Searching for proteins of Mycobacterium avium subspecies paratuberculosis with diagnostic potential by comparative qualitative proteomic analysis of mycobacterial tuberculins

Accurate immunodiagnosis of bovine paratuberculosis is among others hampered by the lack of specific antigens. One of the most frequently used antigen preparations is purified protein derivative (PPD), also known as tuberculin. This crude extract has limitations when used in diagnostic assays due to the presence of cross-reactive antigens. The aim of the current study was to systematically analyze the qualitative protein composition of PPD of the major mycobacterial pathogens.One-dimensional gel electrophoresis followed by tandem mass spectrometry analysis of PPD from Mycobacterium avium subspecies paratuberculosis (MAP), Mycobacterium avium subspecies avium (MAA) and Mycobacterium bovis (MB) identified 156, 95 and 132 proteins, respectively. Comparative sequence analysis led to the selection of a MAP-specific protein (MAP1718c), and finally heterologous expression in Escherichia coli of this and other diagnostic candidate proteins (MAP3515c and MAP1138c (LprG)) enabled evaluation of their immunogenicity. Lymphocyte proliferation responses did not indicate substantial diagnostic potential of the antigens tested. In contrast serum antibody levels for MAP1138c in paratuberculosis infected cows (N = 20) were significantly higher (p < 0.01) than in control animals (N = 20), despite the conserved nature of this protein.In conclusion, this study showed that a combination of proteomics and genomics, starting from complex protein mixtures, present in tuberculins, can reveal novel proteins aiding the development of immunodiagnostics for mycobacterial diseases.     

Management-related risk factors for M. paratuberculosis infection in Michigan, USA, dairy herds

A cross-sectional study was conducted from June through December 1996 to identify management-related risk factors for herd-level M. paratuberculosis infection. Data were collected from 121 participating herds. A two-part questionnaire was administered to gather data on current and previous management practices and herd productivity. A random sample of cows aged ≥24 months was selected from each herd and tested for antibodies to M. paratuberculosis using the IDEXX Antibody ELISA (sensitivity 64%, specificity 96%). A positive herd was one in which ≥2 animals tested positive for antibodies to M. paratuberculosis. A negative herd was one in which no animal tested positive. Herds in which only one animal tested positive were dropped from statistical analysis to reduce the risk of including false-positive herds in the statistical analyses.

There were 80 herds with one or more positive animals and 41 herds with no positive animals in the sample (66% herd-level prevalence). Twenty-six herds (21%) were dropped from further analyses because they had only one positive cow. Twelve herds (10%) were dropped from analysis because of missing data. The resulting sample used for statistical modeling included 46 positive herds and 37 negative herds (55% herd-level prevalence). A multi-variable logistic-regression model was used to evaluate the results. The variable ‘use of an exercise lot for lactating cows' was associated with a three-fold increase in odds of a herd being positive for M. paratuberculosis infection (O.R.=3.01, C.I.=1.03–8.80); ‘cleaning of maternity pens after each use' was associated with a three-fold reduction in odds of a herd being positive for M. paratuberculosis infection (O.R.=0.28, C.I.=0.08–0.89); ‘application of lime to pasture areas in 1993' resulted in a ten-fold decrease in odds of a herd being positive for M. paratuberculosis infection (O.R.=0.10, C.I.=0.02–0.56).     

Mycobacterium avium subsp. paratuberculosis in cow bulk tank milk in Cyprus detected by culture and quantitative IS900 and F57 real-time PCR

The possibility that Mycobacterium avium subsp. paratuberculosis (MAP) plays some role in the development of Crohn's disease in humans is attracting attention to milk and milk products originating from infected animals. In this study, we focused on the detection of MAP in 220 bulk tank milk (BTM) samples from all dairy cattle herds in Cyprus. In total, 63 (28.6%) BTM milk samples were found to be positive for MAP using quantitative real-time PCR assays for IS900 and F57. The presence of MAP in BTM was low, and was assessed to be several tens of MAP cells per one ml of BTM. Milk samples examined by cultivation were found to be negative for MAP in all 220 BTM. In two BTM samples cultivation and subsequent sequencing of 16S rRNA revealed two isolates of M. fortuitum.     

