Humoral immune response of pigs infected with Toxocara cati.

Toxocara cati is one of the causative agents of human toxocariasis. Serological methods are used for diagnosis in paratenic hosts like humans but the humoral immune response triggered by this parasite is unknown. We characterized the humoral immune response to T. cati excretory-secretory antigens (TES) in pigs as animal model during the acute and chronic stages of infection. ELISA and Western Blot techniques were used to determine antibody response. Pigs were experimentally inoculated with 100,000 infective Toxocara cati eggs. Blood was collected at 7, 14, 21 and 28 days post-inoculation (d.p.i.) to assess the acute stage of infection and 90, 120 and 180 d. p.i. for chronic stage analysis. ELISA showed values higher than the cut-off of specific IgM and IgG at 7 d. p.i. with significant differences at 0 and 7 d. p.i. for IgM and at 14, 21 and 28 d. p.i. for IgG in the acute stage. Higher and stable levels were detected in the chronic stage. Western Blot showed bands from 102 to 38 kDa detected by specific IgM and IgG. More immunogenic bands were identified by specific IgG. In the chronic stage of infection a band near 31 kDa was the only band detected by IgM until 150 d. p.i. Specific IgG recognized bands between 102 and 31 kDa. This study demonstrates how the humoral immune response evolves in the acute and chronic stages of infection and provides evidence on the role of the pig as a paratenic host of T. cati.

Evidence of Mycobacterium avium subsp. paratuberculosis binding to albumin: technical and biological implications.

Albumin binding ability is a well-characterized feature of many bacteria. To the best of our knowledge, there are no previous reports about this ability among mycobacteria, even when bovine serum albumin (BSA) is a common component of supplements used for the enrichment of synthetic media for mycobacterial growth in vitro and also of buffers used in laboratory techniques. In this work we explored the albumin binding ability of Mycobacterium avium subsp. paratuberculosis (MAP), a pathogenic bacterium causing a known and relevant ruminant disease worldwide, by immunizing rabbits with MAP (grown in media containing or not BSA) or BSA and conducting ELISA and immunoblot experiments with the obtained sera. As a result, we found that MAP can bind BSA when cultured in a conventional BSA-containing medium and when incubated for a short time in the presence of the protein. We also evaluated the host specificity of MAP interaction with albumin and found a preference for the protein of bovine origin when compared with its horse and rabbit homologs. Considerations about its technical and biological implications are discussed.

Rapid and sensitive method to identify Mycobacterium avium subsp. paratuberculosis in cow's milk by DNA methylase genotyping.

Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, caused by Mycobacterium avium subsp. paratuberculosis. This bacterium is shed primarily through feces of infected cows but can be also excreted in colostrum and milk and might survive pasteurization. Since an association of genomic sequences of M. avium subsp. paratuberculosis in patients with Crohn's disease has been described; it is of interest to rapidly detect M. avium subsp. paratuberculosis in milk for human consumption. IS900 insertion is used as a target for PCR amplification to identify the presence of M. avium subsp. paratuberculosis in biological samples. Two target sequences were selected: IS1 (155 bp) and IS2 (94 bp). These fragments have a 100% identity among all M. avium subsp. paratuberculosis strains sequenced. M. avium subsp. paratuberculosis was specifically concentrated from milk samples by immunomagnetic separation prior to performing PCR. The amplicons were characterized using DNA methylase Genotyping, i.e., the amplicons were methylated with 6-methyl-adenine and digested with restriction enzymes to confirm their identity. The methylated amplicons from 100 CFU of M. avium subsp. paratuberculosis can be visualized in a Western blot format using an anti-6-methyl-adenine monoclonal antibody. The use of DNA methyltransferase genotyping coupled to a scintillation proximity assay allows for the detection of up to 10 CFU of M. avium subsp. paratuberculosis per ml of milk. This test is rapid and sensitive and allows for automation and thus multiple samples can be tested at the same time.

