Longitudinal study of Salmonella shedding in naturally infected finishing pigs.

A 3-year longitudinal study was conducted on a multi-site farrow-to-finish production system. For each of 18 cohorts at three finishing sites, 50 pigs were randomly selected. Faecal samples were collected every 2 weeks for 16 weeks. Salmonella was cultured from 453 (6·6%) of 6836 faecal samples. The pig-level incidence of Salmonella was 20·8% (187/899 pigs). Salmonella prevalence varied between cohorts and within pigs. The adjusted Salmonella prevalence decreased over the finishing period from 6·4% to 0·8%. Intermittent detection of Salmonella was found in more than 50% of pigs that were positive at more than one collection. The finding that the majority of pigs shed intermittently has implications for surveillance and research study design when determining Salmonella status. The variability in shedding over time, as well as between and within cohorts and pigs suggests that there may be time-variant risk factors for Salmonella shedding in swine.

Outbreak of neonatal diarrhea in farmed mink kits (Mustella vison) associated with enterotoxigenic Staphylococcus delphini.

An outbreak of diarrhea on a large commercial mink farm affected 5,000 of 36,000 neonatal mink kits, with 2,000 dying within a 2-week period. Affected kits were severely dehydrated, and their furcoats and paws were covered with yellow- to green-tinged mucoid feces. On necropsy, the small intestines of examined animals were markedly distended by serous to mucoid fluid. Microscopically, there was prominent colonization of the intestinal villar epithelium by gram-positive bacterial cocci in the absence of inflammation and morphologic changes in villous enterocytes. The colonizing bacteria were phenotypically identified as belonging to the Staphylococcus intermedius group of bacteria. This was confirmed by nucleic acid sequence analysis of the 16S ribosomal RNA gene. Further nucleic acid sequencing of polymerase chain reaction (PCR) amplicons from the superoxide dismutase gene and the heat shock protein 60 gene differentiated the isolate as Staphylococcus delphini. Production of staphylococcal enterotoxins A and E was demonstrated with a commercial ELISA-based immunoassay. Sequencing of PCR amplicons confirmed the presence of the enterotoxin E gene, but PCR amplification of the enterotoxin A, B, C, or D genes was not successful. Although direct causation was not confirmed in this study, the authors postulate that the observed hypersecretory diarrhea in these mink kits was the result of colonization of the small intestine by S delphini and subsequent production of enterotoxin.

Experimental inoculation of wild turkeys (Meleagris gallopavo) with Mycobacterium bovis.

Although avian species are known to be susceptible to infection with Mycobacterium spp. organisms, much remains unknown about the susceptibility of birds to infection with M. bovis. The objective of this current study was to determine if wild turkeys (Meleagris gallopavo) can be infected with M. bovis when inoculated by the oral or intratracheal route. Six turkeys were orally inoculated and another six were inoculated via the trachea with a high dose of M. bovis, 1 x 10(5) CFU/ml. Six turkeys were sham-inoculated controls. Two turkeys from each treatment group were sacrificed on days 30, 60, and 90 postinoculation. There were no gross or microscopic lesions consistent with mycobacteriosis in the 23 inoculated turkeys over the 90-day duration of this study. Fecal cultures were also consistently negative for M. bovis when sampled before inoculation and on days 1, 30, and 60 postinoculation. Two intratracheally inoculated turkeys were positive for M. bovis in visceral tissues at 30 days postinoculation. However, this finding was only indicative of passive persistence of mycobacteria in the tissues and not of infection, as there were no attendant lesions or clinical compromise to support infection. Thus, it can be concluded that young wild turkeys are resistant to infection with M. bovis and, therefore, pose minimal threat as reservoir or spillover hosts for this organism.

Cattle and environmental sample-level factors associated with the presence of Salmonella in a multi-state study of conventional and organic dairy farms.

