Association between the level of antibodies in bulk tank milk and bovine respiratory syncytial virus exposure in the herd.

Antibody levels in bulk tank milk (BTM) against bovine respiratory syncytial virus (BRSV) are used to classify BRSV status of herds. The aim of this study was to investigate how these levels correspond with the time at which the herds were infected. Bulk tank milk, individual milk and serum samples from cows and young stock were investigated using an indirect ELISA. Screenings of BTM from 89 dairy herds during two winter seasons revealed a prevalence of positive herds from 82 per cent to 85 per cent. Eleven herds showed a marked increase in antibody levels between two screenings, indicating new infection. However, two of these herds had been free from BRSV for the last five to seven years. Two newly infected herds were monitored for four years and did not appear to get reinfected. Surprisingly, the BTM antibody levels in these herds remained high throughout the study period, but fluctuated significantly. This shows that the levels of antibodies in BTM can remain high for several years, even in herds where reinfection does not occur. BTM serology is a useful tool in the monitoring of infectious diseases in dairy herds, but has limitations as a diagnostic tool for BRSV infections.

Persistence of staphylococcal species and genotypes in the bovine udder.

Staphylococci are a major cause of intramammary infections (IMI) in ruminants. The main aim of this study was to investigate staphylococcal IMI in dairy cattle with emphasis on persistence and distribution of staphylococcal species and genotypes. With a sampling interval of 4-8 weeks, over a year, 4030 samples from 206 cows in 4 herds were collected. Coagulase-negative staphylococci (CNS) and Staphylococcus aureus were detected in 13.2% and 4.2% of the samples, respectively. Selected CNS isolates from quarter milk samples were identified to species level using sodA sequencing. Staphylococcus chromogenes (32%) and Staphylococcus simulans (25%) predominated. The proportion of S. chromogenes was greater in primiparous (52%) than in multiparous cows (12%), while the opposite was the case for Staphylococcus epidermidis (6% and 21%, respectively). Isolates from possibly persistent IMI were selected for pulsed-field gel electrophoresis (PFGE). Six staphylococcal species were found to cause persistent IMI; S. aureus, S. chromogenes, S. simulans, S. epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri. It was shown that several pulsotypes (PTs) within each species were associated with persistent infections, but only a few were spread and caused persistent IMI in multiple cows within a herd. Of special interest was the observation that only one, or a few, strains of each species caused persistent IMI in multiple cows within a same herd. This indicates strain differences with respect to transmissibility and pathogenicity.

Comparison of Staphylococcus aureus genotypes recovered from cases of bovine, ovine, and caprine mastitis.

Staphylococcus aureus is an important pathogen in domestic ruminants. The main objective of this study was to determine the similarity of epidemiologically unrelated S. aureus isolates from bovine, ovine, and caprine mastitis. By pulsed-field gel electrophoresis, 160 different pulsotypes (PTs) were identified among 905 isolates recovered from 588 herds in 12 counties in Norway. Based on estimates of similarity, using an 80% cluster cutoff, the isolates were assigned to 47 clusters. One cluster included 62% of all the isolates and more than 45% of the isolates from each host species. Twenty-three PTs included isolates from more than one host species; these 23 PTs represented 72% of all the isolates. The six most prevalent PTs included isolates from all host species and contained 45% of the bovine isolates, 54% of the ovine isolates, and 37% of the caprine isolates. Antimicrobial susceptibility testing of 373 of the isolates revealed resistance to penicillin in 2.9% and to streptomycin in 2.4%; only 1.9% were resistant to 1 of the other 11 antimicrobials tested. The results of this study suggest that a small number of closely related genotypes are responsible for a great proportion of S. aureus mastitis cases in cows, ewes, and goats in Norway and that these genotypes exhibit little or no host preference among these species. Selection due to antimicrobial resistance appears not to have contributed to the predominance of these genotypes.

Towards single screening tests for brucellosis.

This paper describes an indirect enzyme-linked immunosorbent assay (I-ELISA) and a fluorescence polarisation assay (FPA), each capable of detecting antibody in several species of hosts to smooth and rough members of the genus Brucella. The I-ELISA uses a mixture of smooth lipopolysaccharide (SLPS) and rough lipopolysaccharide (RLPS) as the antigen, and a recombinant protein A/G conjugated with horseradish peroxidase as the detection reagent. When using individually determined cutoff values, the SLPS/RLPS combined-antigen I-ELISA detected antibody in slightly more animals exposed to SLPS or to RLPS than did I-ELISA procedures using each individual antigen separately. Similarly, the assay using combined antigens detected antibody in slightly fewer animals not exposed to Brucella sp. When a universal cutoff of 10% positivity was used (relative to strongly positive control sera of each species), the overall performance index (percentage sensitivity plus percentage specificity) value decreased by 1.0 (from 199.4 to 198.4). In the FPA, it was not possible to use a universal cutoff without significant loss of performance. The overall sensitivity value for the FPA using the combined FPA antigen was 1.0% lower than using the O-polysaccharide (OPS) from SLPS and 9.1% higher than using the core antigen (CORE) from RLPS. When the combined antigen was used, the FPA specificity was slightly higher (1.2%) than from only the OPS, and considerably higher (12.6%) than the CORE. Overall, both the I-ELISA and the FPA with combined antigens were suitable as screening tests for all species of Brucella in the animal species tested.

