Fecal Culture Test Research Articles

Fecal Microbiota Transplantation against Gut Colonization Using a Multidrug-Resistant Organism

Background: Fecal microbiota transplantation against gut colonization using a multidrug-resistant organism is a technique used to treat infections through normalizing the gut microbiota via fecal microbiota transplantation in patients with confirmed colonization by carbapenem-resistant Enterobacteriaceae (CRE) or vancomycin-resistant enterococci (VRE) based on a fecal culture test within the past one week. In this study, we aimed to determine the safety and effectiveness of this technique. Methods: The safety and effectiveness were assessed via a systematic review. A literature search was conducted using five Korean databases, such as KoreaMed, and international databases,including Ovid-MEDLINE, Ovid-EMBASE, and Cochrane Library. Results:Main results are described here. From the studies retrieved using the aforementioned search strategy, the remaining 581 studies were screened using the inclusion and exclusion criteria, resulting in the selection of nine studies for further consideration. In terms of safety, many studies reported deaths and adverse reactions associated with different causes. Fewer studies reported the rate of colonization; however, the effect of colony rate was inconsistent when compared to no treatment group. Additionally, none of the studies assessed the recurrence rate, a decrease in the prevalence of diseases related to infection by multidrug-resistant bacteria, and the quality of life.Conclusion: Fecal bacterial colonization for the decolonization of intestinal multidrug-resistant bacteria was evaluated using a technique that requires further research as there is insufficient literature evidence to validate its safety and efficacy in treating infections through normalizing the intestinal flora of patients with confirmed colonization by CRE or VRE.

ХАРАКТЕРИСТИКА ГАСТРОИНТЕСТИНАЛЬНОГО САЛЬМОНЕЛЛЕЗА У ДЕТЕЙ НИЖНЕГО НОВГОРОДА

The study aims to examine the epidemiological, etiological, and clinical features of gastrointestinal salmonellosis in children living in Nizhny Novgorod. A retrospective analysis of 49 children with gastrointestinal salmonellosis who received treatment in the Infectious Hospital No. 23 in Nizhny Novgorod was conducted. Database was compiled via clinical and laboratory diagnosing methods to assess the age of the patients, the duration of the disease upon admission to the hospital, the results of physical examination and laboratory tests using statistical methods. Most children were 3 years old or older (71.4 %) and admitted in 2–3 days after the onset of the disease (77.6 %). The most common infectious agent was serovar S. enteritidis D (84.8 %), while every 2nd child (46.9 %) had a mixed infection. The severity of the disease was mostly moderate (93.9 %) with the presence of intoxication (87.8 %), febrile fever (65.3 %), abdominal pain syndrome (93.8 %), repeating vomiting (57.7 %). Half of the patients developed grade I exicosis (53 %). astrointestinal symptoms were often combined with leukocyturia (49 %). The fecal culture test was highly informative for verification of specific bacterial agent. Long-term germ fecal excretion was detected in 12.2 % of patients, mostly in children under the age of 3 years (83.3 %). The study provides etiological, clinical and epidemiological features of gastrointestinal salmonellosis in children living in Nizhny Novgorod.

Fecal Microbes Associated with the Outcomes After Esophagectomy in Patients with Esophageal Cancer.

