Droplet digital PCR as alternative to microbiological culture for Mycobacterium tuberculosis complex detection in bovine lymph node tissue samples.

Introduction: Bovine tuberculosis (bTB) caused by Mycobacterium tuberculosis complex (MTC) remains a significant concern for public health. Direct real-time PCR and droplet digital PCR (ddPCR) are proposed as alternative tools to enhance diagnostic precision and efficiency. This study aims to assess the diagnostic performance of a ddPCR assay targeting IS6110 for the detection of MTC DNA in both microbiological culture and fresh lymph node (LN) tissue samples obtained from cattle, in comparison with the established reference standard, the microbiological culture followed by real-time PCR. Methods: The fresh LNs (N=100) were collected each from a different cattle carcass at the slaughterhouse. The limit of detection of ddPCR-IS6110 was set to 101 copies per 20 µl reaction. Results: DdPCR-IS6110 detected 44 out of 49 reference-standard positive samples and yielded negative results in 47 out of 51 reference-standard negative samples, resulting in adjusted sensitivity (Se) and specificity (Sp) of 90.76% [95% confidence interval (CI): 82.58 - 98.96%)], and 100% (95% CI: 100%) respectively. The estimated adjusted false negative rate (FNR) was 9.23% (95% CI: 1.04 - 17.42%) and the false positive rate (FPR) was 0% (95% CI: 0%). When directly applied from fresh bovine LN tissues, ddPCR-IS6110 identified 47 out of 49 reference-standard positive samples as ddPCR-IS6110-positive and 42 out of 51 reference-standard negative samples as ddPCR-IS6110-negative, resulting in adjusted Se and Sp values of 94.80% [95% (CI): 88.52 - 100%] and 100% (95% CI: 100%), respectively. The adjusted FNR was 5.20% (95% CI: 0 - 11.50%) and the FPR was 0% (95% CI: 0%). Noteworthy, ddPCR-IS6110 disclosed as positive 9 samples negative to reference-standard. Discussion: DdPCR-IS6110 proved to be a rapid, highly sensitive, and specific diagnostic tool as an alternative to reference-standard method.

Assessment of the diagnostic performance of intradermal tuberculin test and post-mortem inspection for the diagnosis of bovine tuberculosis according to WOAH guidelines.

Bovine tuberculosis (bTB) constitutes a global challenge for public and animal health with still some deficiencies regarding its diagnosis. This study aimed to estimate the accuracy of the single intradermal tuberculin test (SIT) and post-mortem inspection for different diagnostic objectives following WOAH guidelines. Tissue samples from 59 microbiological culture/PCR-positive and 58 microbiological culture/PCR-negative cattle were evaluated. The diagnostic sensitivity and specificity, the positive and negative probability indices as well as the positive and negative predictive values (PPV and NPV) of each technique were estimated for different pretest probabilities. The SIT with strict interpretation demonstrated moderate precision in confirming the absence of infection in populations historically free of bTB, with a 12.1% rate of false positives, but also detecting positive animals in the early stage of the eradication programs, with a 13.6% rate of false negatives. The diagnostic performance for ruling out bTB was notably high (NPV > 90%) in animals with a pre-test probability (PTP) below 42%. Post-mortem inspection constituted an interesting alternative tool to confirm suspected and positive cases for SIT, particularly in areas with bTB prevalence exceeding 19%, where implementing SIT and eradication measures may be impractical. In these areas, the likelihood that animals with tuberculosis-like lesions are affected by the disease surpasses 90%. Similarly, in herds with a PTP below 25%, the absence of bTB could be confidently ruled out with over 90% certainty. These findings highlight the effectiveness of SIT and post-mortem inspection as valuable techniques for current eradication programs and controlling bTB in high-prevalence areas where molecular techniques may not be feasible.

Optimization of real-time PCR protocols from lymph node bovine tissue for direct detection of Mycobacterium tuberculosis complex.

