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Background: Canine diabetes mellitus (DM) is a common endocrine disease in domestic dogs. A number of pathological mechanisms are thought to contribute to the aetiopathogenesis of relative or absolute insulin deficiency, including immune-mediated destruction of pancreatic beta cells. DM risk varies considerably between different dog breeds, suggesting that genetic factors are involved and contribute susceptibility or protection. Associations of particular dog leucocyte antigen (DLA) class II haplotypes with DM have been identified, but investigations to date have only considered all breeds pooled together. The aim of this study was to analyse an expanded data set so as to identify breed-specific diabetes-associated DLA haplotypes., Methods: The 12 most highly represented breeds in the UK Canine Diabetes Register were selected for study. DLA-typing data from 646 diabetic dogs and 912 breed-matched non-diabetic controls were analysed to enable breed-specific analysis of the DLA. Dogs were genotyped for allelic variation at DLA-DRB1, -DQA1, -DQB1 loci using DNA sequence-based typing. Genotypes from all three loci were combined to reveal three-locus DLA class II haplotypes, which were evaluated for statistical associations with DM. This was performed for each breed individually and for all breeds pooled together., Results: Five dog breeds were identified as having one or more DLA haplotype associated with DM susceptibility or protection. Four DM-associated haplotypes were identified in the Cocker Spaniel breed, of which one haplotype was shared with Border Terriers. In the three breeds known to be at highest risk of DM included in the study (Samoyed, Tibetan Terrier and Cairn Terrier), no DLA haplotypes were found to be associated with DM., Conclusions: Novel DLA associations with DM in specific dog breeds provide further evidence that immune response genes contribute susceptibility to this disease in some cases. It is also apparent that DLA may not be contributing obvious or strong risk for DM in some breeds, including the seven breeds analysed for which no associations were identified.

Digital dermatitis is a painful foot disease compromising welfare in dairy cattle. The disease has a complex multibacterial aetiology, but little is known about its pathogenesis. In this study, gene expression in skin biopsies from five bovine digital dermatitis lesions and five healthy bovine feet was compared using RNA-Seq technology. Differential gene expression was determined after mapping transcripts to the Btau 4.0 genome. Pathway analysis identified gene networks involving differentially expressed transcripts. Bovine digital dermatitis lesions had increased expression of mRNA for α2-macroglobulin-like 1, a protein potentially involved in bacterial immune evasion and bacterial survival. There was increased expression of keratin 6A and interleukin 1β mRNA in bovine digital dermatitis lesions, but reduced expression of most other keratin and keratin-associated genes. There was little evidence of local immune reactions to the bacterial infection present in lesions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Based on results of earlier studies, brain, heart and kidney are most commonly used for West Nile virus (WNV) detection in avian species. Both monoclonal and polyclonal antibodies have been used for the immunohistochemical diagnosis of WNV in these species. Thus far, no studies have been performed to compare the sensitivity and specificity of monoclonal and polyclonal antibodies in detecting WNV in American crows (Corvus brachyrhynchos). Our objectives were to determine 1) the comparative sensitivities of monoclonal and polyclonal antibodies for immunohistochemical (IHC) diagnosis of WNV infection in free-ranging American crows, 2) which organ(s) is/are most suitable for IHC-based diagnosis of WNV, and 3) how real-time RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tissues compared to IHC for the diagnosis of WNV infection., Methods: Various combinations, depending on tissue availability, of sections of heart, kidney, brain, liver, lung, spleen, and small intestine from 85 free-ranging American crows were stained using a rabbit-polyclonal anti-WNV antibody as well as a monoclonal antibody directed against an epitope on Domain III of the E protein of WNV. The staining intensity and the extent of staining were determined for each organ using both antibodies. Real-time RT-PCR on formalin-fixed paraffin-embedded tissues from all 85 crows was performed., Results: Forty-three crows were IHC-positive in at least one of the examined organs with the polyclonal antibody, and of these, only 31 were positive when IHC was performed with the monoclonal antibody. Real-time RT-PCR amplified WNV-specific sequences from tissue extracts of the same 43 crows that were IHC-positive using the polyclonal antibody. All other 42 crows tested negative for WNV with real-time PCR and IHC staining. Both antibodies had a test specificity of 100% when compared to PCR results. The test sensitivity of monoclonal antibody-based IHC staining was only 72%, compared to 100% when using the polyclonal antibody., Conclusion: The most sensitive, readily identified, positively staining organs for IHC are the kidney, liver, lung, spleen, and small intestine. Real-time RT-PCR and IHC staining using a polyclonal antibody on sections of these tissues are highly sensitive diagnostic tests for the detection of WNV in formalin-fixed tissues of American crows.