Although accumulating evidence suggests that an imbalanced gut microbiota leads to cancer progression, few studies demonstrated the implication in patients who underwent oncologic esophagectomy. This study aimed to elucidate the association between gut microbes and the outcomes after oncologic esophagectomy, as well as the host's inflammatory/nutritional status. Overall, 783 consecutive patients who underwent oncologic esophagectomy were eligible. We investigated the microbiota detected by fecal culture tests and then assessed the association between the gut microbiota and patient characteristics, short-term outcomes, and long-term survival. Seventeen different species could be cultivated. We comprehensively examined the impact of each detected microbe on survival. The presence of Bacillus species (Bacillus sp.; 26.8%) was associated with favorable prognosis on overall and cancer-specific survival (p=0.02 and 0.02, respectively). Conversely, the presence of Proteus mirabilis (P. mirabilis; 3.4%) was associated with unfavorable overall and recurrence-free survivals (p=0.02 and < 0.01, respectively). Multivariate analysis showed that the presence of P. mirabilis was one of the independent prognostic factors for poor recurrence-free survival (p<0.01). Patients with Bacillus sp. had lower modified Glasgow prognostic score and better response to preoperative treatment than those without (p=0.01 and 0.03, respectively). Meanwhile, patients withP. mirabiliswere significantly associated with higher systemic inflammation scores and increased postoperative pneumonia incidence than those without (p=0.01 and 0.02, respectively). Preoperative fecal microbiota was associated with the host's inflammatory and nutritional status and may influence the outcomes after oncologic esophagectomy.

Genome-wide association analysis for susceptibility to infection by Mycobacterium avium ssp. paratuberculosis in US Holsteins

Paratuberculosis, or Johne's disease, is a chronic, granulomatous, gastrointestinal tract disease of cattle and other ruminants caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). Control of Johne's disease is based on programs of testing and culling animals positive for infection with MAP and concurrently modifying management to reduce the likelihood of infection. The current study was motivated by the hypothesis that genetic variation in host susceptibility to MAP infection can be dissected and quantifiable associations with genetic markers identified. Two separate GWAS analyses were conducted, the first using 897 genotyped Holstein artificial insemination sires with phenotypes derived from incidence of MAP infection among daughters based on milk ELISA testing records. The second GWAS analysis was a case-control design using US Holstein cows phenotyped for MAP infection by serum ELISA or fecal culture tests. Cases included cows positive for either serum ELISA, fecal culture, or both. Controls consisted of animals negative for all tests conducted. A total of 376 samples (70 cases and 306 controls) from a University of Minnesota Johne's management demonstration project and 184 samples (76 cases and 108 controls) from a Michigan State University study were used. Medium-density (sires) and high-density (cows) genotype data were imputed to full genome sequence for the analyses. Marker-trait associations were analyzed using the single-step (ss)GWAS procedure implemented in the BLUPF90 suite of programs. Evidence of significant genomic contributions for susceptibility to MAP infection were observed on multiple chromosomes. Results were combined across studies in a meta-analysis, and increased support for genomic regions on BTA7 and BTA21 were observed. Gene set enrichment analysis suggested pathways for antigen processing and presentation, antimicrobial peptides and natural killer cell-mediated cytotoxicity are relevant to variation in host susceptibility to MAP infection, among others. Genomic prediction was evaluated using a 5-fold cross-validation, and moderate correlations were observed between genomic breeding value predictions and daughter averages (∼0.43 to 0.53) for MAP infection in testing data sets. These results suggest that genomic selection against susceptibility to MAP infection is feasible in Holstein cattle.

Eficácia de Tithonia diversifolia(Hemsl) A. Gray na inibição do desenvolvimento larval de Haemonchus contortus

Sheep farming is an important economic activity from a commercial and subsistence perspective. However, helminth infections present a large sanitary problem for the production, especially haemonchosis caused by strains resistant to traditional anthelmintics. Here, we evaluated the effects of the stem and leaf of Tithonia diversifolia on the inhibition of Haemonchus contortus larval development. We performed a quantitative fecal culture test with feces of sheep inoculated with larvae tolerant to albendazole. Fecal cultures were treated with leaf or stem powder and were compared to the controls containing distilled water or levamisole. The T. diversifolia leaf and stem powders significantly reduced the production of H. contortus larvae. However, leaf powder was more effective, with 64.95% efficacy in the reduction of larvae production, representing a promising alternative in the integrated control of this nematode.

Bayesian estimation of diagnostic accuracy of fecal culture and PCR-based tests for the detection of Salmonella enterica in California cull dairy cattle.

