Isolation and characterization of staphylococci from external auditory meatus of dogs with or without otitis externa with special reference to Staphylococcus schleiferi subsp. coagulans isolates

Staphylococci were isolated from the external auditory meatus in 14 (48.3%) of 29 dogs affected with otitis externa (OE dogs) and 28 (68.3%) of 41 dogs without OE (non-OE dogs). Twenty-two OE isolates were identified as belonging to 12 species, and 42 non-OE isolates were identified as belonging to 13 species. The predominant species found in both OE and non-OE isolates were S. intermedius, and S. epidermidis. Thirty-eight (59.4%) of 64 isolates were resistant to one or more of the 17 antimicrobial agents tested. Resistance to PCG and ABPC was most frequent. S. schleiferi subsp. coagulans, a recent etiologic agent of canine OE, was isolated from OE and non-OE dogs. All of the 5 S. schleiferi subsp. coagulans isolates showed typical characteristics. No clear difference in the extracellular enzyme or toxin profiles, nor in the PFGE patterns, was demonstrated between the OE and non-OE isolates of S. schleiferi subsp. coagulans. A new PCR primer set specific for 16S rDNA was designed to identify strains of S. schleiferi subsp. coagulans. The amplified fragment was detected in all of the 5 isolates as well as the type strain GA 211 (=JCM 7470) and a reference strain GA 11, but was not detected in any strains of the related species, S. aureus, S. intermedius and S. hyicus. The PCR may allow a simple, rapid and precise identification of S. schleiferi subsp. coagulans, in addition to the standard tube test for free coagulase.     

Microbiological features of Staphylococcus schleiferi subsp. coagulans, isolated from dogs and possible misidentification with other canine coagulase-positive staphylococci

Staphylococcus schleiferi subsp. coagulans has only rarely been isolated and identified from the external auditory meatus of dogs suffering from external otitis. Its morphological and basic biochemical characteristics are of relatively little value for identification, as it phenotypically resembles another coagulase-positive staphylococci (CPS) and, consequently, may be easily misidentified as S. intermedius or even as S. aureus. In the present work, differentiation of S. schleiferi ssp. coagulans was therefore based on specific biochemical and genetic methods. All the strains were evaluated with the following commercial methods: Api Staph System (bioMérieux, Marcy l'Etoil, France), BBL Crystal Identification Systems (Gram-Positive ID Kit and Rapid Gram-Positive ID Kit; Becton Dickinson), and GEN-PROBE AccuProbe, Staphylococcus aureus identification test (bioMérieux). Gram-Positive ID System/GP database includes the broadest range of staphylococcal species and correctly identifies the majority of strains important in veterinary medicine. Therefore, it is an acceptable alternative to conventional methods for identification of canine staphylococcal isolates. Reliable differentiation of S. aureus from S. schleiferi ssp. coagulans and S. intermedius was feasible with AccuProbe for S. aureus, which gave positive results only for S. aureus; all other CPS tested were negative.     

Microbiological Features of Staphylococcus schleiferi subsp. coagulans,Isolated from Dogs and Possible Misidentification with Other Canine Coagulase‐positive Staphylococci

Staphylococcus schleiferi subsp. coagulans has only rarely been isolated and identified from the external auditory meatus of dogs suffering from external otitis. Its morphological and basic biochemical characteristics are of relatively little value for identification, as it phenotypically resembles another coagulase‐positive staphylococci (CPS) and, consequently, may be easily misidentified as S. intermedius or even as S. aureus. In the present work, differentiation of S. schleiferi ssp. coagulans was therefore based on specific biochemical and genetic methods. All the strains were evaluated with the following commercial methods: Api Staph System (bioMérieux, Marcy l'Etoil, France), BBL Crystal Identification Systems (Gram‐Positive ID Kit and Rapid Gram‐Positive ID Kit; Becton Dickinson), and GEN‐PROBE^® AccuProbe, Staphylococcus aureus identification test (bioMérieux). Gram‐Positive ID System/GP database includes the broadest range of staphylococcal species and correctly identifies the majority of strains important in veterinary medicine. Therefore, it is an acceptable alternative to conventional methods for identification of canine staphylococcal isolates. Reliable differentiation of S. aureus from S. schleiferi ssp. coagulans and S. intermedius was feasible with AccuProbe for S. aureus, which gave positive results only for S. aureus; all other CPS tested were negative.     

Antimicrobial resistance in staphylococci from animals with particular reference to bovine Staphylococcus aureus, porcine Staphylococcus hyicus, and canine Staphylococcus intermedius.

Besides their role as commensals on the skin and mucosal surfaces, staphylococci may be involved in a wide variety of diseases in animals. Staphylococcal infections in animals are mainly treated with antimicrobial agents and as a consequence, staphylococci from animal sources have developed and/or acquired resistance to the respective antimicrobial agents. Resistance statistics obtained from national monitoring programmes on staphylococci from cattle and pigs, but also from surveillance studies on staphylococci involved in diseases in dogs are reported and reviewed with regard to their comparability. This review mainly focusses on the genetic basis of antimicrobial resistance in staphylococci of animal origin. Particular attention is paid to resistance to those antimicrobial agents which are most frequently used in veterinary medicine, but also to antimicrobial agents, such as chloramphenicol and mupirocin, which are used in specific cases for the control of staphylococcal infections in pets and companion animals. In addition, plasmids and transposons associated with the respective resistance properties and their ways of spreading between members of the same or different staphylococcal species, but also between staphylococci and other gram-positive bacteria, are described.     

Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections

Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCR-RFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0% (200/263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0% (255/263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system.     

Paratuberculosis with special reference to cattle. A review

Paratuberculosis is a chronic, granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis affecting domestic and wild ruminants. The symptoms of clinical paratuberculosis are chronic diarrhoea and progressive weight loss while subclinically infected animals mainly have decreased production. The infection is widespread throughout the world and causes substantial financial losses for the farming industry. One of the major obstacles in the control of this disease, is the difficulty of identifying subclinically infected animals. This review gives a summary of several aspects of paratuberculosis including clinical importance, pathology, immunology and properties of the infectious agent. Special emphasis will be on the available diagnostic methods, their use and limitations.     

Surveillance of healthy cats and cats with inflammatory skin disease for colonization of the skin by methicillin-resistant coagulase-positive staphylococci and Staphylococcus schleiferi ssp. schleiferi

In this study, bacterial cultures were collected from five sites on each of 50 healthy cats and 48 cats with inflammatory skin disease (ISD), to determine prevalence of carriage and relative frequency of methicillin resistance in coagulase-positive staphylococci and Staphylococcus schleiferi ssp. schleiferi. Latex agglutination testing for penicillin-binding protein 2a (PBP2a) and pulsed field gel electrophoresis (PFGE) were performed on all methicillin-resistant (MR) isolates. Polymerase chain reaction (PCR) for the mecA gene was performed on MR S. intermedius and S. schleiferi isolates. Staphylococcal chromosomal cassette (SCCmec) typing was performed on all MR S. aureus isolates. Coagulase-positive staphylococci and S. schleiferi ssp. schleiferi were isolated from 24 of 48 cats with ISD: Staphylococcus aureus (14 of 24, 58%), Staphylococcus intermedius (11 of 24, 46%), Staphylococcus schleiferi ssp. schleiferi (1 of 24, 4%), and Staphylococcus hyicus (1 of 24, 4%). Prevalence of MR was 7% for S. aureus, 0% for S. intermedius, 100% for S. schleiferi ssp. schleiferi, and 0% for S. hyicus. Coagulase-positive staphylococci were isolated from 17 of 50 healthy cats: S. aureus (10 of 17, 59%), S. intermedius (11 of 17, 65%), and S. schleiferi ssp. coagulans (1 of 17, 6%). Prevalence of MR was 20% for S. aureus, 18% for S. intermedius, and 0% for S. schleiferi ssp. coagulans. All MR isolates were positive for PBP2a via latex agglutination. Methicillin-resistant S. intermedius and S. schleiferi ssp. schleiferi isolates were also positive for the mecA gene via PCR. Methicillin-resistant S. aureus isolates were identified as SCCmec type II. Results of PFGE indicated heterogeneity among isolates. There was no significant difference in staphylococcal isolation or methicillin resistance between study groups. While present, MR coagulase-positive staphylococci are significantly less common in these study populations.     

Electron microscopic observations on canine mixed mammary tumors, with special reference to cytoplasmic filamentous components

Three chief cell types were studied in 8 canine mixed mammary tumors--luminal epithelial cells, myoepithelial cells, and filament cells, the last being the "stellate" cells recognized by light microscopists. Most cytoplasmic filaments in the myoepithelial cells were 5 to 8 nm thick, whereas another type of filaments were scattered, 8 to 10 nm thick. Almost all cytoplasmic filaments in the filament cells were 8 to 10 nm thick. In the adenomatous parts of the tumors, myoepithelial cells were chiefly seen, along with luminal epithelial cells. Filament cells were rarely observed without intercellular accumulation of mucoid. The filament cells were largely confined to the myxomatous and chondromatous parts of the tumors. The presence of cells of transitional forms in adenomatous areas suggested that the filament cells were derived from the myoepithelial cells.     

Immunology of helminth infections, with special reference to immunopathology.

Immune responses resulting in immunity to infection or disease, share the same basic humoral and cellular mechanisms. While immunity to helminth infection has evolved to mediate rapid elimination of the parasite, the strategies evolved by the parasites themselves aim to delay this rejection process and ensure the survival and distribution of their progeny. Ineffective or incomplete immunity results in persistence of parasites or their products within the host tissues, inappropriate or chronic stimulation by parasite antigens, hyper-reactivity and tissue damage or immunopathology. A long standing classification by Gell and Coombs identifies four major types of hypersensitivity responses accounting for most of the immunopathogenesis, three of which are mediated by antibody and one, delayed type hypersensitivity (DTH), by T cells. This paper aims to give a short review of these four classical hypersensitivity reactions with particular reference to infections of large animals with helminth parasites. In addition, in view of the functionally different helper T cell subsets now identified, the existing DTH response is redefined as DTH Type 1 (Th-1 mediated) and two new classes of T cell-dependent DTH responses are proposed; DTH Type II, associated with the Th-2 type cytokines IL-4 and IL-5 and eosinophilic granuloma formation, and DTH Type III, associated with IL-4 and TGF-beta and fibrosis. Finally, some implications of immunopathology on parasite control strategies are discussed.