Search

The objective of this study was to characterize the distribution of Mycobacterium paratuberculosis (Map) in the environment of infected and uninfected Minnesota dairy farms. Eighty herds known to be infected from Minnesota's Johne's Disease Control Program (JDCP) and 28 herds known to be uninfected from Minnesota Voluntary Johne's Disease Herd Status Program (VJDHSP) were sampled. Fecal samples from up to 100 cows in each herd were cultured in pools of 5 cows. Two environmental samples were obtained from each farm from various locations. All samples were tested using bacterial culture for Map. Eighty percent of the JDCP herds had at least one positive pool. Environmental samples were cultured positive in 78% of the JDCP herds. Two (7%) of the VJDHSP herds had one positive pool, and one herd had one positive environmental sample. Environmental samples were cultured positive in cow alleyways (77% of the herds), manure storage (68%), calving area (21%), sick cow pen (18%), water runoff (6%), and postweaned calves areas (3%). There was an association between maximum level of colonies per tube from cow alleyways and manure storage and fecal pool prevalence. Herds with both areas cultured negative were estimated to have 0.3 to 4% fecal pool prevalence. Herds with both areas having a heavy load of bacteria were estimated to have 53 to 73% fecal pool prevalence. The study results indicate that targeted sampling of cow alleyways and manure storage areas appears to be an alternative strategy for herd screening and Johne's infection status assessment and for estimating herd fecal prevalence. [ABSTRACT FROM AUTHOR]

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.

A survey was carried out to verify the sensitivity and specificity of various tests (complement fixation test--CF; agar gel immunodiffusion--AGID; indirect enzyme linked immunosorbent assay--ELISA; immunoblotting--IB) employed in the serological diagnosis of brucellosis caused by Brucella ovis. The tests were executed on 44 blood serum samples of rams coming from B. ovis-free flocks, 75 of B. ovis experimentally infected rams and 1139 from rams living in flocks where B. ovis had been previously isolated. All tests were performed using B. ovis hot saline extract (HS) as antigen. Sensitivity results were 97.4% for IB, 98.68% for CF, 100% for AGID and ELISA; specificity was 100% for all methods. Concordance values were 89.62% (CF-AGID), 78.77% (CF-ELISA), 77.74% (AGID-ELISA), 65.45% (IB-CF), 62.93% (IB-ELISA), 67.24% (IB-AGID). IB identified antibodies to antigenic components with molecular weight of 67, 63, 58, 55, 38, 35, 32, 30, 28, 25, 23, 21, 20-18 kDa (proteins) and 15-12 kDa (rough lipopolysaccharide).

An immunoblotting (IB) technique was developed for the serodiagnosis of brucellosis caused by Brucella ovis. Immunoblotting was performed, using a B. ovis HS (hot saline extract) antigen, on 44 blood serum samples which came from rams belonging to known brucella-free flocks, 114 samples originating from ten experimentally B. ovis infected rams and 100 from rams of naturally B. ovis infected flocks. No bands were noted on any of the 44 serum samples which originated from known negative flocks. Sera from naturally and experimentally infected rams identified antibodies to antigenic components with molecular masses of 67, 63, 58, 55, 38, 35, 32, 30, 28, 25, 23, 21, 20-18 (proteins) and 15-12 (RLPS) kDa.

A dot immunobinding assay (DIA) was developed for the detection of antibodies to Salmonella enteritidis. Western blot analysis of outer membrane proteins from SE identified 2 polypeptides of molecular masses 43 and 46 kD that were specific for S. enteritidis. These 2 polypeptides were utilized as antigens in the DIA. The DIA was tested on sera from chickens experimentally infected with S. enteritidis. Results of the DIA were compared with that of conventional microagglutination and serum plate tests. The DIA was a highly specific and sensitive test that can be useful for screening birds to determine if they are infected with S. enteritidis. Its simplicity, reliability, reproducibility, and speed in interpreting the assay results makes it a useful screening test for flock monitoring.