Epidemiological studies of low prevalence disease problems are often hindered by the high cost of diagnostic testing. The objective of this study was to evaluate PCR screening of both individual and pooled fecal samples from culled dairy cows for the invA gene of Salmonella followed by culture to determine if the sensitivity and specificity were comparable to the results from traditional culture methods applied to individual samples. Cows from six different dairies were sampled in all four seasons. A total of 240 individual cow fecal samples, 24 fecal pools and 24 pools of 24-hour tetrathionate enrichment broth were tested. Diagnostic sensitivity of PCR screening followed by culture of PCR positive or indeterminate samples (i.e PCR-CUL method) was lower than that of culture (CUL) when applied to individual fecal samples (94.8%, 99.5%), however the specificity was comparable (99.6% and 97.7% respectively). For pools of five fecal samples and pools of five, 24 h tetrathionate broth samples, the specificity of both tests were comparable (∼98%); however, their sensitivity was only comparable in pooled fecal samples (∼93%) but greater for culture compared to PCR-CUL in pooled broth samples (∼99% versus ∼93%). Compared to culture results from testing of individual fecal samples, testing pooled fecal samples by culture had a relative sensitivity of 74% and relative specificity of 96%, testing pooled fecal samples by PCR-CUL resulted in relative sensitivity of 90% and relative specificity of 96%. Testing of pooled 24-hour enrichment broth by PCR-CUL increased the relative sensitivity and specificity to 100%. PCR testing followed by culture of positive or indeterminate samples is a time saving alternative to traditional methods. In addition, pooling of samples may be a useful method for decreasing cost if study aims can accommodate a moderate loss of relative sensitivity.

An agent-based model evaluation of economic control strategies for paratuberculosis in a dairy herd

This paper uses an agent-based simulation model to estimate the costs associated with Mycobacterium avium ssp. paratuberculosis (MAP), or Johne's disease, in a milking herd, and to determine the net benefits of implementing various control strategies. The net present value (NPV) of a 1,000-cow milking herd is calculated over 20 yr, parametrized to a representative US commercial herd. The revenues of the herd are generated from sales of milk and culled animals. The costs include all variable and fixed costs necessary to operate a representative 1,000-cow milking herd. We estimate the NPV of the herd with no MAP infection, under an expected endemic infection distribution with no controls, and under an expected endemic infection distribution with various controls. The initial number of cows in a herd with an endemic MAP infection is distributed as 75% susceptible, 13% latent, 9% low MAP shedding, and 3% high MAP shedding. Control strategies include testing using ELISA and fecal culture tests and culling of cows that test positive, and culling based on observable milk production decrease. Results show that culling cows based on test results does not increase the herd's NPV and in most cases decreases NPV due to test costs as well as false positives and negatives with their associated costs (e.g., culling healthy cows and keeping infected cows). Culling consistently low producing cows when MAP is believed to be present in the herd produces higher NPV over the strategy of testing and culling MAP infected animals, and over the case of no MAP control.

Genome-wide association study of susceptibility to infection by Mycobacterium avium subspecies paratuberculosis in Holstein cattle.

Paratuberculosis, or Johne's disease, is a chronic, granulomatous, gastrointestinal tract disease of cattle and other ruminants caused by the bacterium Mycobacterium avium, subspecies paratuberculosis (MAP). Control of Johne's disease is based on programs of testing and culling animals positive for infection with MAP while concurrently modifying management to reduce the likelihood of infection. The current study is motivated by the hypothesis that genetic variation in host susceptibility to MAP infection can be dissected and quantifiable associations with genetic markers identified. For this purpose, a case-control, genome-wide association study was conducted using US Holstein cattle phenotyped for MAP infection using a serum ELISA and/or fecal culture test. Cases included cows positive for either serum ELISA, fecal culture or both. Controls consisted of animals negative for the serum ELISA test or both serum ELISA and fecal culture when both were available. Controls were matched by herd and proximal birth date with cases. A total of 856 cows (451 cases and 405 controls) were used in initial discovery analyses, and an additional 263 cows (159 cases and 104 controls) from the same herds were used as a validation data set. Data were analyzed in a single marker analysis controlling for relatedness of individuals (GRAMMAR-GC) and also in a Bayesian analysis in which multiple marker effects were estimated simultaneously (GenSel). For the latter, effects of non-overlapping 1 Mb marker windows across the genome were estimated. Results from the two discovery analyses were generally concordant; however, discovery results were generally not well supported in analysis of the validation data set. A combined analysis of discovery and validation data sets provided strongest support for SNPs and 1 Mb windows on chromosomes 1, 2, 6, 7, 17 and 29.

