Intradermal reaction, bacterial load and pathological findings in calves experimentally infected with Mycobacterium avium subsp. paratuberculosis isolates from Argentina

INGRATTA, G; COLAVECCHIA, SB.; FERNÁNDEZ, B; JOLLY, A; FORTUNY, M. L.; MINATEL, L.; PAOLICCHI, F.A.; MUNDO, S. L.

Medellin

Congreso; : IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología; 2015

Latin American Association of Immunology

INTRODUCTION: Johne?s disease or paratuberculosis (PTB) is a chronic granulomatous intestinal pathology caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects both, domestic and wild ruminants. It is responsible for heavy economic losses to domestic livestock industries. Transmission occurs through the fecal-oral route and the animals usually become infected in the first months of life. This infectious disease is characterized by a long incubation period (2-5 years) which leads to weight loss, with or without diarrhea, and eventual death. Under natural or experimental infections of MAP, granulomatous lesions are usually found in the mucosal and submucosal regions of jejunum, ileum and associated lymph nodes. Several lesional classifications have been made mostly based in extent and cell types (epithelioid cells, multinucleated giant cells and lymphocytes); both aspects related to the type of immune response elicited against MAP. The infective capability of MAP depends on the strain virulence and the host susceptibility. A balanced between pro- and anti-inflammatory immune response is important for the control of MAP infection. Within the tests routinely employed in cattle, intradermal reaction (IDR) is used to evaluate the specific cellular immune response against bovine purified protein derivative (bPPD) in tuberculosis (TB) diagnosis. Comparative cervical test is much more sensitive and also involves avium purified protein derivative (aPPD). This test is only applied in specific epidemiological situations within the national TB control plan, in order to assess cross-reactions between these mycobacteria as well as the specific cellular response against MAP. Bacteriological culture (fecal and tissue) remains as the gold standard, despite having low sensibility and being time-consuming (18-21 weeks). The use of IS900 fragment length polymorphism (RFLP) and IS1311 PCR-restriction enzyme analysis (PCR-REA) methods, has led to the classification of MAP strains into two main genotypes: sheep isolates (?S-type? or ?type-I and III?) and cattle isolates (also called ?C-type? or ?type II?). Besides the genotypic distinctions between C and S strains of MAP, phenotypic differences have been found in vitro, where infected macrophages exhibit different responses depending on the type of isolate. The differentiation of isolates is essential for a deep understanding of the epidemiology and pathogenesis of MAP. At present, mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR)-typing is one of the most commonly used genotyping technique, able to distinguish between strain types by detecting diversity within a limited region of the genome. Animal infection models have contributed to provide a deeper understanding on the host response to infection, in order to predict the evolution of the disease and to improve diagnostic and control strategies. The aim of this study was to identify the relationship between in vivo diagnosis (IDR and fecal culture) and pathological findings (lesions, bacterial load and tissue culture) in an experimental model of calves challenged with two argentine MAP isolates. MATERIALS AND METHODS: Two MAP isolates genotyped as C-type, and classified as A and C by RFLP, and as INMV 1 by MIRU-VNTR were used. The selection was done taking into account their biological behavior in an in vitro infection model of bovine macrophages (relative level of TNF-alpha and IL-10 induced in Bomac cell line). While MAP A isolate showed an increased IL-10 production, MAP C isolate showed a predominant TNF-alpha secretion. Seven Holstein calves (6-8 weeks old) were infected with two oral doses (250 mg of total wet weight) of MAP isolates A (MA, n=3), C (MC, n=2) or mock infected (MI, n=2) for two consecutive days. At day 160 post-infection (pi) IDR was carried out with 0.1 mL of aPPD, 0.1 mL of bPPD according to Servicio Nacional de calidadagroalimentaria (SENASA) specifications (Res nº 128/2012), using 28-gauge needles. IDR positivity was considered when the difference in skin thickness between the time of inoculation and 72 h later was > 3 mm. At 180 days pi fecal samples were taken and necropsy was performed in order to obtain tissue samples for bacteriological culture, ZiehlNeelsen (ZN) acid fast bacilli (AFB) staining from culture inoculum, MAP bacterial load and histopathology. Organs sampled included: ileocecal valve (icV), ileocecal lymph node (icLN), jejunal mesenteric lymph node (jmLN), distal ileum (dI) and liver (Liv). Two grams of each tissue were processed, decontaminated and cultured on Herrold?s egg yolk medium (HEYM) with mycobactin J (Allied Monitor) for 18 weeks. Results were expressed as colony forming units per tissue gram (CFU/g) and as number of culture-positive tissue/total tissues examined. Fecal samples were processed similarly as described above for tissue samples. MAP identity was confirmed by IS900-PCR from isolated colonies. The formalin-preserved tissues were processed conventionally for haematoxylin/eosin (HE) and ZN staining. Histopathological lesions were identified and quantified for each tissue and isolate. RESULTS: At 160 days pi, all infected calves were asymptomatic and showed positive aPPD reaction., with increases of 21.7 ± 1.15 mm for MA group and 16.1 ± 1.76 mm for MC group (p