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.

Evaluation of the diagnostic accuracy of the serological test for paratuberculosis in cattle according to tuberculosis status.

BACKGROUND: Enzyme-linked immunosorbent assays (ELISAs) are the most widely used diagnostic tools in bovine paratuberculosis (bPTB) control. However, their diagnostic accuracy may be compromised by bovine tuberculosis (bTB) infection, as both diseases share diagnostic targets. METHODS: The bPTB and bTB infection status of 228 animals was determined using microbiological tissue culture as a reference test. The diagnostic performance (sensitivity, specificity, likelihood ratios and predictive values) of the bPTB-ELISA on blood serum samples, taking into account the bPTB animal-level prevalence of the area and the bTB status of the animals, was evaluated. RESULTS: A sensitivity of 40.7% (95% confidence interval [CI]: 27.5%-53.9%) and a specificity of 94.7% (95% CI: 91.4%-98.0%) were obtained for bPTB-ELISA in all animals. A bPTB-ELISA-positive animal would have a post-test probability of 70% or more of being infected in areas with a bPTB prevalence of 23% or more. A negative bPTB-ELISA result, in areas with a bPTB prevalence of 41% or less, would rule out the disease with more than 70% certainty. In bTB-positive animals, sensitivity increased (94.4% [95% CI: 81.4%-100%] vs. 25.1% [95% CI: 11.8%-38.4%]) and specificity decreased (82.6% [95% CI: 71.8%-93.4%] vs. 99.4% [95% CI: 98.0%-99.9%]). The bPTB-ELISA is a good tool to rule out bPTB co-infection in bTB-positive animals, while in bTB-negative animals, it allows confirmation of disease with more than 70% probability if disease prevalence is 6% or more. LIMITATIONS: The observed differences could be enhanced by the effect of frequent application of the intradermal tuberculin test, which was unknown in the animals studied. CONCLUSIONS: These results provide useful guidance for the application and interpretation of ELISA as a tool for bPTB disease control.

Diagnostic performance of faecal and tissue multiplex qPCR IS900/F57 for the detection of Mycobacterium avium subspecies paratuberculosis in cattle.

Mycobacterium avium subspecies paratuberculosis (MAP) is responsible for bovine-paratuberculosis (bPTB), which causes high production losses in cattle. A cross-sectional study was conducted in 228 cattle to evaluate the validity and diagnostic utility of a multiplex real-time PCR (qPCR) on faecal and intestinal samples [ileocaecal valve (ICV) and ileocaecal lymph nodes (ICLN)], using intestinal tissue culture as a reference test. Based on the sensitivity, specificity, and likelihood ratios (LR) obtained, the diagnostic value of faecal qPCR for confirming MAP infection was moderate (sensitivity 50.3%, specificity 93.5%, positive LR 7.8), and low to rule it out (negative LR 0.5). In areas with a prevalence of >23% the credibility of positive results was higher than 70%. In the case of negative results, their credibility was higher than 90% in herds with an infection rate below 19%, so faecal qPCR would be very useful in these areas to certify the absence of infection. For post-mortem diagnosis, qPCR on ICV samples showed good diagnostic accuracy to confirm the disease (sensitivity 71.7%, specificity 93.3%, positive LR 10.8), with a credibility higher than 70% in animals from areas or herds with a prevalence of infection greater than or equal to 18%. The best strategy to rule out the disease was the parallel combination of both tissues (ICV + ICLN) (sensitivity 81.3%, specificity 89.5%, negative LR 0.2) with a credibility of over 95% in animals from areas with an infection prevalence of 0-20%. Faecal and tissues qPCR techniques can be used to monitor bPTB, the interpretation of results, according to epidemiological situation of the herd or area, are shown.

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.

The Role of Histopathology as a Complementary Diagnostic Tool in the Monitoring of Bovine Tuberculosis.