Enzyme immunoassay for the diagnosis of brucellosis: chimeric Protein A-Protein G as a common enzyme labeled detection reagent for sera for different animal species.

A recombinant protein combining the immunoglobulin binding sites of Proteins A and G, conjugated with horseradish peroxidase was used as a universal detection reagent for the assessment of antibodies against Brucella spp. The reagent was applied in an indirect enzyme immunoassay for detection of antibodies to smooth lipopolysaccharide antigen in sera from Brucella spp. exposed and non-exposed cattle, sheep, goats and pigs and to antibodies to rough lipopolysaccharide in sheep, dogs and cattle. The results were similar to those obtained when murine monoclonal antibody-enzyme conjugates were used. An added advantage was that a universal cut-off for all tests using the proteins A and G detection reagent could be established, simplifying diagnostic interpretation of the data.

Rough lipopolysaccharide of Brucella abortus RB51 as a common antigen for serological detection of B. ovis, B. canis, and B. abortus RB51 exposure using indirect enzyme immunoassay and fluorescence polarization assay.

Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA.

Spread of Staphylococcus aureus resistant to penicillin and tetracycline within and between dairy herds.

One hundred and seven bovine isolates of penicillin and tetracycline resistant Staphylococcus aureus, recovered from 25 different dairy herds in various parts of Norway, were characterized using antimicrobial susceptibility testing, multilocus enzyme electrophoresis, ribotyping, plasmid analysis and serotyping of capsular polysaccharide. Forty-one isolates from one particular herd, 37 isolates from 5 herds that used a common pasture and milking parlour in summer and 21 isolates from 12 herds in 8 different counties belonged to the same strain. The remaining 8 isolates, which originated from herds in 5 different counties, were assigned to 6 different strains. Seven out of these 8 isolates had the same plasmid restriction profile. In conclusion, penicillin and tetracycline resistant S. aureus occurring in dairy herds in Norway mainly seems to represent one particular strain that has achieved widespread distribution or belong to one of several different strains carrying identical plasmids.

Antibody responses in sheep vaccinated against Staphylococcus aureus mastitis: a comparison of two experimental vaccines containing different adjuvants.

A double-blind, placebo-controlled study was conducted to compare two vaccines using different adjuvants with regard to their ability to stimulate antibody production against the alpha- and beta-toxins and the exopolysaccharide of Staphylococcus aureus. The vaccines contained identical antigens, consisting of inactivated whole bacteria of two strains of S. aureus in addition to alpha- and beta-toxoid. One vaccine contained mineral oil, while the other used a water-soluble acrylic acid polymer resin (Carbopol) as adjuvant. Saline served as the placebo. One hundred and forty ewes were vaccinated twice before lambing, by subcutaneous injection with vaccine or placebo in the region of the supramammary lymph node, and were observed and sampled over a period of 6 months. The vaccine containing mineral oil as adjuvant induced significantly greater immune responses to the alpha- and beta-toxins than did the vaccine containing Carbopol. The latter vaccine induced higher levels of antibodies to exopolysaccharide. The degree of local adverse reactions did not differ between the two groups. The results indicate differences between the oil-adjuvanted and Carbopol-adjuvanted vaccines with regard to their ability to stimulate antibody production against S. aureus protein antigens in sheep.

Imaging the surface of Staphylococcus aureus by atomic force microscopy.

The surfaces of four strains of Staphylococcus aureus, which differed in their expression of capsular polysaccharides, were examined using atomic force microscopy. The images show that it is possible to get information about surface characteristics of S. aureus using atomic force microscopy (AFM) following simple preparation. Strains Smith Diffuse (serotype 2), Reynolds (serotype 5), Wood-46 (capsule negative) and JL243 (capsule negative) were grown on medium known to promote the expression of capsular polysaccharides. The bacteria were air-dried prior to being imaged using tapping-mode AFM. Differences in the appearance of the bacterial surfaces were evident between the strains. The two capsule-negative strains exhibited a smooth regular surface, as opposed to the mucoid appearance of the two strains having polysaccharide capsules. Moreover, comparison of images of the heavily encapsulated serotype 2 strain and the serotype 5 strain indicates that a type 2 capsule can be distinguished from a type 5 microcapsule.

Staphylococcus aureus capsular polysaccharide type 5 conjugate and whole cell vaccines stimulate antibody responses in cattle.

