Mycobacterial infection influences bone biomarker levels in patients with Crohn's disease.

Patients with Crohn's disease (CD) have higher risk for osteoporosis following decreased level of osteocalcin. We hypothesize that active inflammation following Mycobacterium avium subsp. paratuberculosis (MAP) infection results in elevation of undercarboxylated osteocalcin (ucOC) and downregulation of active osteocalcin in CD patients and cow-disease model (Johne's disease). In this study, we measured ucOC, active osteocalcin, and calcium levels in sera from 42 cattle (21 infected with MAP and 21 healthy cattle), 18 CD patients, and 20 controls. The level of ucOC in MAP+ bovine samples was higher than that in MAP- controls (318 ± 57.2 nmol/mL vs. 289 ± 95.8 nmol/mL, P > 0.05). Consequently, mean calcium level in bovine MAP+ was significantly higher than that in bovine-MAP- samples (9.98 ± 0.998 mg/dL vs. 7.65 ± 2.12 mg/dL, P < 0.05). Also, the level of ucOC was higher in CD-MAP+ than in CD-MAP- (561 ± 23.7 nmol/mL vs. 285 ± 19.6 nmol/mL, P < 0.05). Interestingly, the mean osteocalcin level in MAP+ bovine was lower than that in MAP- bovine (797 ± 162 pg/mL vs. 1190 ± 43 pg/mL) and it was lower in CD-MAP+ than in CD-MAP- infection (1.89 ± 0.184 ng/mL vs. 2.19 ± 0.763 ng/mL) (P < 0.05). The correlation between MAP infection and elevation of sera ucOC, reduction of active osteocalcin and increased calcium supports MAP infection role in CD and complications with osteoporosis.

Evaluation of clinical pathology parameters in fecal PCR-positive or PCR-negative goats for Johne's disease.

Johne's disease (JD) is an economically important infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This study evaluated the differences in various hematological and biochemical parameters between healthy goats and goats with JD. Forty goats were chosen randomly from a herd endemic for JD. A complete physical examination was performed. Blood and fresh fecal samples were collected from each goat. A complete blood cell (CBC) count and a protein electrophoresis were performed. Polymerase chain reaction (PCR) on fecal samples was performed in order to divide goats into two groups: group A "positive PCR on feces"; and group B "control (negative)." A Student's t test was performed for each parameter to verify differences between groups A vs B. Twenty goats were included in each group. Clinical signs likely related to JD were found in the history of 4/40 (10%) goats, while 36/40 (90%) goats were reported to be asymptomatic. CBC and electrophoresis values were within reference intervals in both groups. No differences were found for CBC parameters between the two groups. Values for alpha 1, beta, gamma globulins, and total protein (TP) were statistically higher in group A vs those in group B, while those for albumin and albumin/globulin (A/G) ratio were lower. An increase in TP, hypoalbuminemia, and hypergammaglobulinemia has been reported in group A, while no abnormalities were found concerning CBC. JD-positive goats seem to show earlier clinical pathological alternations than clinical signs. Protein electrophoresis may help the diagnosis of JD in asymptomatic goat herds, acting as an economical screening method.

Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle.

BACKGROUND: Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne's disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne's disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture. RESULTS: Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step. CONCLUSIONS: This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne's disease progression by warranting further research on the presence of MAP in blood.

Early Foetal Loss Correlates Positively with Seroconversion against Mycobacterium avium paratuberculosis in High-Producing Dairy Cows.

This study was designed to examine (i) the seroprevalence of Mycobacterium avium subs paratuberculosis (MAP) in a high-producing dairy herd with clinical symptoms of bovine paratuberculosis, (ii) MAP seroconversion and seronegativation dynamics in the herd and (iii) possible relationships between MAP infection status and herd reproductive performance. One single blood test per cow was performed early post-partum on a monthly basis from day 10-40 post-partum during the first year of the study in 519 cows belonging to a commercial dairy herd. A subset of 111 cows that became pregnant during the study was tested again 60-200 days later during the early foetal period, immediately after the first confirmation of gestation at 58-64 days post-AI. Logistic regression analysis indicated no effect of any independent variable on MAP seropositivity and conception rate 28-34 days post-AI. MAP seropositivity was not a factor affecting the anoestrous, subfertility and early foetal loss rates. In the subset of 111 cows, animals that seroconverted had a 3.9 times greater risk of suffering from early foetal loss (30.3%, 10/33) than the remaining pregnant animals (10.3%, 8/78), (95% confidence interval: 1.11-13.4; p = 0.003). In conclusion, early foetal loss was positively correlated with seroconversion to MAP. Reproductive performance was not impaired by MAP infection.

