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Abstract

Objectives

To determine whether the vaccine Brucella abortus strain RB51 (SRB51) would infect dogs, be shed in urine or feces, or cause placentitis and abortion.

Animals

18 Beagles.

Procedure

Males (n = 3), nonpregnant females (n = 3), and pregnant females (n = 4) were inoculated orally with SRB51; control dogs (n = 2) were fed sterile saline solution. A separate group of pregnant females (n = 5) received SRB51 IV, and their controls (n = 1) received sterile saline solution IV. Dogs were observed twice daily for evidence of abortion. Urine and feces were collected periodically for bacteriologic culture, and blood was collected for bacteriologic culture and serologic analysis. At full gestation (oral and IV inoculated pregnant females) or on postinoculation day 49 (nonpregnant females and males), dogs were euthanatized and samples were collected for bacteriologic culture and microscopic examination.

Results

Abortion was not apparent during the study, and SRB51 was not found in samples of urine or feces from any dog. Strain RB51 was isolated from retropharyngeal lymph nodes from all orally inoculated dogs (9/9). One orally inoculated and 1 IV inoculated pregnant dog had SRB51 in placental tissues. Strain RB51 was also isolated from 1 fetus from the orally inoculated female dog with placentitis, but lesions were not detected in the fetus.

Conclusions and Clinical Relevance

Oral inoculation of nonpregnant female or male dogs with SRB51 did not result in shedding in urine or feces, although oropharyngeal lymph nodes became infected; in pregnant females, it caused infection of the placenta, with resulting placentitis and fetal infection, but abortion was not apparent. Intravenous inoculation resulted in infection of maternal spleen, liver, and placenta; however, fetal infection and abortion were not observed. Infected canine placental membranes or fluids may be a source of infection for other animals and human beings. (Am J Vet Res 1997;58:851–856)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To characterize the early cellular immune response to Mycobacterium avium subsp paratuberculosis ( MAP) infection and evaluate the development of granulomatous inflammation at the SC injection site in experimentally inoculated calves.

Animals—Forty-eight 4-week-old calves.

Procedure—Calves received an SC injection of MAP strain 19698 (n = 25), sterile saline (0.9% NaCl) solution (20), or a commercial paratuberculosis vaccine (3); the inoculation site tissue and associated draining lymph node were excised at postinoculation day (PID) 0 (n = 36), 7 (14), 14 (6), 21 (8), and 60 (32). Sections of inoculation site tissues were evaluated immunohistochemically for T-cell subsets; lymph node mononuclear cells (LNMCs) were assessed for T-cell surface markers and for intracellular interferon-γ via flow cytometry.

Results—At MAP inoculation sites, calves developed mild, focal granulomatous inflammation by PID 7; by PID 60, areas of inflammation contained macrophages with numerous lymphocytes. Compared with control calves, there was increased antigen-specific LNMC proliferation in MAP- and vaccine- inoculated calves at PID 60, although proliferation among lymphocyte subsets was not significantly different between MAP-inoculated and control calves; in vaccine-inoculated calves, CD4+ T-cells predominated. In MAP-inoculated and control calves, antigenspecific interferon-γ production by LNMCs did not differ significantly; vaccine-inoculated calves had marked interferon-γ expression by CD4+ T-cells.

Conclusions and Clinical Relevance—In calves, SC administration of MAP resulted in granulomatous inflammation at inoculation sites and an antigen-specific T-cell proliferative response. Results suggest that this experimental system can be used to reproducibly generate antigen-specific T-cells during MAP infection for functional analysis. (Am J Vet Res 2005;66:474–482)

Full access
in American Journal of Veterinary Research

Summary

Cutaneous reactivity to brucellin was evaluated in 10-month-old heifers vaccinated with low-virulence mutant strains of Brucella abortus and was compared with brucellin reactions in postparturient cows with active brucellosis. In the cows, the cutaneous lesion was characterized microscopically as severe, acute, serofibrinous vasculitis; dermal lesions at 6, 12, 25, and 48 hours after brucellin injection consisted of endothelial activation and perivascular exudation that led to progressive accumulation of fibrin, monocytes, macrophages, and lymphocytes. In vaccinated heifers, cutaneous tests were done, using standard brucellin, brucellin prepared from strain RB51, and the purified brucellar proteins-31K and superoxide dismutase. Negative-control cattle given saline solution, did not have cutaneous reactions. Standard brucellin induced the most marked reactions in vaccinated heifers. Brucellin from rough strain RB51 caused positive reactions in heifers vaccinated with strain 19, but reactions were variable in other groups. Skin lesions induced by purified superoxide dismutase and 31-kd proteins in vaccinated cattle were not acceptable for diagnosis. Marked variability of test responses in vaccinated cattle precludes field use of this test to determine vaccination status.