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as the pro-inflammatory cytokine IFN-γ were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-γ stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-γ. Finally, the results suggest an important role of IL-10 and TGF-β on the profile of NO production which may explain the low NO production in MAP clinically infected cows.     

Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III

Mycobacterium avium subsp. paratuberculosis (MAP) strains with two new IS900 restriction fragment length polymorphism (RFLP) BstEII types intermediate suspected to belong to the MAP Type III group were isolated from migrating sheep in Germany. Such strains have only been sporadically identified in a few studies. For a better understanding of the genomic diversity of MAP with regard to specific host associations, geographic origin, and the discussed classification into Type I, Type II and Type III, these isolates were further characterized.Using IS900-RFLP, the isolates showed unique fingerprint patterns after BstEII-, PstI-, PvuII- and BamHI-digestion which had not been published before. Additionally, using gyrB-PCR-restriction endonuclease analysis (PCR/REA) and mycobacterial interspersed repetitive unit (MIRU)-PCR, the two strains showed differences to known patterns of the Type I as well as the Type II group. Unique genotypes were also obtained with multilocus short sequence repeat (MLSSR) sequencing and MIRU-variable-number tandem-repeat (VNTR) typing.As expected, genomic profiles identical to the Type I and different from the Type II group were detected by IS1311-PCR/REA, IS1311 sequencing as well as by Large Sequence Polymorphism analysis (LSP^A 8, 17, 20, 4-II, and 18).In addition to distinct growth characteristics, the unique genotypes of the studied sheep strains support their affiliation to the assumed third group within the MAP subspecies and suggest the existence of different genotypes within this Type III group. The results could serve as further evidence that Type I and Type III groups are more closely related to each other than to the bovine Type II group.     

Identification of differentially expressed genes in ileum, intestinal lymph node and peripheral blood mononuclear cells of sheep infected with Mycobacterium avium subsp. paratuberculosis using differential display polymerase chain reaction

Discovery of differentially expressed genes aids in understanding molecular mechanisms underpinning normal and pathological states. When studying animals such as sheep where the entire genome has not been characterized, techniques that do not require knowledge of gene sequences are particularly advantageous. We used one such technique, differential display polymerase chain reaction (DD-PCR), to identify genes that had different degrees of expression in response to Mycobacterium avium subsp. paratuberculosis (M. ptb), the organism that causes Johne's disease in ruminants. Differentially expressed genes were validated by quantitative PCR using especially selected reference genes established in this study. Sheep (n = 47) were classified according to history of exposure to M. ptb and infection status by histology and faecal and tissue culture. Differences in levels of gene expression were analyzed using restricted maximum likelihood (REML) in a linear mixed model. Five genes from the ileum and 17 genes from lymph node were differentially expressed in ovine Johne's disease. Expression of seven of these genes was also significantly different in peripheral blood mononuclear cells. Genes identified in association with M. ptb infection had a wide range of functions in pathways including: antigen presentation, signal transduction and cell differentiation, TLR signaling, immune cell activation and chemokine functions, granulomatous inflammation, Th1 suppression and apoptosis.     

Antibody recognition to secreted proteins of Mycobacterium avium subsp. paratuberculosis in sera from infected ruminants

Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46 kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis.     

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated ‘A’, ‘B’, ‘C’ and ‘E’) were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern ‘A’, was found in 46 isolates (75%). The second, pattern ‘E’, included 8 isolates (13%), while the third, pattern ‘B’, included 6 isolates (10%). Pattern ‘C’ was found for only one isolate. All of the deer isolates were classified as pattern ‘A’, while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern ‘A’, was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns ‘B’, ‘C’ and ‘E’, were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.     

