Low temperature and dust favour in vitro survival of Mycoplasma hyopneumoniae: time to revisit indirect transmission in pig housing.

Porcine enzootic pneumonia (EP) caused by Mycoplasma hyopneumoniae adversely affects pig welfare and is associated with major economic losses in the pig industry worldwide. Transmission is predominantly by direct contact, but the role of indirect transmission remains poorly understood. This study examined survival of six M. hyopneumoniae isolates dried onto five different surfaces encountered in pig units and exposed to temperatures of 4, 25 and 37°C for up to 12 days. Survival of the organisms was determined by recovering the organism from the surface material and culturing in Friis broth. Data were analysed by logistic regression to identify factors influencing survival of M. hyopneumoniae. Maximum survival was 8 days for all isolates on at least one surface (except stainless steel) at 4°C and was limited to 2 days at 25 and 37°C. Overall, dust and polypropylene copolymer supported M. hyopneumoniae survival the longest when compared with other surface materials. In conclusion, we have demonstrated that M. hyopneumoniae can survive outside the host for at least 8 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the transmission of Mycoplasma hyopneumoniae and optimizing biosecurity practices are keys to reducing the use of antimicrobial agents to control this pathogen. Direct transmission of the pathogen between pigs is the main route of spread and its lack of cell wall may compromise its resilience outside the host. The results from our study show that M. hyopneumoniae can survive for up to several days on dry surfaces and therefore may have the potential to infect pigs by indirect transmission. Factors influencing the survival of M. hyopneumoniae outside the host are further elucidated.

Development of a skin colonization model in gnotobiotic piglets for the study of the microbial ecology of meticillin-resistant Staphylococcus aureus ST398.

AIMS: Meticillin-resistant Staphylococcus aureus (MRSA) ST398 continues to spread amongst pigs and other domestic animals and man. This highlights the need for models to examine MRSA colonization and investigate control strategies. This study aimed to develop a gnotobiotic pig model and assess the potential of bacterial interference from selected coagulase-negative staphylococci (CNS) against MRSA ST398. METHODS AND RESULTS: Groups of 2-week-old piglets were atraumatically inoculated either with MRSA and/or CNS. Skin and mucosae were swabbed, and bacterial counts compared over a period of 21 days. Piglets developed healthily, and bacterial populations increased similarly for both MRSA and CNS until day 32. On day 37, MRSA counts in groups with CNS reduced significantly compared with MRSA alone (P = 0·03). CONCLUSIONS: The results showed that inoculation of piglet skin with MRSA resulted in spontaneous colonization and that MRSA ST398 has a low pathogenic potential in gnotobiotic piglets. Quantitative bacteriology indicated that initial MRSA colonization was unaffected by concurrent CNS colonization but that interference may occur over a longer period. SIGNIFICANCE AND IMPACT OF THE STUDY: Gnotobiotic piglets provide a reproducible model suitable for bacterial interference studies, which should be further explored as an alternative to antimicrobials in the control of MRSA.

Prevalence of and risk factors for MRSA carriage in companion animals: a survey of dogs, cats and horses.

We investigated the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) carriage in a convenience sample of purposely selected populations of dogs, cats and horses in the Greater London area. Swabs from carriage sites were pooled, enriched and processed by standard bacteriological methods. The presence of nuc and mecA was confirmed for MRSA. Risk factors were investigated among veterinary treatment group animals using exact logistic regression analysis. Twenty-six (1.53%) MRSA carriers were identified in the 1692 animals (15/704 dogs, 8/540 cats, 3/152 horses). Animals presenting for veterinary treatment more frequently carried MRSA than healthy animals (OR 7.27, 95% CI 2.18-24.31, P<0.001). Concurrent carriage of non-MRSA coagulase-positive staphylococci was associated with MRSA carriage (OR 0.088, 95% CI 0.016-0.31, P<0.001); none of the other 13 putative risk factors was significant. MRSA carriage was rare in the selected companion animal populations. The absence of typical risk factors indicates that companion animals act as contaminated vectors rather than as true reservoirs.

