Robert H. Whitlock

ELISA and fecal culture for paratuberculosis (Johne's disease): sensitivity and specificity of each method

The sensitivity and specificity of the ELISA and fecal culture tests for paratuberculosis in dairy cattle are examined. ELISA and fecal culture data from seven dairy herds where both fecal cultures and ELISA testing was done concurrently are included. A cohort of 954 cattle including 697 parturient adults, cultured every 6 months from 10 herds followed over 4 years served as the basis to determine fecal culture sensitivity. The fecal culture technique utilized a 2g sample with centrifugation and double incubation. Of the 954 cattle cohort of all ages (calf to adult) that were fecal sampled on the first herd visit, 79 were culture positive. An additional 131 animals were detected as culture positive over the next seven tests at 6-month intervals. The sensitivity of fecal culture to detect infected cattle on the first sampling was 38%. Of the 697 parturient cattle cohort, 67 were positive on the first fecal culture, while an additional 91 adult cattle were culture positive over the next seven tests, resulting in a sensitivity of 42% on the first culture of the total animals identified as culture positive. Animals culled from the herds prior to being detected as infected and animals always fecal culture negative with culture positive tissues at slaughter are not included in the calculations. Both groups of infected cattle will lower the apparent sensitivity of fecal culture. Infected dairy herds tested concurrently with both fecal culture and ELISA usually resulted in more than twofold positive animals by culture compared to ELISA. The classification of infected cattle by the extent of shedding of Mycobacterium paratuberculosis in the feces helps define the relative proportion of cattle in each group and therefore the likelihood of detection by the ELISA test. ELISA has a higher sensitivity in animals with a heavier bacterial load, i.e. high shedders (75%) compared to low shedders (15%). Repeated testing of infected herds identifies a higher proportion of low shedders which are more likely to be ELISA negative. Thus, the sensitivity of the ELISA test decreases with repeated herd testing over time, since heavy shedders will be culled first from the herds.

Preclinical and Clinical Manifestations of Paratuberculosis (Including Pathology)

The term Johnes disease immediately brings to mind an image of an emaciated, debilitated ruminant with bottlejaw and fluid, pipestream diarrhea. However, the clinical case is merely the tip of the iceberg in terms of the total number of infected animals on the farm. If the clinically affected animal was born on the farm, a minimum of 25 other animals are probably infected and less than 30% of those will be detectable by currently available tests. Infected animals in the early stages of the disease progress slowly over a period of months or several years to stages that may be detected by antigen detection tests and antibody-based diagnostic tests. The duration of each stage of infection depends on age at the time of exposure and the dose of organisms ingested. The lack of tests sensitive enough to detect infected animals in the earliest stages of disease is a serious obstacle to eradication of Johnes disease. The animals with overt infection may be culled from the herd immediately on detection. However, the animals that are shedding organisms below the threshold of detectability and have not mounted an immune response remain behind to contaminate the environment and infect herdmates.

Evaluation of Five Antibody Detection Tests for Diagnosis of Bovine Paratuberculosis

ABSTRACT Five diagnostic tests based on enzyme-linked immunosorbent assay (ELISA) technology for bovine paratuberculosis were evaluated by using individual serum or milk samples from 359 dairy cattle in seven paratuberculosis-free herds and 2,094 dairy cattle in seven Mycobacterium paratuberculosis-infected dairy herds. Three independent laboratories using three different culture procedures completed fecal cultures for M. paratuberculosis on these cattle and found 417 cows to be shedding M. paratuberculosis in their feces. An animal that was fecal culture positive for M. paratuberculosis by any of the three laboratories was considered a confirmed case of infection. The specificity of three ELISAs (two on serum and one on milk) was ≥99.8%. The specificity of the remaining two ELISAs, both done on serum, was 94.9 and 84.7%. Four of the five ELISAs evaluated produced similar sensitivity in detecting fecal culture-positive cattle (27.8 to 28.9%). Serum ELISA “D” had the lowest specificity (84.7%) and the highest sensitivity (44.5%), but if the cutoff value defining a positive test was changed from 125 to 250% (of the positive control) the sensitivity and specificity, 31.8 and 97.5%, respectively, were comparable to those of the other four assays. If the case definition for M. paratuberculosis infection was based on the culture results of a single laboratory instead of the combined results of three laboratories, ELISA sensitivity estimates were 45.7 to 50.0%. With the exception of ELISA D, assay agreement was high (kappa 0.66 to 0.85) for categorical assay interpretations (positive or negative), but linear regression of quantitative results showed low correlation coefficients (r2 = 0.40 to 0.68) due to the fact that ELISA results for some cows were high in one assay but low in another assay. Likelihood ratio analysis showed a direct relationship between the magnitude of ELISA result and the odds of a cow shedding M. paratuberculosis in its feces. If used judiciously and interpreted quantitatively, these ELISAs are useful tools in support of paratuberculosis control programs in dairy herds.

Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay for the Diagnosis of Paratuberculosis in Dairy Cattle

The performance of a commercially available ELISA for detection of antibodies to Mycobacterium paratuberculosis was evaluated using sera from 1,146 cows. Samples were from uninfected cattle, infected subclinical cattle shedding low numbers of organism in feces, subclinical heavy shedders, clinical cases, and randomly selected cattle in a slaughterhouse survey for paratuberculosis. The overall sensitivity of the test, using the manufacturers recommended cutoff was 45% ± 4.8%, and the specificity was 99% ± 0.9%. The ELISA result was significantly correlated with the number of colonies of M. paratuberculosis detected by fecal culturing. The sensitivity of the test was highest for clinical cases of paratuberculosis (87% ± 8.4%), and lowest for subclinical, light-shedding cattle (15% ± 6.6%). Changing the cutoff point did not improve performance of the test. Evaluating ELISA results with a kinetics-based method reduced plate-to-plate variation in results but did not improve performance of the test based on receiver-operating characteristic curve analysis.

Parallel faecal and organ Mycobacterium avium subsp. paratuberculosis culture of different productivity types of cattle.

Faecal (at least 3 months before slaughtering) and organ examinations were carried out in 611 animals (497 dairy, 69 dual-purpose and 44 beef cattle) originating from eight paratuberculosis infected cattle herds. The diagnosis in cattle was established by routine intestinal culture (ileum and the adjacent lymph nodes) after slaughter. In selected 132 animals, post-mortem intensive culture was performed on tissue samples collected from the gastrointestinal tract (duodenum, jejunum, ileum, ileocecal valve, caecum, rectum) and the corresponding lymph nodes, submandibular, retropharyngeal, tracheobronchial, liver and supramammary lymph nodes, kidney, liver and spleen. In 251 (41.1%) of all 611 animals, Mycobacterium avium subspecies paratuberculosis could be isolated from the faeces; in 164 (65.7%) out of 251 shedding animals the infection was detected in the ileum and adjacent lymph nodes. The detection of M. paratuberculosis by routine intestinal culture of faecal culture positive animals varied from 46.0% in animals shedding 1 CFU (colony forming unit), to 94.7% in massive shedders. On the contrary, M. paratuberculosis was detected by routine intestinal culture in 92 (25.5%) of the 360 faecal culture negative animals. Shedding animals had significantly higher (P<0.01) number of organisms in their organs than non-shedding animals. During the intensive tissue cultivation from selected 132 animals, 72 (54.5%) of them were positive. For the negative animals, no significant difference was found between the detection rate in organs examined after slaughter with routine and intensive method. However, in the subgroup of tissue culture positive animals a highly significant difference (P<0.01) was found by intensive examination (83.0%) compared with the routine examination (60.4%). Out of 72 tissue culture positive animals 73.6% of them harboured M. paratuberculosis in the gastrointestinal tract, 16.7% in the gastrointestinal tract and the parenchymatous organs, tracheobronchial and mandibular lymph nodes. The rest of the 9.7% of the infection was detected in the lymph nodes of head and lungs. Our study concerning the distribution of M. paratuberculosis by intensive examinations revealed a minimum effect of breed and production type on localisation of the agent. Thus, the results suggest that in case of an active infection, M. paratuberculosis can be localised in different organs of animals irrespective of their breed or production type.

