Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), can cause considerable economic losses in affected herds. Early diagnosis of JD is hampered by the chronic nature of the disease with a slow subclincal progression. The aim of the present study was to challenge the hypothesis that lymphatic fluid is of diagnostic value in the early stages of the disease. Lymphatic fluid from 122 animals was collected and tested for MAP by nested PCR for IS900 and compared to the results of testing for MAP in feces (culture), blood and milk (ELISA) in 110 of these samples. MAP was detected by PCR in 27.1% of the lymph samples. Agreement between the tests was poor: 6.9% of the lymph positive cows were also positive in all other tests applied, and 69.0% had negative results in fecal culture, blood and milk ELISA. Resampling of 25 cows after 8 to 12 and 16 to 20 months revealed 20.0% lymph positive animals at the first, 5.5% at the second and 27.8% at the third sampling, respectively. Only one cow showed positive lymph-PCR results at more than one sampling date. Lymph-positive cows had a 7.2 times greater likelihood of being culled within 8 to 12 months after sampling, compared to negative cows, mainly due to other health issues than JD. It can be concluded, that lymphatic fluid might be promising for the detection of early MAP-infection in cows, but further studies to elucidate the potential of this diagnostic approach are needed.
The objective of the current study was to evaluate the feasibility of lymph collection from the bovine udder and to investigate if the lymphatic fluid might be of diagnostic value in cows infected with Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of paratuberculosis. Lymph fluid collection was attempted from 58 cows, and the reactions of the cows as well as the level of difficulty of the procedure were recorded in 56 animals. Lymph samples (51 in total) were tested for the presence of MAP by nested polymerase chain reaction. Collection of the lymphatic fluid caused no or mild signs of discomfort in 94.6% of the cows; in 51.8% of cows, lymphatic fluid was attained on the first attempt, while sample collection was unsuccessful in 12.1%. Mycobacterium avium subsp. paratuberculosis was detected in 43.1% of all lymph samples. The bacterium was present in 66.7% of cows with clinical Johnes disease, in 42.8% of asymptomatic cows with a positive or suspicious enzyme-linked immunosorbent assay (ELISA) result in blood, and in 38.7% of cows with a negative ELISA result in blood. The present study shows that the procedure was well tolerated by most cows and can easily be performed on farm. The current report of the isolation of MAP from lymph fluid suggests that the present approach could be used for the early detection of Johnes disease in cattle.
Bovine anaplasmosis (BA) is a hemoparasitic disease of great importance in cattle within the tropical and subtropical regions of the world. Control programs for BA require accurate diagnostic assays but validation can be challenging because the true disease status of all animals is frequently not known with certainty. The objective of this study was to estimate the accuracy of assays for detection of Anaplasma marginale infection in lactating dairy cattle of Puerto Rico using Bayesian methods without a perfect reference test. There were 2,331 cattle with complete diagnostic results sampled from 79 herds, and the prevalence of BA was estimated as 22% (95% probability interval [PI]: 19-25%). The sensitivity (Se) and specificity (Sp) of a major surface protein 5 competitive enzyme-linked immunosorbent assay (MSP-5 cELISA) were estimated as 99% (95% PI: 96-100%) and 89% (95% PI: 87-92%), respectively. The Se and Sp of a quantitative polymerase chain reaction (qPCR) were 67% (95% PI: 60-74%) and 99% (95% PI: 99-100%). The Se and Sp of a card agglutination test were 34% (95% PI: 29-39%) and 99% (95% PI: 99-100%). Area under the receiver-operating characteristic curve for the MSP-5 cELISA was 0.748 (95% PI: 0.71-0.79). The MSP-5 cELISA appears to be the test of choice for screening cattle for subclinical BA based on the high estimated Se, rapidity of results, relative low cost, and ease of standardization.