The diagnosis of bovine tuberculosis (bTB) is based on the single intradermal tuberculin test (SIT), interferon gamma, and compulsory slaughter of reactor animals. Culture and PCR from fresh tissue are regarded as gold standard techniques for post-mortem confirmation, with the former being time-consuming and presenting moderate to low sensitivity and the latter presenting promising results. Histopathology has the advantage to identify and categorize lesions in both reactor and non-reactor animals. Therefore, this study aims to highlight the role of histopathology in the systematic diagnosis of bTB to shorten the time to disclose positive animals. Blood (212) and lymph node (681) samples were collected for serological, bacteriological, and histopathological analyses from a total of 230 cattle subjected to the Spanish bTB eradication program. Seventy-one lymph nodes and 59 cattle yielded a positive result to bacteriology, with 59 lymph nodes and 48 cattle presenting a positive result in real-time PCR from fresh tissue. Roughly 19% (40/212) of sera samples gave a positive result to ELISA. Tuberculosis-like lesions (TBLs) were observed in 11.9% (81/681) of the lymph nodes and 30.9% (71/230) of cattle. Noteworthy, TBLs were evidenced in 18 out of 83 SIT- and real-time PCR and bacteriology negative animals, with 11/18 disclosing a positive result to Ziehl-Neelsen technique and two of them to ddPCR from paraffin blocks targeting IS6110. Six out of these 11 ZN+ corresponded with mesenteric LN and were confirmed positive to paratuberculosis. Histopathology yielded a sensitivity of 91.3% (CI95 83.2-99.4%) and a specificity of 84.4% (CI95 78.6-89.3%) with good agreement (κ = 0.626) when compared with real-time PCR. Our results confirm that histopathology allows a rapid confirmation of real-time PCR and bacteriology, emphasizing its contribution to bTB control and monitoring.

Real-Time PCR Validation for Mycobacterium tuberculosis Complex Detection Targeting IS6110 Directly From Bovine Lymph Nodes.

Rapid and accurate diagnostic tools, such as Real-Time PCR (qPCR), need to be implemented as a confirmatory test in the framework of bovine tuberculosis (bTB) surveillance and control programs, shortening the turnaround time to confirm bTB infection. The present study aimed to evaluate a direct qPCR from fresh tissue samples targeting the insertion sequence IS6110 using individually homogenized bovine lymph nodes compared with microbiological culture. Retropharyngeal, tracheobronchial, and mesenteric lymph nodes fresh tissue samples (n = 687) were collected from 230 different cattle carcasses at the slaughterhouse. Only 23 of the 230 examined animals showed tuberculosis-like lesions, with 62 of 230 considered as positive. Among these 62 animals, 61 resulted as culture-positive, whereas 48 were qPCR-positive. Thus, this qPCR targeting IS6110 showed an apparent diagnostic sensitivity and specificity values of 77.1% [95% confidence interval (CI): 66.5-87.6%] and 99.4% (95% CI: 98.3-100.6%), respectively, and a positive predictive value of 97.9% (95% CI: 93.9-102.0%) and negative predictive value of 92.3% (95% CI: 88.4-96.2%). Positive and negative likelihood ratios were 130.2 and 0.2, respectively, and the agreement between microbiological culture and this qPCR was almost perfect (κ = 0.82). These results highlight this qPCR targeting IS6110 as a suitable complementary method to confirm bTB in animals with either tuberculosis-like lesions or non-tuberculosis-like lesions, decreasing the number of samples subjected to microbiological culture and, hence, its overall associated costs and the turnaround time (under 48 h) to confirm bTB infection. Besides, sampling mesenteric lymph node, which is uncommonly sampled, together with tracheobronchial and retropharyngeal ones, is advisable during postmortem inspection in bTB surveillance programs at the slaughterhouse, especially in areas with a low bTB prevalence scenario.

Histopathological and microbiological study of porcine lymphadenitis: contributions to diagnosis and control of the disease.

Tuberculosis like lesions (TBL) in free-range pigs are characterised by presenting a marked heterogeneity in pathology and microbiology features, with a notorious role of Mycobacterium tuberculosis complex (MTC), Trueperella pyogenes and different Streptococcus species. However, the capacity of these microorganism to spread to different organic cavities leading to a generalised disease is unknown. Therefore, this study evaluated the organic distribution of these agents in free-range pig carcasses whole condemned due to generalised TBL.A total of 37 totally condemned animals were analysed, and samples of lymph nodes and organs were obtained (n = 262) and subjected to histopathological and microbiological examination. In addition, T. pyogenes and streptococci species were further characterised by PFGE analysis. Two different patterns were evidenced with lack or occasional lesions in superficial inguinal (SILN) and popliteal (PLN) lymph nodes and advanced lesions in submandibular (SLN) (35/36) and gastrohepatic (GHLN) (33/35) lymph nodes (stages III and IV). Early stage granulomas (stage I and II) prevailed in lungs (16/20), liver (14/31) and spleen (7/18). The microbiological analysis revealed that MTC, detected by qPCR, was present in 31 out of 37 animals and 90 (90/262) samples. In 26 out of the 31 pigs, MTC was detected from two or more organs. SLN (24/31) and GHLN (19/31) were the MTC+ organs most frequently detected, with 29 out of 31 MTC+ pigs detected as positive in one or both samples, which points out that both lymph nodes must be included in the sampling of surveillance programs. Other pathogens, such as T. pyogenes and Streptococcus spp., were also involved in generalised lymphadenitis, being frequently isolated from SLN and other organs, such as liver (T. pyogenes), tonsils or lung (Streptococcus spp.). A wide genetic diversity among streptococci was observed, showing the ubiquitous character of these pathogens, however, the isolation of a single clone of T. pyogenes from different organic locations from animals with generalised TBL was a common finding of this study, highlighting that the role of this pathogen in porcine lymphadenitis may be underestimated. These results should be considered in future studies on the pathogenesis and control of porcine lymphadenitis.