Short communication: Heritability estimates for susceptibility to Mycobacterium avium subspecies paratuberculosis infection defined by ELISA and fecal culture test results in Jersey cattle

Paratuberculosis (Johne’s disease), an enteric disorder in ruminants caused by Mycobacterium avium ssp. paratuberculosis, causes economic losses in excess of $200 million annually to the US dairy industry. Costly diagnostic testing, cumbersome control programs, incurability, and ineffective vaccination all make M. avium ssp. paratuberculosis susceptibility a good candidate for genetic studies and genetic selection a potentially useful adjunct to management-based control programs. No report has been published for heritability of susceptibility to M. avium ssp. paratuberculosis infection in Jersey cattle. The objective of this study was to estimate variance components and heritability for susceptibility to M. avium ssp. paratuberculosis infection in US Jersey cattle. Data consisted of complete serum ELISA and partial fecal culture results on a total of 2,861 Jersey cows from 23 commercial herds throughout the United States after editing. Four M. avium ssp. paratuberculosis susceptibility phenotypes were defined using (1) ELISA sample-to-positive ratios as a continuous trait, (2) ELISA results as a binary trait (positive=1, negative=0), (3) ELISA results as an ordered categorical trait, and (4) a combined test in which ELISA and fecal culture results were both taken into account in a binary analysis. Three statistical models, including linear, binary threshold, and ordered threshold sire models, were used to analyze the data. All analyses were executed using the restricted maximum likelihood method in ASReml 3 software. The heritability estimates were low to moderate and ranged from 0.08 (±0.03) to 0.27 (±0.11) based on different trait definitions. The nonzero heritability indicates that susceptibility to M. avium ssp. paratuberculosis infection in Jersey cattle is influenced by genetic factors. Therefore, selection of the least susceptible animals could decrease genetic predisposition to M. avium ssp. paratuberculosis infection in Jersey populations in future generations.

Genome-Wide Association Analysis and Genomic Prediction of Mycobacterium avium Subspecies paratuberculosis Infection in US Jersey Cattle

Paratuberculosis (Johne’s disease), an enteric disorder in ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), causes economic losses in excess of $200 million annually to the US dairy industry. To identify genomic regions underlying susceptibility to MAP infection in Jersey cattle, a case-control genome-wide association study (GWAS) was performed. Blood and fecal samples were collected from ∼5,000 mature cows in 30 commercial Jersey herds from across the US. Discovery data consisted of 450 cases and 439 controls genotyped with the Illumina BovineSNP50 BeadChip. Cases were animals with positive ELISA and fecal culture (FC) results. Controls were animals negative to both ELISA and FC tests that matched cases on birth date and herd. Validation data consisted of 180 animals including 90 cases (positive to FC) and 90 controls (negative to ELISA and FC), selected from discovery herds and genotyped by Illumina BovineLD BeadChip (∼7K SNPs). Two analytical approaches were used: single-marker GWAS using the GRAMMAR-GC method and Bayesian variable selection (Bayes C) using GenSel software. GRAMMAR-GC identified one SNP on BTA7 at 68 megabases (Mb) surpassing a significance threshold of 5×10−5. ARS-BFGL-NGS-11887 on BTA23 (27.7 Mb) accounted for the highest percentage of genetic variance (3.3%) in the Bayes C analysis. SNPs identified in common by GRAMMAR-GC and Bayes C in both discovery and combined data were mapped to BTA23 (27, 29 and 44 Mb), 3 (100, 101, 106 and 107 Mb) and 17 (57 Mb). Correspondence between results of GRAMMAR-GC and Bayes C was high (70–80% of most significant SNPs in common). These SNPs could potentially be associated with causal variants underlying susceptibility to MAP infection in Jersey cattle. Predictive performance of the model developed by Bayes C for prediction of infection status of animals in validation set was low (55% probability of correct ranking of paired case and control samples).