Modeling Paratuberculosis in Laboratory Animals, Cells, or Tissues: A Focus on Their Applications for Pathogenesis, Diagnosis, Vaccines, and Therapy Studies.

Paratuberculosis is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. Paratuberculosis that affects a wide variety of domestic and wild animals. It is considered one of the diseases with the highest economic impact on the ruminant industry. Despite many efforts and intensive research, paratuberculosis control still remains controversial, and the existing diagnostic and immunoprophylactic tools have great limitations. Thus, models play a crucial role in understanding the pathogenesis of infection and disease, and in testing novel vaccine candidates. Ruminant animal models can be restricted by several reasons, related to space requirements, the cost of the animals, and the maintenance of the facilities. Therefore, we review the potential and limitations of the different experimental approaches currently used in paratuberculosis research, focusing on laboratory animals and cell-based models. The aim of this review is to offer a vision of the models that have been used, and what has been achieved or discovered with each one, so that the reader can choose the best model to answer their scientific questions and prove their hypotheses. Also, we bring forward new approaches that we consider worth exploring in the near future.

Early-stage findings in an experimental calf model infected with Argentinean isolates of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is an important pathogen that causes granulomatous enteritis known as Johne's disease or paratuberculosis (PTB). In this study an experimental model of calves infected with Argentinean isolates of MAP for 180 days was used to provide more data of the early PTB stages. Calves were challenged by oral route with MAP strain IS900-RFLPA (MA; n = 3), MAP strain IS900-RFLPC (MC; n = 2) or mock infected (MI; n = 2), and response to infection was evaluated through peripheral cytokine expression, MAP tissue distribution and histopathological early-stage findings. Specific and varied levels of IFN-γ were only detected at 80 days post-infection in infected calves. These data indicate that specific IFN-γ is not a useful indicator for early detection of MAP infection in our calf model. At 110 days post-infection, TNF-α expression was higher than IL-10 in 4 of the 5 infected animals and a significant decrease of TNF-α expression was detected in infected vs. non-infected calves. All calves challenged were identified as infected by mesenteric lymph node tissue culture and real time IS900 PCR. In addition, for lymph nodes samples, the agreement between these techniques was almost perfect (κ = 0.86). Colonization of tissues and levels of tissue infection varied between individuals. Evidence of early MAP dissemination to extraintestinal tissues such as the liver was detected by culture in one animal (MAP strain IS900-RFLPA). In both groups microgranulomatous lesions were observed predominantly in the lymph nodes, with giant cells present only in the MA group. In summary, the findings described herein may indicate that local MAP strains induced specific immune responses with particularities that could suggest differences in their biological behavior. Further studies should be carried out in order to obtain an in-depth understanding of the influence of MAP strains in host-pathogen interactions and the outcome of disease.

Anthropogenic Infection of Domestic Cats With SARS-CoV-2 Alpha Variant B.1.1.7 Lineage in Buenos Aires.

SARS-CoV-2 reverse zoonosis, particularly to domestic animals, and the potential role of infected animals in perpetuating the spread of the virus is an issue of increasing concern. In this case report, we identified the natural infection of two cats by SARS-CoV-2, in Argentina, whose owner had been previously infected by SARS-CoV-2. Viral genetic material was detected in feline oropharyngeal (OP) and rectal (R) swab by RT-qPCR, and sequence analysis revealed that the virus infecting the owner and one cat were genetically similar. The alpha variant (B.1.1.7 lineage) was identified with a unique additional mutation, strongly suggesting human-to-cat route of transmission. This study reinforces the One Health concept and the importance of integrating human, animal, and environmental perspectives to promptly address relevant health issues.

Early IgG2 in calves experimentally infected with Mycobacterium avium subsp. paratuberculosis.