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.

Supplementing feed with Pediococcus acidilactici reduces Campylobacter load in finishing pigs.

BACKGROUND: Pigs are asymptomatic carriers of foodborne bacteria, such as Salmonella enterica and Campylobacter species, which can pose a risk to human health. New strategies to control bacteria burden before reaching the slaughterhouse are necessary. This study evaluated the effect of Pediococcus acidilactici on performance parameters and on the burden of foodborne pathogens, that have subsequent implications on food quality and safety, in free-range finishing pigs at the slaughterhouse. METHODS: Pigs were randomly allocated and blocked by weight into control group (control diet) and treated group (control diet supplemented with P acidilactici) 31 days before slaughter. Weight and average daily gain were recorded and changes in faecal microbiota were determined at the beginning and at the end of the study. RESULTS: No changes were observed in performance parameters. No statistically significant differences were observed when comparing between treated and control animals at the beginning or at the end of the study. However, a significant decrease was detected in the counts of Campylobacter species in treated animals between day 0 and day 31 (4.86-3.40 log colony-forming units/g; P=0.002). CONCLUSION: This study indicates that supplementation with P acidilactici represents a useful approach to control Campylobacter species load in free-range finishing pigs before slaughter.

Antimicrobial susceptibility of Trueperella pyogenes isolated from food-producing ruminants.

A total of 96 Trueperella pyogenes isolates, an opportunistic pathogen of food-producing ruminants, obtained from cattle (n = 34), sheep (n = 35) and goats (n = 27), and identified by Real Time PCR (qPCR), were analysed to determine the susceptibility to 12 antimicrobials commonly used in livestock, using a broth microdilution. The Minimal Inhibitory Concentration (MIC) distribution was unimodal for half of the antimicrobials tested with the exception of apramycin, gentamicin, streptomycin, oxytetracycline, tylosin, and erythromycin all of which showed bimodal MIC distributions. Low MIC90 values for penicillin, amoxicillin, ceftiofur, enrofloxacin, and gentamicin (<1 µg/ml) were obtained, suggesting that these antimicrobials would be the most effective first line empiric treatment for T. pyogenes infections in livestock. Furthermore, according to the specific T. pyogenes breakpoints for penicillin, sulfamethoxazole/trimethoprim and erythromycin, 93.7 % of isolates were susceptible to penicillin and 77.2 % to erythromycin, whereas 92.7 % were non-susceptible to sulfamethoxazole/trimethoprim. Significant differences were observed in the MIC distribution of almost all antimicrobials, except enrofloxacin, tylosin and erythromycin against cattle, sheep or goat isolates, although all antimicrobials showed similar MIC90 values, except apramycin and oxytetracycline that showed higher values when tested against cattle isolates. These data provide interesting information on the antimicrobials of choice for the treatment of infections caused by T. pyogenes in ruminants.

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.

Antimicrobial susceptibility and genetic characterization of Trueperella pyogenes isolates from pigs reared under intensive and extensive farming practices.