Longitudinal data collection of Mycobacterium avium subspecies Paratuberculosis infections in dairy herds: the value of precise field data.

Longitudinal infection data on Mycobacterium avium subspecies paratuberculosis (MAP) was collected on three dairy farms in Northeastern United States during approximately 10 years. Precise data on animal characteristics and animal location within farm were collected on these farms. Cows were followed over time with regard to MAP status during biannual fecal and serum sampling and quarterly serum sampling. Approximately 13 000 serum samples, 6500 fecal samples and 2000 tissue samples were collected during these years. Prevalence of positive samples was 1.4% for serological samples, 2.2% in fecal samples and 16.7% in tissue samples. Infection dynamics of MAP was studied and resulted in a number of potential changes in our understanding of MAP infection dynamics. First, a high prevalence of MAP infection was observed in these herds due to lifetime follow up of cows, including slaughter. Second, two distinctly different infection patterns were observed, so called non-progressors and progressors. Non-progressors were characterized by intermittent and low shedding of MAP bacteria and a virtual absence of a humoral immune response. Progressors were characterized by continuous and progressive shedding and a clearly detectable and progressive humoral immune response. Strain typing of MAP isolates on the three farms identified on two of three farms a dominant strain type, indicating that some strains are more successful in terms of transmission and infection progression. Continuous high quality longitudinal data collection turned out to be an essential tool in our understanding of pathobiology and epidemiology of MAP infections in dairy herds.

Evaluation of performance of bacterial culture of feces and serum ELISA across stages of Johne's disease in cattle using a Bayesian latent class model.

The objective of this study was to evaluate the performance of bacterial culture of feces and serum ELISA to correctly identify cows with Mycobacterium avium ssp. paratuberculosis (MAP) at heavy, light, and non-fecal-shedding levels. A total of 29,785 parallel test results from bacterial culture of feces and serum ELISA were collected from 17 dairy herds in Minnesota, Pennsylvania, and Colorado. Samples were obtained from adult cows from dairy herds enrolled for up to 10 yr in the National Johne's Disease Demonstration Herd Project. A Bayesian latent class model was fitted to estimate the probabilities that bacterial culture of feces (using 72-h sedimentation or 30-min centrifugation methods) and serum ELISA results correctly identified cows as high positive, low positive, or negative given that cows were heavy, light, and non-shedders, respectively. The model assumed that no gold standard test was available and conditional independency existed between diagnostic tests. The estimated conditional probabilities that bacterial culture of feces correctly identified heavy shedders, light shedders, and non-shedders were 70.9, 32.0, and 98.5%, respectively. The same values for the serum ELISA were 60.6, 18.7, and 99.5%, respectively. Differences in diagnostic test performance were observed among states. These results improve the interpretation of results from bacterial culture of feces and serum ELISA for detection of MAP and MAP antibody (respectively), which can support on-farm infection control decisions and can be used to evaluate disease-testing strategies, taking into account the accuracy of these tests.

A preliminary survey of the seroprevalence of Mycobacterium avium subspecies paratuberculosis in Mongolian cattle.

Johne's disease is a chronic infection with Mycobacterium avium susp. paratuberculosis (MAP), which causes huge economic losses to cattle industry. The seroprevalence of MAP in cattle of Mongolian was estimated by an ELISA assay using 356 serum samples which were collected from eleven provinces and Ulaanbaatar city. Out of these samples, 3 (0.84%) were found to be seropositive for MAP, originating from Tsenkher sum of Arkhangai province, Murun sum of Khuvsgul province, and Bornuur sum of Tuv province in Mongolia. This study represents first conformation of Johne's disease in Mongolian cattle. These findings provide vital information that can be used for the planning and execution of control measures for Johne's disease in the Mongolian cattle industry.