Free access
in American Journal of Veterinary Research

SUMMARY

Objective

To determine the influence of brucellosis vaccination on tumor necrosis factor-α (TNF-α) con-centrations in pregnant cattle and the possible role of the bovine placenta in TNF-α production.

Animals

Polled Hereford heifers obtained from a nonvaccinated, brucellosis-free herd and bred at 16 to 27 months at age. All cattle were seronegative for Brucella abortus by results of the standard tube agglutination test.

Procedure

At 6 months' gestation, cattle were vaccinated IV with B abortus strain RB51 (n = 10), SC with B abortus strain RB51 (n = 5), or SC with B abortus strain 19 (n = 5); controls received pyrogen-free saline solution SC (n = 2). Blood samples were collected periodically for TNF-α assays. At necropsy, 8 to 12 weeks after vaccination, placental fluids and fetal blood were collected for TNF-α analysis and placental tissues were collected for immunohistochemical detection of TNF-α.

Results

Radioimmunoassays indicated no increase in TNF-α concentration in blood from IV or SC vaccinated cattle, compared with controls. Similarly, TNF-α concentrations in amniotic and allantoic fluids from SC vaccinated cattle were not different from values for controls. Although only IV vaccinated cattle developed placentitis, immunohistochemical analysis for TNF-α revealed increased immunoreactivity within placental trophoblastic epithelial cells of SC and IV vaccinated cattle.

Conclusions

SC vaccination for prevention of brucellosis, using recommended adult dosages, does not result in increase of TNF-α concentration in plasma, serum, or placental fluids; however, vaccination of pregnant cattle stimulates trophoblastic epithelial cells to express TNF-α, although the physiologic and quantitative importance of this expression remains unknown. (Am J Vet Res 1998;59:153–156)

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

SUMMARY

Uptake, transfer to rough endoplasmic reticulum, and intracellular growth of Brucella abortus were studied in Vero cells treated with endocytic and metabolic inhibitors. Infection of Vero cells was suppressed when inhibitors of energy metabolism (iodoacetate, dinitrophenol), receptor-mediated endocytosis (monodansylcadaverine, amantadine, methylamine), or endosomal acidification (chloroquine, ammonium chloride, monensin) were added to the inoculum. Inhibition was not observed when these drugs were added after the inoculation period. Infection of Vero cells by B abortus was inhibited by dibutyryl-cyclic adenosine monophosphate and Vibrio cholerae enterotoxin, but was stimulated by dibutyryl-cyclic guanosine monophosphate and Escherichia coli heat-stable enterotoxin a. Uptake of B abortus by Vero cells was not prevented by colchicine, but was abolished by cytochalasin B. Uptake of heat-killed B abortus and noninvasive E coli was similar to that of viable brucellae. Intracellular growth of B abortus was not affected by cycloheximide. Results indicate that: B abortus may be internalized by a receptor-mediated phagocytic process; transfer of B abortus from phagosomes to rough endoplasmic reticulum may require endosomal acidification; and replication of B abortus within the rough endoplasmic reticulum may not depend on protein synthesis by the host cell.

Free access
in American Journal of Veterinary Research

SUMMARY

Tissues from cattle that died of experimentally induced mucosal disease (n = 3), naturally acquired mucosal disease (n = 6), or naturally acquired chronic bovine viral diarrhea (n = 4) were examined. Consistent findings were lymphocytic depletion of lymphoid tissues, degeneration of myenteric ganglion cells, and mild adrenalitis. Intracytoplasmic viral antigen was detected in myenteric ganglia and in endocrine glandular cells. Noncytopathic virus was isolated from all cattle, and cytopathic virus was isolated from 12 of 13 cattle.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To develop a method for inducing acute leptospirosis in dogs.

Animals—31 nine-week-old female Beagles.

Procedure—Beagles were randomly assigned to 2 inoculation groups or a control group. Dogs were inoculated on 3 successive days by conjunctival instillation of 5 X 107 cells of Leptospira kirschneri serovar grippotyphosa strain 82 (12 dogs) or strain RM 52 (14 dogs). Control dogs (n = 5) were similarly inoculated with sterile leptospiral culture media. Clinical signs, clinicopathologic variables, anti-leptospiral antibody titers, and evidence of leptospires in tissues and body fluids were evaluated. Dogs were euthanatized and necropsied on days 7, 14, 22, or 28 after inoculation or as required because of severe illness.

