Evaluation of a virulent strain of Mycobacterium avium subsp. Paratuberculosis used as a heat-killed vaccine.

Bovine paratuberculosis is one of the most important chronic infectious diseases in livestock. This disease is difficult to control because of its inefficient management (test and cull strategy and inadequate biosecurity). Thus, the development of an effective vaccine is essential. In this study, we evaluated a local virulent strain (6611) of Mycobacterium avium subsp. paratuberculosis as an inactivated vaccine in comparison with the Silirum vaccine in mouse model and cattle. Regarding the mice model, only the groups vaccinated with 6611 showed lower colony forming unit (CFU) counts with a lower lesion score in the liver in comparison to the control group at 6 and 12 weeks post-challenge (wpc). The immune response was predominantly humoral (IgG1), although both vaccinated groups presented a cellular response with IFNγ production as well, but the 6611 group had also significant production of IL-2, IL-6, IL-17a, TNF, and IL-10. In cattle, the 6611 vaccinated group was the only one that maintained significant antibody values at the end of the trial, with significant production of IgG2 and IFNγ. No PPDb reactor was detected in the vaccinated animals, according to the intradermal caudal fold tuberculin test. Our results indicate that the 6611 local strain protected mice from challenge with a virulent strain, by inducing a humoral and cellular immune response. In the bovine, the natural host, the evaluated vaccine also induced humoral and cellular immune responses, with higher levels of CD4 + CD25+ and CD8 + CD25+ T cells populations than the commercial vaccine. Despite the encouraging results obtained in this study, an experimental challenge trial in cattle is mandatory to evaluate the efficacy of our candidate vaccine in the main host.

Effect of the deletion of lprG and p55 genes in the K10 strain of Mycobacterium avium subspecies paratuberculosis.

The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.

Genetic diversity of Mycobacterium avium sp. paratuberculosis by mycobacterial interspersed repetitive Unit-Variable number tandem repeat and multi-locus short-sequence repeat one-sentence summary: Genetic diversity of Mycobacterium avium sp. paratuberculosis isolates.

Background: Paratuberculosis is an enteric disease caused by Mycobacterium avium sp. paratuberculosis (MAP) that affects mainly ruminant producing losses to the livestock industry. Many molecular epidemiological methods have been used to discriminate MAP isolates. Method: The aim of this study was to describe the genetic diversity of the Argentinean MAP isolates using a combination of two molecular systems, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) ("automated and "non-automated") and the multi-locus short-sequence repeat (MLSSR) system. Results: Thirty-two isolates were identified as MAP of C type by IS900 polymerase chain reaction (PCA) and IS1311 PCA-restriction enzyme analysis. The main patterns found by both MIRU-VNTR systems were INMV1 (54.5%), INMV2 (24.2%) and INMV11 (9.1%). The INMV5, INMV8 and INMV16 were represented with one isolate each (3.0%). Only 4 MIRU-VNTR loci were polymorphic. Conclusion: Those isolates sharing the same INMV patterns were analyzed by MLSSR, being locus 2 the most polymorphic one showing isolates with 9, 10, 11, and more than 11 "G" repeats. Besides, the global discriminatory power among isolates could be increased using both techniques. Based on these results, a short version of the "automated" MIRU-VNTR could be used as a screening tool to group isolates genetically related and subsequently perform the SSR using locus 2 on those isolates sharing the same INMV pattern.

Development and Validation of a Novel ELISA for the Specific Detection of Antibodies against Mycobacterium avium Subspecies paratuberculosis Based on a Chimeric Polyprotein.