Seroprevalence and risk factors of Mycobacterium avium subspecies paratuberculosis infection in domestic sika deer in China.

Paratuberculosis or Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic infectious granulomatous enteritis of ruminants and other animals, which has a worldwide occurrence, but little is known of MAP infection in domestic sika deer in Jilin Province, China. The objective of the present investigation was to examine seroprevalence and risk factors of MAP infection in Jilin Province. Serum samples collected from 1400 sika deer from 16 sika deer herds were collected in the 4 districts of the province between May 2013 and August 2014 and were tested independently for the presence of antibodies against MAP. A total of 247 (17.64 %) sika deer tested positive for MAP antibodies using a commercially available enzyme immunoassay kit. The management level of farm and collecting region of sika deer was the main risk factor associated with MAP infection. The present study revealed the seroprevalence of MAP infection in sika deer in Jilin Province, China, which provided the baseline data for taking comprehensive countermeasures and measures in effectively preventing and controlling MAP infection in sika deer.

Increased blood-circulating interferon-γ, interleukin-17, and osteopontin levels in bovine paratuberculosis.

Paratuberculosis-infected cattle initially develop an effective cell-mediated immune response that declines as the disease progresses. Blood is one of best sources for characterizing the inflammatory status of infected cows and for studying mediators related to chronic diseases. The aim of this study was to evaluate the cow-level association between blood cytokine concentration, the influence of serum on immune cell proliferation, and dairy cows naturally infected with Mycobacterium avium ssp. paratuberculosis (MAP). Positive animals (n=41) from 19 herds were selected on the basis of 2 positive fecal culture results and divided into 2 groups: single-positive, or serum ELISA-negative cows (n=32), and double-positive, or cows that gave positive results for both mycobacterial culture and serum ELISA (n=9). Negative animals (n=39) were selected from paratuberculosis-negative herds in which at least 80% of the animals had been diagnosed as negative by fecal culture and ELISA and that did not produce positive results during the 2-yr study. Analysis of plasma levels of the cytokines IL-4, IL-10, IL-17, IFN-γ, and osteopontin was performed, revealing distinct patterns. The ELISA-positive cows with MAP shedding had similar plasma concentrations of IL-4 and IL-10 but elevated levels of IFN-γ, IL-17, and osteopontin, which is indicative of inflammatory disease in these subclinical positive cows. In vitro MAP infection of bovine macrophages showed increased gene expression of tumor necrosis factor-α, IL-1ß, IL-6, IL-23, and transforming growth factor-ß as early as 6h postinfection for all of the cytokines involved in the establishment of a T-helper type-17 immune response. To determine the systemic influence of serum on immune cell functions, lymphoproliferation assays were also performed in presence of JD serum. The serum from shedding cows showed 15% less proliferation. These results indicate that infected cows have a lower systemic capacity to maintain a protective immune response and that, as the disease progresses, an emerging T-helper type-17 immune response is established.

Serological studies to determine the occurrence of Johne's disease and mycoplasma infection in the Northern-East Polish population of European bison (Bison bonasus).

A serological study of twenty three European bison (Bison bonasus) derived from Northern-East Poland for the seroprevalence of Mycobacterium avium subsp. paratuberculosis, Mycoplasma bovis, Mycoplasma mycoides subsp. mycoides SC, Mycoplasma agalactiae and Mycoplasma capricolum subsp. capripneumoniae was conducted. Only specific antibodies to M. bovis were detected in two animals (8.7%) which were connected with the clinical signs and macroscopic anatomopathological lesions.

Comparison of two serological diagnosis tests for bovine paratuberculosis.

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.

Herd-level true seroprevalence of caseous lymphadenitis and paratuberculosis in the goat population of Poland.

