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.

An Outbreak of SARS-CoV-2 in Captive Armadillos Associated with Gamma Variant in Argentina.

The current pandemic produced by SARS-CoV-2 and its variants represent an example of the one health concept in which humans and animals are components of the same epidemiologic chain. Animal reservoirs of these viruses are thus the focus of surveillance programs, to monitor their circulation and evolution in potentially new hosts and reservoirs. In this work, we report the detection of the SARS-CoV-2 Gamma variant infection in four specimens of Chaetophractus villosus (big hairy armadillo/armadillo peludo) in Argentina. In addition to the finding of a new wildlife species susceptible to SARS-CoV-2 infection, the identification of the Gamma variant three months after its last detection in humans in Argentina is a noteworthy result, which can be due to alternative non-exclusive scenarios, such as unidentified viral reservoirs, unrecognized circulation in humans or species-specific variation in incubation periods.

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.

Antibodies from healthy or paratuberculosis infected cows have different effects on Mycobacterium avium subspecies paratuberculosis invasion in a calf ileal loop model.

In this work, we used a calf ileal loop model to evaluate whether the preincubation of Mycobacterium avium subspecies paratuberculosis (MAP) with antibodies from healthy, MAP-positive or Lipoarabinomannan (LAM) immunized cows could affect the results of infection after 3.5 h. Bacterial load in tissue was assessed by Ziehl-Neelsen and by culture for each loop. MAP was detectable in all infected loops after 3.5 h.p.i.; although the presence of antibodies from MAP-positive cows significantly reduced bacterial load in loops as compared with antibodies from healthy donors (by Ziehl-Neelsen and culture, p-value < 0.003 and 0.0203, respectively). A possible direct effect of antibodies on MAP viability was shown to be not significant. Severity of histopathologic changes induced by MAP infection also varied according to the pretreatment: MAP induced less changes when inoculated in the presence of antibodies from MAP-positive cows as compared with antibodies from healthy donors. Overall, our results show that the presence of antibodies from MAP-positive cows reduced MAP invasion and consequent early histological changes in this ileal short-term loop model. These results may suggest a protective role of antibodies in the response against MAP at the portal of entry in cattle.

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.

Evidence of a pro-apoptotic effect of specific antibodies in a bovine macrophage model of infection with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous enteritis in ruminants. Understanding the protective immune response following infection is crucial to improve the diagnosis and the development of vaccines against this disease. The goal of this work was to assess whether specific antibodies were able to modulate the macrophage response to MAP infection by evaluating apoptosis and TNF-α secretion in an in vitro model. Sera from healthy (n=2), MAP-infected (n=3) and lipoarabinomannan (LAM)-immunized (n=3) bovines were evaluated. LAM was chosen as immunogen due to its relevant role in mycobacterial pathogenesis. We demonstrated by two different techniques (Acridine Orange/Ethidium Bromide microscopy and Annexin V/7-Amino-Actinomycin D flow cytometry) that the immune sera from both, MAP-infected and LAM-immunized bovines, significantly increased macrophage apoptosis in infected cultures. Comparable levels of apoptosis were detected when MAP was pre-incubated with purified specific antibodies instead of whole serum. Furthermore, this effect was accompanied by a significantly higher secretion of TNF-α. These results strongly suggest that specific antibodies could limit the impact of MAP on the apoptosis of bovine cells. This work would contribute to elucidate the role of the specific antibody response in bovine JD and its prevention.

Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25(+) cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25(+) cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved.

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.

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.