Companion animals: a reservoir for methicillin-resistant Staphylococcus aureus in the community?

This article reviews the literature on the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in dogs, cats and horses. Over the past 10 years, MRSA has emerged as an important pathogen in veterinary medicine, especially in countries with a high MRSA burden in human hospitals. During the same period, community-associated MRSA (CA-MRSA) infections in humans without apparent links to healthcare facilities have increased dramatically. Although animal infections occur outside human hospitals, significant epidemiological, clinical and genetic differences exist between CA-MRSA in humans and the majority of MRSA infections in the different animal species. The recognition of MRSA in animals has raised concern over their role as potential reservoirs or vectors for human MRSA infection in the community. However, available data on MRSA transmission between humans and companion animals are limited and the public health impact of such transmission needs to be the subject of more detailed epidemiological studies.

In vitro activity of fusidic acid and mupirocin against coagulase-positive staphylococci from pets.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) and multiresistant Staphylococcus pseudintermedius (MRSP) have emerged as important pathogens in animal infections. Associated therapeutic problems and the zoonotic potential of staphylococci have renewed interest in topical antibiotics for treatment and carrier decolonization. Fusidic acid and mupirocin are used topically in humans and animals but resistant strains isolated from people are increasing. This study investigates the in vitro activity of fusidic acid and mupirocin against coagulase-positive staphylococci from pets. METHODS: A collection of 287 staphylococci was examined, comprising 102 MRSA, 102 methicillin-susceptible S. aureus, 71 S. pseudintermedius and 12 MRSP from canine and feline infections and carrier sites isolated in the UK and Germany. MICs were determined by the agar dilution method according to CLSI (formerly NCCLS) standards. RESULTS: The majority (89.7%) of all MICs were

What has changed in canine pyoderma? A narrative review.

Canine pyoderma is a common presentation in small animal practice and frequently leads to prescription of systemic antimicrobial agents. A good foundation of knowledge on pyoderma was established during the 1970s and 1980s, when treatment of infection provided relatively few challenges. However, the ability to treat canine pyoderma effectively is now limited substantially by the emergence of multidrug-resistant, methicillin-resistant staphylococci (MRS) and, in some countries, by restrictions on antimicrobial prescribing for pets. The threat from rising antimicrobial resistance and the zoonotic potential of MRS add a new dimension of public health implications to the management of canine pyoderma and necessitate a revisit and the search for new best management strategies. This narrative review focusses on the impact of MRS on how canine pyoderma is managed and how traditional treatment recommendations need to be updated in the interest of good antimicrobial stewardship. Background information on clinical characteristics, pathogens, and appropriate clinical and microbiological diagnostic techniques, are reviewed in so far as they can support early identification of multidrug-resistant pathogens. The potential of new approaches for the control and treatment of bacterial skin infections is examined and the role of owner education and hygiene is highlighted. Dogs with pyoderma offer opportunities for good antimicrobial stewardship by making use of the unique accessibility of the skin through cytology, bacterial culture and topical therapy. In order to achieve long term success and to limit the spread of multidrug resistance, there is a need to focus on identification and correction of underlying diseases that trigger pyoderma in order to avoid repeated treatment.

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

Alopecia in pomeranians and miniature poodles in association with high urinary corticoid:creatinine ratios and resistance to glucocorticoid feedback.

The adrenocortical function of pomeranians and miniature poodles with alopecia was tested by serial measurements of the urinary corticoid:creatinine ratio (uccr) and by an oral low-dose dexamethasone suppression test (lddst) and uccr measurements. In most of the dogs there was day-to-day variation in the uccrs of the 10 sequential urine samples, often with values above or below the upper limit of the range of healthy control dogs. In 22 alopecic pomeranians the basal uccrs were significantly higher than in 18 non-alopecic pomeranians, and the values of both groups were significantly higher than those of 88 healthy pet dogs. The uccrs of 12 alopecic miniature poodles were significantly higher than those of healthy dogs. In 12 alopecic pomeranians and eight alopecic miniature poodles the oral lddst revealed increased resistance to dexamethasone. In six non-alopecic pomeranians the uccrs after the administration of dexamethasone were not significantly different from those in seven healthy dogs at the same time. In an oral high-dose dexamethasone suppression test, using 0.1 mg dexamethasone/kg bodyweight, the uccrs of seven alopecic pomeranians and five alopecic miniature poodles decreased to low levels.

