Estudo da presença de Mycobacterium avium subespécie paratuberculosis numa exploração leiteira no Litoral Centro de Portugal Continental

A Paratuberculose, conhecida também como Doença de Johne, é uma afeção infecciosa que provoca uma enterite granulomatosa crónica causada pelo agente Mycobacterium avium subespécie paratuberculosis (Map). É uma doença de carácter contagioso, de distribuição mundial, atingindo os mamíferos, particularmente os pequenos e grandes ruminantes, equinos, suínos, búfalos, coelhos, etc. Tem um grande impacto económico, nomeadamente na redução da produção leiteira, na redução dos teores de proteína no leite, na susceptibilidade a outras doenças, no refugo de animais e no aumento dos custos na sanidade. Pensa-se que também terá impacto a nível de Saúde Pública uma vez que o Map pode estar associado á doença de Crohn em humanos em que o leite cru, leite em pó ou leite pasteurizado podem ser os veículos de transmissão, porém não existem estudos suficientes para sustentar este acontecimento. A técnica de diagnóstico “Gold Standard” é a cultura microbiológica de fezes, no entanto é um procedimento muito demorado podendo levar até 4 meses para se observarem as colónias bacterianas, uma vez que o seu crescimento é muito lento. Deste modo, existem outros testes, tais como o PCR e ELISA, com elevada especificidade para o agente que fornecem resultados mais rápidos e que permitem minimizar os falsos-positivos, apesar da sua reduzida sensibilidade (por volta dos 50%). Este estudo é baseado na observação das instalações da exploração e consequente comparação com parâmetros analisados e documentados em literatura. Para além disso, são utilizadas informações fornecidas pelo Software da exploração com o intuito de estudar a incidência da Paratuberculose, ao longo dos anos, de modo a comprovar se as medidas de maneio e de higiene adotadas pela exploração afetada contribuem ou não de alguma maneira para o controlo/erradicaçãoda doença de Johne.

Mycobacterium avium subspecies paratuberculosis and Crohn's disease: a systematic review and meta-analysis

This systematic review assesses the evidence for an association between Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's disease. We analysed 28 case-control studies comparing MAP in patients with Crohn's disease with individuals free of inflammatory bowel disease (IBD) or patients with ulcerative colitis. Compared with individuals free of IBD, the pooled odds ratio (OR) from studies using PCR in tissue samples was 7.01 (95% CI 3.95-12.4) and was 1.72 (1.02-2.90) in studies using ELISA in serum. ORs were similar for comparisons with ulcerative colitis patients (PCR, 4.13 [1.57-10.9]; ELISA, 1.88 [1.26-2.81]). The association of MAP with Crohn's disease seems to be specific, but its role in the aetiology of Crohn's disease remains to be defined.

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

BACKGROUND: Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP. RESULTS: By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%). CONCLUSIONS: There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease

Sensitive and specific detection of viable Mycobacterium avium subsp. paratuberculosis in raw milk by the Peptide-mediated magnetic separation (PMS)-phage assay

This study describes further validation of a previously described Peptide-mediated magnetic separation (PMS)-Phage assay, and its application to test raw cows’ milk for presence of viable Mycobacterium avium subsp. paratuberculosis (MAP). The inclusivity and exclusivity of the PMS-phage assay were initially assessed, before the 50% limit of detection (LOD50) was determined and compared with those of PMS-qPCR (targeting both IS900 and f57) and PMS-culture. These methods were then applied in parallel to test 146 individual milk samples and 22 bulk tank milk samples from Johne’s affected herds. Viable MAP were detected by the PMS-phage assay in 31 (21.2%) of 146 individual milk samples (mean plaque count of 228.1 PFU/50 ml, range 6-948 PFU/50 ml), and 13 (59.1%) of 22 bulk tank milks (mean plaque count of 136.83 PFU/50 ml, range 18-695 PFU/50 ml). In contrast, only 7 (9.1%) of 77 individual milks and 10 (45.4%) of 22 bulk tank milks tested PMS-qPCR positive, and 17 (11.6%) of 146 individual milks and 11 (50%) of 22 bulk tank milks tested PMS-culture positive. The mean 50% limits of detection (LOD50) of the PMS-phage, PMS-IS900 qPCR and PMS-f57 qPCR assays, determined by testing MAP-spiked milk, were 0.93, 135.63 and 297.35 MAP CFU/50 ml milk, respectively. Collectively, these results demonstrate that, in our laboratory, the PMS-phage assay is a sensitive and specific method to quickly detect the presence of viable MAP cells in milk. However, due to its complicated, multi-step nature, the method would not be a suitable MAP screening method for the dairy industry.

Análisis del potencial epizoótico de cepas de Mycobacterium avium subsp. paratuberculosis aisladas de diversos hospedadores empleando modelos de infección in vitro

299 p.

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BACKGROUND Mycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission. RESULTS 164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900--restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property. CONCLUSION The results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

Investigation of the impact of simulated commercial centrifugation and microfiltration conditions on levels of Mycobacterium avium subsp. paratuberculosis in milk

The potential for physical removal of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) from milk by centrifugation and microfiltration was investigated by simulating commercial processing conditions in the laboratory by means of a microcentrifuge and syringe filters, respectively. Results indicated that both centrifugation of preheated milk (60 degrees C) at 7000 x g for 10 s, and microfiltration through a filter of pore size 1.2 mu m, were capable of removing up to 95-99.9% of M. paratuberculosis cells from spiked whole milk and Middlebrook 7H9 broth suspensions, respectively. Centrifugation and microfiltration may therefore have potential application within the dairy industry as pretreatments to reduce M. paratuberculosis contamination of raw milk.

Antibacterial activities of naturally occurring compounds against Mycobacterium avium subsp. paratuberculosis.

The antibacterial activities of 18 naturally occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols, and other plant extracts) against three strains of Mycobacterium avium subsp. paratuberculosis (a bovine isolate [NCTC 8578], a raw-milk isolate [806R], and a human isolate [ATCC 43015]) were evaluated using a macrobroth susceptibility testing method. M. avium subsp. paratuberculosis was grown in 4 ml Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase, 0.05% Tween 80 (or 0.2% glycerol), and 2 µg/ml mycobactin J supplemented with five concentrations of each test compound. The changes in the optical densities of the cultures at 600 nm as a measure of CFU were recorded at intervals over an incubation period of 42 days at 37°C. Six of the compounds were found to inhibit the growth of M. avium subsp. paratuberculosis. The most effective compound was trans-cinnamaldehyde, with a MIC of 25.9 µg/ml, followed by cinnamon oil (26.2 µg/ml), oregano oil (68.2 µg/ml), carvacrol (72.2 µg/ml), 2,5-dihydroxybenzaldehyde (74 µg/ml), and 2-hydroxy-5-methoxybenzaldehyde (90.4 µg/ml). With the exception of carvacrol, a phenolic compound, three of the four most active compounds are aldehydes, suggesting that the structure of the phenolic group or the aldehyde group may be important to the antibacterial activity. No difference in compound activity was observed between the three M. avium subsp. paratuberculosis strains studied. Possible mechanisms of the antimicrobial effects are discussed.