Phenotype definition is a main point in genome-wide association studies for bovine Mycobacterium avium ssp. paratuberculosis infection status

Paratuberculosis caused by Mycobacterium avium ssp. paratuberculosis (MAP) causes economic losses and is present in dairy herds worldwide. Different studies used different diagnostic tests to detect infection status and are the basis of genome-wide association (GWA) studies with inconsistent results. Therefore, the aim of this study was to identify and compare genomic regions associated with MAP susceptibility in the same cohort of cattle using different diagnostic tests. The GWA study was performed in German Holsteins within a case-control assay using 305 cows tested for MAP by fecal culture and additional with four different commercial ELISA-tests. Genotyping was performed with the Illumina Bovine SNP50 BeadChip. The results using fecal culture or ELISA test led to the identification of different genetic loci. Two single-nucleotide polymorphisms showed significant association with the ELISA-status. However, no significant association for MAP infection could be confirmed. Our results show that the definition of the MAP-phenotype has an important impact on the outcome of GWA studies for paratuberculosis.

Evidence of birth seasonality and clustering of Mycobacterium avium subspecies paratuberculosis infection in US dairy herds

Paratuberculosis (Johne's disease) is a contagious intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). In cattle, young calves are at the highest risk for acquiring the infection which occurs mainly through ingestion of MAP from contaminated milk, colostrum and feces or environmental contacts. Data consisted of birth dates and ELISA results of 8000 mature cows from 24 Jersey herds from throughout the US and 4 Wisconsin Holstein herds. Some herds also had complete fecal culture (FC) results. The first infection (case) definition (CD1) relied on only ELISA results. A second case definition (CD2) was used in which results of both ELISA and FC tests were considered: animals testing positive to either test were considered “test-positives” and cows testing negative to ELISA or to both ELISA and FC were regarded as “test-negatives”. Objective one was to assess seasonality in birth of MAP-infected animals. The effects of age, breed, herd and season of birth (expressed as the sine and cosine functions of birth days within year) were examined using logistic regression. Age was significantly associated with the MAP infection status of dairy cows for both CDs (OR=1.11; 95% CI 1.09, 1.14; P<0.0001 for CD1; OR=1.16; 95% CI 1.08, 1.24; P<0.0001 for CD2). Season of birth had a significant effect on the risk of MAP infection based on CD1 (OR=0.79; 95% CI 0.71, 0.89; P<0.001 for cosine of birth days) with a peak in summer and a trough in winter based on the fitted model. Objective two was to assess whether test-positive animals were randomly distributed or were clustered by date of birth within herds. A temporal cluster analysis approach (scan statistic) implemented in SaTScan software was used for each case definition to detect clusters of birth cohorts using birthdates. Results identified significant clustering of MAP infection cases for CD1 in multiple herds (P<0.05). These results necessitate matching cases and controls of MAP infection on their birth dates to control for non-uniform exposure to MAP in paratuberculosis case–control genome wide association studies, candidate gene studies or in on-farm disease intervention trials.

