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Mycoplasma hyorhinis is an important pathogen of swine that can often occur as a respiratory coinfection with viral pathogens, but can also cause arthritis and polyserositis in infected animals. To date, no assay is available to assess the serologic response to M. hyorhinis vaccines, to our knowledge. We used recombinantly expressed M. hyorhinis p37 protein to monitor the magnitude of the IgG response in vaccinated animals. The assay was able to distinguish animals vaccinated with M. hyorhinis from those vaccinated with the other important Mycoplasma species: M. hyopneumoniae and M. hyosynoviae. When formulated with an ideal adjuvant, inactivated vaccines designed to protect animals against M. hyorhinis induced a measurable and dose-dependent antibody response against the p37 protein. Additionally, the protein appears to be highly conserved between strains of M. hyorhinis isolated in the United States. The specificity of the assay as well as the conservation and immunogenicity of the p37 protein make it an ideal candidate antigen for use in measuring the immune response against M. hyorhinis after vaccination in weaned pigs.

Haemophilus parasuis is a normal commensal of the upper respiratory tract of healthy pigs. However, in conjunction with stress and/or viral infections, or in immunocompromised animals, H. parasuis can transform into a pathogen causing Glasser's disease, which is typically characterized by fibrinous polyserositis, polyarthritis, meningitis, and sometimes acute pneumonia and septicemia. H. parasuis serotype 5 is highly virulent and more frequently isolated from respiratory and systemic infection in pigs. Recently Newport Laboratories isolated highly virulent H. parasuis serotype 4 strains from the tissues of diseased pigs. This study was undertaken to identify the genes responsible for H. parasuis serotype 4 virulence. To achieve this objective we performed genome-wide association studies (GWAS) across two virulent and three avirulent H. parasuis serotype 4 strains., (Copyright © 2014 Lawrence et al.)

Since the outbreak of porcine epidemic diarrhea virus (PEDV) in May 2013, U.S. swine producers have lost almost five million baby pigs. In an attempt to understand the evolution of PEDV in the United States and possibly develop a control strategy, we compared the genome sequences of a PEDV strain isolated from an infected piglet against its in vitro adapted version. The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line. The sequence analysis of the native PEDV strain and in vitro passaged virus shows that the cell culture adaptation specifically modifies PEDV spike protein whereas the open reading frame 1a/b (ORF1a/b)-encoded polyprotein, the nucleoprotein, NS3B (ORF3), and membrane and envelope proteins remain unchanged., (Copyright © 2014 Lawrence et al.)

Mycoplasma hyosynoviae is a Gram-negative bacterium that can cause debilitating arthritis in swine. Currently, there are no M. hyosynoviae genome sequences in the GenBank database, which makes it impossible to understand its pathogenesis, nutrition, or colonization characteristics, or to devise an effective strategy for its control. Here, we report the genome sequences of seven strains of M. hyosynoviae. Within each genome, several virulence factors were identified that may prove important in the pathogenesis of M. hyosynoviae-mediated arthritis and serve as potential virulence markers that may be critical in vaccine development., (Copyright © 2014 Bumgardner et al.)

Mannheimia haemolytica is a Gram-negative bacterium and the principal etiological agent associated mostly with bovine respiratory disease complex. However, we report here the sequence of a strain with the novel A1/A6-cross-reactive serotype, strain PKL10, isolated from white-tailed deer. PKL10 was isolated from the spleen of farmed white-tailed deer showing clinical signs of pneumonia. The genome structure of PKL10 is dramatically different from that of previously sequenced isolates, which was demonstrated by genome alignments. In addition, the coding sequences in PKL10 share approximately 86% sequence identity with the coding sequences in other fully sequenced M. haemolytica strains. This suggests that PKL10 is a novel Mannheimia species.

In vivo, neutralizing antibodies are critical for viral clearance. A high-throughput serum neutralization (HTSN) assay was developed to antigenically categorize Swine influenza virus (SIV) isolates. Uncategorized viruses were tested using a panel of antisera representing the H3N2 SIV subtypes and the results expressed as a serum neutralization ratio. Antisera were generated against contemporary isolates representing circulating H3N2 SIV subtypes (clusters I, III, IV). Reference viruses and the corresponding antisera were evaluated using traditional hemagglutination inhibition (HI) and the HTSN assays and good correlation (r = 0.84) was observed between the 2 tests. Categorical clustering of 40 recent (2008-2009) SIV isolates was assessed using the HTSN assay. The H3N2 SIV isolates with amino acid similarity >97% to the commonly used H3N2 cluster IV reference strain A/Swine/Ontario/33853/2005 (ON05) showed strong reactivity with cluster IV antisera. Isolates with <97% amino acid similarity to ON05 sporadically or completely failed to react with any antiserum. A cluster of 3 isolates with weak reaction with cluster III antiserum may be a potential emerging cluster of H3N2 with moderate genetic similarity to cluster II H3N2 (93% similarity). Potential uses of the HTSN assay include identification of broadly cross-reactive or antigenically distinct SIV isolates for use in vaccine virus selection or as part of surveillance efforts monitoring antigenic drift.