The objective of this study was to evaluate associations between cattle-level factors and environmental samples with the isolation of Salmonella from dairy farms in Minnesota, Wisconsin, Michigan, and New York. The study farms included 129 conventional and organic farms enrolled without regard to previous history of Salmonella infection. Herds were sampled at two-month intervals over a one-year period. Cattle groups more likely to be associated with Salmonella shedding (compared to preweaned calves) were cows designated as sick by farm personnel (OR=2.5, 95% CI: 1.7, 3.7), cows within 14 days of calving (OR=1.8, 95% CI: 1.1, 2.8), and cows due for culling within 14 days (OR=1.9, 95% CI: 1.0, 3.4). State of origin was also associated with the presence of Salmonella in samples from cattle and the farm environment; Midwestern states were more likely to have Salmonella-positive samples compared to New York. Cattle treated with antimicrobials within 14 days of sampling were more likely to be Salmonella-negative compared with nontreated cattle (OR=2.0, 95% CI: 1.1, 3.4). Farms with at least 100 cows were more likely to have Salmonella-positive cattle compared with smaller farms (OR=2.6, 95% CI: 1.4, 4.6). Season was associated with Salmonella shedding in cattle, and compared to the winter period, summer had the highest odds for shedding (OR=2.4, 95% CI: 1.5, 3.7), followed by fall (OR=1.9, 95% CI: 1.2, 3.1) and spring (OR=1.8, 95% CI: 1.2, 2.6). Environmental samples significantly more likely to be Salmonella-positive (compared to bulk tank milk) included, in descending order, samples from sick pens (OR=7.4, 95% CI: 3.4, 15.8), manure storage areas (OR=6.4, 95% CI: 3.5, 11.7), maternity pens (OR=4.2, 95% CI: 2.2, 8.1), haircoats of cows due to be culled (OR=3.9, 95% CI: 2.2, 7.7), milk filters (OR=3.3, 95% CI: 1.8, 6.0), cow waterers (OR=2.8, 95% CI: 1.4, 5.7), calf pens (OR=2.7, 95% CI: 1.3, 5.3), and bird droppings from cow housing (OR=2.4, 95% CI: 1.3, 4.4). Parity, stage of lactation, and calf age were not associated with Salmonella shedding.

Resistance of Mallard ducks (Anas platyrhynchos) to experimental inoculation with Mycobacterium bovis.

The purpose of this study was to investigate whether mallard ducks (Anas platyrhynchos) are susceptible to infection with Mycobacterium bovis by either oral or intratracheal inoculation and to assess their potential role in the spread of bovine tuberculosis. Six ducks were orally inoculated with 1.0 x 10(5) colony-forming units of M. bovis, six ducks were intratracheally inoculated with the same dose, and six ducks served as sham-inoculated controls. The study length was 90 days postinoculation, with samples of two birds from each group necropsied at 30-day intervals. Both fecal and tissue samples were collected for mycobacterial culture. None of the inoculated ducks shed M. bovis in their feces at any culture point (days 1, 30, and 60) during the study. No evidence of illness or weight loss was present during the course of the study, and only one duck had M. bovis isolated from any tissue, although there were no associated microscopic lesions. Mallard ducks were highly resistant to infection with M. bovis following high-dose inoculation and did not shed the organism in their feces. This study was conducted using high-dose inoculation; therefore, it appears that ducks are unlikely to play any significant role in the transmission of M. bovis between infected and uninfected mammalian hosts.

Cloning of the pfaP gene of Leptospira borgpetersenii.

A lambda gt11 library constructed with Leptospira borgpetersenii DNA was screened with monoclonal antibodies (mAb) recognizing a periplasmic flagella-associated protein. A plaque expressing a fusion protein (lambda F15) which reacted with the mAb was isolated and the nucleotide sequence analyzed. The deduced amino-acid (aa) sequence indicates that the pfaP gene belongs to a group of bacterial genes whose products share aa sequence and possibly functional homologies with sppA, an Escherichia coli signal peptidase-encoding gene.

Nucleic acid probe characterizes Leptospira interrogans serovars by restriction fragment length polymorphisms.