Bayesian analysis of longitudinal Johne's disease diagnostic data without a gold standard test

A Bayesian methodology was developed based on a latent change-point model to evaluate the performance of milk ELISA and fecal culture tests for longitudinal Johne's disease diagnostic data. The situation of no perfect reference test was considered; that is, no “gold standard.” A change-point process with a Weibull survival hazard function was used to model the progression of the hidden disease status. The model adjusted for the fixed effects of covariate variables and random effects of subject on the diagnostic testing procedure. Markov chain Monte Carlo methods were used to compute the posterior estimates of the model parameters that provide the basis for inference concerning the accuracy of the diagnostic procedure. Based on the Bayesian approach, the posterior probability distribution of the change-point onset time can be obtained and used as a criterion for infection diagnosis. An application is presented to an analysis of ELISA and fecal culture test outcomes in the diagnostic testing of paratuberculosis (Johne's disease) for a Danish longitudinal study from January 2000 to March 2003. The posterior probability criterion based on the Bayesian model with 4 repeated observations has an area under the receiver operating characteristic curve (AUC) of 0.984, and is superior to the raw ELISA (AUC=0.911) and fecal culture (sensitivity=0.358, specificity=0.980) tests for Johne's disease diagnosis.

Absorbed EVELISA: A Diagnostic Test with Improved Specificity for Johne's Disease in Cattle

The use of enzyme-linked immunosorbent assays (ELISAs) is recommended for Johne's disease (JD) control in dairy herds. In 2006, we developed a novel ELISA test for JD, named EVELISA (ELISA using ethanol extract of Mycobacterium avium subsp. paratuberculosis), which showed higher sensitivity than commercial ELISA tests. To further investigate the performance of EVELISA, we obtained 38 serum samples from cattle in a JD-free herd with suspected cases of serological false-positive reactions. When these samples were tested using the EVELISA and a commercial ELISA test, more than 70% of the samples were falsely identified as JD positive. Antibodies in the serum samples reacted strongly with antigens of various environmental mycobacteria, suggesting the presence of cross-reactive antibodies in the samples. The possible cross reactions in the EVELISA were inhibited markedly by the use of Mycobacterium phlei antigens for antibody absorption. When these samples were tested, 8 samples were classified as positive for JD by the EVELISA with the antibody absorption, whereas 27 samples were classified as positive for JD by the commercial ELISA. For an estimation of tentative sensitivity and specificity, the ELISA tests were performed on 38 serum samples from JD-negative herds with no suspected cases of serological false-positive reaction and 68 samples from cattle diagnosed as positive for M. avium subsp. paratuberculosis infection by fecal culture test. Sensitivity and specificity of the EVELISA with preabsorption of serum with M. phlei ("ethanol vortex absorbed-ELISA" or EVA-ELISA) were estimated to be 97.1% and 100%, respectively, whereas those of the commercial ELISA were 48.5% and 97.4%, respectively. Further, in 85 fecal culture-negative cattle in JD-positive herds, higher sensitivity of the EVA-ELISA than the commercial ELISA was demonstrated by a Bayesian analysis. This study indicates that the EVA-ELISA may form a basis for a sensitive diagnostic test with a higher level of specificity than that of the current commercial ELISA test.

Evaluation of Diagnostic Tests for Johne's Disease (Mycobacterium Avium Subspecies Paratuberculosis) in New Zealand Dairy Cows

To quantify the sensitivity and specificity of a serum enzyme-linked immunosorbent assay (ELISA) and fecal culture (FC) tests and to estimate the prevalence of Johne's disease (JD) in New Zealand dairy herds using Bayesian methods, 4 New Zealand dairy herds were tested simultaneously by ELISA and FC 5 times over 3 lactations. Test results were dichotomized. A Bayesian regression model was developed that considered test sensitivity as a function of the covariates parity, lactation stage, and prevalence of JD, which is expected to vary between herds. It was applied to a cross-sectional subset of the data and the full, repeated measures data set. Results were compared with frequentist pseudo gold standard results of the full data. Using the regression model, sensitivity of the ELISA was higher in older animals, but the sensitivity of the FC test showed no trend across age groups. Both FC and ELISA sensitivity were lower in late lactation. Estimated prevalence was lower and FC sensitivity higher when analyzing the complete data. The regression model enabled a more accurate diagnosis of JD to be made because it incorporated cow-specific information in the diagnosis, such as age and lactation stage. The model also enabled the incorporation of previous test results for an individual when diagnosing disease. The trends in results from the regression model support the current understanding of the disease process. The advantage of repeated testing of individuals in the assessment of test performance is discussed in the current study.

