Mycobacterium avium
subsp.
paratuberculosis
(MAP) is the causal agent of paratuberculosis (PTBC), a chronic infectious granulomatous enteritis of ruminants. The PTBC diagnosis with commercial ELISA ...has limitations in sensitivity and specificity, and its results depend on the state of progress of the disease. This research aimed to evaluate two different ELISAs: (a) an “
in-house
” ELISA with a sonicated antigen obtained from a MAP I47 strain, and (b) a commercial ELISA. In total, the evaluated sample consisted of 394 bovine serum samples from 12 farms in Argentina with high (5–9%) and low (≤ 0.05%) prevalence of PTBC. The evaluation of the new antigen (2.5 µg/mL) was against a 1:50 dilution of the
M. phlei
faced sera. The cut-off point, sensitivity, and specificity determinations of both techniques were by ROC curve analysis. The area under the curve for the I47 ELISA was 0.9 (CI 95%, 0.93–0.97). With a cut-off point of 8.8%, the sensitivity was 84.3% and the specificity 96.6%. The agreement between both techniques was 0.7 (CI 95%, 0.6–0.8). These results indicate a high discriminative capacity to differentiate positive and negative bovine sera of MAP infection with the I47 ELISA. This result would represent an advantage to dispense with the imported kit.
ESAT-6, CFP-10 and EspC are virulence factors that have been extensively assayed for bovine and human tuberculosis diagnosis due their potent T-cell inducing activities. While polymorphisms of ESAT-6 ...and CFP-10 were analyzed, with the description of CFP-10 variants in M. tuberculosis, this fact has not been explored in M. bovis field isolates. The coding sequences of esxA (ESAT-6), esxB (CFP-10) and mb3645c (EspC) from 58 M. bovis strains exhibiting genomic variability (spoligotyping) were analyzed. Two genes –esxA and esxB – remained invariant while mb3645c exhibited one synonymous polymorphism (G to A mutation, position 66bp) in one isolate, compared to M. bovis AF2122/97 reference strain. All isolates exhibited a synonymous nucleotide polymorphism simultaneously (G to A mutation, position 255bp), compared to M. tuberculosis H37Rv reference strain. This study confirms the high conservation for ESAT-6, CFP-10 and EspC in local M. bovis field isolates and reinforce the use of these three antigens in the diagnosis of bovine tuberculosis. Further studies should be performed to globally confirm these findings.
Bovine tuberculosis (bTB) is caused by Mycobacterium bovis and disseminated worldwide. In Argentina, the highest prevalence occurs in dairy areas. BoLA DRB3.2 is related to the adaptive immunity in ...mycobacterial infections. Genetic polymorphisms of this marker have been associated with resistance or susceptibility to bovine diseases. We evaluated the association between BoLA DRB3.2 polymorphisms and bTB pathology scores in dairy and beef cattle breeds of Argentina. Most bovines exhibited visible lesions compatible with tuberculosis and, furthermore, 150 (85.7%) were also positive by bacteriology. A pathology index showed a variable degree of disease, from 3 to 76 (median pathology score = 9 (IQR: 7–15)). Thirty-five BoLA DRB3.2 alleles were identified with an associated frequency from 16% to 0.3%, distributed 73% (n = 128) in heterozygosis and 27% (n = 47) in homozygosis, with 12 BoLA DRB3.2 alleles (*0101, *1101, *1501, *0201, *2707 *1001, *1002, *1201, *14011, *0501 *0902 and *0701) representing the 74.7% of the population variability. A functional analysis grouped them in 4 out of 5 clusters (A-D), suggesting a functional overlapping. Among the 90 identified genotypes, *1101/*1101, *1101/*1501 and *0101/*0101 were the most frequent (10%, 8.9% and 8.9%, respectively). No association was detected between the pathology scores and a specific DRB3.2 allele (p > .05). Animals infected with M. bovis spoligotype SB0153 showed a significantly higher pathology score than those affected by the spoligotype SB0145 (p = .018). Furthermore, the Aberdeen Angus breed exhibited highest pathological scores (p < .0001), which were associated with disseminated lesion, thus suggesting that the host component could be important to the disease progression.
•BoLA DRB3.2 alleles were not associated to tuberculous pathological score.•Cattle breed was associated to the tuberculous pathology score.•Genomic variability of BoLA DBR3.2 alleles was higher than the functional.•M. bovis strain, cattle category and geographic area influence disease progression.