Red-backed voles (Clethrionomys gapperi) were live trapped in northern St. Louis County, Minnesota (USA), in late September and October 1988 and experimentally inoculated with Borrelia burgdorferi. Spirochetes were isolated from most animals 14 and 28 days following inoculation. Thus, red-backed voles exposed to B. burgdorferi were susceptible to infection and could be a reservoir host, along with chipmunks (Tamias striatus) and other small rodents, in areas where white-footed mouse (Peromyscus leucopus) populations are low. No evidence of clinical disease was noted in any infected voles.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to monitor virus shedding associated with experimental infection with a field strain of swine influenza in pigs. The assay consisted of a monoclonal anti-nucleoprotein capture antibody and a biotinylated rabbit anti-influenza A (H1N1) sandwich antibody. The antigen-capture system was capable of detecting as little as 1 ng/ml purified virus. The ELISA system surpassed egg cultivation procedures in the detection of low levels of shedding virus. Egg cultivation procedures indicated that most viral shedding had ceased by day 10 postinfection. In contrast, antigen-capture ELISA still showed an ongoing presence of viral antigen. A virus-capture ELISA, using this capture-sandwich antibody system, is equivalent in sensitivity to conventional egg inoculation procedures for the detection of the early phases of virus shedding. The automative potential of an ELISA-based system coupled with a substantially reduced assay time requirement give this virus-capture ELISA a distinct advantage over other cell culture or egg-based diagnostic techniques.

An ELISA-based method to estimate hemagglutination-inhibition (HI) titer was developed. Subtype specificity was obtained by using purified H1 and H3 hemagglutinin antigens. Using the linear relation that exists between ELISA and HI methods, regression lines for H1N1- and H3N2-monospecific porcine antisera were constructed. Approximation of actual HI titer could be obtained from insertion of ELISA values into the appropriate regression line. The HI estimations were within 50% of the actual measured HI value 84% of the time. In young pigs that had suckled immune sows, use of this ELISA revealed estimated HI titer > 320 at 2 and 4 weeks of age. After a typical farm outbreak of influenza A/swine (H1N1), estimated HI titers remain high for 4 to 6 months. Sub-type-specific estimation of the distribution frequency of positive influenza A (H1N1 or H3N2) results for sera from swine in regional herds indicated that 31.3 and 7.4% of the swine tested were positive (HI > 41) for H1N1 and H3N2, respectively. From these observations, we conclude that in many circumstances, an ELISA-based HI estimation method could be used as a substitute for the HI test.

To determine the seroprevalence of Lyme disease in gray wolves (Canis lupus) from various counties of Minnesota and Wisconsin (USA), 589 serum samples were collected from 528 wolves from 1972 to 1989. An indirect fluorescent antibody (IFA) test was used to detect the presence of antibodies against Borrelia burgdorferi. Titers of greater than or equal to 1:100 were considered positive. Results were confirmed by testing a few selected sera by Western blotting. Of the 589 sera tested, 15 (3%) had IFA titers of greater than or equal to 1:100. Three of the positive samples were collected from Douglas County in Wisconsin and twelve were from Minnesota counties. This study indicates that wolves are exposed to B. burgdorferi and are susceptible to Lyme disease.

The immunogenicity of leptospires cultivated in modified bovine albumin-polysorbate 80 medium and those cultivated in protein-free medium were quantitatively evaluated in the dog. Vaccine preparations, whole cell or outer envelope, prevented leptospiremia; however, kidney culture data revealed that 1 of 4 dogs vaccinated with 0.1 to 1 mg of whole cell prepared from leptospires cultivated in the modified bovine albumin-polysorbate 80 medium was positive for Leptospira, whereas dogs vaccinated with whole cells prepared in protein-free medium were not. Dogs vaccinated with greater than or equal to 0.5 mg of outer envelope were refractory to infection after challenge exposure.

After the observation of 2 horses with uveitis on a horse farm in the Minnesota River valley, 100 horses from this geographic area were given ophthalmologic examinations and were evaluated serologically for leptospirosis. A statistically significant (P less than 0.001) association was observed between the finding of antibodies against Leptospira interrogans serovar pomona and uveitis.