Restriction endonuclease analysis (REA) of genomic DNA can discriminate between many Leptospira interrogans serovars. However, several serovars have similar restriction endonuclease digestion patterns which prohibits accurate identification. This investigation expands previous REA studies of L. interrogans to include serovars in serogroup Tarassovi. Most serovars in this serogroup had characteristic digestion patterns by which they could be identified. However, four of the serovars in this serogroup had similar digestion patterns, thus preventing serovar identification by REA alone. To discriminate between these serovars REA was supplemented with Southern blot analysis. The DNA from each serovar showed similar but unique patterns when hybridized with a probe synthesized from a repetitive sequence element cloned from L. interrogans serovar hardjo type hardjo-bovis. The applicability of this technique to characterize other serogroups was assessed. One hundred sixty six of 190 serovars screened by Southern blot analysis contained sequences which hybridized with the repetitive element probe under conditions of relaxed stringency. These results suggest that Southern blot analysis using this probe will be a valuable supplement for typing L. interrogans.

Evaluation of an enzyme immunoassay for diagnosis of bovine leptospirosis caused by Leptospira interrogans serovar hardjo type hardjo-bovis.

Sensitivity and specificity of 4 different antigen preparations from Leptospira interrogans serovar hardjo were compared in an enzyme immunoassay for detection of antibodies against serovar hardjo type hardjo-bovis in serum. Two antigens prepared using detergents showed serogroup cross-reactivity. A mechanically extracted membrane and a lipopolysaccharide antigen showed a high degree of leptospiral serogroup specificity. The lipopolysaccharide antigen was the most suitable antigen for detection of anti-hardjo antibodies. Enzyme immunoassay was more sensitive than the microscopic agglutination test for detecting antibodies in serum from experimentally and naturally infected cattle. It was not possible to differentiate vaccinated from infected animals or to detect a secondary immune response in vaccinated animals that were subsequently infected.

Distribution of lesions in cattle infected with Mycobacterium bovis.

Detailed postmortem examinations were conducted on 30 cattle from a dairy herd with bovine tuberculosis to determine the distribution of lesions in Mycobacterium bovis-infected cattle. Twenty-four different tissue specimens from each animal were examined for gross lesions and collected for bacteriologic culturing and histologic examination. Tuberculosis was confirmed in 15 cattle with evidence of infection in 1 or more of the following tissues: medial retropharyngeal, parotid, tracheobronchial, mediastinal, caudal deep cervical, and subiliac lymph nodes; palatine tonsil; and lung. Gross and histologic lesions were present most frequently in lymph nodes of the thoracic region. Mycobacterium bovis was isolated from 3 cattle that had no gross lesions of tuberculosis. One animal had lesions only in the subiliac lymph node, which is not routinely examined during slaughter surveillance. Results of this study indicate that not all cattle infected with M. bovis have visible lesions of tuberculosis in sites that are routinely inspected. These findings are important because detection of gross lesions of tuberculosis during inspection of carcasses at slaughter is the primary method for detection of tuberculous cattle and herds in the United States.

Reproductive failure associated with Leptospira interrogans serovar bratislava infection of swine.

Specimens from 17 swine herds experiencing reproductive failure were examined for Leptospira interrogans serovar bratislava. Clinical signs observed in these herds included stillborn pigs, weak neonatal pigs, and abortion. Diagnostic tests used to determine L. interrogans serovar bratislava infection were bacteriologic culture, serologic assays to detect antibodies, and immunofluorescence. Examination of fetal serum for antibodies against serovar bratislava and a fluorescent antibody test were the most practical diagnostic procedures.

Pseudorabies virus infection of six- and ten-day-old porcine embryos.

Nine susceptible gilts were exposed to pseudorabies virus (PrV) by intrauterine inoculation immediately after breeding. Embryos were collected from each of three gilts on days 3, 6, and 10 following exposure to PrV. The number of embryos collected from each gilt was compared with the number of corpora lutea (CL). On days 6 and 10, there were substantially fewer embryos collected than there were CL. The embryos were examined for the presence of viral particles by electron microscopy. PrV was observed in embryos collected at 6 and 10 days following exposure of the gilts. The fluids used to flush the embryos from the uterus during collection were tested for PrV by virus isolation and direct fluorescent antibody procedures. PrV was isolated from the uterine-flush fluids of one of three gilts at each time of embryo collection.