Bovine tuberculosis (bTB) is a zoonotic disease and a global health problem that is subjected to obligatory eradication programs in the European Union. Microbiological culture is an imperfect technique for bTB diagnosis. This study aims to compare and validate two DNA isolation protocols and three different specific DNA targets, IS6110, IS4, and mpb70, to confirm Mycobacterium tuberculosis complex (MTC) infection by real-time PCR directly from fresh tissue samples. Fresh lymph node samples were collected from 81 cattle carcasses at the slaughterhouse. A comparison of both extraction protocols was performed with IS6110-real-time PCR, showing an adjusted sensitivity (SE) of 78.34% and 95.9% for protocols 1 and 2, respectively, while the specificity (SP) was 100% in both cases. Afterward, the comparison between IS4 and mpb70 targets was performed from the samples extracted with protocol 2, obtaining an adjusted SE of 90.87% and 83.3%, respectively, and an SP of 100% in both cases. The positive likelihood ratio was ∞ for the three targets, and the negative likelihood ratio was 0.04, 0.091, and 0.16 for IS6110, IS4, and mpb70, respectively. Negative predictive values were ≥90%, ≥85%, and ≥80% for real-time PCR targeting IS6110, IS4, and mpb70, respectively, when the true prevalence is ≤60%, and the positive predictive value is 100% in any scenario of true prevalence. According to these results, the DNA extraction protocol 2 and real-time PCR targeting IS6110 or IS4 could be potential first-choice molecular assays to detect MTC directly in fresh bovine tissue samples. IMPORTANCE Bovine tuberculosis (bTB), a chronic infectious and zoonotic disease caused by Mycobacterium tuberculosis complex (MTC), is considered a neglected disease of global importance, causing a detrimental impact on public health, particularly in developing countries where tuberculosis remains a major health problem. However, debate around the efficacy of control measures is still an ongoing matter of concern, with poor diagnostic performance being considered one of the most relevant factors involved in the failure to eradicate the disease since many truly infected animals will be misclassified as bTB-free. This study highlights a DNA extraction protocol and real-time PCR targeting IS6110 or IS4 as potential first-choice molecular assays to detect MTC directly in fresh bovine tissue samples, providing rapid, highly sensitive, and specific diagnostic tools as an alternative to microbiology, which could take up to 3 months to complete, shortening the turnaround time for decision makers to be promptly informed.

Pre-test probability and likelihood ratios for clinical findings in canine leishmaniasis.

Canine leishmaniasis is a parasitic zoonosis mainly caused by L. infantum; an obligate intracellular protozoan transmitted by haematophagous insects of the genus Phlebotomus, which affects dogs and wild canids. The clinical implications of this disease are highly variable, since infected animals may remain asymptomatic (absence of observable clinical signs) or present a wide spectrum of clinical alterations and degrees of severity, including the death of the animal. Symptoms such as lymphadenomegaly, alopecia, weight loss, keratoconjunctivitis and onychogryphosis are usually the first diagnostic reference available. The objectives of this study are to evaluate the validity (sensitivity, specificity and likelihood ratios) and diagnostic utility (pre-test probability) of the clinical signs commonly associated with canine leishmaniasis based on the prevalence in the area and to explore the combination of symptoms that best predicts the diagnosis of canine leishmaniasis. It is a matched case-control study in the canine population of southern Spain based on the comparison of the findings collected in the clinical history and the results of the LeisSCAN quantitative ELISA. A total of 39 cases and 78 controls were analysed. Approximately 80% of the infected animals showed signs compatible with the disease. The most frequent alterations were cutaneous (64.1%), systemic (51.3%) and oculo-nasal (30.7%). The most useful signs to support this diagnosis were alopecia and epistaxis (LR+ 6.69 and 6.0, respectively) (pre-test leishmaniasis probability is ≥70% for prevalence ≥28% when alopecia or epistaxis is present), followed by lameness (LR+ 5.0). The combinations of signs that showed greater validity were alopecia with hyperkeratosis of the snout and alopecia with onychogryphosis (LR+ > 10). None of the observed signs or their combinations resulted useful to rule out the diagnosis (LR- 0.55 to 1.15). The results found show notable differences in the diagnostic value of the clinical signs, individually and in combination, so we believe that medical decisions should be based on their diagnostic validity (LR+) and the estimation of the pre-test and post-test probability.