Bovine paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The optimization of detection tests specific for MAP is crucial to improve PTB control. In this work, we aimed to develop and validate a diagnostic tool based on an ELISA to specifically detect anti-MAP antibodies from bovine serum samples. For that purpose, we designed a recombinant polyprotein containing four specific antigens from MAP and optimized the ELISA. The validation consisted of the assessment of 10 sera from PTB-infected and healthy bovines with different OD values. The diagnostic performance of the polyprotein-ELISA was evaluated by testing 130 bovine serum samples (47 healthy, 48 MAP-infected, and 35 M. bovis-infected bovines). The ELISA using the polyprotein yielded an area under the ROC curve (AUC) of 0.9912 (95% CI, 0.9758-1.007; P < 0.0001). Moreover, for this ELISA, the cut-off selected from the ROC curve based on the point with a sensitivity of 95.56% (95% CI, 0.8485-0.9946) and specificity of 97.92 (95% CI, 0.8893-0.9995) was 0.3328. Similar results were obtained with an ELISA using the commercial Paratuberculosis Protoplasmatic Antigen (PPA). However, the ELISA with the polyprotein antigen showed a better performance against sera from animals infected with Mycobacterium bovis compared to the ELISA with PPA: lower cross-reactivity (2.85% versus 25.71%). These results demonstrate a very low cross-reactivity of the polyprotein with antibodies present in serum samples from animals infected with M. bovis. The designed polyprotein and the validated ELISA could be very useful for the specific identification of MAP-infected animals in herds.

Tipificación molecular de Mycobacterium avium subsp. paratuberculosis en lecherías de Antioquia, Colombia

RESUMEN Objetivo. Determinar la diversidad molecular de Mycobacterium avium subsp. paratuberculosis (MAP) en muestras ambientales de hatos lecheros colombianos. Materiales y métodos. Las muestras ambientales de 25 hatos lecheros positivos a MAP por IS900-qPCR se cultivaron por duplicado en medio de yema de huevo de Herrold con micobactina J para obtener aislamientos. Las colonias sospechosas fueron confirmadas para MAP por IS900-qPCR. El ADN positivo se subtipó utilizando técnicas de unidades micobacterialess repetitivas intercaladas - número variable de repeticiones en tándem (MIRU-VNTR) y técnicas de repeticiones de multilocus de secuencia corta (MLSSR) para analizar las diferencias genéticas entre los aislamientos. Resultados. El subtipado reveló dos genotipos diferentes por MIRU-VNTR (INMV 2 e INMV 36). La técnica de MLSSR se realizó para aumentar el poder discriminatorio de lo obtenido por MIRU-VNTR, pero no se observaron diferencias entre los aislamientos recuperados. Conclusiones. El presente estudio representa un enfoque importante para el conocimiento del estatus epidemiológico de MAP en la población de estudio.

ABSTRACT Objective. To determine Mycobacterium avium subsp. paratuberculosis (MAP) molecular diversity in environmental samples from Colombian dairy herds. Materials and methods. Environmental samples from 25 IS900-qPCR MAP-positive dairy herds were cultured by duplicate in Herrold's egg yolk medium with mycobactin J to obtain isolates. Suspicious colonies were confirmed by MAP-IS900-qPCR. Positive DNA was sub-typed using mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) and multilocus short sequence repeats (MLSSR) techniques to analyze the genetic differences between the isolates. Results. Sub-typing revealed two different genotypes by MIRU-VNTR (INMV 2 and INMV 36). MLSSR technique was carried out to increase the discriminatory power from what was obtained by MIRU-VNTR, but no differences were observed among the recovered isolates. Conclusions. The present study represents an important approach to the knowledge on MAP epidemiological status in the study population.

Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis.

Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.

Protection efficacy of Argentinian isolates of Mycobacterium avium subsp. paratuberculosis with different genotypes and virulence in a murine model.