Keratoconjunctivitis outbreaks occur each summer in reindeer (Rangifer tarandus tarandus) herds in western Alaska, USA. This condition has not been well characterized nor has a definitive primary etiologic agent been identified. We evaluated the eyes of 660 calves near Nome, Alaska, between 29 June and 14 July 2005. Clinical signs of keratoconjunctivitis were observed in 26/660 calves (3.9%). Samples were collected from the conjunctival sac of both affected (n=22) and unaffected (n=24) animals for bacterial culture, enzyme-linked immunosorbent assay testing for Chlamydophila psittaci, and for polymerase chain reaction assays for Mycoplasma and Moraxella spp. No primary bacterial or viral etiologic agent(s) were isolated or identified. The cause of keratoconjunctivitis among reindeer calves was not determined, but it could involve an anaerobic bacteria, a difficult-to-isolate viral agent, stress associated with repeated handling, ocular foreign bodies, exposure to corral dust or arthropods, or a combination.

Objective: To determine factors associated with implementation and use of an on-farm system for bacteriologic culture of milk from cows with lowgrade mastitis, including information on how producers used the on-farm bacteriologic culture system to guide antimicrobial selection practices and the resulting impact on patterns of antimicrobial use., Design: Retrospective cohort study., Sample Population: Producers of 81 dairy farms., Procedure: Farms that used an on-farm system for bacteriologic culture of milk from January 2001 to July 2003 were surveyed., Results: Over half of those producers continuing to use the on-farm culture delayed antimicrobial treatment pending results of bacteriologic culture. Most other producers initiated empirical antimicrobial treatment while bacteriologic culture results were pending. Several barriers to the use of an on-farm system were identified. Significant reductions in rates of antimicrobial use were detected when comparing antimicrobial use rates before and during use of the on-farm system. Most producers chose to treat cows with mastitis caused by gram-positive pathogens with antimicrobials, whereas treatment choices for cows with mastitis caused by gram-negative bacteria and in cases in which no growth was detected varied., Conclusions and Clinical Relevance: Readily available results permit antimicrobial selections to be made on the basis of the causative agent of mastitis. Adoption of an on-farm system for bacteriologic culture of milk may result in significant reductions in the percentage of cows treated with antimicrobials. Decreasing antimicrobial use may have several benefits including preventing unnecessary discarding of milk, decreasing the potential for drug residues in milk, and improving treatment outcomes as a result of targeted treatments.

Objective: To develop a simple system for scoring hygiene in dairy cattle and determine whether hygiene scores were associated with individual cow somatic cell scores (SCSs)., Design: Observational study., Animals: 1,191 cows., Procedure: With the aid of a chart containing line drawings and descriptive text, hygiene scores ranging from 1 (clean) to 5 (dirty) were assigned for 5 body areas: tail head, thigh (lateral aspect), abdomen (ventral aspect), udder, and hind limbs (lower portion). To determine repeatability, hygiene scores were assigned to 75 cows twice by 4 experienced evaluators. To determine accuracy and ease of use, hygiene scores assigned by 14 college students to 23 cows were compared with scores assigned by 2 faculty members. To determine association with SCSs, hygiene scores were assigned to each of 1,093 cows by a single observer., Results: Mean correlation coefficients for hygiene scores assigned twice by 4 experienced evaluators were > or = 0.884, indicating high repeatability. Students indicated that the scoring system was easy to use, and mean correlation coefficient for student and faculty member scores was 0.804. Hygiene scores for the tail head, thigh (lateral aspect), and abdomen (ventral aspect) were not significantly associated with SCS. However, hygiene scores for the udder and hind limbs (lower portion) and udder-hind limb composite scores were significantly associated with SCS, with SCS increasing as scores increased., Conclusions and Clinical Relevance: Results suggest that the hygiene scoring system was repeatable, accurate, and easy to use. However, only hygiene scores for the udder and hind limbs and the udder-hind limb composite score were significantly associated with SCS.

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.

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.

