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.

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.

Virulence potential of Listeria monocytogenes strains recovered from pigs in Spain.

BACKGROUND: Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis, an infectious disease in animals and people, with pigs acting as asymptomatic reservoirs. In August 2019 an outbreak associated with the consumption of pork meat caused 222 human cases of listeriosis in Spain. Determining the diversity as well as the virulence potential of strains from pigs is important to public health. METHODS: The behaviour of 23 L monocytogenes strains recovered from pig tonsils, meat and skin was compared by studying (1) internalin A, internalin B, listeriolysin O, actin assembly-inducing protein and PrfA expression levels, and (2) their invasion and intracellular growth in eukaryotic cells. RESULTS: Marked differences were found in the expression of the selected virulence factors and the invasion and intracellular replication phenotypes of L monocytogenes strains. Strains obtained from meat samples and belonging to serotype 1/2a did not have internalin A anchored to the peptidoglycan. Some strains expressed higher levels of the studied virulence factors and invaded and replicated intracellularly more efficiently than an epidemic L monocytogenes reference strain (F2365). CONCLUSION: This study demonstrates the presence of virulent L monocytogenes strains with virulent potential in pigs, with valuable implications in veterinary medicine and food safety.

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 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.

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.

Evaluation of five serologic assays for bovine tuberculosis surveillance in domestic free-range pigs from southern Spain.

In countries where bovine tuberculosis (bTB) is still prevalent the contact among different animal species in extensive systems contributes to the circulation of Mycobacterium bovis (M. bovis) and other members of the Mycobacterium tuberculosis complex (MTC). Thus, free-range pigs can develop subclinical infections and may contribute to disease spread to bovine and wildlife. Serodiagnosis has been proposed as a screening tool for detecting infected pig herds; however, the value of this method to obtain an accurate diagnosis in this species is still not clear. In this study, sensitivity (Se) and specificity (Sp) estimates of four ELISAs and a lateral flow immunochromatographic antibody assay based on different M. bovis antigens, including MPB70 and MPB83 proteins, were evaluated in naturally infected domestic free-range pigs. For this purpose, submandibular lymph nodes and blood samples from 217 pigs from both TB-infected and historically negative farms were sampled at slaughterhouse and analysed by gross examination, histopathology, bacteriological culture and qPCR. Se and Sp estimates of the 5 evaluated assays ranged from 66.1% to 78% (CI95 from 52.6 to 87.7%) and from 98.9% to 100% (CI95 from 93.8 to 100%), respectively. Results of our study suggest that all the evaluated assays could be used as a first screening tool to conduct bTB surveillance in domestic pigs at population level; however, animals from seropositive herds should later be surveyed by other methods in order to reduce false negative results.

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.

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.

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.

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.

Septicaemic pasteurellosis in free-range pigs associated with an unusual biovar 13 of Pasteurella multocida.

Biochemical profiles, PFGE typing and MLST analysis were used to investigate an outbreak of septicaemic pasteurellosis in a free-range pig farm in Spain. Signs of coughing, dyspnoea and a visible inflammation of the ventral area of the neck (jowl), which acquired a cyanotic and necrotic appearance, were the characteristic findings in affected animals, associated with a high morbidity (70%) and case mortality (95%). Diffuse, haemorrhagic and fibrinous pleuroneumonia and acute, focally extensive and haemorrhagic myositis and panniculitis were observed in the histopathological analysis from three analyzed animals. Pasteurella multocida subsp. multocida, capsular type B, biovar 13 was isolated in pure culture from lung, submandibular tissue (jowl), liver, spleen and kidney tissue from diseased pigs. After PFGE typing, all P. multocida isolates displayed undistinguishable macrorestriction patterns with Bsp120I restriction enzyme demonstrating that the infection was caused by a single strain. With the multihost P. multocida MLST database, all P. multocida isolates were assigned to the new sequence type ST47 which was highly related with other bovine isolates of P. multocida type B associated with haemorrhagic septicaemia. This is the first description of an outbreak of septicaemic pasteurellosis in free-range pigs associated with P. multocida type B of the unusual biovar 13. The communication and complete diagnosis of cases of swine septicaemia and the possible role of pigs as reservoirs of this new pathogen must be evaluated to determine the importance of this disease for pigs.