Paratuberculosis is a chronic disease caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease causes economic losses and, therefore, it is imperative to follow proper control strategies, which should include an effective vaccine. Several strategies have assessed the virulence and immune response of Map strains that could be used as a vaccine. This study evaluates the degree of virulence, immune response, and protection of Argentinian strains of Map with different genotype in a murine model. Four local isolates (Cattle type) with different genotypes (analyzed by MIRU-VNTR and SSRs) were selected and evaluated in a virulence assay in BALB/c mice. This assay allowed us to differentiate virulent and low-virulence Map strains. The less virulent strains (1543/481 and A162) failed to induce a significant production of the proinflammatory cytokine IFNg, whereas the virulent strain 6611 established infection along with a proinflammatory immune response. On the other hand, the virulent strain 1347/498 was efficient in establishing a persistent infection, but failed to promote an important Th1 response compared with 6611 at the evaluated time. We selected the low-virulence strain 1543/498 as a live vaccine and the virulent strain 6611 as a live and inactivated vaccine in a protection assay in mice. Strain 1543/481 failed to protect the animals from challenge, whereas strain 6611, in its live and inactivated form, significantly reduced the CFUs count in the infected mice, although they had different immunological response profiles. The inactivated virulent strain 6611 is a potential vaccine candidate against paratuberculosis to be tested in cattle.

Identification of the Apa protein secreted by Mycobacterium avium subsp. paratuberculosis as a novel fecal biomarker for Johne's disease in cattle.

Paratuberculosis (PTB) or Johne's disease is a chronic intestinal infection of ruminants, caused by Mycobacterium avium subsp. paratuberculosis. The shedding of mycobacteria in the feces starts at the initial stages and increases with disease progression, suggesting that antigens secreted by mycobacteria could be excreted in the feces. Previously, we demonstrated that the alanine and proline-rich antigen (Apa), a secretory antigen of Map, could be detected in the intestine of cows with PTB using a monoclonal antibody. In this study, we verified whether this protein can be found in consistently detectable levels in the feces of cattle with PTB. Feces were obtained from cows with Johne's disease confirmed by laboratory tests, cows with suspected PTB based on seropositivity and from PTB-free control cows. Samples were immunoprecipitated using anti-Apa monoclonal antibody and analyzed by immunoblot. The Apa was detected as a 60/70 kDa doublet band in all samples obtained from animals with laboratory-confirmed disease and in a substantial proportion of seropositive asymptomatic animals, but not in the control samples. Additionally, the antigen was detected in the feces of animals with Johne's disease by ELISA. This study strongly suggests that Apa is a potential fecal biomarker of Johne's disease that could serve for immunodiagnosis.

Identification of the Apa protein secreted by Mycobacterium avium subsp. paratuberculosis as a novel fecal biomarker for Johne's disease in cattle

Paratuberculosis (PTB) or Johne's disease is a chronic intestinal infection of ruminants, caused by Mycobacterium avium subsp. paratuberculosis. The shedding of mycobacteria in the feces starts at the initial stages and increases with disease progression, suggesting that antigens secreted by mycobacteria could be excreted in the feces. Previously, we demonstrated that the alanine and proline-rich antigen (Apa), a secretory antigen of Map, could be detected in the intestine of cows with PTB using a monoclonal antibody. In this study, we verified whether this protein can be found in consistently detectable levels in the feces of cattle with PTB. Feces were obtained from cows with Johne's disease confirmed by laboratory tests, cows with suspected PTB based on seropositivity and from PTB-free control cows. Samples were immunoprecipitated using anti-Apa monoclonal antibody and analyzed by immunoblot. The Apa was detected as a 60/70 kDa doublet band in all samples obtained from animals with laboratory-confirmed disease and in a substantial proportion of seropositive asymptomatic animals, but not in the control samples. Additionally, the antigen was detected in the feces of animals with Johne's disease by ELISA. This study strongly suggests that Apa is a potential fecal biomarker of Johne's disease that could serve for immunodiagnosis.