A large-scale study was carried out in the Polish goat population in 2014-2021 to determine the herd-level true seroprevalence (HTP) of caseous lymphadenitis (CLA) caused by Corynebacterium pseudotuberculosis (Cp) and paratuberculosis (PTB) caused by Mycobacterium avium ssp. paratuberculosis (Map). Two-stage cluster sampling was applied to herds counting at least 20 adult goats (aged >1 year) and in each herd all males and 10-13 females were tested. At least one seropositive goat regardless of its sex was necessary to consider the herd as infected. HTP was estimated using the Bayesian approach with the Gibbs sampler in the EpiTools and reported as the median and 95 % credibility interval (95 % CrI). A total of 1282 adult goats from 86 herds were serologically tested using two commercial ELISAs (Cp-ELISA and Map-ELISA). At least 1 seropositive result of Cp-ELISA and Map-ELISA was obtained in 73/86 herds (84.9 %) and 40/86 herds (46.5 %), respectively. HTP of CLA was estimated at 73.3 % (95 % CrI: 65.0 %, 80.4 %) and HTP of PTB was estimated at 42.9 % (95 % CrI: 25.8 %, 58.0 %). There was a significant positive association between the occurrence of CLA and PTB in the herds (odds ratio 6.0, 95 % confidence interval: 1.2, 28.8; p = 0.010). Probability of the seropositive result for PTB was also significantly higher in Cp-seropositive goats than in Cp-seronegative goats (odds ratio 3.9, 95 % confidence interval: 2.4, 6.3; p < 0.001) which could indicate either a higher risk of co-infection or a higher rate of false positive results for PTB in Cp-positive goats. To investigate this issue, optical densities obtained in Map-ELISA were compared between Cp-positive and Cp-negative goats and results of Map-ELISA were adjusted accordingly. Map-negative sera from Cp-positive goats turned out to have significantly higher optical densities than Map-negative sera from Cp-negative goats (p < 0.001). After the adjustment, the herd-level apparent seroprevalence of PTB was 41.9 % (36/86 herds) so it still fell within the 95 % CrI of HTP of PTB calculated before the adjustment. Concluding, CLA appears to be widespread in the Polish goat population. In many of them it may be subclinical at the moment, however will likely emerge in the future as the disease follows cyclic pattern in Poland. On the other hand, given the total lack of clinical PTB in Polish goats, an explanation for a high HTP of PTB remains unclear and warrants further studies using tests of higher analytical specificity than ELISA.

Postexposure subunit vaccination against chronic enteric mycobacterial infection in a natural host.

The control of chronic bacterial diseases with high prevalence in areas of endemicity would strongly benefit from availability of postexposure vaccines. The development of these vaccines against mycobacterial infections, such as (para)tuberculosis, is hampered by lack of experience in natural hosts. Paratuberculosis in cattle is both a mycobacterial disease of worldwide importance and a natural host model for mycobacterial infections in general. The present study showed beneficial effects of therapeutic heat shock protein 70 (Hsp70) vaccination in cattle with naturally acquired chronic infection with Mycobacterium avium subsp. paratuberculosis. Vaccination-induced protection was associated with antibody responses, rather than with induction of specific T helper 1 cells. Targeted therapeutic postexposure vaccination complementary to selective use of antibiotics could be an effective approach for control of chronic mycobacterial infections.

Mycobacterium avium subsp. paratuberculosis and multiple sclerosis in Sardinian patients: epidemiology and clinical features.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is an infectious factor recently found in association with multiple sclerosis (MS) in Sardinia. OBJECTIVES: The objectives of this study were to confirm this association and evaluate its role in clinical features. METHODS: A total of 436 patients and 264 healthy controls (HCs) were included. We examined the blood of each individual for MAPDNA and MAP2694 antibodies using IS900-specific PCR and ELISA, respectively. Differences in MAP presence between the MS group and HCs were evaluated. In MS patients, we considered: gender, age, age at onset, duration of disease, course, EDSS, therapy, relapse/steroids at study time, and oligoclonal bands (OBs). RESULTS: MAPDNA and MAP2694 antibodies were detected in 68 MS and six HCs (p = 1.14 × 10(-11)), and 123 MS and 10 HCs (p = 2.59 × 10(-23)), respectively. OBs were found with reduced frequency in MAP-positive patients (OR = 0.52; p = 0.02). MAP2694 antibodies were detected more in patients receiving MS treatments (OR = 2.26; p = 0.01), and MAPDNA in subjects on steroids (OR = 2.65; p = 0.02). CONCLUSION: Our study confirmed the association of MAP and MS in Sardinia. The low OB frequency in MAP patients suggests a peripheral role as a trigger in autoimmunity. MAP positivity might be influenced by steroids and MS therapy. Studies in other populations are needed to confirm the role of MAP in MS.