Utility assessment of an Enzyme-linked immunosorbent assay for detection of subclinical cases of caseous lymphadenitis in small ruminant flocks.

The actual prevalence of CLA (caseous lymphadenitis) in small ruminant flocks is underestimated in many countries, and because it is not a notifiable disease, it will continue to spread without data and information about its real economic impact. The difficulty in the accurate identification of the causative agent in internal subclinical cases allows the disease to spread within and between flocks. This research intends to assess the utility of an ELISA (enzyme-linked immunosorbent assay) test in the detection of internal subclinical cases of CLA in farms and to simultaneously add data on the seroprevalence of the disease in Portugal. Sera from 756 small ruminants, 70% sheep (528/756) and 30% goats (228/756) were screened for antibodies against Corynebacterium pseudotuberculosis using the ELISA technique based on a recombinant phospholipase D (ELITEST CLA # CK105A® ). The animals showing internal lesions (n êžŠ 58) were sampled for the identification of the aetiological agent. In this investigation, the prevalence of CLA was 34% (258/756), with the ELISA test showing a low specificity (78%) and high sensitivity (100%). The proof was able to detect 57% (13/23) of subclinical cases of CLA confirmed by postmortem examination and conventional PCR (polymerase chain reaction). The results also reveal that goats have a higher propensity for the disease, and dairy farms and non-extensive production units appear to be more susceptible to CLA. This research clarifies an actual problem and pointed out the importance of CLA in small ruminant herds in Portugal. Finally seems to demonstrate that the ELISA test is a good diagnostic tool for use in CLA eradication programmes.

Search of Potential Vaccine Candidates against Trueperella pyogenes Infections through Proteomic and Bioinformatic Analysis.

Trueperella pyogenes is an opportunistic pathogen, responsible for important infections in pigs and significant economic losses in swine production. To date, there are no available commercial vaccines to control diseases caused by this bacterium. In this work, we performed a comparative proteomic analysis of 15 T. pyogenes clinical isolates, by "shaving" live cells, followed by LC-MS/MS, aiming at the identification of the whole set of surface proteins (i.e., the "pan-surfome") as a source of antigens to be tested in further studies as putative vaccine candidates, or used in diagnostic tools. A total of 140 surface proteins were detected, comprising 25 cell wall proteins, 10 secreted proteins, 23 lipoproteins and 82 membrane proteins. After describing the "pan-surfome", the identified proteins were ranked in three different groups based on the following criteria: to be (i) surface-exposed, (ii) highly conserved and (iii) widely distributed among different isolates. Two cell wall proteins, three lipoproteins, four secreted and seven membrane proteins were identified in more than 70% of the studied strains, were highly expressed and highly conserved. These proteins are potential candidates, alone or in combination, to obtain effective vaccines against T. pyogenes or to be used in the diagnosis of this pathogen.

Reduced Susceptibility of Salmonella Typhimurium Strains to Oregano Essential Oil and Enrofloxacin: An In Vitro Assay.