Dairy heifers were immunized subcutaneously with one of four different vaccines which contained preparations of Staphylococcus aureus capsular polysaccharide type 5 (CP5) and a mineral oil adjuvant, or received a placebo containing saline and adjuvant. The vaccine containing a CP5-human serum albumin conjugate (CP5-HSA) and the vaccine with formaldehyde inactivated whole cells expressing CP5, both elicited strong anti-CP5 antibody responses. After two injections three weeks apart and a third injection 10 months later, the mean level and duration of the anti-CP5 antibody response was significantly higher in the whole cell group. No differences were found between the two groups with regard to the relative proportion of IgG subclasses, and the antibody responses to the polysaccharide were composed of both the IgG1 and IgG2. Vaccines containing only free CP5 or CP5 mixed with HSA produced weak and transient humoral immune responses. Only animals vaccinated with the whole cell vaccine or the conjugate vaccine showed responses to CP5 in a lymphocyte proliferation assay conducted one year after the third vaccination. This study indicates that CP5 expressed on the surface of formaldehyde inactivated whole cells, emulsified in an oil adjuvant, gives a strong and long lasting immune response in cattle. The use of conjugation technology, although effective, might not be necessary in order to achieve an immune response against S. aureus CP5 in cattle.

Characterisation of isolates of Staphylococcus aureus from acute, chronic and subclinical mastitis in cows in Norway.

Eighty-six Staphylococcus aureus isolates from cases of bovine mastitis were characterised biochemically and with respect to serotype, multilocus enzyme electrophoresis genotypes, antibiotic sensitivity, and production of enterotoxins A through D (SEA-D) and toxic shock syndrome toxin-1 (TSST-1). The samples were obtained from 81 different cows from 79 Norwegian dairy herds in 10 different counties in southern Norway. There was an equal representation of isolates from cases of acute, chronic and subclinical mastitis. Multilocus enzyme electrophoresis using 13 genetic loci showed that 69 of 86 isolates had the same electrophoretic type. This common electrophoretic type comprised isolates that differed in the expression of other phenotypical characteristics studied. Fifty-eight percent of the isolates produced one or more enterotoxins, predominantly a combination of SEC and TSST-1. Capsular serotyping revealed that 95% of the isolates belonged to serotype 8. No correlation was found between the factors studied and the clinical classification of mastitis. It appears that the majority of S. aureus isolates recovered from cases of bovine mastitis in Norway are genetically closely related and express common phenotypical characteristics.

Genetic and serologic evaluation of capsule production by bovine mammary isolates of Staphylococcus aureus and other Staphylococcus spp. from Europe and the United States.

Bovine mastitis caused by Staphylococcus aureus is responsible for major economic losses to the dairy industry, and more-effective therapeutic or preventive approaches are sorely needed. The predominance of staphylococcal capsular polysaccharide types 5 and 8 among human isolates from many sources is well documented, but there seems to be a greater variation in the distribution of capsular serotypes among isolates from cows. A total of 636 isolates of S. aureus from cases of bovine mastitis in Sweden, Denmark, Finland, Iceland, Ireland, and the United States were investigated for production of capsular polysaccharide types 5 and 8. Approximately half of all the European isolates tested were of serotype 8, although variation among countries and among isolates of clinical and subclinical origin was observed. Sweden had the highest frequency (87%) of serotypeable isolates, and Finland had the lowest (48%). Capsule types 5 and 8 accounted for only 42% of the U.S. isolates tested. A few isolates showed weak reactivity with CP5 antiserum in a colony blot assay, and an enzyme-linked immunosorbent assay inhibition method confirmed that the levels of capsule produced by these strains were <10% of those produced by control strains. Fifty isolates that failed to react with capsular antisera all possessed the genes for production of capsular polysaccharide type 5 or 8. These results underscore the variability in capsule production by bovine isolates of S. aureus from different geographic regions. This information is important for the rational design of a capsule-based vaccine to prevent S. aureus bovine mastitis.

Capsule expression by bovine isolates of Staphylococcus aureus from Argentina: genetic and epidemiologic analyses.

Staphylococcus aureus is an important cause of bovine mastitis worldwide, and effective preventive or therapeutic modalities are lacking. Although most human S. aureus isolates produce capsular polysaccharides (CPs), few reports have described the prevalence of capsules on bovine isolates. This information is important for the rational design of a vaccine for the prevention of staphylococcal mastitis. We serotyped 195 S. aureus strains isolated between 1989 and 1997 from the milk of mastitic cows in Argentina. Only 14 (7.1%) of the strains were serotype 5, and all were recovered between 1989 and 1992. Thirteen serotype 8 strains were identified, and 12 of these were isolated between 1991 and 1994. The remaining 168 isolates were nonreactive (NR) with CP serotype 5 (CP5)- or CP8-specific antibodies. Hybridization studies performed with genomic DNA from eight NR strains revealed that only three of them carried the capsule genes. Pulsed-field gel electrophoresis (PFGE) performed with 127 of the 195 S. aureus isolates revealed that most (86%) strains belonged to one of four major PFGE groups. Although 8 of 14 CP5 isolates showed a common PFGE pattern (arbitrarily defined as A1), 31 other A1 isolates from the same time period (1989 to 1992) were not CP5 positive. In contrast, only nine PFGE type B3 isolates were recovered between 1990 and 1994, and eight of these were positive for CP8 (P < 0.0003). The results of this study underscore the variability in capsule expression by S. aureus strains isolated from different geographical regions and cast doubt on the roles of CP5 and CP8 in the pathogenesis and immunoprophylaxis of bovine mastitis in Argentina.