The humoral immune response of dogs vaccinated with leptospiral pentavalent outer envelope and whole culture vaccines was monitored with the microscopic agglutination (MA) test and leptospiricidal activity (LA) test for a 2-year period. The leptospiral serovars in the vaccines was canicola, icterohaemorrhagiae, grippotyphosa, pomona, and hardjo. The LA test was markedly more sensitive than the MA test for detecting anti-Leptospira antibodies and was least noticeable with anti-pomona antibodies. Both Igm and IgG were produced in similar amounts in the vaccinated dogs, and both classes of immunoglobulins were reactive in the MA and LA tests. The IgM class of antibodies was slightly more reactive in the MA test, whereas IgG antibodies were somewhat more reactive in the LA test. A direct correlation between protective antibodies, as determined by the hamster passive-protection test, and antibodies reactive in the MA and LA tests was observed.

The immunogenicity of leptospiral whole cell (WC) and outer envelope (OE) vaccines from virulent and avirulent strains was compared in hamsters. The 50% protective dose against death (PD(50D)) and kidney infection (PD(50K)) was evaluated for serotypes icterohaemorrhagiae, canicola, pomona, and grippotyphosa. The OE and WC of virulent strains possessed greater immunogenicity than the OE and WC of avirulent strains. More vaccine (<0.1 to 11.0 mug [dry weight] per hamster) was required for the PD(50K) than for the PD(50D) (<0.1 to 1.7 mug [dry weight] per hamster). The OE from virulent strains of these four serotypes plus hardjo were combined to form a pentavalent OE vaccine which proved to be satisfactory in immunogenic potency. Duration of immunity was not evaluated.

The distribution of Ixodes dammini in Minnesota was studied by collecting adult ticks from hunting dogs during the grouse seasons in September and October of 1985 and 1986. The tick was most frequently found in the east-central part of the state. Borrelia spp. were observed by immunofluorescence in 10% of the ticks. The locations where ticks were found coincide with the primary endemic areas for Lyme disease in the state.

The purpose of this study was to determine the effects of Leptospira interrogans serovar hardio on fertility in cattle. Twenty seronegative mature dairy cows were assigned to two groups. Group I (challenged cows) was bred by a seronegative bull followed by intrauterine infusion (within 30 minutes) of Leptospira interrogans serovar hardjo. Group II was bred by the same bull followed by intrauterine infusion of 5 ml of sterile culture medium. Blood samples were collected at two-day intervals to monitor serum antibody titers. Daily blood cultures for 10 days and weekly urine cultures for five weeks were performed to monitor the animals for leptospiremia and leptospiuria. Cows were slaughtered 35 days post-breeding, and their reproductive tracts were examined. All animals remained clinically normal following intrauterine challenge. There was no difference in pregnancy rates (Group I, 7/10; Group II, 6/10). All embryos, reproductive tracts, and kidneys appeared normal. A microscopic agglutination test (MA) showed that 4 of 10 challenged cows developed serum antibody titers between 8 and 20 days after challenge. However, on the basis of the hamster passive protection test, all challenged cows had serum antibodies present. All blood and urine cultures were negative through the experimental period, as were the final kidney and uterine cultures. In a second experiment, six seronegative cows were infused with killed microorganisms immediately after insemination. Results of a microscopic agglutination test and a hamster passive protection test indicated that these cows did not develop humoral antibodies against serovar hardjo. These results indicated that intrauterine inoculation of Leptospira interrogans serovar hardjo (hamster-adapted strain) following breeding did not affect pregnancy rates despite an intrauterine challenge which caused the development of humoral antibodies.

The humoral immune response of cattle vaccinated with leptospiral pentavalent outer envelope and whole culture vaccines was monitored with the microscopic agglutination (MA) test and the leptospiricidal activity (LA) test for a 2.5-year period. The serovars present in the vaccines were canicola, icterohaemorrhagiae, grippotyphosa, pomona, and hardjo. The LA test had greater sensitivity than the MA test for detecting antibodies to the hardjo, canicola, and icterohaemorrhagiae components of the pentavalent vaccine and was approximately equal in sensitivity to the MA test for detecting antibodies to the grippotyphosa and pomona components of the leptospiral vaccine. Both IgM and IgG were produced in similar amounts in the immunized cattle, and both classes were reactive in the MA and the LA tests. The IgM class of antibodies was more reactive in the MA test, whereas the IgG class of antibodies was more reactive in the LA test. Using the MA test as the criterion of evaluating, the immunogenic potency of the individual vaccine components was found to be canicola more than icterohaemorrhagiae more than grippotyphosa more than pomona more than hardjo. A direct correlation between protective antibodies, as determined by the hamster passive protection test, and antibodies reactive in the MA and the LA tests was observed.