Antimicrobial use Guidelines for Treatment of Respiratory Tract Disease in Dogs and Cats: Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Diseases.

Respiratory tract disease can be associated with primary or secondary bacterial infections in dogs and cats and is a common reason for use and potential misuse, improper use, and overuse of antimicrobials. There is a lack of comprehensive treatment guidelines such as those that are available for human medicine. Accordingly, the International Society for Companion Animal Infectious Diseases convened a Working Group of clinical microbiologists, pharmacologists, and internists to share experiences, examine scientific data, review clinical trials, and develop these guidelines to assist veterinarians in making antimicrobial treatment choices for use in the management of bacterial respiratory diseases in dogs and cats.

Total and relative deficiency of gut mucosal IgA in German shepherd dogs demonstrated by faecal analysis.

The concentration of immunoglobulins in faecal extracts was investigated as a method of assessing the production of immunoglobulins by the gut mucosa of 137 dogs. There were significant correlations between the concentrations in faecal extracts and the concentrations produced in duodenal organ cultures. Seventy-six German shepherd dogs had significantly lower median immunoglobulin A (IgA) concentrations in their faecal extracts than 63 controls of various breeds. Sixteen of the German shepherd dogs had IgA concentrations below the 95 per cent confidence limit of the control population and six had no demonstrable faecal IgA. The faecal concentrations of immunoglobulin G and albumin were significantly higher in the German shepherd dogs than in the controls, but their immunoglobulin M concentrations were similar.

The use of trilostane for the treatment of alopecia X in Alaskan malamutes.

Three Alaskan malamutes with hair loss and slightly elevated blood concentrations of 17-hydroxyprogesterone after stimulation with adrenocorticotropic hormone (ACTH) were treated with trilostane. Trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, was given twice daily at a dose of 3.0 to 3.6 mg/kg per day orally for 4 to 6 months. Routine ACTH stimulation tests were performed over 8 months to evaluate the degree of adrenal function suppression. Treatment with trilostane led to complete hair regrowth in all three dogs within 6 months. No adverse effects associated with trilostane were recognized.

Survey of dermatophytes on clinically normal cats in the southeast of England.

OBJECTIVES: To report the incidence of dermatophytes on the hair coat of asymptomatic cats in the southeast of England. Asymptomatic cats are often blamed for transmission of dermatophytes between animals and humans. This study may help to clarify whether cats are responsible for the increase in fungal infections among the human population. METHODS: A total of 169 clinically healthy cats without any dermatological signs were sampled using the Mackenzie brush technique and cultured for dermatophytes. Thirty cats were from a closed colony and 139 were feral or from domestic households in the southeast of England. RESULTS: The incidence of Microsporum canis and Trichophyton mentagrophytes in household and feral cats was 2.16 per cent for each dermatophyte. This survey shows little difference in the isolation rates of M. canis between the southeast and southwest of England, which was reported on in 1994. CLINICAL SIGNIFICANCE: Given the low number of dermatophytes isolated, asymptomatic cats are unlikely to be responsible for the increasing incidence of human infection. Asymptomatic carriers with T. mentagrophytes in the hair coat have not been previously reported and may need to be considered when treating humans with trichophytosis.

Increasing antimicrobial resistance in clinical isolates of Staphylococcus intermedius group bacteria and emergence of MRSP in the UK.