Several European animal nutrition companies have incorporated essential oils (EOs) into animal feed as a result of the prohibition of antibiotics to promote animal growth. Previous studies of EOs have highlighted the absence of bacterial resistance for these substances, although most of the published works focus on studying their tolerance to subinhibitory doses. For this study, oregano essential oil (OEO) was chosen for its proven inhibitory and bactericidal activity. This study is an in vitro assay of the possible induction of Salmonella enterica serovar Typhimurium strains with reduced susceptibility to OEO by mutation, seeking to calculate the mutant prevention concentration (MPC) since this is an important measurement for the control Salmonella's resistance to fluoroquinolones such as enrofloxacin (ENR), the treatment of choice for this infection. To establish the MPC, we used a bacterial inoculum ≥109 colony-forming unit (CFU)/mL and examined the bases for points of resistance to ENR and mutations of target genes of the quinolone resistance determining region (QRDR). The three strains of Salmonella Typhimurium used in this study showed an MPC of four times the minimum inhibitory concentration (MIC) for ENR. In all cases, strains with reduced susceptibility to ENR were obtained, although none reached the point of resistance. The QRDR characterization region was in all cases of wild type (wt). Two of the strains tested with OEO grew at a concentration of 1 × MIC, which could be strains with reduced susceptibility, associated with mutation or not. In this case, the MPC was 2 × MIC. Once isolated and identified as Salmonella Typhimurium, the MIC against OEO of all strains obtained in the induction test indicated a possible reduction in susceptibility. However, the result obtained for both strains coincided with MIC of the original strains, rejecting a priori such a reduced susceptibility of Salmonella Typhimurium to OEO.

Paratuberculosis in dairy goat flocks from southern Spain: risk factors associated with seroprevalence.

BACKGROUND: Paratuberculosis (PTB) is a chronic, enteric wasting disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), with a worldwide distribution. Andalusia, located in southern Spain, is one of the European regions with the highest goat census and the highest milk production; however, current data on the prevalence of MAP in this species are not available. METHODS: A cross-sectional study was performed to determine the seroprevalence and risk factors associated with PTB in dairy goat flocks from southern Spain. A total of 3312 serum samples were collected from 48 flocks located in three different geographical areas. Health and productive parameters were surveyed during the visit to the herds. RESULTS: A total of 511 goats were seropositive, with overall true seroprevalence of 22.54 per cent (95 per cent confidence interval (CI95) 21.12-23.97). Of the goat herds, 87.50 per cent (CI9578.14-96.98) were seropositive. The intraherd seroprevalence was 25.43±31.71, distributed as follows: 22 flocks with a seroprevalence under 10 per cent; 18 flocks between 10 per cent and 50 per cent; and eight flocks with a frequency over 50 per cent. Multivariate logistic regression showed significant association between PTB seropositivity and the following variables: intensive production system, lack of management by batches, inappropriate ventilationandseropositivity tocaprinearthritisencephalitisvirus (CAEV). CONCLUSIONS: The results indicate a widespread PTB infection in goat herds in southern Spain. Thus, control programmes must include management and sanitary measures to reduce the prevalence. Further experimental studies are necessary to determine the influence of CAEV-PTB coinfection on immune status.

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.

Competencias y contenidos de salud pública en los programas del Grado en Veterinaria en las universidades españolas / Public health competencies and contents in Spanish university degree programmes of Veterinary Medicine

Objetivo: Consensuar las competencias profesionales de salud pública que deben adquirir los/las estudiantes en el Grado en Veterinaria y los contenidos fundamentales que deben incluir los programas de salud pública según el criterio de docentes de salud pública del Grado en Veterinaria representantes de distintas universidades españolas. Métodos: Se organizó la 3ª Reunión del Foro de Profesorado Universitario de Salud Pública en la Facultad de Veterinaria de la Universidad de Córdoba (12-13 de enero de 2016). Participaron 42 docentes de nueve universidades españolas con Grado en Veterinaria y se distribuyeron en cinco grupos durante tres sesiones de trabajo para identificar y clasificar las competencias propias del grado, proponer contenidos de salud pública para las competencias identificadas, y organizar los contenidos en bloques temáticos. Los resultados se discutieron en distintas sesiones plenarias hasta alcanzar acuerdos. Resultados: El mayor número de competencias identificadas corresponde a actividades de las funciones «Valorar las necesidades de salud de la población» y «Desarrollar políticas de salud». El programa resultante incluye contenidos básicos organizados en cinco bloques: 1) Fundamentos de salud pública; 2) Estudio e investigación en salud pública; 3) Producción, sanidad animal y medio ambiente; 4) Seguridad alimentaria; y 5) Educación sanitaria y comunicación. Conclusiones: Los acuerdos alcanzados pueden ser un buen punto de partida para orientar una propuesta formativa en salud pública del grado para los futuros profesionales de veterinaria

