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Abstract

Objective

To examine effects of in utero inoculation with a mutagen-attenuated Rift Valley fever virus (RVFV) vaccine (RVF MP-12) on fetal bovids and to assess the safety and efficacy of calfhood vaccination with RVF MP-12.

Animals

18 pregnant Hereford and Hereford-type cows in the third or fifth month of gestation, their progeny, and 25 calves from cows immunized with RVF MP-12 during pregnancy.

Procedure

Bovine fetuses were inoculated, via laparotomy, with 1 ml of RVF MP-12 containing 5 log10 plaque-forming units (PFU) of virus. Blood was obtained from newborn calves prior to their ingestion of colostrum. Immune-naive calves and calves born to RVF MP-12-vaccinated dams, ranging in age from 2 to 45 days, were vaccinated with RVF MP-12, and some were later challenge exposed with 1 ml of 5.7 log10 PFU of virulent RVFV strain ZH-501. Cows were monitored for viremia and antibody responses and for hematologic and serum biochemical alterations through parturition or abortion.

Results

Surviving in utero-vaccinated calves were healthy, with no noticeable defects. Except for 1 vaccine-inoculated fetus that died on postinoculation day 21, all in utero-vaccinated fetuses had serum neutralizing antibody titer ≥ 1:20 at the time of delivery. All dams of in utero-vaccinated fetuses also developed neutralizing antibody titer. Calves born to cows vaccinated during gestation did not have antibody at birth, and all but 1 quickly acquired colostral antibody. Postparturient inoculation of immune-naive calves and calves with colostral antibodies resulted in no untoward effects, and all calves with detectable neutralizing antibodies were protected against virulent virus challenge exposure.

Conclusions

Fetal death and abortion would be rare even if fetuses were exposed to RVF MP-12. The trauma and complications associated with in utero inoculation do not make this a practical method of immunization. RVF MP-12 was safe, immunogenic, and protective in calves as young as 2 days of age. (Am J Vet Res 1997;58:1110–1114)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To characterize cytokine profiles and lymphocyte subpopulations in lung parenchyma and bronchoalveolar lavage (BAL) fluid from normal bovine lungs.

Animals

Eight 12- to 18-month-old cattle.

Procedure

Cell populations in BAL fluid and collagenase-digested lung parenchyma were analyzed by flow cytometry and monoclonal antibodies. Proportions of total cell populations were determined, using Giemsa-stained cytospots. Distribution of lymphocytes within the lung parenchyma was analyzed by immunohistochemistry, and cytokine mRNA species in the parenchyma were characterized by use of reverse transcriptase-polymerase chain reaction analysis.

Results

Cytokine profiles indicated high amounts of mRNA for interleukins 6 and 10 and transforming growth factor β. In the BAL fluid and lung parenchyma, macrophages were the predominant cell type, although the proportion was lower in the parenchyma. Lymphocytes made up approximately 3% of both cell populations. Common to both lung compartments was the predominance of CD2+ and γδ T cells over B lymphocytes. There were more CD8+ T cells than CD4+ T cells in both compartments. The γδ cells made up approximately 9% of the lymphocyte populations. Two-color flow cytometry revealed CD8+ γδ T cell and CD8+CD5 populations that were unique to BAL fluid. In the BAL fluid and parenchyma, most CD4+ and CD8+T cells expressed high amounts of CD44, a characteristic of memory T cells. The γδ T cells were CD44lo, as were B cells in the lung parenchyma. The B cells from BAL fluid expressed high amounts of CD44. Immunohistologic analysis of lung tissue revealed bronchus-associated lymphoid tissue structures with distinctive germinal center organization of B cells encompassed by CD4+ T cells.

Conclusions

Results provided normal values for comparison with those of other species and with the bovine respiratory tract response to disease. (Am J Vet Res 1997;58:969–975)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate, under field conditions, the immunogenicity of 2 pseudorabies virus (PRV) vaccines (each with deletion of the gene for glycoprotein G [gG], and 1 with an additional deletion for glycoprotein E [gE]), particularly in the presence of maternal antibodies, and to investigate the effect of vaccination schedules in overcoming maternal antibody interference with vaccination.