Efficacy of monensin sodium for the reduction of fecal shedding of Mycobacterium avium subsp. paratuberculosis in infected dairy cattle

Reducing the quantity of Mycobacterium avium subsp. paratuberculosis (MAP) being shed by cows with Johne's disease should decrease the risk of spread of this disease to young stock. Previous work has suggested that monensin sodium decreases the pathologic lesions associated with Johne's disease, but the impact on shedding of viable MAP remains unknown. After serologic screening of 32 dairy herds in southwestern Ontario, 228 cows from 13 of these herds were enrolled into a randomized clinical trial. Fecal culture and PCR were used to identify 114 cows as potential fecal shedders, while another 114 cows were enrolled as ELISA negative, herd and parity matched controls. All cows were randomized to receive either a monensin controlled release capsule (CRC) or a placebo capsule. Serial fecal and blood samples were collected for fecal culture and serum ELISA testing over a 98-day period. On day 98 of the study, treatments were switched for all cows continuing in the trial. These remaining cows were followed for another 98 days with a similar sampling protocol. Mixed effect models were used to measure the impact of treatment on the number of colony forming units identified on fecal cultures over time. During the first 98 days of the study, cows treated with a monensin CRC were found to shed 3.4 cfu per tube less than placebo treated cows (P = 0.05). The serum ELISA S/P ratio was reduced by 1.39 units in cows given monensin (P = 0.06). However, treatment with monensin did not reduce the odds of testing positive on serology. Only the cows shedding MAP on day 0 were found to have a reduced odds of testing positive on fecal culture when treated with monensin (OR = 0.27; P = 0.03). Monensin sodium administered to infected animals at 335 mg/day marginally reduced fecal shedding of MAP in mature dairy cattle, but the biological significance of this reduction is unknown.     

Risk management of paratuberculosis in dairy herds

Risk management of paratuberculosis (Johne's disease) in a dairy herd requires an assessment of the likelihood of paratuberculosis occurring in the herd, the economic impact of paratuberculosis on the herd and an evaluation of measures that can be taken to reduce this likelihood and impact.The likelihood of paratuberculosis occurring in the herd is related to the regional herd-level prevalence of paratuberculosis and the herd management (e.g., introducing animals from other herds). The economic impact of paratuberculosis includes production losses due to subclinical and clinical cases, losses due to increased replacement of animals and costs of control measures. Furthermore, a reduction of the price of milk from infected herds might result from consumer concerns about the zoonotic potential of paratuberculosis.Measures that reduce the likelihood of paratuberculosis occurring in a herd and its impact include preventive management measures (e.g., closed herd management and an effective separation of susceptible young stock from adult cattle), test-and-cull schemes for known infected herds and quality assurance schemes for test-negative herds. Quality assurance schemes for test-negative herds, such as schemes for 'low-Map bulk milk' and 'Map-free' herds, aim at safeguarding or increasing the profitability of these herds.Keys to success of risk management of paratuberculosis include realistic expectations of the results of paratuberculosis control, quality assurance and control programmes that are appreciated by farmers and incentives for farmers to participate in such programmes.     

Transmission parameters of Mycobacterium avium subspecies paratuberculosis infections in a dairy herd going through a control program

A Johne's disease control program, including stringent management practices and a test-and-cull program (whole-herd fecal-samples taken twice a year), was implemented on a medium-sized Pennsylvania dairy farm that was suffering losses from clinical Johne's disease. The data that emerged from the control program, combined with birthdates, culling dates, lactation information and pedigrees, yielded an extensive longitudinal dataset. The dataset was processed through SAS 9.1 for statistical analysis; herd-level disease dynamics and dam-to-daughter transmission parameters were calculated. After the implementation of the program in 1984, prevalence dropped dramatically from 60% to less than 20% in 1989. After an apparent prevalence peak (25%) in 1991 due to improved test sensitivity, prevalence maintained a plateau of 10% from 1996 to 2000. After the implementation of the program, 9.5% of the offspring from test-negative dams and 26.8% of the offspring from known-infected dams became infected with Mycobacterium avium subspecies paratuberculosis (Map) (χ² = 14.7; p = 0.0001). Calves born shortly following the calving of an infected dam and calves growing up with a future high shedder were more likely to be infected compared to calves without this risk profile. It was concluded that, after the implementation of the control program, the most important causes of infections of susceptible calves were their own dams or infected animals which had calved recently.     