Identification of the Apa protein secreted by Mycobacterium avium subsp. paratuberculosis as a novel fecal biomarker for Johne's disease in cattle

Paratuberculosis (PTB) or Johne's disease is a chronic intestinal infection of ruminants, caused by Mycobacterium avium subsp. paratuberculosis. The shedding of mycobacteria in the feces starts at the initial stages and increases with disease progression, suggesting that antigens secreted by mycobacteria could be excreted in the feces. Previously, we demonstrated that the alanine and proline-rich antigen (Apa), a secretory antigen of Map, could be detected in the intestine of cows with PTB using a monoclonal antibody. In this study, we verified whether this protein can be found in consistently detectable levels in the feces of cattle with PTB. Feces were obtained from cows with Johne's disease confirmed by laboratory tests, cows with suspected PTB based on seropositivity and from PTB-free control cows. Samples were immunoprecipitated using anti-Apa monoclonal antibody and analyzed by immunoblot. The Apa was detected as a 60/70 kDa doublet band in all samples obtained from animals with laboratory-confirmed disease and in a substantial proportion of seropositive asymptomatic animals, but not in the control samples. Additionally, the antigen was detected in the feces of animals with Johne's disease by ELISA. This study strongly suggests that Apa is a potential fecal biomarker of Johne's disease that could serve for immunodiagnosis.

Clinical Relevance, Diversity and Genetic Variability of Different Species within the Genus Mycobacterium

Introduction: The term non-tuberculous mycobacteria (NTM) includes different ambient species capable of sickening humans and/or animals, even by means of a potential zoonotic transmission. Objectives: To determine: The clinical importance of several species within the genus Mycobacterium and the genetic diversity of the M. avium complex (MAC), the in vitro bacterial sensitivity and the success of the specific treatment. Materials and Methods: Collection of clinical and epidemiologic data and information about isolates of the 2009-2016 period; molecular identification of the isolates; determination of the in vitro bacterial sensitivity and genetic diversity of the MAC; treatment evaluation. Results: 225 mycobacteriosis cases were diagnosed, with a stable prevalence of ≈6% per year and 22 recovered species: 4 rapidly growing species isolated from 66 patients and 18 slowly growing species. The MAC was isolated in 95 cases, M. avium hominissuis - 40 cases, M. intracellulare - 51 cases, M. chimaera - 3 cases and M. colombiense - 1 case. We observed a greater probability of getting sick from M. intracellulare in patients previously treated for tuberculosis (TB). HIV-positive patients had a greater risk of falling ill from M. avium hominissuis. Aminoglycosides, fluoroquinolones and macrolides were the most active drugs against most NTM. Approximately half of the cases healed. Conclusions: M. intracellulare, M. aviumhominissuis with great genetic variability and M. abscessus were the most commonly found pathogens. The cases of TB+NTM mixed disease were an important finding. For treating these patients, it was necessary to add second line drugs to the therapeutic regimen for TB; and most of them healed

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis.

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.

Description of a novel adhesin of Mycobacterium avium subsp. paratuberculosis.

The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the µM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding.

Evaluation of cocktails with recombinant proteins of Mycobacterium bovis for a specific diagnosis of bovine tuberculosis.

The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.

Characterization of a Mycobacterium avium subsp. avium operon associated with virulence and drug detoxification.

The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo.

Search for Mycobacterium avium Subspecies paratuberculosis Antigens for the Diagnosis of Paratuberculosis.

The aim of this study was to evaluate a wide panel of antigens of Mycobacterium avium subsp. paratuberculosis (MAP) to select candidates for the diagnosis of paratuberculosis (PTB). A total of 54 recombinant proteins were spotted onto nitrocellulose membranes and exposed to sera from animals with PTB (n = 25), healthy animals (n = 10), and animals experimentally infected with M. bovis (n = 8). This initial screening allowed us to select seven antigens: MAP 2513, MAP 1693, MAP 2020, MAP 0038, MAP 1272, MAP 0209c, and MAP 0210c, which reacted with sera from animals with PTB and showed little cross-reactivity with sera from healthy animals and animals experimentally infected with M. bovis. The second step was to evaluate the antigen cocktail of these seven antigens by ELISA. For this evaluation, we used sera from animals with PTB (n = 25), healthy animals (n = 26), and animals experimentally infected with M. bovis (n = 17). Using ELISA, the cocktail of the seven selected MAP antigens reacted with sera from 18 of the 25 animals with PTB and did not exhibit cross-reactivity with healthy animals and only low reactivity with animals with bovine tuberculosis. The combined application of these antigens could form part of a test which may help in the diagnosis of PTB.

