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Veterinary Medicine International
Volume 2011, Article ID 352561, 12 pages
Diagnosis and Molecular Characterization of Mycobacterium avium subsp. paratuberculosis from Dairy Cows in Colombia
1Institute of Veterinary Food Sciences, Justus Liebig University Giessen, Frankfurter Straße 92, 35392 Giessen, Germany
2Escuela de Medicina Veterinaria (Grupo Centauro), Facultad de Ciencias Agrarias, Universidad de Antioquia, Carrera 75, No. 65-87, Medellín, Colombia
3Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany
Received 15 December 2010; Revised 29 January 2011; Accepted 16 March 2011
Academic Editor: Jesse M. Hostetter
Copyright © 2011 J. A. Fernández-Silva et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The objective of this study was the serological, bacteriological and molecular diagnosis, as well as the molecular characterization of Mycobacterium avium subsp. paratuberculosis (Map) in adult cows of five Colombian dairy herds. Serum samples were tested by an indirect absorbed enzyme–linked immunosorbent assay (ELISA-C). All fecal samples were tested by pooled culture. After that, fecal samples of Map positive pools were tested individually by culture and polymerase chain reaction (PCR). In one herd, slurry and tissue samples from one animal were also taken and tested by PCR and culture. Map isolates were analyzed by the Multilocus Short Sequence Repeat (MLSSR) and the Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) methods. ELISA produced positive results in 1.8% (6/329) of the animals and 40% (2/5) of the herds. Four fecal, two tissue, and two slurry samples from a herd were Map positive by culture and PCR. MLSSR and MIRU-VNTR revealed two different strain profiles among eight Map isolates recovered. This study reports the first molecular characterization of Map in one dairy herd in Colombia, the limitations for individual diagnosis of subclinical Map infections in cattle, and the usefulness of pooled fecal samples and environmental sampling for Map diagnosis.
1. Introduction
Mycobacterium avium subsp. paratuberculosis (Map) is a slow growing, mycobactin-dependent acid fast bacterium that causes paratuberculosis or Johne's disease in cattle [1]. Paratuberculosis produces a considerable economic impact on the cattle industry, especially on milk production and body condition. Map has also been related to the chronic human enteritis known as Crohn's disease, but this relationship still remains controversial [2].
Enzyme-linked immunosorbent assay (ELISA), bacteriological cultivation of fecal samples, and polymerase chain reaction (PCR) are test widely used for the antemortem diagnosis of paratuberculosis in cattle herds [3–5]. On the other hand, bacteriological culture of pooled fecal samples and environmental sampling are cost-effective methods to classify herds as Map infected [6, 7]. In addition, sampling all adult cattle in every herd, environmental sampling, serial testing, and the use of two to three diagnostic tests have been recommended for herd screening and to increase the accuracy of Map diagnosis [5, 8].
Strain differentiation of Map is very useful to understand the origin of the infections and the disease transmission dynamics, to design more adequate control measures, and to improve diagnosis rates and the development of vaccines [9]. Although paratuberculosis is a notifiable disease in Colombia, the lack of an official national control program and some limitations of the diagnostic tests have contributed to the reduced local information on the disease.
The objective of this study was the serological, bacteriological, and molecular diagnosis of Map in adult cows of five Colombian dairy herds, as well as the molecular characterization of Map isolates and the comparison of results with similar studies.
2. Materials and Methods
2.1. Selection of Herds
Between November and December of 2009, five dairy herds were selected to be examined for Map (Table 1 and Figure 1). Of these five herds, four herds (herds 1, 2, 3, and 4) tested ELISA and PCR positive but culture negative for Map in a previous cross-sectional study in 2007 [10]. In this study, 13 herds without previous history of paratuberculosis and one herd (herd 1 of the present study) with previous diagnosis of Johne's disease [11] were examined. In every herd, only a sample (between 19 and 29 randomly selected adult cows, depending on the herd population) was tested. Serological testing was initially carried out to all sampled animals using a commercial lipoarabinomannan- (LAM-) based indirect ELISA test without preabsorption (Svanovir Para-TB Ab ELISA Kit, Uppsala, Sweden) (ELISA-A). For confirmation of positives samples by ELISA-A, a commercial indirect ELISA test based on detection of antibodies to protoplasmic Map antigens, including a pre-absorption step with Mycobacterium phlei (M. phlei) (ELISA paratuberculosis antibody verification, Institut Pourquier, Montpellier, France) (ELISA-B) was used. For the PCR diagnosis, two methods targeted to F57 and ISMav2, and to IS900, respectively, were applied [10].