Cow-level association between serum 25-hydroxyvitamin D concentration and Mycobacterium avium subspecies paratuberculosis antibody seropositivity: a pilot study.

Vitamin D deficiency has been associated with various human diseases. Therefore, the objective of this study was to evaluate the cow-level association between serum 25-hydroxyvitamin D [25(OH)D] concentration and Mycobacterium avium ssp. paratuberculosis (MAP) seropositivity of dairy cows, adjusting for diet, breed, hair coat color, stage of lactation, reproductive status, and cow age. The sera of 80 MAP antibody ELISA-positive and 80 test-negative herd mates from 5 Minnesota dairy herds were analyzed for 25(OH)D and 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. The cows' age, production records, and hair coat color were recorded. Additionally, feed samples were obtained and analyzed for vitamin D(2) and vitamin D(3) content. A linear mixed model was used to identify potential predictors for serum 25(OH)D concentration, accounting for herd of origin. The majority of rations analyzed had over 22,000 IU of vitamin D/day (maximum: 52,000 I U/d) and the study cows' average serum 25(OH)D concentration was 62.5 ± 13.8 ng/mL. Serum ELISA-positive cows had, on average, 5.3 ng/mL lower 25(OH)D serum levels than test-negative herd mates. The reproductive status of cows was also associated with the 25(OH)D levels, with fresh cows having the lowest serum concentration. In this cross-sectional study, a temporal or causal association between MAP antibody ELISA status and serum 25(OH)D concentration could not be evaluated. In addition, the high levels of vitamin D in the rations of participating farms and the average 25(OH)D serum concentration suggest that additional supplementation with vitamin D in the ration is likely to be ineffective.

[Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis]. / Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of yIFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.

Bovine paratuberculosis: a review of the advantages and disadvantages of different diagnostic tests.

Paratuberculosis (PTB), or Johne's disease, is a chronic infectious granulomatous enteritis of ruminants, caused by Mycobacterium avium subspecies paratuberculosis (Map). It is characterized by diarrhea and progressive cachexia, which may cause the death of the animal. Calves are the most susceptible to infection. Infected animals excrete Map mainly by the feces. PTB is endemic worldwide, with high prevalence levels, strong economic impact and public health relevance because of its possible association with Crohn's disease. Although the current reference diagnostic test is identification of Map in the bacterial culture, there are different diagnostic tests to identify infected individuals and/or herds. The sensitivity and specificity of these tests vary according to the stage of the disease in the animals to be evaluated. The correct choice and application of each of these diagnostic tests will ensure their success and may allow to establish a control program. The aim of this work is to review and discuss the different diagnostic tests used in the detection of Map-infected animals, focusing on their advantages and disadvantages.

Effect of management practices on paratuberculosis prevalence in Danish dairy herds.