Dogs inoculated intranasally with a live avirulent Bordetella bronchiseptica vaccine were monitored for the development of resistance to experimentally induced infectious tracheobronchitis (canine cough). Dogs were challenge exposed with a virulent strains of B bronchiseptica at various times after they were vaccinated. Clinical protection was detectable as early as 48 hours. At postvaccination days 4, 5, and 14, 56%, 83%, and 95% protection was observed. Humoral immunoglobulin (Ig) titers ranged from 1:8.6 on day 0 to 1:147 on postvaccination day 21. In the monitoring of B bronchiseptica-specific secretory IgA by indirect immunofluorescence, titers appeared as early as day 4 after vaccination. The IgA titers ranged from 1:16 on day 4 to 1: 1,024 on day 21. The appearance of IgA titers correlated with the development of resistance to clinical infection.

The complement subcomponent, C1q, was isolated from serum obtained from clinically normal dogs, using a rapid 2-step process involving affinity chromatography. Yield of C1q ranged from 8 to 10 mg/L of serum. Hemolytically active C1q had 3 protein bands after sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and formed a single line of identity with rabbit anti-canine C1q. The amino acid composition of canine C1q was similar to that of human C1q and contained a high percentage of glycine. Isolated canine C1q was iodinated, and the fluid-phase binding assay was used to detect circulating immune complexes in dogs with systemic lupus erythematosus and rheumatoid arthritis.

The immunogenicity of 2 leptospiral cell structural components, the outer envelope (OE) and protoplasmic cylinder (PC), as well as the leptospire whole cell (WC), was compared in hamsters and dogs. The 50% protective dose for hamsters against death (PD50D) and kidney infection (PD50k) was evaluated for Leptospira interrogans serovars canicola, icterohaemorrhagiae, pomona, and grippotyphosa. All 3 immunogens had similar PD50D values. However, the PD50K values for OE and WC vaccines ranged from 0.05 to 0.80 micrograms (dry weight), whereas the PC vaccines ranged from 7.0 to 14.0 micrograms. Cellular and humoral responses of dogs to serovar canicola WC, OE, and PC vaccines were monitored for 21 weeks. Little difference was observed among the canine humoral responses to the different preparations. Protoplasmic cylinder vaccines sensitized the greater population of lymphoid cells followed by OE and WC. Cross-reactivity was greater in the blastogenic response of lymphoid cell populations than in the humoral response.

Lyme disease spirochetes were isolated from the kidneys of two Peromyscus spp. trapped in Minnesota in September and October 1983. No spirochetes were isolated from white-tailed deer (Odocoileus virginianus), red backed voles (Clethrionomys gapperi), or shrews (Sorexy cinereus and Blarina brevicauda). This is the first report of the isolation of the Lyme disease spirochete from the midwestern United States and isolations from these animals, which were free of ticks, suggest that the Lyme disease spirochete may persist in animal organs for months.

Swine serologically negative for anti-Leptospira antibodies were given 2 doses of a pentavalent vaccine (3 weeks between doses) prepared from Leptospira serovars canicola, icterohaemorrhagiae, hardjo, pomona, and grip-potyphosa (0.2 mg/serovar/dose). Leptospires used for vaccinal production were cultivated in a protein-free medium or in a bovine albumin-containing medium. All vaccinated swine had demonstrable antibody titers within 1 week of the initial vaccination. Peak microscopic agglutination titers were between 256 and 1,024 after the 2nd vaccinal dose was given. After challenge exposure with serovar canicola, control swine had titers of at least 13,653 and the vaccinated swine had titers of 3,403 to 8,192, depending on the vaccine. Leptospiremia and kidney infections were not detected in any canicola Moulton immunized swine, but did appear in control swine. The Al(OH)3 adjuvant had no obvious influence of any of the vaccinal titers.