The diagnosis of the early stages of paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a cumbersome task. In this study, an experimental Map-infection model of calves was used to improve the knowledge of early antibody response and to evaluate different in-house ELISAs in the detection of subclinical paratuberculosis. Calves were challenged with Map strain IS900-RFLPA (n = 3) or Map strain IS900-RFLPC (n = 2) (Argentinean isolated strains) or mock infected (n = 3), and their specific humoral response was evaluated. The diagnostic ELISA (IgG against Map protoplasmic antigen; PPA) could not detect the infection throughout the experimental period (180 days post-infection; dpi), whereas the IgG2/PPA-ELISA was able to identify infected calves at least once during the experiment. In addition, the use of crude Map extract detected most of the infections from 60 dpi onwards. Antibodies were also characterized by immunoblot: IgG2-reactivity to antigens of molecular weight lower than 50 kDa was detected in all infected calves. The experimental Map-infection model of calves used allows the study of the early humoral immune response in paratuberculosis. The evaluation of IgG2 specific to antigens lighter than 50 kDa emerges as an interesting alternative in calves naturally infected with paratuberculosis.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR.

The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900-PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 10(1) CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

Bovine response to lipoarabinomannan vaccination and challenge with Mycobacterium paratuberculosis.

This study aimed to evaluate the immune response in bovines following immunization with a mycobaterial Lipoarabinomannan extract (LAMe) and the effect of Map challenge. LAMe vaccine induced specific antibody levels that diminished after the challenge and affected Map excretion at least for 100 days thereafter.

Detection of Bovine IgG Isotypes in a PPA-ELISA for Johne's Disease Diagnosis in Infected Herds.

Johne's Disease or Paratuberculosis is a chronic granulomatous enteritis disease affecting ruminants. Detection of subclinically infected animals is difficult, hampering the control of this disease. The aim of this work was to evaluate the performance of detection of IgG isotypes in a PPA-ELISA to improve the recognition of cattle naturally infected with Map in different stages. A total of 108 animals from Tuberculosis-free herds were grouped as follows: exposed (n = 30), subclinically infected (n = 26), clinically infected (n = 14), and healthy controls (n = 38). Receiver-operating characteristic (ROC) curves of isotypes/PPA-ELISAs were constructed and areas under the curves were compared to evaluate the performance of each test. Our study demonstrated that the conventional PPA-ELISA (detecting IgG) is the best to identify clinically infected animals with high sensitivity (92.9%) and specificity (100%). Meanwhile, IgG2/PPA-ELISA improved the number of subclinically infected cattle detected as compared with conventional IgG/PPA-ELISA (53.8 versus 23.1%). In addition, it had the maximum sensitivity (65.0%, taking into account all Map-infected cattle). In conclusion, the combination of IgG and IgG2/PPA-ELISAs may improve the identification of Map-infected cattle in different stages of disease. The usefulness of IgG2 detection in serological tests for Johne's Disease diagnosis should be further evaluated.

Antibodies Induced by Lipoarabinomannan in Bovines: Characterization and Effects on the Interaction between Mycobacterium Avium Subsp. Paratuberculosis and Macrophages In Vitro.

Lipoarabinomannan (LAM) is a major glycolipidic antigen on the mycobacterial envelope. The aim of this study was to characterize the humoral immune response induced by immunization with a LAM extract in bovines and to evaluate the role of the generated antibodies in the in vitro infection of macrophages with Mycobacterium avium subsp. paratuberculosis (MAP). Sera from fourteen calves immunized with LAM extract or PBS emulsified in Freund's Incomplete Adjuvant and from five paratuberculosis-infected bovines were studied. LAM-immunized calves developed specific antibodies with IgG1 as the predominant isotype. Serum immunoglobulins were isolated and their effect was examined in MAP ingestion and viability assays using a bovine macrophage cell line. Our results show that the antibodies generated by LAM immunization significantly increase MAP ingestion and reduce its intracellular viability, suggesting an active role in this model.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests ( 0.20), and the proportions of agreement were 0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.(AU)