Trueperella pyogenes is an opportunistic pathogen associated with a variety of diseases and responsible for important economic losses for pig production. Minimal Inhibitory Concentration (MIC) and Pulsed Field Gel Electrophoresis (PFGE) typing analysis were used to determine the MIC distribution and to genetically characterize a total of 180 T. pyogenes isolates obtained from slaughtered pigs reared under intensive (TpIN, n = 89) and extensive (TpEX, n = 91) farming practices. Low MIC90 values for penicillin and amoxicillin (0.008 and 0.06 µg/ml, respectively), ceftiofur, gentamicin and enrofloxacin (1 µg/ml, respectively) were obtained, so they could be of choice for the empiric treatment of T. pyogenes infections. Except for the penicillin, amoxicillin and ceftiofur, a statistically significant difference was observed in the MIC distribution of all antimicrobials analysed between TpIN and TpEX isolates. Also, MIC90 values were higher in TpIN than in TpEX isolates for neomycin and streptomycin (32 µg/ml vs 8 µg/ml), sulfamethoxazole/trimethoprim (30.4/1.6 µg/ml vs 1.90/0.10 µg/ml) and tylosin (≥1024 µg/ml vs 1 µg/ml). A relatively lower genetic diversity was detected in TpIN in comparison with TpEX isolates (GD 0.42 and GD 0.47, respectively). All isolates were distributed in three clusters (A, B, C). TpIN isolates were statistically associated with cluster A (P = 0.0002; OR 3.21; CI95 1.74-5.93), whereas the TpEX were distributed throughout the dendrogram, showing more genetic diversity. These data suggest that the antimicrobial susceptibility and genetic variability of the T. pyogenes isolates could be influenced by the management systems.

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.

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.

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.

Characterization of the immune response and evaluation of the protective capacity of rSsnA against Streptococcus suis infection in pigs.

The efforts made to develop vaccines against Streptococcus suis have failed because of lack of common antigens cross-reactive against different serotypes of this species. The cell wall-anchored proteins can be good vaccine candidates due to their high expression and accessibility to antibodies, among these, a cell-wall protein, DNA-nuclease (SsnA), present in most of the S. suis serotypes and clinical isolates collected from infected pigs, was selected. An experimental challenge against S. suis serotype 2 in a pig model was used to validate the efficacy of recombinant SsnA combined with aluminium hydroxide plus Quil A as adjuvants, previously tested in mice by our research group with good results. In our study, clinical characteristics, bacterial load and spread, haematological and immunological parameters and the antibody response, including the opsonophagocytosis analysis of the sera were evaluated. Moreover the composition of peripheral blood leukocyte populations was studied in infected animals. The results show that the immunization of piglets with rSsnA elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are challenged with a virulent strain in our conventional vaccination model. Further studies are necessary to evaluate the use of rSsnA as a vaccine candidate for swine.

A new recombinant SsnA protein combined with aluminum hydroxide protects mouse against Streptococcus suis.

An experimental challenge in a mouse model was used to select the most effective adjuvant in a vaccine formulation with the surface-anchored DNA-nuclease (SsnA). We used a protocol based on clinical, histopathological, bacterial kinetics and immune response against S. suis serotype 2 in infected animals. The three adjuvants used, aluminum hydroxide (ALOH), incomplete Freund's adjuvant (FIA), oil-in-water adjuvant (OW) showed a protective effect against death by S. suis serotype 2 in this mouse model, although aluminum hydroxide revealed as the best option. Subsequently, in a second experimental assay, we showed that a recombinant SsnA protein combined with ALOH as adjuvant allowed a significant decrease of clinical and lesional findings in animals, faster reduction of the bacteria from organs and a highest humoral response against S. suis after 3 days post-infection. The results show that this combination (rSsnA+AlOH) could be a good vaccine formulation against S. suis, although further studies are necessary to evaluate their use for swine and human species.

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.

Exploring the pan-surfome of Streptococcus suis: looking for common protein antigens.

Streptococcus suis is a swine and human pathogen for which no commercial vaccine is still available. Conserved and broadly distributed surface proteins have become the chosen targets for the development of efficacious vaccines that could overcome the problems of non-heterologous protection of bacterins or capsule polysaccharide-based vaccines. In this work, we have analyzed by proteomics a collection of 39 strains obtained from infected pigs. The isolates belonged to 19 of the most prevalent serotypes during the last years. We have applied the "shaving" approach to define the "pan-surfome" or the set of both common and unique surface proteins identified in such strains. This set was constituted by 113 proteins. We have categorized them for their potential for further vaccination studies, based on their distribution among strains and their a priori accessibility to antibodies. According to these criteria, the cell-wall protein SsnA appears to be the best candidate from this list, as it was that with the widest distribution among the analyzed pathogen types, it showed to be highly immunogenic and highly accessible to antibodies, as demonstrated by flow cytometry.

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.