Paratuberculosis (Johnes disease) imposes a significant problem to the world dairy and beef industries and today is considered a potential zoonosis. The disease is caused by Mycobacterium avium subsp. paratuberculosis and is characterized by progressive weight loss and profuse diarrhoea. Susceptibility to infection is suspected to have a genetic component, and moderated values for heritability of infection have been reported. Interferon gamma is an inducible cytokine with a crucial role in the innate host response to intracellular bacteria. Toll-like receptors are trans-membrane structures responsible for coordination of innate and adaptive immune responses. The solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1) gene plays an important role in innate immunity, preventing bacterial growth in macrophages during the initial stages of infection. The objective of this candidate gene case-control study was to characterize the distribution of polymorphisms in three candidate genes related to the immune function; interferon gamma (BoIFNG), toll-like receptor 4 (TLR4), and SLC11A1 genes and to test their role as potential risk factors for paratuberculosis infection in cattle. The statistical analysis demonstrated significant differences in allelic frequencies between cases and controls for BoIFNG-SNP(1)2781 and SLC11A1 microsatellites, indicating a significant association between infection and variant alleles. In the analysis of genotypes, a significant association was also found between infection status and BoIFNG-SNP(1)2781 and SLC11A1-275-279-281 microsatellites. However, when variables such as breed and age were included in the multivariate logistic regression analysis, a tendency toward statistical significance for the effect of polymorphisms in the odds of infection was only found for alleles SLC11A1-275 and 279.
Paratuberculosis represents a major problem in farmed ruminants and at the present is considered a potential zoonosis. The disease is caused by Mycobacterium avium subsp. paratuberculosis, and susceptibility to infection is suspected to have a genetic component. Caspase recruitment domain 15 (CARD15) gene encodes for a cytosolic protein implicated in bacterial recognition during innate immunity. Crohns disease (CD) is an idiopathic inflammatory bowel disease in humans comparable in many features to bovine paratuberculosis involving an abnormal mucosal immune response. The association between mutations in the CARD15 gene and increased risk of Crohns disease has been described. The objective of this candidate gene case-control study was to characterize the distribution of three polymorphisms in the bovine CARD15 gene and test their association with paratuberculosis infection in cattle. Three previously reported single nucleotide polymorphisms (E2[-32] intron 1; 2197/C733R and 3020/Q1007L) were screened for the study population (431 adult cows). The statistical analysis resulted in significant differences in allelic frequencies between cases and controls for SNP2197/C733R (P<0.001), indicating a significant association between infection and variant allele. In the analysis of genotypes, a significant association was also found between SNP2197/C733R and infection status (P<0.0001); cows with the heterozygous genotype were 3.35 times more likely to be infected than cows with the reference genotype (P=0.01). Results suggest a role for CARD15 gene in the susceptibility of cattle to paratuberculosis infection. These data contribute to the understanding of paratuberculosis, suggest new similarities with Crohns disease and provide new information for the control of bovine paratuberculosis.
Paratuberculosis (Johnes disease), caused by Mycobacterium avium subsp. paratuberculosis, is an important disease for bovines, although its genetic basis is poorly understood. In this study, three candidate genes were typed to study the associations between single nucleotide polymorphisms (SNPs) and paratuberculosis susceptibility (measured in a 1 or 0 form) at the haplotype level. A significant risk haplotype, constructed by a variant allele (C) at the first SNP and a common allele (A) at the second SNP, within the CARD15 gene was detected to trigger genetic effects on paratuberculosis infection in an overdominace manner. Marginally significant haplotypes were identified for the other two genes. The results obtained will provide scientific guidance about the selection and prediction of resistance types in bovines.
A prevalence study was conducted to survey tick larvae populations in Puerto Rico (PR), compare the number of infested sites with Rhipicephalus (Boophilus) microplus larvae between the wet and dry season, and assess the associations of ecologic factors on the presence of R. microplus larvae. Ninety-six sites were selected using a GIS-based sampling method. Each site was sampled twice; the first sampling was performed during the dry season (March 4-18, 2007) and the second sampling during the wet season (August 13-26, 2007). Sites were sampled using a tick drag with a 1-m(2) white flannel cloth along a 50-m straight course. Only 2 tick species were identified. In the dry season, 15 sites (0.16, 95 % CI = 0.09-0.24) were identified with R. microplus larvae (n = 606) and 9 sites (0.09, 95 % CI = 0.04-0.17) with Dermacentor (Anocentor) nitens larvae (n = 779), whereas in the wet season 5 sites (0.05, 95 % CI = 0.02-0.12) were identified with R. microplus (n = 94), and 5 sites (0.05 %, 95 % CI = 0.02-0.12) with D. nitens (n = 275). Difference in the number of infested sites with R. microplus was significant (P = 0.031) between the 2 seasons. Factors associated with the presence of R. microplus larvae in PR were wind speed of >4.0 km/h (OR = 0.07, 95 % CI = 0.01-0.63), more than 25 % bushes and shrubs on the site (OR = 11, 95 % CI = 1.6-71), and presence of cattle on the site (OR = 26, 95 % CI = 3.4-188).
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