To better understand the transmission and epidemiology of human listeriosis, 647 Listeria monocytogenes isolates obtained from human listeriosis cases in four U.S. locations (Michigan, Ohio, New York State, and New York City) over 61 months (1998 to 2003) were characterized by automated EcoRI ribotyping. A total of 65 ribotypes were differentiated among the characterized isolates; 393, 227, and 24 isolates were classified into lineages I, II, and III, respectively, and 3 isolates were not classified to lineage. The three most common ribotypes (responsible for 39% of all cases) represented L. monocytogenes epidemic clones, each of which had previously been linked to at least two human listeriosis outbreaks. Categorical analyses revealed that ribotypes and lineages were nonrandomly distributed among the four locations. Temporal cluster analysis of cases identified 13 statistically significant temporal subtype clusters, which represented 26% of all cases. Three of these clusters matched previously described human listeriosis outbreaks. Isolates involved in clusters belonged to nine ribotypes. Four, eight, and one cluster were caused by lineages I, II, and III, respectively. The two largest clusters were both caused by the epidemic clone representing ribotype DUP-1044A. Categorical analyses revealed no significant associations between lineage or ribotype and clinical manifestation (central nervous system infection, septicemia, fetal infection, or other infection) or disease outcome (fatal or not fatal). Although human listeriosis cases are caused by isolates belonging to a diversity of EcoRI ribotypes, specific lineage I epidemic clones cause a large number of human listeriosis cases. Many human listeriosis cases can be grouped into statistically significant temporal clusters, including widely distributed and region-specific clusters associated with isolates of various ribotypes. L. monocytogenes lineages and EcoRI ribotypes do not appear to differ in their likelihood of causing different clinical manifestations or mortality.

The Michigan Department of Community Health (MDCH) reported 644 laboratory positive human cases of West Nile Virus (WNV) in the 2002 outbreak in the US, of which 559 cases presented with either meningitis or encephalitis. The first line test utilized for diagnosis of WNV infection was the immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (MAC-ELISA). We continued testing for WNV even during winter months of the year 2002-2003 due to the awareness of other modes of WNV transmission (blood transfusion, organ transplantation, transplacental, breast milk, and occupational) as well as concern for people traveling to endemic areas. As a result of year-round testing for WNV infections during 2002-2003, we detected WNV IgM-specific antibodies in cerebrospinal fluid (CSF) specimens from three patients persisting for 110, 141, and 199 days post acute phase infection in patients with central nervous system (CNS) disease. This is a new observation and there is no published data on the persistence of WNV IgM antibodies in CSF specimens beyond 47 days. Thus, it is important to note that the presence of WNV IgM class antibodies may not always reflect acute phase infection with this virus.

Since the initial report of West Nile virus in the northeastern United States in 1999, the virus has spread rapidly westward and southward across the country. In the summer of 2002, several midwestern states reported increased cases of neurologic disease and mortality associated with West Nile virus infection in various native North American owl species. This report summarizes the clinical and pathologic findings for 13 captive and free-ranging owls. Affected species were all in the family Strigidae and included seven snowy owls (Nyctea scandiaca), four great-horned owls (Bubo virginianus), a barred owl (Strix varia), and a short-eared owl (Asio flammeus). Neurologic signs identified included head tilt, uncoordinated flight, paralysis, tremors, and seizures. Owls that died were screened for flaviviral proteins by immunohistochemical staining of formalin-fixed tissues, followed by specific polymerase chain reaction assay to confirm West Nile virus with fresh tissues when available. Microscopic lesions were widespread, involving brain, heart, liver, kidney, and spleen, and were typically nonsuppurative with infiltration by predominantly lymphocytes and plasma cells. Lesions in owls were much more severe than those previously reported in corvids such as crows, which are considered highly susceptible to infection and are routinely used as sentinel species for monitoring for the presence and spread of West Nile virus. This report is the first detailed description of the pathology of West Nile virus infection in Strigiformes and indicates that this bird family is susceptible to natural infection with West Nile virus.

This report describes a new, inexpensive procedure for the rapid and efficient purification of Sarcocystis neurona sporocysts from opossum small intestine. S. neurona sporocysts were purified using a discontinuous potassium bromide density gradient. The procedure provides a source of sporocyst wall and sporozoites required for reliable biochemical characterization and for immunological studies directed at characterizing antigens responsible for immunological responses by the host. The examined isolates were identified as S. neurona using random amplified polymorphic DNA primers and restriction endonuclease digestion assays. This method allows the collection of large numbers of highly purified S. neurona sporocysts without loss of sporocyst viability as indicated by propidium iodide permeability and cell culture infectivity assays. In addition, this technique might also be used for sporocyst purification of other Sarcocystis spp.

Objective: To determine whether cats exposed at a residence were infected with Mycobacterium bovis, whether the tuberculin skin test can identify cats infected with M bovis, and whether an ELISA could identify tuberculosis-infected cats., Animals: 20 domestic cats exposed to a cat with laboratory-confirmed disseminated M bovis infection., Procedure: Cats were administered a tuberculin skin test and monitored for 72 hours. Blood and fecal samples were collected. Cats were then euthanatized, and postmortem examinations were performed. Tissues were examined grossly and histologically for signs of mycobacteriosis. Pooled tissue samples and fecal samples were submitted for mycobacterial culture. Blood samples were examined for evidence of tuberculosis by use of a comparative ELISA., Results: 4 cats had positive responses for the ELISA, and 2 cats had suspicious responses. All tuberculin skin tests yielded negative results. No gross or histologic lesions of tuberculosis were detected in any tissues, and mycobacteria were not isolated from tissues or feces obtained from the 20 cats., Conclusions and Clinical Relevance: All cats that had positive or suspicious responses for the ELISA were offspring of the cat with tuberculosis. Evidence of tuberculosis was not seen in other cats at the residence, the owner, or the attending veterinarian. The most likely source of tuberculosis for the infected cat was through the consumption of M bovis-infected wildlife carcasses or offal. Because M bovis is endemic in wildlife in northeastern Michigan, there is a risk of exposure to tuberculosis in companion animals, their owners, and attending veterinarians.