Identification of leptospires of the Pomona and Grippotyphosa serogroups isolated from cattle in Zimbabwe.

Two strains of the genus Leptospira, isolated from kidneys of oxen slaughtered in Zimbabwe, one belonging to serogroup Pomona (strain SBF 8) and the other to serogroup Grippotyphosa (strain SBF 32), were identified by using cross-agglutinin absorption, monoclonal antibody, restriction fragment length polymorphism and polymerase chain reaction analyses. The identification of the two strains was equivocal. Strain SBF 8 showed a close similarity to both serovars mozdok and proechimys by cross-agglutinin absorption tests and to serovar pomona by monoclonal antibody analysis, but had a distinct DNA restriction pattern. Strain SBF 32 showed a close antigenic similarity to serovars ratnapura, grippotyphosa and valbuzzi by the cross-agglutinin absorption test, and to serovar ratnapura by monoclonal antibody analysis but also had a distinct DNA restriction pattern. Both strains SBF 8 and SBF 32 reacted as members of species Leptospira kirschneri by the polymerase chain reaction. It is concluded that strains SBF 8 and SBF 32 represent new genetic strains in the Pomona and Grippotyphosa groups, respectively.

Effects of exogenous recombinant interleukin-12 on immune responses and protection against Brucella abortus in a murine model.

This study determined if murine interleukin-12 (IL-12) would influence immunity in mice vaccinated with live or killed Brucella abortus strain RB51 (SRB51). Mice received live or gamma-irradiated SRB51 bacteria alone, or with IL-12 (0.5 or 1.0 microg, 2x or 3x), whereas other mice received saline or IL-12 alone. Post-vaccination antibody responses to live or killed SRB51 and clearance of live SRB51 from splenic tissue were not influenced by IL-12 treatments. Mice were challenged at 12 weeks with 4 x 10(4) cfu of B. abortus strain 2308 (S2308) and were euthanized 2 weeks later. The highest IL-12 treatment increased (P < 0.05) post-challenge antibody responses when co-administered with killed SRB51. Co-administration of 1.0 microg of IL-12 with live SRB51, but not killed SRB51, reduced (P < 0.05) S2308 colonization of splenic tissues. Our data suggest that although IL-12 may augment protective immunity induced by live SRB51, it does not influence protection induced by vaccination with killed SRB51.

The adjuvant effect of a single dose of interleukin-12 on murine immune responses to live or killed Brucella abortus strain RB51.

This study was designed to determine if a single 0.5 microg administration of recombinant murine interleukin-12 (IL-12) would influence immune responses of mice vaccinated with live or killed Brucella abortus strain RB51 (SRB51). Mice were vaccinated intraperitoneally with 5 x 10(8) cfu of live or gamma-irradiated SRB51 bacteria alone, or in combination with 0.5 microg of IL-12. Control mice received saline or 0.5 microg of IL-12. Serologic responses and spleen weights after vaccination were greater in mice vaccinated with live SRB51 when compared to mice receiving killed SRB51 or control treatments. Administration of a single dose of IL-12 as a vaccine adjuvant did not influence immune responses, clearance of live SRB51, or resistance against B. abortus strain 2308 (S2308) challenge. The results of this study suggest that a single administration of 0.5 microg of IL-12 at the time of vaccination does not have significant adjuvant effects on vaccine-induced immune responses against live or killed Brucella.

Effects of exogenous iron on Escherichia coli septicemia of turkeys.

The effect of inoculation with Escherichia coli on serum iron concentrations of turkeys and the effect of exogenous iron, as ferric ammonium citrate, on E coli septicemia in turkeys were determined. Inoculation of air sacs with E coli produced hypoferremia in 18-day-old turkeys. Administration of iron with E coli significantly (P less than 0.01) increased mortality, frequency and degree of bacteremia, and severity of lesions in inoculated turkeys, compared with those in turkeys given E coli but not given iron. Similar results were seen whether iron was inoculated at the same location as E coli or at a different location.

Preliminary evaluation of antimicrobial agents for treatment of Leptospira interrogans serovar pomona infection in hamsters and swine.