Identification of Mycobacterium avium ssp. paratuberculosis infected dairy herds by environmental sampling

In herds with known prevalence (P) use of environmental sampling (ES) to detect Mycobacterium avium ssp. paratuberculosis (MAP) infected cattle herds was proofed in relation to P. In 31 MAP-infected free stall dairy herds and 15 non-infected herds P was defined by annually repeated whole herd testing by fecal culture (34 877 individual samples). Eight infected herds had a very low (> 0-2%), 14 a low (> 2-5%), four a medium (> 5-10%), and five a high P (> 10%). A mean number of nine environmental samples per herd were collected from the floor of lactating cows, milking, calving and sick cow areas and the crossover to the calf area. After twelve weeks cultivation on HEYM-medium with and without mycobactin positive samples were further characterized by PCR. All non-infected herds (100%) showed negative and 22 (71%) of the infected herds positive results in ES. Nine infected herds with negative ES results had a low P (0.04-4,04%). Proportion of positive ES depended on P and on sampling areas with 53.3% positive results in lactating cow areas and 45.2% in milking areas. For P > 5%, ES in these two areas caused a positive herd status; herds with P < 5% required sampling in the other areas too. The ES method has a herd sensitivity of 87% for dairy herds with P > 2% and provides an efficient tool to determine MAP infection status or herd prevalence.     

Factor analysis of a Johne's disease risk assessment questionnaire with evaluation of factor scores and a subset of original questions as predictors of observed clinical paratuberculosis

Factor analysis was used to examine the interrelationships among 38 variables collected as part of a Johne's disease risk assessment questionnaire completed in 2002 on 815 U.S. dairy operations. Eleven factors were extracted, accounting for two-thirds of the variance encountered in the original variables. Responses to many of the risk assessment questions were closely related. Standardized scores on the 11 factors were calculated for operations providing complete information, and were evaluated as predictors in a model-based logistic regression analysis with the outcome being whether operations had observed one or more cows with clinical signs suggestive of paratuberculosis during the previous year. A logistic regression model was also used to evaluate the predictive ability of a reduced subset of approximately one-third of the original variables that was selected to represent the derived factors. The performance of both sets of predictors was comparable with respect to goodness-of-fit and predictive ability. In conclusion, the length of the current risk assessment instrument could be reduced considerably without a substantial loss of information by removing or combining questions that are strongly correlated.     

The contribution of molecular biology to Mycobacteriumavium subspecies Paratuberculosis research

Molecular biology has contributed to our knowledge and understanding of the structure of Mycobacteriumavium subspecies paratuberculosis and has been particularly useful in determining those components that elicit immune responses in the host or discriminate M. avium paratuberculosis from other closely related environmental mycobacteria. As such, it has made a significant impact in the field of diagnosis, and has been instrumental in the development of specific and sensitive diagnostic tests. The next decade will see exciting new developments in paratuberculosis research as a consequence of substantial advances made in the construction of gene transfer systems in mycobacteria. These will provide opportunities for applying new strategies to determine the genetic basis for pathogenesis and the mechanisms of drug resistance and will offer new prospects for the rational design of efficient vaccines.     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

Flock-level factors associated with the risk of Mycobacterium avium subsp. paratuberculosis (MAP) infection in Greek dairy goat flocks

In this cross-sectional study we identified flock-level risk factors for Mycobacterium avium subsp. paratuberculosis (MAP) infection, in Greek dairy goat flocks. We collected 1599 milk samples from does that were at the last stage of lactation in 58 randomly selected dairy goat flocks, during May to September 2012. The collected samples were tested with a commercial milk ELISA (IdexxPourquier, Montpellier, France) and the results were interpreted at a cut-off that optimized the accuracy of the diagnostic process. For the analysis of the data we used Bayesian models that adjusted for the imperfect Se and Sp of the milk-ELISA. Flock was included as a random effect. Does in flocks that used common water troughs and communal grazing grounds had 4.6 [95% credible interval (CI): 1.5; 17.4] times higher odds of being MAP-infected compared to does in flocks that had no contact with other flocks. Does of flocks supplied with surface water from either streams or shallow wells had 3.7 (1.4; 10.4) times higher odds of being infected compared to those in flocks watered by underground and piped water sources. When kids were spending equal to or more than 10 h per day with their dams they had 2.6 (1.1; 6.4) times higher odds of being MAP infected compared to kids that were separated from their dams for less than 10 h per day. Finally, does in flocks that continuously used the same anti-parasitic compound had 2.2 (1.0; 4.6) times higher odds of MAP infection compared to those in flocks alternating anti-parasitic compounds. These results should be considered in the development of a nationwide future control program fοr caprine paratuberculosis in Greece.     