Frequencies of antimicrobial resistance were determined amongst 14,555 clinical Staphylococcus intermedius group (SIG) isolates from UK dogs and cats to estimate resistance trends and quantify the occurrence of meticillin-resistant Staphylococcus pseudintermedius (MRSP). Reports from two diagnostic laboratories (13,313 general submissions, 1242 referral centre only submissions) were analysed retrospectively (2003/2006-2012). MRSP were defined by phenotypic resistance to meticillin and concurrent broad ß-lactam resistance; a subset was confirmed genetically (SIG-specific nuc and mecA). Trends were analysed by Cochran-Armitage test. Resistance remained below 10 per cent for cefalexin, amoxicillin-clavulanic acid and the fluoroquinolones. Increasing resistance trends were seen in both laboratories for ampicillin/amoxicillin (both P<0.001), cefovecin (both P<0.046) and enrofloxacin (both P<0.02). Resistance to cefalexin increased over time in referral hospital isolates (P<0.001) to clindamycin (P=0.01) and trimethoprim-sulfamethoxazole (P=0.001) amongst general laboratory submissions. Overall, 106 MRSP were isolated (0.7 per cent of submissions) including 32 (2.6 per cent of submissions, all genetically confirmed) from the referral centre population (inter-laboratory difference P<0.001). Against a background of widely susceptible SIG isolates, a new trend of increasing resistance to important antimicrobials was identified overtime and the emergence of MRSP from UK clinical cases was confirmed. Attention to responsible use of antibacterial therapy in small animal practice is urgently needed.

Synthesis and serotonin receptor affinities of a series of enantiomers of alpha-methyltryptamines: evidence for the binding conformation of tryptamines at serotonin 5-HT1B receptors.

A procedure for the preparation of optically pure alpha-methyltryptamines (AMTs) from substituted indoles was developed. The key step in the sequence was the reductive amination of substituted indole-2-propanones with the commercially available pure enantiomers of alpha-methylbenzylamine, followed by the chromatographic separation of the resulting pair of diastereomeric amines by preparative centrifugal (Chromatotron) chromatography. Catalytic N-debenzylation then afforded the pure AMT enantiomers. Optical purity was established by chiral HPLC analysis of the 2-naphthoylamide derivatives. An improved procedure for the preparation of indole-2-propanones was also developed. To probe structure-activity relationships of serotonin receptors, affinities of the alpha-methyltryptamine enantiomers were then measured at the 5-HT2 antagonist receptor subtype, with displacement of [3H]ketanserin, and were estimated at the 5-HT1B receptor, with displacement of [3H]serotonin, respectively, in rat frontal cortex homogenates. Enantioselectivity at the receptor subtypes varied, depending on aromatic substituents. For a 5-hydroxy or 5-methoxy, the S enantiomer had higher affinity or was equipotent to the R enantiomer. This selectivity at [3H]serotonin binding sites was reversed for 4-oxygenated alpha-methyltryptamines, where a 4-hydroxy or 4-methoxy did not enhance affinity over the unsubstituted compounds. These results can be explained, for the [3H]serotonin displacement data, if the binding conformation is one where the ethylamine side chain is trans and lying in a plane perpendicular to the indole ring plane.

Effects of certain hallucinogenic amphetamine analogues on the release of [3H]serotonin from rat brain synaptosomes.

The enantiomers of 3,4-(methylenedioxy)amphetamine (MDA), p-methoxyamphetamine (PMA), and N-Me-MDA (MDMA), along with their alpha, alpha-dimethylated derivatives, were evaluated for an effect on the release of [3H]serotonin from rat whole brain synaptosomes. The amphetamine isomers were all potent in inducing the release of [3H]serotonin at bath concentrations of 1 and 10 micrometers but were inactive at 0.1 micrometers. No significant difference in isomer potency was observed at the 10 micrometers concentration. However, at 1 micrometer the (+) isomer of MDMA was more effective in inducing release than was the (-) isomer. Since it is the (+) isomer which is clinically active, this result suggests that transmitter release may play a role in the biological activity of MDMA. By contrast, the alpha, alpha-dimethyl compounds were not effective in releasing serotonin, even at the highest bath concentration.