OBJECTIVE: To evaluate antimicrobial agents for treatment of models of acute and persistent leptospirosis caused by Leptospira interrogans serovar pomona. DESIGN: Randomized trials comparing dosages and regimens of various antimicrobial agents for treatment of acute and persistent leptospirosis. ANIMALS: 245 Golden hamsters to model acute leptospirosis and 121 mixed-breed swine to model persistent leptospirosis. PROCEDURE: Hamsters and swine were inoculated with L interrogans serovar pomona. Antimicrobial agents were given to hamsters for 3 or 5 days after inoculation, with necropsy at 14 days after inoculation. Swine were treated for 1, 3, or 5 days beginning at 3 weeks after inoculation, and were necropsied 7 to 10 days after completion of antimicrobial agent treatment. Hamster tissue and swine tissue and urine specimens were examined by culture, fluorescent antibody testing, and histologic examination for presence of leptospires. RESULTS: All untreated control hamsters became infected and manifested clinical signs and lesions of acute leptospirosis. Leptospires were not detected in hamsters treated with dihydrostreptomycin/penicillin G (25 mg/kg of body weight). Administration of ampicillin at all dosages reduced the number of hamsters infected, as confirmed at necropsy; the other agents tested required dosages greater than label recommendations to reduce the number infected. All untreated control swine became infected and shed leptospires in urine through the time of necropsy. Leptospires were not detected in kidneys or urine of swine treated with dihydrostreptomycin/penicillin G (25 mg/kg) for 1, 3, or 5 days, or in swine treated with oxytetracycline (40 mg/kg for 3 or 5 days), tylosin (44 mg/kg for 5 days), or erythromycin (25 mg/kg for 5 days). Treatment with ceftiofur and ampicillin was not effective in elimination of L interrogans serovar pomona in swine. CONCLUSIONS: Dihydrostreptomycin/penicillin G is effective for treatment of acute and persistent leptospirosis. Differences between the effectiveness of antimicrobial agents in the acute and persistent model of leptospirosis emphasize the importance of using the appropriate model for treatment evaluation. Antimicrobial agents evaluated for treatment of persistent leptospirosis in swine required the use of dosages above those recommended by the manufacturer. CLINICAL RELEVANCE: Use of antimicrobial agents at extra-label dosages for treatment of persistent leptospirosis may cause residue problems in food animals; however, these regimens may be useful for treatment of breeding stock or animals destined for import/export.

Use of a monovalent leptospiral vaccine to prevent renal colonization and urinary shedding in cattle exposed to Leptospira borgpetersenii serovar hardjo.

OBJECTIVE: To determine whether a monovalent Leptospira borgpetersenii serovar hardjo (type hardjobovis) vaccine commercially available in Australia, New Zealand, Ireland, and the United Kingdom would protect cattle from renal colonization and urinary shedding when exposed to a US strain of Leptospira borgpetersenii serovar hardjo. ANIMALS: 24 Hereford heifers that lacked detectable antibodies against serovar hardjo. PROCEDURE: Heifers received 2 doses, 4 weeks apart, of the commercial hardjo vaccine (n = 8) or a monovalent US reference hardjo vaccine (8) or were not vaccinated (controls; 8). Heifers were challenged 16 weeks later by intraperitoneal inoculation or conjunctival instillation. Serum antibody titers were measured weekly, and urine samples were examined for leptospires. Heifers were euthanatized 11 to 14 weeks after challenge, and kidney tissue was examined for evidence of colonization. RESULTS: All 8 heifers vaccinated with the reference vaccine were found to be shedding leptospires in their urine and had evidence of renal colonization. All 4 control heifers challenged by conjunctival instillation and 2 of 4 control heifers challenged by intraperitoneal inoculation shed leptospires in their urine, and all 8 had evidence of renal colonization. In contrast, leptospires were not detected in the urine or tissues of any of the 8 heifers that received the commercial hardjo vaccine. Heifers that received the commercial hardjo vaccine had significantly higher antibody titers than did heifers that received the reference vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cattle that received 2 doses of the commercial hardjo vaccine were protected against renal colonization and urinary shedding when challenged with L borgpetersenii serovar hardjo strain 203 four months after vaccination.