A 110-kDa Borrelia burgdorferi fusion protein, Escherichia coli expressing the fusion protein, transformed E. coli lacking the fusion protein insert, and lyophilized whole B. burgdorferi bacteria were compared for immunogenicity in C3H/He mice. Immunized mice were challenged with a variety of isolates from the United States or the European isolate P/Gau 3 weeks following the last inoculation. An average of 76.7% of the mice immunized with 25 micrograms of lyophilized whole B. burgdorferi cells were protected from infection, while 60% of the mice immunized with the 110-kDa fusion protein were protected. Whole E. coli bacteria expressing the fusion protein protected 57.7% of immunized mice against experimental challenge. Lower levels of protection occurred in mice challenged with the European isolate than in those challenged with isolates originating from the United States. These results demonstrate the potential of the 110-kDa fusion protein for use as a component of a subunit vaccine for prevention of Lyme borreliosis.

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.

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.

EcoRI-digested DNA from Borrelia burgdorferi was ligated into the dephosphorylated vector pWR590 and transformed into Escherichia coli DH5 alpha. When the gene library was screened, 20 clones reacted with pooled dog sera with high titers (immunofluorescent antibody titer, greater than or equal to 1,280) to this spirochete. One clone expressed a 110-kDa antigen that reacted strongly with the high-titered pooled sera from dogs with Lyme borreliosis and serum from goats immunized with B. burgdorferi. The 110-kDa protein was serum from goats immunized with B. burgdorferi. The 110-kDa protein was expressed with and without isopropyl-beta-D-thiogalactosidase, indicating the protein is not a fusion protein with beta-galactosidase. Monospecific antisera to the 110-kDa antigen recognized a 75-kDa Borrelia protein. Of the sera that reacted with B. burgdorferi by immunoblotting; 57, 100, and 83% of human, dog, and horse serum samples, respectively, reacted with the 110-kDa protein. Sera from individuals that tested negative with a B. burgdorferi lysate with immunoblotting showed no reaction with the 110-kDa protein. The 110-kDa antigen appears to be useful for the diagnosis of Lyme borreliosis.

The activity of ceftriaxone, a newly developed cephalosporin, against early cutaneous infections with Treponema pallidum in rabbits was compared with that of equimolar doses of penicillin G. Activity was related to the time required for cutaneous lesions to become dark-field negative, serological response, and the disappearance of T. pallidum from the popliteal lymph nodes. Both antibiotics were very effective in the treatment of syphilis in this animal model. The 50% curative dose for penicillin G was 0.8 mumol/kg (0.29 mg or 480 U/kg) and for ceftriaxone, it was 1.45 mumol/kg (0.96 mg/kg). Overall, ceftriaxone was slightly less effective than penicillin G was. Transmission and scanning electron microscopy studies of testicular aspirates obtained from rabbits treated with ceftriaxone revealed alterations in the treponeme surface which apparently resulted in changes in cell permeability and morphology.

The testicular fluid and serum from rabbits infected intratesticularly with Treponema pallidum inhibited the mitogenic response of normal rabbit peripheral blood lymphocytes to concanavalin A. Mucopolysaccharide material present in the testicular fluid and serum was associated with the lymphocyte-inhibitory activity. Degradation of the mucopolysaccharide material with hyaluronidase resulted in the loss of the inhibitory activity of testicular fluid and serum of T. pallidu-infected rabbits.

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 hemolytic and sphingomyelinase C activities of supernatants of cultures of Leptospira interrogans serovar pomona tended to copurify when isoelectric fractionation was carried out. Both activities focused primarily at pH 8.1. Considered in conjunction with other circumstantial evidence, the results led to the conclusion that sphingomyelinase C is responsible for hemolysis.

A protein-free medium composed of charcoal-detoxified Tweens (polysorbates), vitamins B12 and B1, inorganic salts, and organic buffer is described that supports the growth and subculture of pathogenic and saprophytic Leptospira. Growth was initiated from small inocula, and cell densities of 10(9) organisms per ml were attained. Antigenicity and immunogenicity of Leptospira cultivated in this medium were similar to those of cells cultivated in serum-containing media. The protein-free medium was converted to a low-protein medium by the addition of 0.1% bovine serum albumin.

The synthesis of complex lipids by Borrelia hermsi while growing in Kelly medium was investigated by labeling cultures with D-[14C]glucose or [14C]palmitic acid. Labeled glucose was incorporated into phosphatidyl choline, phosphatidyl glycerol, monogalactosyl diglyceride, cholesterol glucoside, and acylated cholesteryl glucoside. The fatty acid composition reflected that of the medium suggesting that this spirochete directly incorporates acyl chains unaltered from its external milieu. Furthermore, the distribution of labeled carbon between acyl groups (lipid-soluble) and water-soluble moieties indicates that there is no metabolic exchange between these two pools. The relationship of B. hermsi to other spirochetes is discussed in terms of lipid metabolism as a generic characteristic.