Results—Clinical signs in infected dogs included conjunctivitis, lethargy, diarrhea, dehydration, vomiting, and icterus. Consistent clinicopathologic alterations included azotemia, hyperphosphatemia, increased anion gap, hyperbilirubinemia, and an increase in alkaline phosphatase activity. Leptospires were cultured from the kidneys (11/12), urine (6/9), aqueous humor (9/12), blood (12/12), and liver (12/12) of dogs inoculated with strain 82. Only 3 of 14 dogs became infected after inoculation with strain RM 52. Histopathologic lesions in infected dogs included interstitial nephritis, renal tubular degeneration and necrosis, pulmonary hemorrhage, and hepatic edema and perivasculitis.

Conclusions and Clinical Relevance—Conjunctival exposure to L kirschneri serovar grippotyphosa strain 82 resulted in acute leptospirosis in all inoculated dogs, but only 3 of 14 dogs inoculated with strain RM 52 became infected. This method of infection by serovar grippotyphosa can be used to study the pathogenesis and prevention of leptospirosis in dogs. (Am J Vet Res 2004;65:1100–1107)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To determine the ability of Brucella abortus strain RB51 to induce placentitis and abortion in bison after SC vaccination.

Animals

10 pregnant bison cows, 3 to 10 years old and at 3 to 8 months' gestation.

Procedure

Pregnant bison cows on a Montana ranch were vaccinated SC with 109 colony-forming units of B abortus strain RB51. Two cows, identified prior to the study, were euthanatized and examined 5 weeks after vaccination to obtain optimal histologic samples of placenta. Other cows were euthanatized and examined after abortion. After euthanasia, tissue specimens were collected for histologic and immunohistochemical evaluation. Tissue and fluid specimens for bacteriologic culture were also collected during necropsy.

Results

Of 8 cows, 2 aborted at 68 and 107 days after vaccination. Aborting cows had endometritis. Strain RB51 was isolated from reproductive tissues and supramammary lymph nodes. Fetal lesions were not seen; however, fetal bronchial lymph nodes and amniotic fluid contained strain RB51. Cows examined 5 weeks after vaccination had placentitis and endometritis, with numerous bacteria within trophoblastic epithelial cells that were immunoreactive for strain RB51 antigen. Strain RB51 was isolated from placentomes and numerous lymph nodes. Fetal lesions were not seen 5 weeks after vaccination; however, strain RB51 was isolated from numerous lymph nodes and lung, allantoic fluid, and rectal swab specimens.

Conclusions

The vaccine candidate B abortus RB51 has tropism for the bison placenta, and can cause placentitis, which induces abortion in pregnant bison. The vaccine dose used was similar to that being tested in cattle, but may not be appropriate for pregnant bison. (Am J Vet Res 1996;57:1604–1607)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To establish that female calves vaccinated with Brucella abortus strain RB51 at 3, 5, and 7 months of age are protected against infection and abortion when challenged exposed during their first pregnancy.

Animals

Polled Hereford heifer calves obtained from a brucellosis-free herd.

Procedure

Calves were inoculated SC at 3, 5, or 7 months of age with strain RB51 (n = 26), strain 19 (n = 16), or sterile saline solution (n = 15). Calves were bred at 16 to 17 months of age and challenged exposed during the first pregnancy with virulent B abortus strain 2308.

Results

After vaccination, none of the heifers given strain RB51 developed serum antibodies that reacted in the standard tube agglutination test, but reacted in a dotblot assay, using RB51 antigen. B abortus was cultured from biopsy specimens of superficial cervical lymph nodes in the RB51 and S19 vaccinates at 10 weeks, but not at 12 weeks after vaccination. All 4 heifers that had been vaccinated with RB51 at 3 months of age were protected against infection and abortion when challenged exposed. Vaccination at 5 and 7 months of age gave equivalent protection. Heifers given strain 19 were 95% protected and controls (given saline solution) had a high incidence of infection and abortion.

Conclusions

Strain RB51 is protective at doses comparable to those of strain 19 in calves 3 to 10 months of age.

Clinical Relevance

Immunogenicity and failure to induce antibodies that interfere with the serologic diagnosis of field infections of B abortus make strain RB51 an effective vaccine. (Am J Vet Res 1996;57:1153—1156)

Free access
in American Journal of Veterinary Research