Antimicrobial Resistance and Distribution of Staphylococcus spp. Pulsotypes Isolated from Goat and Sheep Bulk Tank Milk in Southern Spain.

Bulk tank milk from 58 dairy goat and sheep flocks located in southern Spain was examined to determine the prevalence and distribution of Staphylococci. A total of 45 isolates were obtained and characterized to determine the species, antimicrobial resistance profile, and genetic similitude by pulse-field gel electrophoresis (PFGE) using SmaI. Staphylococcus aureus isolates were confirmed by polymerase chain reaction (PCR) analysis of nuc, and resistance to methicillin was determined by PCR analysis of mecA. A total of 10 different staphylococcal species were identified, 22.2% and 77.8% of which were coagulase positive and negative, respectively. Twenty-two (48.89%) isolates were resistant to at least one antimicrobial agent. Higher antimicrobial resistance values were obtained against tetracycline (28.9%) and penicillin (22.2%). Two isolates (S. aureus and Staphylococcus lentus) were resistant to cefoxitin; however, none of the 45 isolates harbored mecA. Thirty pulsotypes were detected by PFGE. Interestingly, some isolates of S. aureus, S. lentus, Staphylococcus simulans, and Staphylococcus caprae showed high genetic similarity (>80%). These data suggest that genetically similar staphylococcal isolates circulate among goat and sheep dairy herds, and their different resistance patterns could be influenced by the management systems used.

Distribution and tracking of Clostridium difficile and Clostridium perfringens in a free-range pig abattoir and processing plant.

The presence and genetic diversity of Clostridium difficile and C. perfringens along the slaughtering process of pigs reared in a free-range system was assessed. A total of 270 samples from trucks, lairage, slaughter line and quartering were analyzed, and recovered isolates were toxinotyped and genotyped. C. difficile and C. perfringens were retrieved from 14.4% and 12.6% of samples, respectively. The highest percentage of positive samples for C. difficile was detected in trucks (80%) whereas C. perfringens was more prevalent in cecal and colonic samples obtained in the slaughter line (85% and 45%, respectively). C. difficile isolates (n = 105) were classified into 17 PCR ribotypes (including 010, 078, and 126) and 95 AFLP genotypes. C. perfringens isolates (n = 85) belonged to toxinotypes A (94.1%) and C (5.9%) and were classified into 80 AFLP genotypes. The same genotypes of C. difficile and C. perfringens were isolated from different pigs and occasionally from environmental samples, suggesting a risk of contaminated meat products.

Comparative immunosecretome analysis of prevalent Streptococcus suis serotypes.

Streptococcus suis is a major Gram-positive swine pathogen associated with a wide variety of diseases in pigs. The efforts made to develop vaccines against this pathogen have failed because of lack of common cross-reactive antigens against different serotypes. Nowadays the interest has moved to surface and secreted proteins, as they have the highest chances to raise an effective immune response because they are in direct contact with host cells and are really exposed and accessible to antibodies. In this work, we have performed a comparative immunosecretomic approach to identify a set of immunoreactive secreted proteins common to the most prevalent serotypes of S. suis. Among the 67 proteins identified, three (SSU0020, SSU0934, and SSU0215) were those predicted extracellular proteins most widely found within the studied serotypes. These immunoreactive proteins may be interesting targets for future vaccine development as they could provide possible cross-reactivity among different serotypes of this pathogen.

Survival of selected foodborne pathogens on dry cured pork loins.

The safety of ready-to-eat products such as cured pork loins must be guaranteed by the food industry. In the present study, the efficacy of the dry curing process of pork loins obtained from free-range pigs in the reduction of three of the most important foodborne pathogens is analysed. A total of 28 pork loin segments, with an average weight of 0.57±0.12kg, were divided into four groups with three being inoculated by immersion with 7logCFU/ml of either Salmonella Typhimurium, Campylobacter coli or Listeria innocua and the last one inoculated by immersion with sterile medium (control group). The loin segments were treated with a seasoning mixture of curing agents and spices, packed in a synthetic sausage casing and cured for 64days. Microbiological analysis, pH and water activity (aw) were assessed at four stages. The values of pH and aw decreased with curing time as expected. S. Typhimurium and C. coli dropped significantly (3.28 and 2.14 log units, respectively), but limited reduction of L. innocua (0.84 log unit) was observed along the curing process. In our study, three factors were considered critical: the initial concentration of the bacteria, the progressive reduction of pH and the reduction of aw values. Our results encourage performing periodic analysis at different stages of the manufacturing of dry cured pork loins to ensure the absence of the three evaluated foodborne pathogens.

Multi-Etiological Nature of Tuberculosis-Like Lesions in Condemned Pigs at the Slaughterhouse.

