropneumoniae cellular vaccine is principally the result of immunity to capsular antigens and that a weak cellular immune response occurs as compared with that induced by infection with H. pleuropneumoniae.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC260200  PMID: 3490442
11.  Vaccine potential of Haemophilus pleuropneumoniae oligosaccharide-tetanus toxoid conjugates. 
Infection and Immunity  1986;54(2):583-586.
Oligosaccharides of smooth-type lipopolysaccharide (LPS) and oligosaccharides of rough-type LPS were isolated from Haemophilus pleuropneumoniae and conjugated to tetanus toxoid by reductive amination. The antigenic and immunogenic characteristics of the oligosaccharides, the oligosaccharide-tetanus toxoid conjugates, and the LPS of H. pleuropneumoniae were determined by passive hemagglutination, enzyme-linked immunosorbent assay, and inhibition enzyme-linked immunosorbent assay with antisera produced by immunization of rabbits and pigs. The findings were compared with the immunologic response induced by immunization of pigs with an H. pleuropneumoniae whole-cell vaccine and by infection of pigs with viable H. pleuropneumoniae. The results show that conjugation of isolated oligosaccharides of H. pleuropneumoniae to tetanus toxoid improves the immunogenicity of the oligosaccharides without significantly altering their antigenic character. These findings extend the understanding of the immunobiology of H. pleuropneumoniae infection in pigs and suggest the potential of purified oligosaccharides as vaccines to prevent porcine pleuropneumonia.
PMCID: PMC260201  PMID: 3770954
12.  Mechanisms involved in protection provided by immunization against core lipopolysaccharides of Escherichia coli J5 from lethal Haemophilus pleuropneumoniae infections in swine. 
Infection and Immunity  1986;53(2):298-304.
In an investigation of the potential protective effects of immunity against common lipopolysaccharide core antigens of gram-negative bacteria during a severe gram-negative infection in the natural host, we induced Haemophilus pleuropneumoniae infections in weanling pigs immunized with a vaccine of an Rc mutant of Escherichia coli (strain J5). To help define the mechanism involved in J5-mediated protection, we compared the clinical, hematologic, bacteriologic, and serologic responses following an H. pleuropneumoniae infection in J5-immunized pigs with those following an H. pleuropneumoniae infection in nonimmunized control animals. As a result of an intranasal inoculation, all of the control animals and the J5-immunized animals were infected with H. pleuropneumoniae. However, while 80% (4 of 5) of the nonimmunized pigs died within 24 h as a result of the infection, no deaths occurred in the J5-immunized animals. In the immunized group, J5 titers dropped during the acute stages of the infection and rebounded to well above the prechallenge levels during convalescence. The J5 titer also increased in the single surviving control animal. These findings suggest that antibodies against common subsurface components of gram-negative bacterial cell walls correlate with protection from an otherwise lethal challenge of H. pleuropneumoniae but do not prevent infection. Important growth-phase-dependent antigenic changes have been recognized to occur during the growth of H. pleuropneumoniae in cultures (R. Nielson, Nord. Veterinaermed. 28:337-348, 1976). In a study of these changes and during an inquiry into the mechanism of J5 antibody-mediated protection, measured quantities of H. pleuropneumoniae were removed from a broth culture at hourly intervals and used to absorb hyperimmune equine J5 antiserum. Significantly greater amounts of J5-specific antibodies were absorbed during the log phase of bacterial growth than during the early or late phase. The availability of epitopes recognized by J5 antibodies appears to be closely related to the rate of bacterial multiplication. The results of these experiments suggest a mechanism of protection provided by increased immunity to E. coli J5 during gram-negative infections.
PMCID: PMC260874  PMID: 3525407
13.  Simple procedure for preparation of bluetongue virus and epizootic hemorrhagic disease virus antigens for agar gel immunodiffusion. 
Journal of Clinical Microbiology  1983;18(6):1310-1313.
A simplified procedure was developed for preparing soluble antigen from two related orbiviruses, bluetongue and epizootic hemorrhagic disease viruses, for agar gel immunodiffusion. The antigens gave excellent results in both micro-agar gel diffusion (agar gel precipitin) and macro-agar gel diffusion (bluetongue immunodiffusion). Minor modification in the spatial arrangement of reference antisera, commonly utilized in the agar gel immunodiffusion tests, was employed to reduce the possible development of false-positive reactions. The procedures for antigen preparation were inexpensive and did not require elaborate filtration or high-speed centrifugation. Stability of antigen preparations at 5 degrees C was excellent (in excess of 3 years for bluetongue virus and 2 years for epizootic hemorrhagic disease virus).
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PMCID: PMC272898  PMID: 6140269
14.  Immunity to rotavirus in conventional neonatal calves. 
Journal of Clinical Microbiology  1982;16(5):935-942.
The local and systemic humoral immune responses to rotavirus were studied in six conventional neonatal calves. Attenuated bovine rotavirus was administered either orally or directly into an isolated intestinal loop. The parameters monitored were neutralizing rotavirus antibody in serum, immunofluorescent and neutralizing rotavirus antibody in intestinal loop washings, and rotavirus antibody-producing cells in intestinal mucosa. An antibody response was observed in the serum and intestinal secretions from one calf only. Viral replication was not detected in the isolated intestinal loop. Rotavirus antibody-producing cells were found in the intestinal mucosa of five calves. Double staining revealed that most of these cells produced antibody of the immunoglobulin A class. The conclusions were: (i) a previously described system to detect rotavirus antibody-producing cells can be used to study immune responses in neonatal calves, (ii) the class or subclass of antibody in rotavirus antibody-producing cells can be determined by double immunofluorescent staining, (iii) neonatal calves respond to rotavirus inoculation with a local immunoglobulin A response, and (iv) most of the rotavirus antibody-producing cells are located in the mucosa of the proximal small intestine.
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PMCID: PMC272505  PMID: 6296197

Results 1-14 (14)