Sample Population

Two cohorts of 105 growing pigs each on a PRV-seronegative commercial swine farm where breeding stock had been vaccinated with a PRV vaccine containing deletions of genes for gG and gE.

Procedure

Within each cohort, pigs were randomly assigned to 1 of 7 treatment groups. For each vaccine, vaccination was done at 8, 12, or 8 and 12 weeks of age. One group remained unvaccinated. Blood and nasal swab specimens were obtained at 8, 10, 12, 14, and 16 weeks of age, and the immune response was measured, by use of an ELISA.

Results

In cohort 1, where prevalence of maternal antibodies at 8 weeks of age was lower, an immune response lasting until 16 weeks of age was induced in most pigs by either vaccine. In cohort 2, where prevalence of maternal antibodies at 8 weeks of age was higher, the gG-gE- vaccine elicited a lower immune response in the presence of maternal antibodies than did the gG- vaccine after single vaccination at 8 weeks of age. This maternal antibody interference with the response to vaccination was evident in serum and nasal mucosal antibodies.

Conclusions

The gE deletion decreases the immunogenicity of PRV vaccine in the presence of maternal antibodies. Although evidence of maternal antibody interference for the gG- vaccine existed, its immunogencity was diminished less in the presence of maternal antibodies than that of the gG-gE- vaccine. (Am J Vet Res 1997;58:976–984)

Free access
in American Journal of Veterinary Research

Abstract

Objectives

To evaluate the safety and efficacy of a live canarypox virus recombinant-canine distemper virus (CDV) combination vaccine against virulent CDV challenge exposure, and to document lack of interference among the other modified-live virus (MLV) components.

Animals

33 specific-pathogen-free (SPF) Beagle pups (7 to 10 weeks old).

Procedure

A canarypox virus recombinant-CDV combination vaccine was tested for safety and efficacy along with MLV components (canine adenovirus type 2, canine coronavirus, canine parainfluenza virus, and canine parvovirus) in 26 SPF Beagle pups. The combination vaccine was rehydrated with either Leptospira canicola-L icterohaemorrhagiae combination bacterin (vaccine 1) or sterile diluent (vaccine 2). An additional group of 7 seronegative SPF pups received the control MLV components devoid of the combination vaccine (vaccine 3). Two vaccinations were administered 21 days apart, either IM or SC. The dose of the combination vaccine used to inoculate these pups was 40 times lower than the recommended commercial dose. At 21 days after the booster vaccination, all pups were challenge exposed with a virulent CDV strain, then were observed for 21 days to record morbidity and mortality.

Results

Adverse local or generalized reactions were not induced by vaccinations. All vaccinates seroconverted to CDV. Serum antibody titers to MLV components were not different, with or without inclusion of the combination vaccine. After challenge exposure, morbidity and mortality in vaccinates were 0% (0/26); in control dogs, values were 100% morbidity and 86% mortality (6/7). Brain impression smear slides made from all dogs that did not survive challenge exposure were CDV positive by use of a direct fluorescein isothiocyanate method.

Conclusions

The canarypox virus-CDV combination vaccine, administered SC or IM, is a safe product that elicits CDV seroconversion, does not interfere with other vaccine components, and protects vaccinated pups against virulent CDV challenge exposure. (Am J Vet Res 1997;58:833–836)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether recombinant porcine somatotropin (PST) or chromium picolinate (CrP) affected cytokine production and metabolism in swine after endotoxin challenge exposure.

Animals

20 Poland China × Landrace pigs, 5/group.

Procedure

Pigs were given CrP-supplemented feed at body weight of 20 kg; PST treatment began at 60 kg, and both treatments continued through body weight of 90 kg. At 90 kg, pigs were challenge exposed with 20 μg of lipopolysaccharide (LPS)/kg of body weight. Blood samples were obtained at various times through 24 hours after LPS challenge exposure.