Accuracy assessment and screening of a dairy herd with paratuberculosis by three different ELISAs.

Although the culture of Mycobacterium avium subsp. paratuberculosisis is the gold standard for the diagnosis of paratuberculosis, this bacterium is difficult to grow. In contrast, serological tests like ELISAs are inexpensive, rapid, and easy to perform. The aims of this study were to evaluate the accuracy of three different ELISAs: one with the commercial antigen PPA-3, another one with L5P (a recently described lipopentapeptide), and a third one with an in-house antigen whole cell lysates (WCL) of M. avium (MAA) strain D4ER (Study 1), and to compare them with other tests for paratuberculosis (PTB) diagnosis (Study 2). In Study 1, the sensitivities of the three ELISAs tested were 74.1%, 37% and 74.1%, respectively, whereas their specificities were 98.9%, 100% and 100%, respectively. In Study 2, we compared the three above-mentioned ELISAs with the intradermal reaction test using Avian PPD (PPDa) and fecal culture associated with Ziehl-Neelsen stain and PCR tests, in a dairy herd with 4.6% of cows with clinical signs of PTB. The results showed that fecal samples from 14 cows (16%) were culture-positive and that fecal samples from nine cows (10%) were PPDa-positive. Most of these animals (culture-positive and PPDa-positive) were detected as positive with any of the three ELISAs tested. Serological results showed that 31% of the animals were positive to ELISA-PPA-3, 17% to ELISA-L5P and 42.5% to ELISA-WCL. The combination of these three ELISAs identified 50.6% of the animals as positive in the infected herd. In particular, the results show that the locally developed ELISA seems to be useful for identifying many infected animals in a herd.

An applied printing immunoassay with recombinant Nc-SAG1 for detection of antibodies to Neospora caninum in cattle.

Neospora caninum is a protozoan parasite that causes an important reproductive disease in cattle. Neospora caninum surface antigen 1 (Nc-SAG1) is an immunodominant candidate for the development of a diagnostic reagent for neosporosis. The current study describes the development and evaluation of an antigen print immunoassay (APIA) with recombinant Nc-SAG1 for the detection of specific antibodies to N. caninum in cattle. The concordance between APIA and a commercial enzyme-linked immunosorbent assay (ELISA) was evaluated with 232 serum samples from experimentally and naturally infected cattle. Sixty-one (26.7%) samples were positive for antibodies to N. caninum by ELISA and 58 (25.4%) by APIA. The new assay had a sensitivity of 85% and a specificity of 96%. These results, along with the potential of APIA to evolve into a multiple antigen detection format, suggest that this method would be a reliable diagnostic test for detection of antibodies to N. caninum in cattle.

Detection of Mycobacterium avium subsp. paratuberculosis by a Direct In Situ PCR Method.

In situ detection of Mycobacterium avium subsp. paratuberculosis is useful for diagnosis and research of paratuberculosis. The aim of this paper was to detect this agent in formalin-fixed, paraffin-embedded tissue samples by a direct in situ PCR. The technique was performed on ileum or ileocaecal lymph node samples from 8 naturally infected cattle and 1 healthy calf, by using p89 and p92 primers for amplification of IS900 sequence. Moderate positive signal was detected in all positive samples and not in negative control, but tissues resulted were affected in many cases due to the enzymatic treatment and the high temperature exposition. Although the technique was useful for Map detection, the signal was lower than immunohistochemistry probably because of the fixation process. In one case, signal was higher, which might be due to the detection of spheroplasts. Thus, the described method should be recommended when others resulted negative or for spheroplasts detection.