A p37-based ELISA used to monitor anti- Mycoplasma hyorhinis IgG in serum from pigs immunized with inactivated M. hyorhinis vaccines.

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.

Genome-Wide Association Studies of Virulent and Avirulent Haemophilus parasuis Serotype 4 Strains.

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.

Genome Sequences of Seven Mycoplasma hyosynoviae Strains Isolated from the Joint Tissue of Infected Swine (Sus scrofa).

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.

Genome sequences of porcine epidemic diarrhea virus: in vivo and in vitro phenotypes.

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.

Genome Sequence of a Presumptive Mannheimia haemolytica Strain with an A1/A6-Cross-Reactive Serotype from a White-Tailed Deer (Odocoileus virginianus).

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.

Antigenic categorization of contemporary H3N2 Swine influenza virus isolates using a high-throughput serum neutralization assay.

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.

Preliminary studies on the etiology of keratoconjunctivitis in reindeer (Rangifer tarandus tarandus) calves in Alaska.

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.

Evaluation of the use of an on-farm system for bacteriologic culture of milk from cows with low-grade mastitis.

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.

Association between hygiene scores and somatic cell scores in dairy cattle.

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.

Experimental infection of dogs with Borrelia burgdorferi sensu stricto using Ixodes scapularis ticks artificially infected by capillary feeding.

Specific pathogen-free dogs were experimentally infected with Borrelia burgdorferi sensu stricto using nymphal or adult female Ixodes scapularis ticks artificially infected with spirochetes by capillary feeding. The ticks were capillary fed B. burgdorferi isolate 610, previously isolated from a dog with Lyme disease and grown in BSK medium. This isolate induced clinical signs in the dogs similar to those for dogs infested with ticks naturally infected with B. burgdorferi. Adult ticks were more efficient than nymphs in transmitting spirochetes to the dogs. One of five dogs infested with nymphal ticks capillary fed B. burgdorferi was skin biopsy culture and serologically positive, and demonstrated lameness. In contrast, all five dogs infested with adult female ticks that had been capillary fed with B. burgdorferi were culture and serologically positive, with one dog developing lameness. The immunoblot profiles of dogs challenged with female ticks infected by capillary feeding (8 weeks post challenge) were similar to immunoblots (4 weeks post challenge) from dogs challenged with naturally infected females collected in the field. These studies demonstrated that B. burgdorferi cultured in BSK medium can be capillary fed to either nymphal or adult female ticks under laboratory controlled conditions for the purpose of transmitting the spirochete to dogs during the tick's blood meal. This tick infection system would be useful for a controlled and defined challenge of vaccinated and non-vaccinated dogs for proper evaluation of vaccine efficacy, which is difficult to achieve using field-collected ticks. Furthermore, this system may also be useful for investigation of the pathogenesis of Lyme disease, evaluation of the pathogenicity of new isolates of B. burgdorferi, or evaluation of antibiotic therapy.