Tuberculosis-like lesions (TBL) in pigs have been associated with microorganisms other than mycobacteria. In this work a histopathological and microbiological evaluation of TBL in pigs is shown. A total of 352 samples belonging to 171 pigs totally condemned at slaughterhouse due to generalized TBL were sampled and selected for analysis. Pyogranulomatous (56.2%) and granulomatous lesions (20.2%) were observed in all analysed organs. Most of the granulomas observed in both lymph nodes and lungs belonged to more advanced stages of development (stages III and IV) whereas in the liver and the spleen most of lesions belonged to intermediate stages (stages II and III). Different microorganisms were simultaneously detected from TBL in the 42.7% of the animals. Mycobacterium tuberculosis complex (MTC) (38%), coryneform bacteria (40.3%) and streptococci (28.1%) were the main groups of microorganisms detected after bacteriological analysis, with Trueperella pyogenes and Streptococcus suis as the most frequently isolated species. Mycobacteria belonging to MTC were the most frequently detected pathogens in granulomatous and pyogranulomatous lesions in submandibular lymph nodes (32.7%) and coryneform bacteria were the microorganisms more frequently isolated from lungs (25.9%) and spleen samples (37.2%). These results may provide new insights into the pathogenesis and diagnosis of this pathology. The importance of coryneform bacteria and streptococci in such processes must be evaluated in future studies.

High prevalence of the epidemic Clostridium difficile PCR ribotype 078 in Iberian free-range pigs.

Previous studies in intensively raised piglets have detected a high prevalence of the epidemic Clostridium difficile PCR ribotype 078. In this article we present a longitudinal survey of C. difficile colonisation in a population of Iberian pigs reared under a free-range system. A total of 160 faecal samples from 20 piglets belonging to different litters were obtained by weekly sampling. C. difficile was recovered from samples collected at different times throughout the survey from a 90% of piglets, resulting in an overall prevalence of 25.6% in the studied samples. Most positive samples (75.6%) came from ≤ 15-day animals, but some piglets shed C. difficile even on day +50. All isolates were ribotype 078, harboured toxin-encoding genes and showed in vitro resistance to several fluoroquinolones. A majority of isolates (80.5%) were also high-level resistant to ertapenem, and four metronidazole heteroresistant isolates (9.8%) were detected. In conclusion, Iberian free-range pigs can be a potential reservoir of epidemic antimicrobial-resistant strains of C. difficile, showing a prevalence rate similar to that found for intensively raised animals.

Salmonella prevalence and characterization in a free-range pig processing plant: tracking in trucks, lairage, slaughter line and quartering.

New consumer tendencies are focused on products derived from systems which allow both a high animal welfare condition and a high food safety level. However, sometimes animal welfare regulations make the adoption of adequate bio-security measures difficult, representing a barrier for animal health and food safety. Thus the aim of this study was to determine the prevalence of Salmonella at different points of the pig slaughtering process (Trucks, Lairage, Slaughter line and Quartering, TLSQ) from pigs reared in free-range systems. From eight samplings a total of 126 Salmonella isolates out of 1160 different samples were recovered (10.86%). The highest percentage of isolates was detected at the points of pre-scalding (29/80, 36.25%), trucks (13/56, 23.21%), cecal contents (17/80, 21.25%), tonsils (14/80, 17.50%), ileocecal lymph nodes (13/80, 16.25%) and lairage (9/64, 14.06%). Furthermore, eighteen isolates were obtained from different environmental samples from slaughter line and quartering plant (knives and surface of tables) (5.63%) and three isolates at the quartering plant samples (ham, shoulder and loin) (3.75%). Fourteen different serotypes were isolated: Bredeney, Rissen, Derby, Typhimurium, Montevideo, Israel, Anatum, Emek, Monophasic Salmonella Typhimurium (mST), Choleraesuis, Durban, Kentucky, London and Sandiego. S. Typhimurium phage types U311, 193, 104b and UT were identified. Moreover, mST strain was phage typed as U311. From TLSQ1, TLSQ2 and TLSQ4, different strains of S. Derby, S. Rissen and S. Bredeney serotypes were isolated from pig and environmental samples, pointing to a potential cross contamination. Molecular typing (Pulsed-Field Gel Electrophoresis, PFGE) of these strains confirmed the cross contamination. In the remaining samplings, different serotypes were obtained in each sampled point of the chain, assuming that the isolated serotypes belonged to different epidemiological origins. Our results show the isolation of different serotypes of Salmonella spp. from both pigs and environmental samples, which constitutes a great risk for the contamination of pork from free-range pigs both prior and post slaughter. These data support the intensification of the cleaning and disinfection in the pre-slaughter environment (i.e. trucks, lairage), especially when a higher workload is present, as well as the inclusion of new strategies to decrease or eliminate the risk of Salmonella spp. infection or recontamination from the environment in pork from organic or eco-friendly systems.