A voluntary risk-based control program on paratuberculosis in dairy cattle was initiated in Denmark in 2006. Cows were categorized as high-risk (antibody-positive at least once within the last 3 tests) or low-risk animals based on the results of 3 to 4 annual milk ELISA detecting Mycobacterium avium ssp. paratuberculosis (MAP)-specific antibodies. High-risk animals require management practices aimed at decreasing calf exposure to MAP-contaminated colostrum and milk, and feces originating from these cows. Moreover, repeated test-positive cows are recommended for slaughter before next calving. The objective was to assess the effect of different management practices on the prevalence of MAP-specific antibodies. A questionnaire on management practices was distributed to 1,261 participating herds in December 2008. A total of 1,092 (87%) herd managers returned the questionnaire. Repeated prevalence data from 1,081 herds were available for a period up to 4.25 yr after the first test round. The changes in the prevalence of MAP-specific antibodies from the start of interventions were assessed using a hierarchical logistic model, where different management practices were assessed: a) culling of repeated test-positive cows, b) separation of high-risk from low-risk cows in calving areas, c) cleaning of calving areas after high-risk cows calved, d) removal of calves born to high-risk dams within 2h after calving, e) use of colostrum for feeding of heifer calves from low-risk cows only, f) use of waste milk for feeding of heifer calves from low-risk cows only, g) herd size, and h) proportion of purchased animals. Multivariable analyses suggested that only the proportion of purchased animals (>15% purchased animals as well as 0 to 15% purchased animals compared with no purchased animals in the herd), culling of repeated test-positive animals, and use of waste milk from specific cow groups influenced the decrease in prevalence of MAP-specific antibodies. The control program has been running for just 4.25 yr, and it is assumed that the full effect of the risk-based management practices will only be observed after 4 to 8 yr. Therefore, lack of association between some practices and decrease in prevalence may be a reflection of a short study period. Furthermore, decreases in the prevalence of MAP-specific antibodies may not reflect discontinued transmission of MAP in all age groups.

Serological investigation and genotyping of Mycobacterium avium subsp. paratuberculosis in sheep and goats in Inner Mongolia, China.

Paratuberculosis a contagious and chronic disease in domestic and wild ruminants, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Typical clinical signs include intractable diarrhea, progressive emaciation, proliferative enteropathy, and mesenteric lymphadenitis. Paratuberculosis is endemic to many parts of the world and responsible for considerable economic losses. In this study, different types of paratuberculosis and MAP in sheep and goats were investigated in Inner Mongolia, a northern province in China contiguous with two countries and eight other provinces. A total of 4434 serum samples were collected from six cities in the western, central, and eastern regions of Inner Mongolia and analyzed using the ELISA test. In addition, tissue samples were collected from seven animals that were suspected to be infected with MAP. Finally, these tissues samples were analyzed by histopathological examination followed by polymerase chain reaction (PCR), IS1311 PCR-restriction enzyme analysis (PCR-REA), and a sequence analysis of five genes. Among all 4434 ruminant serum samples collected from the six cities in the western, central, and eastern regions of Inner Mongolia, 7.60% (337/4434) measured positive for the MAP antibody. The proportions of positive MAP antibody results for serum samples collected in the western, central, and eastern regions were 5.10% (105/2058), 6.63% (85/1282), and 13.44% (147/1094), respectively. For the seven suspected infected animals selected from the herd with the highest rate of positivity, the gross pathology and histopathology of the necropsied animals were found to be consistent with the pathological features of paratuberculosis. The PCR analysis further confirmed the diagnosis of paratuberculosis. The rest of the results demonstrated that herds of sheep and goats in Inner Mongolia were infected with both MAP type II and type III. To the best of our knowledge, this is the first study of the two subtypes of MAP strains in sheep and goats in Inner Mongolia.

Development of rELISA using novel markers for the diagnosis of paratuberculosis.

Paratuberculosis is one of the complex livestock infections whose control has largely been hampered due to the lack of efficacious diagnostics. Present study optimized plate ELISA assay for the diagnosis and screening of paratuberculosis using recombinant secretory proteins. Five secretory antigens (2677c, 3547c, 4308c, 1693c, and 2168c) were produced in the recombinant system using the E. coli host and used for the optimization of the assay. These proteins were selected because of their prior proven specificity and antigenicity as humoral immunity markers. The assay was first optimized using traditional ELISA reader and then the performance was evaluated using a handheld ELISA reader. Findings were identical in both traditional ELISA reader as well as handheld ELISA reader. Optimized ELISA was found reproducible using different batches of the recombinant antigens as well as in terms of the inter and intra assay %CV values. The present ELISA has a sensitivity and specificity of 91.6% and 100%, respectively. Also, rELISA revealed AUCROC and Youden index J of 0.95 and 0.91, respectively. In conclusion, assay conditions of MAP-recombinant protein-based ELISA were optimized and the optimized ELISA ODs can be read using portable handheld ELISA reader. Thereby, opening a future window to develop assay for onsite testing.