Objective: To reach a consensus among public health faculty from various Spanish universities about the core public health competencies that should be integrated into the Veterinary Medicine degree training. Methods: The 3rd Forum of University Professors of Public Health was held at the School of Veterinary Medicine of the University of Cordoba (12-13 January 2016). Forty-two university professors and lecturers from nine Spanish universities with veterinary degrees participated in the forum. They were divided into five working groups during three working sessions to identify and classify core public health competencies for the Veterinary Medicine degree, propose public health contents for the identified competencies and organize such contents in thematic blocks. The results were discussed in different plenary sessions. Results: The highest number of core competencies was identified in the activities related to the following public health functions: «Assessment of the population's health needs» and «Developing health policies». The final programme included basic contents organized into five units: 1) Fundamentals of public health; 2) Study and research in public health; 3) Production, animal health and environment; 4) Food security; and 5) Health education. Conclusions: The public health core competencies and contents identified in this Forum may be considered as a starting point to update public health training programmes for future veterinary professionals

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.

Susceptibility Distribution to Essential Oils of Salmonella enterica Strains Involved in Animal and Public Health and Comparison of the Typhimurium and Enteritidis Serotypes.

To determine the distribution of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of essential oils (EOs) of cinnamon (Cinnamomum zeylanicum), clove (Eugenia caryophyllata), oregano (Origanum vulgare), common thyme (Thymus vulgaris), and red thyme (Thymus zygis) against Salmonella enterica, double serial dilutions of each EO were challenged with 85 Salmonella strains belonging to 23 serotypes of animal origin. The results showed the bactericidal character of the EOs tested against S. enterica, highlighting the oregano with MIC50 and MBC50 of 3.12 × 10-4 g/mL, and MIC90 and MBC90 of 6.25 × 10-4 g/mL. When comparing the Salmonella Typhimurium and Salmonella Enteritidis serotypes susceptibility, we observed a significantly higher sensitivity of Typhimurium to clove and Enteritidis to cinnamon. In addition, Typhimurium isolates with significantly higher MIC and MBC values for all the EOs tested were found, suggesting the existence of a possible resistance profile. The results of this study provide relevant data for the potential of EOs as antibacterials, although they highlight the need to continue bacterial sensitivity distribution studies and consider the differences detected for future in vivo studies.

Antimicrobial activity of selected essential oils against Streptococcus suis isolated from pigs.

The inhibitory potential by contact and vapor of basil, cinnamon, clove, peppermint, oregano, rosemary, common thyme, and red thyme essential oils (EOs) against 20 strains of Streptococcus suis was determined by the disk diffusion test. The broth microdilution method was used to determine the minimal inhibitory and minimal bactericidal concentration (MIC and MBC) of the four selected oils. Furthermore, the bactericidal power (ratio MBC/MIC) was calculated. The EOs with the major potential in the disk diffusion method were red thyme, common thyme, oregano, and cinnamon (∅ mean 16.5-34.2 mm), whereas cinnamon did not show vapor activity. In the microdilution test, all the EOs showed notable antimicrobial activity (MIC90 and MBC90 312.5-625 µg·ml-1 ) and a strong bactericidal power (ratio = 1). This is the first study that selects essential oils against S. suis. New studies about the possible synergic effect of EOs with antibiotics and about toxicity and efficacy in in vivo conditions are recommended.

[Public health competencies and contents in Spanish university degree programmes of Veterinary Medicine]. / Competencias y contenidos de salud pública en los programas del Grado en Veterinaria en las universidades españolas.