Comparison of three techniques to detect Leptospira interrogans serovar hardjo type hardjo-bovis in bovine urine.

Nucleic acid hybridization, bacteriologic culture, and a fluorescent antibody test were compared for detection of Leptospira interrogans serovar hardjo type hardjo-bovis in bovine urine. Seventy-five urine samples were collected from pregnant cows challenge exposed with type hardjo-bovis. Twenty samples were collected from steers not exposed to hardjo-bovis. Sediments from each sample were examined, using fluorescent antibodies and a repetitive sequence element nucleic acid probe, to detect the presence of leptospires. Urine samples were processed for bacteriologic culture, using standard techniques. Under laboratory conditions typically used for these techniques, leptospires were detected in 60 of 75 urine samples from challenge exposed cows by nucleic acid hybridization, in 24 samples by fluorescent antibody test, and in 13 samples by bacteriologic culture. Leptospires were not detected in the urine of steers not exposed to hardjo-bovis.

Effect of vaccination with a pentavalent leptospiral vaccine containing Leptospira interrogans serovar hardjo type hardjo-bovis on type hardjo-bovis infection of cattle.

Effectiveness of 2 pentavalent leptospiral vaccines containing Leptospira interrogans serovar hardjo was evaluated for protection of steers from infection with serovar hardjo type hardjo-bovis. The hardjo component of 1 vaccine was prepared from serovar hardjo type hardjoprajitno. The hardjo component of the other vaccine was prepared from serovar hardjo type hardjo-bovis. Two steers were vaccinated once and 4 steers were vaccinated twice with the pentavalent vaccine containing type hardjoprajitno. Four steers were vaccinated once and 4 steers were vaccinated twice with the pentavalent vaccine containing type hardjo-bovis. Four steers were maintained as non-vaccinated controls. Steers given vaccine containing type hardjo-bovis developed higher mean serum microscopic agglutination titers against serovar hardjo than steers given vaccine containing hardjoprajitno. Six months after the first vaccination, all steers were challenge-exposed on 3 occasions by conjunctival instillation of 10(7) serovar hardjo type hardjo-bovis organisms, and on 1 occasion by conjunctival instillation of urine from a steer shedding hardjo-bovis. All control and all vaccinated steers became infected and shed serovar hardjo type hardjo-bovis in the urine. Lesions were detected in kidneys of 3 of 4 nonvaccinated control steers, 5 of 6 steers given hardjoprajitno vaccine, and 6 of 8 steers given hardjo-bovis vaccine. Leptospires were detected in kidneys of 4 of 4 control steers and 13 of 14 vaccinated steers.

Effect of vaccination with a monovalent Leptospira interrogans serovar hardjo type hardjo-bovis vaccine on type hardjo-bovis infection of cattle.

Effectiveness of 2 concentrations of a monovalent vaccine containing Leptospira interrogans serovar hardjo type hardjo-bovis was evaluated for protection of heifers from infection with type hardjo-bovis. Nine heifers were given 2 doses of low-dose vaccine (8.32 x 10(8) cells/dose); 9 heifers were given 2 doses of high-dose vaccine (8.32 x 10(9) cells/dose); and 1 steer and 1 heifer were maintained as nonvaccinated controls. Groups of vaccinated cattle were challenge-exposed with serovar hardjo type hardjo-bovis at 7 (n = 6), 11 (n = 6), or 15 (n = 6) weeks after completion of vaccination. All cattle were challenge-exposed by conjunctival instillation of 1 x 10(5) hardjo-bovis cells on 3 consecutive days. Both control and all vaccinated cattle became infected and shed serovar hardjo type hardjo-bovis in their urine. Leptospires were detected in 15 of 16 (94%) urine samples from control cattle and in 124 of 143 (87%) samples from vaccinated cattle. Leptospires were detected in kidneys of 17 of 18 vaccinated cattle and 2 of 2 control cattle and in the uterus or oviducts of 13 of 18 vaccinates and the 1 control heifer.