Although approximately 95% of disease caused by nontyphoidal salmonella is transmitted by foodborne vehicles, four documented salmonella outbreaks in the 1990s have been traced to contact with young poultry. No environmental studies of source hatcheries were completed. This case-control study was performed by comparing culture-confirmed Salmonella Infantis in Michigan residents, identified between May and July 1999, with two age- and neighbourhood-matched controls. Eighty environmental and bird tissue samples were collected from an implicated hatchery; all salmonella isolates underwent pulsed-field gel electrophoresis (PFGE) analysis. The study included 19 case-patients sharing the same PFGE subtype and 37 matched controls. Within 5 days before illness onset, 74% of case-patients resided in households raising young poultry compared with 16% of controls (matched OR 19.5; 95% CI 2.9, 378.1). Eight hatchery samples yielded Salmonella Infantis with PFGE subtypes matching the patients' isolates. This investigation identified birds from a single hatchery as the source of human illness and confirmed the link by matching PFGE patterns from humans, birds and the hatchery environment. Subsequent public health interventions reduced, but did not eliminate, transmission of poultry-associated salmonellosis. Five additional PFGE-linked cases were identified in Spring 2000, necessitating quarantine of the hatchery for depopulation, cleaning and disinfection.

A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.

Background: Bronchoscopy-related transmission of Mycobacterium tuberculosis is rarely reported. In August 1999, five M tuberculosis-positive bronchial washing culture findings were noted in patients who underwent bronchoscopy in July in a hospital that reported only eight M tuberculosis-positive culture findings from 1995 to 1998, prompting further investigation., Methods: A case was defined as a M tuberculosis-positive culture finding from specimens obtained from patients who underwent bronchoscopy during January to August of 1999. Bronchoscopy and laboratory records, procedures, and practices were reviewed. M tuberculosis isolates were compared using restriction fragment length polymorphism (RFLP) analysis., Results: During July 1999, 19 bronchoscopic procedures were performed in 19 patients. Bronchial washing specimens for mycobacterial culture were obtained from 18 patients. Ten cases were identified. Two case patients, including the index patient, had signs and symptoms of active tuberculosis prior to bronchoscopy. M tuberculosis infections developed in two more case patients despite starting a standard four-drug antituberculous regimen within 3 weeks after bronchoscopy. Six case patients had positive culture findings but no evidence of infection. All M tuberculosis isolates were antituberculosis-drug susceptible, and all but one were indistinguishable by RFLP analysis. Three bronchoscopes were used during the outbreak period; one bronchoscope was used in 9 of the 10 case patients (relative risk, 8.1; 95% confidence interval, 1.3 to 52). A hole was discovered in the sheath of this bronchoscope. Leak testing, a critical step in bronchoscope reprocessing, was not routinely performed at this institution., Conclusions: M tuberculosis contamination of the bronchoscope occurred during the index patient's procedure. The hole in the sheath provided access to a space that was difficult to mechanically clean and chemically disinfect. The reprocessing recommendations of bronchoscope manufacturers, including leak testing after each use, should be closely followed.

Objectives: To determine the cause of a coagulase-negative staphylococcal outbreak and to identify risk factors for surgical-site infections among patients following Medtronic Freestyle bioprosthesis implantation., Design: Retrospective case-control study., Setting: An 800-bed university referral center., Patients: The cohort of 64 patients undergoing Freestyle valve replacement from September 1998 to December 1998., Results: Seven patients developed infection (10.9% vs 1.1% during the preceding 8 months), including two with mediastinitis and five with endocarditis. There were no statistically significant differences between cases and controls with respect to age, gender, weight, underlying illness, preoperative hospital stay, duration of surgery, time on bypass, central venous catheter duration, National Nosocomial Infection Surveillance risk index, New York Heart Association class, albumin, or antibiotic prophylaxis. However, only three cases were documented to have received vancomycin prophylaxis. Of all staff evaluated, only surgical resident A was significantly associated with infection (odds ratio, 7.68; 95% confidence interval, 1.3-44.1; P=.02) Pulsed-field gel electrophoresis patterns on Staphylococcus epidermidis isolates from four of the six cases were identical. These cases were performed on different days. Surgical resident Awas the only staff member present in the operating room for all cases caused by the epidemic strain. This S epidermidis strain, however, was not isolated from operating room staff., Conclusion: A surgical resident was significantly associated with infection. However, the cause of this outbreak was likely multifactorial. Changes occurring during the investigation included institution of vancomycin as routine prophylaxis and modification of surgical technique, which contributed to the resolution of the outbreak.