Overcoming Limited Access to Virus Infection Rapid Testing: Development of a Lateral Flow Test for SARS-CoV-2 with Locally Available Resources.

The COVID-19 pandemic highlighted testing inequities in developing countries. Lack of lateral flow test (LFT) manufacturing capacity was a major COVID-19 response bottleneck in low- and middle-income regions. Here we report the development of an open-access LFT for SARS-CoV-2 detection comparable to commercial tests that requires only locally available supplies. The main critical resource is a locally developed horse polyclonal antibody (pAb) whose sensitivity and selectivity are greatly enhanced by affinity purification. We demonstrate that these Abs can perform similarly to commercial monoclonal antibodies (mAbs), as well as mAbs and other pAbs developed against the same antigen. We report a workflow for test optimization using nasopharyngeal swabs collected for RT-qPCR, spiked with the inactivated virus to determine analytical performance characteristics as the limit of detection, among others. Our final prototype showed a performance similar to available tests (sensitivity of 83.3% compared to RT-qPCR, and 90.9% compared to commercial antigen tests). Finally, we discuss the possibility and the challenges of utilizing affinity-purified pAbs as an alternative for the local development of antigen tests in an outbreak context and as a tool to address inequalities in access to rapid tests.

Bovine paratuberculosis: a review of the advantages and disadvantages of different diagnostic tests.

Paratuberculosis (PTB), or Johne's disease, is a chronic infectious granulomatous enteritis of ruminants, caused by Mycobacterium avium subspecies paratuberculosis (Map). It is characterized by diarrhea and progressive cachexia, which may cause the death of the animal. Calves are the most susceptible to infection. Infected animals excrete Map mainly by the feces. PTB is endemic worldwide, with high prevalence levels, strong economic impact and public health relevance because of its possible association with Crohn's disease. Although the current reference diagnostic test is identification of Map in the bacterial culture, there are different diagnostic tests to identify infected individuals and/or herds. The sensitivity and specificity of these tests vary according to the stage of the disease in the animals to be evaluated. The correct choice and application of each of these diagnostic tests will ensure their success and may allow to establish a control program. The aim of this work is to review and discuss the different diagnostic tests used in the detection of Map-infected animals, focusing on their advantages and disadvantages.

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.

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 IgG1 antibodies against Mycobacterium avium subsp. paratuberculosis protein p34-cx improve association of bacteria and macrophages.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic granulomatous enteric disease in cattle. Among molecular components of Map, protein p34 was identified as specific and immunodominant for bovine B cells. In order to determine if specific antibodies could influence the course of Map pathogenesis, the interaction between bacteria and bovine macrophages was studied. Bovine polyclonal antibodies from 3 calves vaccinated with protein p34-cx, 6 calves vaccinated with heat-killed Map, 8 naturally infected, and 3 healthy calves -as negative controls- were used. Specific anti-Map, -p34-cx and -PPA-3 antibodies were evaluated and isotype characterized. Infected and Map vaccinated animals showed similar IgG1 and IgG2 response against Map whole bacteria. When p34-cx was used as the antigen, mainly IgG1 and IgG3 were detected in infected and only IgG1 in p34-cx vaccinated animals. Bovine polyclonal antibodies from three animals of each category were isolated and affinity purified through Map and p34-cx columns. The effect of these antibodies in association with Map and a transformed bovine peritoneal macrophage's cell line (Bov-Mac) as well as activation of NF-kappaB transcription factor was studied. Our results show that association of Map significantly increased in vitro after pretreatment with bovine anti-Map or anti-p34-cx antibodies obtained from vaccinated or infected cattle when compared with those of controls. Improved activation of NF-kappaB was detected in macrophages that ingested Map opsonized with either anti-Map or anti-p34-cx specific antibodies of infected or vaccinated calves, suggesting that both anti-Map and IgG1 anti-p34-cx antibodies support Map-macrophage interactions.