The importance of culling in Johne's disease control

Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection and results in economic losses in the dairy industry. To control MAP transmission in herds, test-based culling has been recommended and immediate culling of high shedding animals is typically implemented. In this study, we quantified the effects of MAP control in US dairy herds, using the basic reproduction ratio R 0 . The effectiveness of culling strategies was evaluated for good and poor herd management (low- and high-transmission rates, respectively) by a phase diagram approach. To establish a quantitative relationship between culling rates and test properties, we defined the average detection times for low and high shedding animals. The effects of various culling strategies and test characteristics, such as test sensitivity, test turnaround time, and testing interval, were analyzed. To understand the overall effect of model parameters on R 0 , we performed global uncertainty and sensitivity analyses. We also evaluated the effectiveness of culling only high shedding animals by comparing three test methods (fecal culture, fecal polymerase chain reaction, PCR, and enzyme-linked immunosorbent assay, ELISA). Our study shows that, in the case of good herd management, culling of only high shedding animals may be effective in controlling MAP transmission. However, in the case of poor management, in addition to immediate culling of high shedding animals, culling of low shedding animals (based on the fecal culture test) will be necessary. Culling of low shedding animals may be delayed 6–12 months, however, if a shorter testing interval is applied. This study suggests that if farmers prefer culling only high shedding animals, faster MAP detection tests (such as the fecal PCR and ELISA) of higher sensitivity should be applied with high testing frequency, particularly on farms with poor management. Culling of infectious animals with a longer testing interval is generally not effective to control MAP.

Age at Occurrence of Mycobacterium avium Subspecies paratuberculosis in Naturally Infected Dairy Cows

Paratuberculosis is a chronic infection of ruminants and other species caused by Mycobacterium avium ssp. paratuberculosis (Map). Establishing test strategies for paratuberculosis will require insight into the temporal aspects of certainty with a given test. In this study, the age at which cows tested positive by ELISA and fecal culture (FC) was investigated by use of time-to-event analyses. The effects of herd, parity, and shedding group were evaluated at the age of test-positive ELISA and FC, respectively. Finally, the test frequency was investigated for the probability of cows being tested ELISA-positive. Milk and fecal samples were collected repeatedly over a 3-yr period from 1,776 Danish dairy cows from 8 herds. The milk samples were tested for the presence of antibodies by using an ELISA, and an FC test was used for detection of Map. Repeated ELISA testing detected 98 and 95% of cows classified as high and low shedders, respectively, suggesting that most infected cows develop antibodies. Among the high shedders, 50% were positive before 4.3 yr of age (quartiles 1 to 3: 3.4 to 5.7 yr of age). Repeated FC detected only 72% of the cows that were ELISA-positive, and 50% of the ELISA-positive cows were detected by FC at 7.6 yr of age. The age with the highest probability of testing positive was determined as the interval with the steepest slope in the survival probability plots. The highest probability of testing positive by ELISA was from 2.5 to 4.5 yr of age. The highest probability of testing positive by FC was from 2.5 to 5.5 yr of age. For both ELISA and FC, testing positive was highest in the first 300 d in milk. For cows younger than 4 yr of age, monthly testing with ELISA, compared with testing every 2 yr, could increase the probability of detecting cows with antibodies by 19%. In older cows, there were no apparent differences in the probability of testing positive by monthly sampling compared with sampling every second year. Therefore, for older animals the effect of more frequent sampling would be for early detection rather than to obtain additional information. Cows shedding high numbers of Map will produce antibodies, although not necessarily concomitantly with the shedding. These antibodies can be detected by ELISA with a test strategy that is different for younger and older cows. We suggest testing younger cows more frequently than older cows and that testing should be done prior to 350 d in milk.