OBJECTIVE: To reach a consensus among public health faculty from various Spanish universities about the core public health competencies that should be integrated into the Veterinary Medicine degree training. METHODS: The 3rd Forum of University Professors of Public Health was held at the School of Veterinary Medicine of the University of Cordoba (12-13 January 2016). Forty-two university professors and lecturers from nine Spanish universities with veterinary degrees participated in the forum. They were divided into five working groups during three working sessions to identify and classify core public health competencies for the Veterinary Medicine degree, propose public health contents for the identified competencies and organize such contents in thematic blocks. The results were discussed in different plenary sessions. RESULTS: The highest number of core competencies was identified in the activities related to the following public health functions: «Assessment of the population's health needs¼ and «Developing health policies¼. The final programme included basic contents organized into five units: 1) Fundamentals of public health; 2) Study and research in public health; 3) Production, animal health and environment; 4) Food security; and 5) Health education. CONCLUSIONS: The public health core competencies and contents identified in this Forum may be considered as a starting point to update public health training programmes for future veterinary professionals.

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.

Combination of Antimicrobials and Essential Oils as an Alternative for the Control of Salmonella enterica Multiresistant Strains Related to Foodborne Disease.

Due to the increase in bacterial resistance to antimicrobials (AMBs) commonly used in veterinary and human medicine, the new strategies for controlling zoonoses focus on the study of natural products with demonstrated AMB activity, such as essential oils (EOs). The aim of this study was to evaluate the in vitro effect of the combination of enrofloxacin (ENR), ceftiofur (CEF), and trimethoprim-sulfamethoxazole (SXT) with cinnamon, clove, oregano, and red thyme EOs against multiple drug-resistant strains of Salmonella enterica. The minimum inhibitory concentration (MIC) of each product was determined by microdilution and "Checkerboard" methods and their combined effect was evaluated against 15 strains of S. enterica. The results were interpreted by the calculation of fractional inhibitory concentration (FIC) and their respective indexes (FICI). Significant susceptibility of all strains to the four EOs was observed. The results showed a synergistic effect between EOs and AMBs tested, highlighting the upper percentage of total synergies of the SXT with the four EOs (FICI ≤0.5 in 60% of assays), and the most effective combination being the one of ENR and cinnamon. The MIC of cinnamon was reduced from 1250 to 312.5 µg/mL and the MIC of ENR from 2 to 0.031 µg/mL. There was no antagonism in the tested combinations (AMBs-EOs). Our results support the combined use of EOs and AMBs for the control of multiresistant strains of S. enterica with a reduction of the minimum effective dose of AMBs and their adverse effects.

Evaluation of rapid methods for diagnosis of tuberculosis in slaughtered free-range pigs.

Free-range pigs can be infected by Mycobacterium tuberculosis complex (MTC) and may contribute to the spread of bovine tuberculosis (bTB). In the present study, the diagnostic values of bacteriological culture, a duplex real-time quantitative PCR and an antibody ELISA were evaluated in an abattoir study of submandibular lymph nodes and serum samples from 73 pigs with and without lesions consistent with bTB. The duplex qPCR was an accurate method for diagnosis of TB in pigs (specificity 100%; sensitivity 80%). Combining qPCR with histopathology improved sensitivity and had very good concordance (κ = 0.94) with the reference method. Serological results suggest that the antibody ELISA can be used for monitoring herds but not individuals.

Comparison of two biochemical methods for identifying Corynebacterium pseudotuberculosis isolated from sheep and goats.

The biochemical pattern of Cowan and Steel (BPCS) was compared with a commercial biochemical strip for the identification of Corynebacterium pseudotuberculosis isolated from small ruminants. On 16S rRNA gene sequencing, 40/78 coryneform isolates from the lymph nodes of sheep and goats with lesions resembling caseous lymphadenitis were identified as C. pseudotuberculosis. The sensitivities of the BPCS and the commercial biochemical strip relative to 16S rRNA sequencing were 80% and 85%, and their specificities were 92.1% and 94.7%, respectively; the level of agreement between the BPCS and the commercial biochemical strip was high (κ=0.82). Likelihood ratios for positive and negative results were 10.0 and 0.22 for the BPCS, and 16.0 and 0.16 for the commercial biochemical strip, respectively. These results indicate that the BPCS and the commercial biochemical strip are both useful for identifying C. pseudotuberculosis in veterinary microbiology laboratories.

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.