To study the deficiency of minerals and its relationship with Paratuberculosis, blood, serum, and fecal samples were obtained from 75 adult bovines without clinical symptoms of the disease and from ...two bovines with clinical symptoms of the disease, from two beef herds with a previous history of Paratuberculosis in the Province of Buenos Aires, Argentina. Serum samples were processed by ELISA and feces were cultured in Herrolds medium. Copper, zinc and iron in serum were quantified by spectrophotometry and selenium was measured by the activity of glutathione peroxidase. We also determined copper, zinc, iron and molybdenum concentrations in pastures and the concentration of sulfate in water. Mycobacterium avium subsp paratuberculosis (Map) was isolated from 17.3% of fecal samples of asymptomatic animals and from the fecal samples from the two animals with clinical symptoms. All the Map-positive animals were also ELISA-positive or suspect, and among them, 84.6% presented low or marginal values of selenium and 69.2% presented low or marginal values of copper. The two animals with clinical symptoms, and isolation of Map from feces and organs were selenium-deficient and had the lowest activity of glutathione peroxidase of all the animals from both herds. All the animals negative to Map in feces and negative to ELISA had normal values of Se, while 13.8% of animals with positive ELISA or suspect and culture negative presented low levels of Se. Half of the animals that were negative both for ELISA and culture in feces were deficient in copper but none of them presented low values of selenium. The content of molybdenum and iron in pasture was high, 2.5 ppm and 1.13 ppm in one herd and 2.5 ppm and 2.02 ppm in the other, respectively, whereas the copper:molybdenum ratio was 1.5 and 5.2, respectively. These results do not confirm an interaction between imbalances of the micronutrients and clinical Paratuberculosis, but show evidence of the relationship between selenium deficiencies in animals with Map infection and ELISA positive results.
No data concerning the presence of caprine paratuberculosis (PTBC) in Argentina are currently available. In this work, a Saanen dairy goat herd located in Buenos Aires province was sampled and tested ...for PTBC. Feces and blood samples were collected from 210 goats. Sera were analyzed by ELISA and fecal samples were pooled for culture. Necropsy was performed in two affected adult goats. Colonies were characterized and genotyped by IS900 PCR and IS1311 PCR-REA, respectively. Gross pathology and histopathology of necropsied goats were consistent with PTBC. Bacteriology, serology, and PCR confirmed the diagnosis. The Mycobacterium avium subsp. paratuberculosis isolates showed a restriction pattern characteristic of type C strains. This is the first report on the isolation and molecular characterization of M. avium subsp. paratuberculosis in dairy goats in Argentina.
Johne's disease or paratuberculosis is widespread in almost all countries and remains difficult to eradicate. Nowadays, diagnosis of
Mycobacterium avium subsp.
paratuberculosis (MPTB) infection is ...one of the main concerns. In this work, we evaluated the expression, biochemical properties and antigenicity of the Apa antigen, encoded by the gene annotated as MAP1569, in the MPTB genome. We confirmed its expression in MPTB and its glycosylation by the ConA binding assay. Although the MPTB-Apa is not an immunodominant antigen, MPTB-infected cattle showed a strong humoral response to recombinant Apa by Western blot and ELISA. Milk was also a suitable sample to be tested by ELISA. We comparatively analysed the humoral cross-reactivity to the Apa from MPTB (MPTB-Apa) and the orthologue from
Mycobacterium tuberculosis (MT-Apa, identical to that from
Mycobacterium bovis) in both infected and control cows. Response of
M. bovis- and MPTB-infected animals against MT-Apa was similar (
P
=
0.6985) but the response of the
M. bovis-infected ones to MPTB-Apa was differential, being significantly diminished (
P
<
0.0001). Although 6 out 45 animals from MPTB-infected herds responded to MPTB-Apa stimulation in the IFNγ release assay, we found no significant differences when compared infected herds with non-infected ones (
P
=
0.34). This antigen, in contrast to bovine Purified Protein Derivative (PPDb), was strongly represented in avian PPD (PPDa), as shown by the recognition of BALB/c mice hyperimmune sera against MPTB-Apa by Dot-blot immunoassay. We therefore demonstrated the antigenicity of Apa in MPTB-infected animals and a differential response to the recombinant antigen when compared to
M. bovis-infected animals. These traits herein described, added to the usefulness of milk samples to detect IgG anti-Apa, could be important for routine screening in dairy cattle, considering a multiantigenic approach to overcome the lack of immunodominance.