Risk factors associated with the antimicrobial resistance of staphylococci in canine pyoderma.

This study reports the susceptibility to antimicrobial agents of staphylococci (n=105) isolated from dogs, and the factors associated with this resistance. The study animals were 23 healthy dogs (group A), 24 with first-time pyoderma (group B), and 27 with recurrent pyoderma that had undergone long-term antibiotic treatment (group C). Staphylococci were more commonly isolated from the pyoderma-affected than the healthy dogs (p<0.0001). Some 78% of the isolates were resistant to at least one antimicrobial agent. Resistance to amoxicillin-clavulanate, cephalosporins (OR 4.29, 95% CI [1.15, 16.3] respectively), enrofloxacin (OR 9.47, 95% CI [1.53, 58.5]) and ciprofloxacin (OR 79.7 95% CI [3.26, 1947.4]) was more common among group C isolates. Some 32% of all the isolates were multiresistant (MR) and 10.4% were methicillin-resistant (MRS). The probability of isolating MRS staphylococci in group C increased by a factor of four (95% CI [1.18, 17.9]) compared to A plus B. Multi-resistant (MR) isolates were obtained more commonly from urban than rural dogs (OR 3.79, 95% CI [1.09, 13.17]). All the MRS staphylococci encountered were obtained from urban dogs and more commonly from male dogs (p=0.07). This study shows that dogs bred in urban habitat, with a history of antibiotic therapy in the past year represents significant risk of being carriers of isolates resistant to methicillin (MRS) and other antimicrobials. These factors should be considered before applying an antimicrobial treatment in veterinary clinics.

Factors affecting the seroprevalence of lagovirus infection in wild rabbits (Oryctolagus cuniculus) in Southern Spain.

Cross-sectional studies were carried out on wild rabbit (Oryctolagus cuniculus) populations in Southern Spain to assess the prevalence of lagovirus infection and to identify potentially associated risk factors. A total of 619 blood and 487 liver samples from wild rabbits were collected from seven hunting areas with different Mediterranean ecosystems. Logistic regression analysis was used to assess associations between seropositivity and an extensive set of variables. The seroprevalence was 29.2% (95% CI: 25.6-32.8) and lagoviruses were not detected in liver samples. Logistic regression indicated that seropositivity to lagoviruses was associated with seropositivity to myxomatosis, wild rabbit density, the existence of artificial feeding sites, mean maximum monthly temperatures of 20-30 °C, and annual accumulated rainfall of >600 mm.

Prevalence and antimicrobial susceptibility of Salmonella infections in free-range pigs.

The prevalence of Salmonella spp. infection was determined in 67 free-range pig herds in southern Spain. Microbiological assessment was performed on ileocolic lymph nodes collected at slaughter according to ISO 6579:2002 procedures. Overall, 33% of herds were infected and the prevalence of infection was 5.3%. Salmonella spp. serovars most frequently isolated were Anatum and Typhimurium, although uncommon serovars such as Hessarek and Mikawasima were also detected. Isolates were tested against 16 antimicrobial agents and exhibited resistance to streptomycin (46%), tetracycline (30%), sulphonamides (25%) and ampicillin (23%) by the break-point method. Multi-drug resistance, defined as resistance to ≥ 4 antimicrobials, was 36%.

Influence of recovery method and microbial contamination on the response to freezing-thawing in ibex (Capra pyrenaica) epididymal spermatozoa.

The method of sperm recovery may influence the initial quality of sperm samples and their response to freezing-thawing. The aim of the present work was to compare two methods for collecting epididymal spermatozoa in order to improve the quality of recovered sperm and reduce possible contamination. Testes were obtained from 23 legally hunted, adult ibex males. The sperm mass of the right epididymis was collected by small longitudinal and transverse cuts made in the cauda epididymidis. The sperm mass of the left epididymis was collected by retrograde flushing from the vas deferens to the cauda epididymidis (using a cannula), employing a Tris, citric acid, glucose, egg yolk-based medium. The flushing method recovered more spermatozoa (P<0.001) than the cutting method. After freezing-thawing, greater acrosomes damage (P<0.001) and more morphological abnormalities (P<0.05) were seen among the sperm cells recovered by the cutting method than among those obtained by retrograde flushing. The method of sperm recovery did not, however, influence the microbial contamination rate. In frozen-thawed samples that were microbially contaminated, motility was significantly reduced (P<0.05) and membrane integrity tended to be poorer (P=0.06). In conclusion, retrograde flushing is recommended for ibex sperm collection since it would appear that microbial contamination is no more of a problem than that encountered with the cutting method, while a larger number of sperm cells more resistant to freezing-thawing can be obtained.