Results

In all pigs not given PST, glucose concentration decreased 2 to 4 hours after LPS. In PST-treated pigs, blood glucose concentration was decreased at 6 to 8 hours after LPS. Plasma insulin concentration paralleled changes in glucose concentration. Nonesterified fatty acid concentration was high 2 to 24 hours after LPS in pigs not given PST and at 6 to 24 h in PST-treated pigs. Plasma urea nitrogen concentration was high at 6 to 24 hours after LPS in pigs not given PST. The urea nitrogen values in PST-treated pigs were lower at all times. Serum aspartate transaminase activity was high 6 to 24 hours after LPS in pigs not given PST, whereas PST treatment prevented the increase in this enzyme activity. In untreated (PST) pigs, plasma bilirubin (total and direct) concentrations were high 4 to 8 hours after LPS and returned to normal at 24 hours. The PST- and CrP-treated pigs maintained normal plasma bilirubin concentrations. Interleukin 6 activity was unaffected by CrP and PST treatments. Treatment with CrP and PST decreased the tumor necrosis factor α response to LPS, compared with that in control pigs.

Conclusions

PST, and to a lesser extent CrP, provide protection against the adverse metabolic effects of LPS-induced septic shock. (Am J Vet Res 1997;58: 594–600)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To investigate the ability of rinderpest virus (RPV) antigens, expressed in pox virus vectors, to protect against canine distemper virus (CDV) infection in ferrets.

Animals

Ferrets (Mustela putorius; n = 27) with no previous exposure to CDV.

Procedure

Ferrets were inoculated intradermally with recombinant vaccinia viruses expressing the H gene of RPV, the F gene of RPV, the H and F genes of RPV, or a fowlpox virus recombinant expressing both genes. Two ferrets were vaccinated SC with CDV vaccine as positive controls, and 1 group was left unvaccinated as a negative control. Blood was obtained from ferrets biweekly; antibody titer to RPV was detected by ELISA, and CDV antibody titer was measured by serum neutralization testing and ELISA.

Results

Partial protection was seen in all groups, with vRVFH vaccination being the most protective (60%).

Conclusions and Clinical Relevance

A single inoculation with a vaccinia virus expressing the H and F genes of RPV was able to protect 60% of the vaccinated ferrets challenge exposed with a high dose of CDV. These results indicate the ability of RPV antigens expressed by vaccinia virus to protect ferrets against a related morbillivirus. Further, they document the safety and efficacy of a recombinant vaccinia virus vaccine for ferrets. Such vaccines may be useful given the susceptibility of ferrets to CDV and the problem of maternal antibody interfering with vaccination of young animals. (Am J Vet Res 1997;58:590–593)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To purify complement component C3 from bovine serum, characterize and analyze NH2-terminal amino acid sequences from its various cleavage products, and do cross-species homology comparisons.

Animals

2 healthy lactating Holstein cows, and 2 healthy adult female New Zealand White rabbits.

Procedure

Bovine C3 was isolated from serum, and was cleaved to C3b. The resulting protein was analyzed to determine apparent molecular mass of resulting protein segments. Bands were electroblotted onto a membrane and excised, then NH2-terminal amino acid sequences were determined.

Results

The C3 preparation consisted of 6 segments, with molecular mass of 30, 40 (2 bands, a and b), 70, 75, and 115 kd. Via sequence comparisons, the 115-kd band was identified as the α chain; the 75-kd segment was determined to be the NH2-terminal portion of α chain; the 70-kd piece was identified as the intact β chain; and the two 40-kd bands are believed to be located at the C-terminal portion of the α chain, at the cleavage site that yields C3f. The 30-kd band is the NH2-terminal portion of the α chain (minus the C3a segment). Sequence analysis of each band revealed a high degree of homology with human, rat, mouse, and horse C3. Polyclonal antibodies raised in rabbits yielded sera that reacted to the purified sample in manner similar to that of commercially available antibodies.

Conclusions

The purified preparation contained intact C3, C3b, and the degradation products iC3b and C3c, which had high sequence homology with those of other species. The C3a and C3d, and C3g segments of the protein were not detected and may have been lost during the purification, lyophilization, or transfer steps. Structure and cleavage characteristics of bovine C3 can be used to better understand immune responses to bacterial pathogens in the mammary gland. (Am J Vet Res 1997;58:585–589)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether the respiratory burst of neutrophils from bovine blood and milk can be analyzed by use of a fluorometric resazurin reduction assay.

Sample Population

Neutrophils were obtained from EDTA-anticoagulated blood of 7 dairy cows. Neutrophils also were isolated from milk samples of a cow intramammarily challenge exposed with Escherichia coli lipopolysaccharide.