Genetic Variation of Mycobacterium avium ssp. paratuberculosis Infection in US Holsteins

The objective of this study was to estimate genetic variability of Mycobacterium avium ssp. paratuberculosis infection in US Holsteins. Blood and fecal samples were collected primarily from daughters of 12 bulls in their second or third lactation. Routine disease testing of the sires documented that they were not infected. Herds without a “suspect” or positive ELISA (sample/positive ratio≥0.10) or positive fecal culture test were deleted from the data set. The remaining 4,603 cows from 238 herds and 46 sires were used to estimate heritability of M. paratuberculosis infection. Heritability was estimated with 3 Johne's disease diagnostic tests: 1) fecal culture alone, 2) serum antibody ELISA alone, and 3) both tests (combined) with a positive animal defined as all animals with either a positive fecal culture or ELISA test. Four statistical models were used to estimate heritability: 1) linear (ELISA), 2) threshold (fecal culture and combined), 3) ordered threshold (ELISA), and 4) bivariate linear-threshold (ELISA-fecal culture). A sire model and Bayesian approach using Markov chain Monte Carlo methods were used in each case. Heritability of infection based on the fecal culture test was 0.153 [posterior standard deviation (PSD) = 0.115]. Heritability with the ELISA was 0.159 (PSD = 0.090) with a linear model and 0.091 (PSD = 0.053) with an ordered threshold model. Heritability of the combined tests was 0.102 (PSD = 0.066). Heritability estimates of fecal culture and ELISA with the bivariate model varied slightly from estimates obtained with the univariate models (0.125 and 0.183, respectively), with a corresponding increase in precision (PSD = 0.096 and 0.082, respectively). This study demonstrates that exploitable genetic variation exists in dairy cattle for M. paratuberculosis infection susceptibility.

New Method of Serological Testing forMycobacterium aviumsubsp.paratuberculosis(Johne's Disease) by Flow Cytometry

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups: (1) sera from a JD-free farm; (2) sera from JD-positive farms that had tested negative by ELISA; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD.

Inflammatory Cytokine Gene Expression in Different Types of Granulomatous Lesions during Asymptomatic Stages of Bovine Paratuberculosis

The granulomatous lesions in bovine paratuberculosis have been classified into two types, i.e., the lepromatous type and the tuberculoid type. To clarify the immunopathologic mechanisms at the site of infection, we compared inflammatory cytokine gene expression between the two types of lesions. Samples were obtained from noninfected control cows (n = 5) and naturally infected cows (n = 7) that were diagnosed by enzyme-linked immunosorbent assay (ELISA) and fecal culture test. Although none of the infected cows showed clinical signs, tuberculoid lesions were observed in five cows (tuberculoid group) and lepromatous lesions in two cows (lepromatous group). Among the cytokines examined by reverse transcription-polymerase chain reaction (RT-PCR), Th2-type cytokines interleukin-4 (IL-4) and IL-10, and Th1-type cytokine IL-2 were expressed more significantly in the lepromatous group than in the tuberculoid (P < 0.01) and noninfected groups (P < 0.05). No statistical differences were observed in the expression of interferon-gamma, IL-1 beta, TNF-alpha, and GM-CSF among lepromatous, tuberculoid, and noninfected groups. Expression of proinflammatory cytokine IL-12 mRNA, however, did not differ among the three groups; IL-18 was expressed at lower levels in the lepromatous group than in the tuberculoid group and the noninfected group (P < 0.0001). Moreover, the number of cells in which IL-18 mRNAs were detected by in situ hybridization was markedly decreased in the lepromatous group. These results indicate that the formation of lepromatous-type lesions or tuberculoid-type lesions may be influenced by alterations in Th1/Th2-type cytokine production and that IL-18 may play an important role in a Th1-to-Th2 switch in paratuberculosis.