Prevalence and distribution of paratuberculosis (Johne's disease) in cattle herds in Ireland

A simple random survey was conducted in Ireland during 2005 to estimate the ELISA-prevalence of paratuberculosis, commonly called Johne's disease (JD), in the cattle population. Serum samples were collected from all 20,322 females/breeding bulls over 12 months-of-age in 639 herds. All samples were tested using a commercially available absorbed ELISA. The overall prevalence of infected herds, based on the presence of at least one ELISA-positive animal, was 21.4% (95% CI 18.4%-24.9%). Herd prevalence levels amongst dairy herds (mean 31.5%; 95% CI: 24.6%, 39.3%) was higher than among beef herds (mean 17.9%; 95% CI: 14.6%-21.8%). However, the animal level prevalence was similar. The true prevalence among all animals tested, was calculated to be 2.86% (95%CI: 2.76, 2.97) and for animals >= 2 yrs, it was 3.30% (95%CI: 3.17, 3.43). For animals in beef herds, true prevalence was 3.09% (95%CI: 2.93, 3.24), and for those in dairy herds, 2.74% (95%CI: 2.59, 2.90). The majority of herds had only one ELISA-positive infected animal. Only 6.4% (95% CI 4.7%-8.7%) of all herds had more than one ELISA-positive infected animal; 13.3% (CI 8.7%-19.7%) of dairy herds ranging from two to eight ELISA-positive infected animals; and, 3.9% beef herds (CI 2.4%-6.2%) ranging from two to five ELISA-positive infected animals. The true prevalence of herds infected and shedding Mycobacterium avium subspecies paratuberculosis is estimated to be 9.5% for all herd types; 20.6% for dairy herds; and 7.6% for beef herds. If ELISA positive animals <2-years-of-age are excluded, the true herd prevalene reduces to: 9.3% for all herd types; 19.6% for dairy herds; and 6.3% for beef herds based on a test specificity (Sp) of 99.8% and test sensitivity (Se) (i.e., ability to detect culture-positive, infected animals shedding at any level) of 27.8-28.9%.     

Prevalence and associated management factors of Cryptosporidium shedding in 50 Swedish dairy herds

Cryptosporidium parvum is a protozoan parasite causing diarrhoea in young calves. This cross-sectional study was performed to estimate the prevalence of Cryptosporidium infected herds in a sample of Swedish dairy herds and to identify potential risk factors associated with shedding of oocysts. Fifty dairy herds, selected by stratified random sampling, were included. The herds were visited once during the indoor seasons of 2005–2006 and 2006–2007. Faecal samples were collected from 10 calves, 10 young stock and 5 cows in each herd. Clinical observations of sampled animals and environmental status were recorded, and farmers were interviewed about management procedures. Faecal samples were cleaned by sodium chloride flotation and detection of oocysts was made by epifluorescence microscopy. Cryptosporidium parvum-like oocysts were found in 96% of the herds. Prevalence was 52% in calves, 29% in young stock and 5.6% in cows. Three two-day-old calves shed oocysts. Cryptosporidium andersoni was found in seven animals from four different herds. Factors associated with prevalence of shedders among sampled animals in a herd were age at weaning, cleaning of single calf pens, placing of young stock, system for moving young stock, and year of sampling. Factors associated with shedding in calves were age, placing of young stock, routines for moving young stock and time calf stays with the cow. The only significant factor in young stock was age. In cows, number of calves in the herd and type of farming (organic vs. conventional) affected shedding.