Procedure

The respiratory burst of neutrophils was analyzed in parallel, using the conventional luminol-enhanced luminometric procedure and a novel fluorometric procedure with resazurin as the fluorogenic substrate. Opsonized zymosan and phorbol myristate acetate were used as stimulants. The mechanism of the fluorescent response was analyzed, using metabolic inhibitors to various cell functions. Luminometry and fluorometry were carried out in parallel, using microtitration tray-reading instruments.

Results

Stimulation of neutrophils induced resazurin reduction to resorufin and a fluorescent response. The luminescent response was transient, but the fluorescent response (build-up of fluorescent resorufin) was cumulative. Therefore, a single end-point measurement can be used for the fluorometric assay.

Conclusions

The proposed fluorometric microtitration tray technology is simple and has a high throughput capacity. The fluorometric and luminometric assays seem to have similar potential in the analysis of phagocyte functions. (Am J Vet Res 1997;58:601–607)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine the safety and immunogenicity of Brucella abortus strain RB51 as a vaccine in pregnant cattle.

Animals

12 Polled Hereford heifers obtained from a brucellosis-free herd and bred on site at 16 months of age to a brucellosis-free bull.

Procedure

Pregnant heifers were vaccinated at 6 months’ gestation with 109 colony-forming units of B abortus strain RB51 (n = 5), 3 × 108 colony-forming units of B abortus strain 19 (n = 5), or sterile pyrogen-free saline solution (n = 2). Samples were periodically collected for serologic testing and lymphocyte blastogenesis assays. At full gestation, heifers were euthanatized and specimens were collected for bacteriologic culture, histologic analysis, and lymphocyte blastogenesis assay, using various antigenic stimuli.

Results

None of the strain RB51- or strain 19-vaccinates aborted or had gross or microscopic lesions at necropsy that were consistent with brucellosis. Maternal blood mononuclear cells from strain RB51- and strain 19-vaccinates had proliferative responses to γ-irradiated strain RB51 and strain 19 that were greater than responses by cells from nonvaccinated controls. In contrast, maternal superficial cervical lymph node cells from strain 19-vaccinates had proliferative responses to γ-irradiated strain RB51 or strain 19 bacteria greater than those of cells from RB51-vaccinates and nonvaccinated controls. None of the heifers vaccinated with strain RB51 developed antibodies detected by use of the standard tube agglutination test, but all developed antibodies to strain RB51 that reacted in a dot ELISA, using irradiated strain RB51 as antigen.

Conclusions

Pregnant cattle can be safely vaccinated with strain RB51 without subsequent abortion or placentitis. Furthermore, strain RB51 is immunogenic in pregnant cattle, resulting in humoral and cell-mediated immune responses, but does not interfere with serologic diagnosis of field infections. (Am J Vet Res 1997;58:472–477)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To compare the ability of 6 commercially available multicomponent canine vaccines to stimulate antibody production in pups with variable amounts of maternally derived canine parvovirus (CPV) antibody and to induce protective immunity against challenge exposure.

Animals

Sixty-three 5- to 6-week-old Beagle pups with passively acquired CPV antibody titer between 1:20 and 1:320.

Procedure

9 pups were assigned to each of 6 vaccine groups and 1 control group. Eight pups in each group were inoculated with vaccine or saline solution twice, with 3 weeks between administrations. The ninth pup served as an uninoculated contact control. Serum samples were obtained weekly and tested for CPV antibody by hemagglutination-inhibition assay. All pups were challenge exposed with virulent CPV-2a and CPV-2b at 14 to 15 weeks of age.

Results

3 of the vaccines failed to provide protective immunity against challenge exposure because all pups in these groups became infected and most died. A fourth vaccine protected against death, but not infection and disease. Two of the 6 vaccines induced an immune response that was protective against infection and disease.

Conclusion and Clinical Relevance

Substantial differences existed among commercial vaccines available in 1994 in their ability to immunize pups with maternally derived CPV antibody. These differences caused many vaccinated pups to be susceptible to CPV disease for variable periods because some vaccines failed to immunize. Importantly, all 4 of the vaccines that performed poorly have recently been replaced by more effective products so that the 6 vaccines now perform similarly. (Am J Vet Res 1997;58: 360-363)

Free access
in American Journal of Veterinary Research