Characterisation of Staphylococcus intermedius isolates from canine pyoderma and from healthy carriers by SDS-PAGE of exoproteins, immunoblotting and restriction endonuclease digest analysis.

Ten Staphylococcus intermedius isolates from cases of canine pyoderma and 10 from healthy carriers were examined by SDS-PAGE of exoproteins, immunoblotting and restriction endonuclease digest analysis. Similarities between banding patterns of the isolates were calculated as Dice coefficients for all three methods. For SDS-PAGE and immunoblotting, no significant differentiation was found between the pyoderma and "healthy" groups. Analysis of DNA digested with BglII indicated that S. intermedius is genetically heterogeneous; Dice coefficients for the pyoderma group were distinct from those for the healthy group (p < 0.001), and cluster analysis confirmed that the pyoderma isolates (9) formed a group separate from the majority (6 of 9) of the normal isolates.

Capillary gel electrophoresis and antisense therapeutics. Analysis of DNA analogs.

Capillary gel electrophoresis (CGE) has become an effective tool for the analysis of antisense oligonucleotides. As these compounds begin to show promise in the pharmaceutical field, CGE is often used to determine the quality of chemically synthesized DNA analogs, which are presently being studied as potential antisense therapeutics. The demand for gel capillaries to process high resolving power and provide statistically meaningful data has indirectly provided a better understanding of what is required to denature single-stranded oligonucleotides. For CGE to be useful for the analysis of oligonucleotides in general, an internal standard is often employed; however, apart from being a strictly quantitative tool, CGE has the capability to be useful in a wide range of applications within the field of antisense therapeutics. CGE can be used in conjunction with HPLC to determine an effective method for the purification of crude oligonucleotide solutions. It has also proven useful in determining whether or not a DNA analog can promote the ribonuclease H-mediated hydrolysis of RNA. An understanding of the interactions between antisense oligonucleotides and nucleases in general is critical for determining how antisense oligonucleotides function within a biological system.

Cytotoxic activity of Staphylococcus hyicus.

Culture supernatants from a number of Staphylococcus hyicus strains caused toxic effects to both murine fibroblast and porcine keratinocyte cells in culture. The extent of cytotoxicity was shown to differ between strains and may provide an indication of strain virulence. Purification of cytotoxic activity produced by S. hyicus (strain P119) using preparative isoelectric-focussing demonstrated it to be cytolytic, haemolytic and non-proteolytic. The cytotoxin demonstrates certain properties in common with the delta haemolysin of Staphylococcus aureus.

Sequential production of specific antibodies in serum and at the skin surface of cattle following intradermal vaccination with Dermatophilus congolensis.

Studies of the temporal changes in specific antibody titres to D. congolensis in serum and washings from inoculated and uninoculated skin indicate that production of IgG1 and IgG2 antibody at the skin surface is a transudative process related to serum antibody concentration. Transport of IgM is likely to be based on a similar mechanism but that of IgA appears to depend on a local secretory process.

Canine atopic disease: the prevalence of positive intradermal skin tests at two sites in the north and south of Great Britain.

Results of intradermal skin test responses to the same panel of 53 allergens were compared in 118 dogs with atopic disease presented at two geographical centres, Edinburgh (87 cases) and London (31 cases). The allergens most commonly positive at both centres were human dander and Dermatophagoides farinae, but positive tests to all of the allergens used occurred in at least one case. The mean number of allergens to which positive tests resulted in atopic dogs was 5.126 (Edinburgh) and 5.129 (London). The majority of animals were sensitive to allergens from more than one group. A significantly higher number of positive reactors to house dust allergen was observed at London than at Edinburgh (P < 0.05), while a significantly higher number of positive reactions to grass pollens was detected at Edinburgh than in London (P < 0.05). Sensitivity to Dermatophagoides pteronyssinus, in the absence of sensitivity to D. farinae, was uncommon and therefore both of these mite allergens should be incorporated in intradermal skin testing panels in Great Britain.