A whole genome association analysis identifies loci associated with Mycobacterium avium subsp. paratuberculosis infection status in US holstein cattle

The purpose of this study was to identify loci associated with Mycobacterium avium subspecies paratuberculosis (Map) infection status in US Holsteins using the Illumina BovineSNP50 BeadChip whole genome single nucleotide polymorphism (SNP) assay. Two hundred forty-five cows from dairies in New York, Pennsylvania and Vermont enrolled in longitudinal herd studies between January 1999 and November 2007 were assessed for the presence of Map in both faecal and tissue samples. An animal was considered tissue infected if any sample contained at least one colony forming unit of Map per gram of tissue (CFU/g) and the same definition was employed for faecal samples. Each animal was genotyped with the Illumina BovineSNP50 BeadChip and after quality assurance filtering, 218 animals and 45 683 SNPs remained. We sought to identify loci associated with four different case/control classifications: presence of Map in the tissue, presence of Map in faeces, presence of Map in both tissue and faeces and presence of Map in tissue but not faeces. A case-control genome wide association study was conducted to test the four different classifications of Map infection status (cases) when compared with a Map-negative control group (control). Regions on chromosomes 1, 5, 7, 8, 16, 21 and 23 were identified with moderate significance (P < 5 x 10(-5)). Two regions, one on chromosome 3 (near EDN2) and another on chromosome 9 (no positional gene candidates), were identified with a high level of association to the presence of Map in tissue and both tissue and faeces respectively (P < 5 x 10(-7), genome-wide Bonferonni P < 0.05).

Evaluation of a rapid fecal PCR test for detection of Mycobacterium avium subsp. paratuberculosis in dairy cattle.

ABSTRACT A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assays specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-“gold standard” analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI] = 98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI = 24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle.

Efficacy of commercial and field-strain Mycobacterium paratuberculosis vaccinations with recombinant IL-12 in a bovine experimental infection model

The efficacy of commercial (Strain 18) and field-isolate paratuberculosis vaccine preparations was investigated. The effect of prior exposure to Mycobacterium paratuberculosis and the adjuvant effect of rIL-12 on vaccine efficacy were also tested. Both Strain 18 and field-isolate vaccines induced strong local, systemic and enteric IFN-gamma responses. A significant reduction in mycobacterial colonization was observed when calves were vaccinated with the field-isolate prior to challenge, but not following vaccination with Strain 18 vaccine. Vaccination with rIL-12 prevented infection in some calves but its overall effect on IFN-gamma response and total mycobacterial load was not statistically significant. Efficacy of paratuberculosis vaccines may be enhanced if calves are vaccinated prior to M. paratuberculosis exposure with field-isolate vaccine instead of Strain 18 vaccine currently in use.

A longitudinal study on the impact of Johne's disease status on milk production in individual cows

Longitudinal data from 3 commercial dairy herds in the northeast United States were collected from 2004 to 2007. Johnes disease status, as indicated by Mycobacterium avium ssp. paratuberculosis infection levels, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues at slaughter. Milk production data were collected from the Dairy Herd Improvement Association. The effect of Johnes disease status on milk production was analyzed using a mixed linear model with an autocorrelation random effect structure. Infected animals produced more milk than uninfected cows before they began shedding M. avium ssp. paratuberculosis. Cows infected with M. avium ssp. paratuberculosis had monthly decreases of 0.05 to 1 kg in daily milk production relative to uninfected animals, with greater decreases in progressive disease categories. Animals with fecal culture results of >30 cfu/g produced approximately 4 kg less milk per day compared with uninfected cows. These results will be valuable in calculating the economic effect of Johnes disease.

Type C botulism in dairy cattle from feed contaminated with a dead cat

Four hundred twenty-seven of 441 adult Holstein dairy cattle from a 1,200-cow dairy died over a 1-week period during early spring 1998. Affected animals were from 4 late lactation pens, one of which included the bull string. Signs included weakness, recumbency, watery diarrhea, and death. Eighty animals from the 4 pens were dead approximately 8 hours after the first ill cows were noted. Affected cows would collapse on stimulation and extend all 4 limbs with moderate rigidity. Several lacked lingual tonus and had abdominal breathing patterns. The animals had been fed a load of total mixed ration that included a rotten bale of oat hay containing a dead cat. No common toxicants were identified, and pathologic examination revealed no consistent lesions. Testing of tissue from the cat carcass found in the feed sample using mouse protection bioassay identified the presence of type C botulinum toxin. Samples of feed, tissue from affected animals, cat tissue from feed, milk, and serum were also tested using an enzyme-linked immunosorbent assay (ELISA) specific for type C botulinum. Two samples of rumen contents were tested and found to be positive for botulism by ELISA, and 1 of 3 liver samples had a weak positive finding. No botulinum toxin was found in milk or sera using the ELISA.