Abstract Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis or Johne’s disease (JD), a chronic enteritis disease that affects cattle and other animal species ...and has been linked to Crohn’s disease, a chronic inflammatory bowel disease in humans. MAP could resist pasteurization conditions and has been isolated from retail milk in several countries. The aims were to identify MAP and other mycobacteria in retail milk and dairy products in Argentina and to assess the product quality through Mesophilic Total Aerobic Bacteria (TAB) count. Three hundred and eighty-four samples of retail milk were tested for 24 months. All samples were negative for mycobacteria growth. However, 1.56% of the samples were positive for MAP identification by IS900-PCR. The TAB count was higher than the limits established by the Alimentary Argentinian Code (AAC) in 4.95% of the samples. Mycobacteria other than MAP were not detected either by culture or PCR. The MAP positive PCR from retail milk samples would indicate that they could come from dairy farms with JD and suggests that pasteurized milk or dairy products are not significant sources of human exposure to MAP in Argentina. Some milk samples exceeded the limits established by the AAC for TAB counts, indicating that commercialized milk could be processed and stored incorrectly.
The purpose of this study was to determine the viability of Mycobacterium avium subsp. paratuberculosis (MAP), Escherichia coli (E. coli), and Salmonella Enteritidis (S. Enteritidis) during ...preparation and refrigerated storage of yogurt. Three yogurts were prepared using pasteurized commercial milk. Each yogurt was artificially contaminated with (1) MAP, (2) E. coli + S. Enteritidis, and (3) MAP + E. coli + S. Enteritidis. Samples were taken during and after the fermentation process until day 20 after inoculation. MAP was not detected during their preparation and short-term storage but was recuperated after starting at 180 min after inoculation storage. Live bacterial counts of E. coli, and S. Enteritidis increased during the first 24 hours, followed by a slight decrease towards the end of the study. In this study it was shown how MAP, E. coli, and S. Enteritidis resisted the acidic conditions generated during the preparation of yogurt and low storage temperatures. This work contributes to current knowledge regarding survival of MAP, E. coli, and S. Enteritidis during preparation and refrigerated storage of yogurt and emphasizes the need to improve hygiene measures to ensure the absence of these pathogenic microorganisms in dairy products.
Mycobacterium avium subesp. paratuberculosis (MAP) es el agente etiológico de la paratuberculosis o la enfermedad de Johne (JD), una enteritis crónica que afecta al ganado vacuno y otras especies ...animales que se la vincula con la enfermedad de Crohn, una afección intestinal inflamatoria crónica en humanos. MAP podría resistir las condiciones de pasteurización y se lo aisló de la leche al por menor en varios países. Los objetivos fueron identificar MAP y otras micobacterias en leche y productos lácteos al por menor en Argentina y evaluar la calidad de estos a través del conteo de bacterias aerobias totales mesófilas (TAB). Se analizaron 384 muestras de leche de venta minorista durante 24 meses. Todas las muestras resultaron negativas en cuanto al crecimiento de micobacterias. Sin embargo, el 1.56% de las muestras dieron positivas en la identificación de MAP por IS900-PCR. El recuento de TAB superó los límites establecidos por el Código Alimentario Argentino (CAA) en el 4.95% de las muestras. No se detectaron micobacterias distintas de MAP ni por cultivo ni por PCR. La PCR positiva en MAP resultante de muestras de leche al por menor indicaría que podrían provenir de granjas lecheras con JD y sugiere que la leche pasteurizada o los productos lácteos no son fuentes significativas de exposición humana a MAP en Argentina. Algunas muestras de leche excedieron los límites establecidos por la AAC para los recuentos de TAB, lo cual indica que la leche comercializada podría procesarse y almacenarse incorrectamente.
All members of
Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of
M. avium complex and their ...preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among
M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21
M. avium subsp.
hominissuis and 26
M. avium subsp.
paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21
M. avium subsp.
hominissuis with 16 different IS
1245 RFLP and four different profiles with PCR-restriction analysis of
hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish
M. avium subsp.
paratuberculosis isolates with five different IS
900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate
M. avium subsp.
hominissuis but not
M. avium subsp.
paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between
M. avium subsp.
hominissuis and
M. avium subsp.
paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.
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