The four herds previously tested in 2007 and selected in the present study (herds 1, 2, 3, and 4) have never followed any structured or consistent control program for prevention or control of paratuberculosis before the first study in 2007, or in the period between both studies. However, culling of animals with nonresponsive diseases (including animals with compatible signs of Johne's disease in herd 1) or low productive or reproductive performance was done permanently. The remaining herd (herd 5) had a cow with weight loss and nonresponsive diarrhea compatible with paratuberculosis, but did not have a history of Johne's disease or a previous diagnosis of Map.
From the herds tested, only herd 2 has purchased animals before the first study in 2007 and between both studies. Herd 5 has purchased animals in the last two years before the sampling of the present study. Between herd 3 and herd 4, which belong to the same farmer, cattle exchange occurs usually. All herds raise their own replacement heifers (Table 1).
2.2. Collection of Samples
Serum and fecal samples were taken from all adult dairy cows (≥2 years) in every herd. In herd 2, 110 cows were sampled for feces, but only 53 of them were sampled for serum due to reluctance of farmer to sample all animals. In one herd (herd 1) that had slurry pit collecting liquid manure and wastewater from the herd's milking parlor, slurry samples were additionally taken from three different places of the pit. From one animal of the same herd (herd 1), a section of thick intestine (colon) and a mesenteric lymph node were obtained after euthanasia and necropsy due to advanced clinical symptoms compatible with Johne's disease. Information about age was collected from all animals with exception of six animals, from which farmers did not have available data at the moment of sampling.
Serum samples () were tested with an ELISA test based on detection of antibodies to Map extract (ID Screen Paratuberculosis Indirect, IDVET, Montpellier, France) (ELISA-C). This test included a pre-absorption step with M. phlei. ELISA-C was not carried out in duplicate due to economical reasons. A herd was considered positive if at least one animal tested positive by ELISA-C.
2.4. Bacteriological Culture
Fecal samples () were examined on the basis of a strategic pooling procedure. Fecal samples were sorted on the basis of birth order of the animals, and 2 g of feces from each cow was mixed at the laboratory into pooled fecal samples of 8–12 cows per pool. After this, 3 g of the pooled fecal sample was decontaminated with 0.75% (w/v) Hexadecylpyridinium Chloride solution (0.75% HPC) for 24 h, according to standard procedures [12]. Briefly, 3 g of feces were added to a 50 mL sterile tube (Sarstedt, Nümbrecht, Germany) containing 30 mL of a 0.75% HPC. This suspension was manually mixed by shaking and vortexting and let in vertical position for 5 min at room temperature to allow the precipitation and sedimentation of big particles. Approximately 20 mL of the upper portion of the supernatant was transfer to another 50 mL sterile tube in which the whole suspension was agitated for 30 min by 200 U/min. Tubes were placed in vertical position in the dark for 24 h at room temperature. Decontaminated pooled fecal samples were centrifuged at 900 ×g during 30 min, supernatant was discarded, and two Herrold's Egg Yolk Agar medium (HEYM) slants, supplemented with mycobactin J (Prepared Culture Media, Becton Dickinson, Heidelberg, Germany) were inoculated with 300 μL of the decontaminated pellet [12]. The slants were incubated at 37°C for a maximum of 20 weeks and checked at 1-2-week intervals.
Slurry samples were also pooled, decontaminated, and inoculated as described above for fecal samples. If HEYM slants inoculated with pooled fecal or slurry samples showed mycobacterial growth, single fecal and slurry samples were cultured individually. The individual samples from a negative pool were assumed negative and not tested individually, except for the fecal samples from ELISA-C-positive animals of herd 2, which were cultured individually regardless of their culture results.
Tissue samples (colon and mesenteric lymph node) were prepared, decontaminated, and inoculated in duplicate onto HEYM slants [12]. Briefly, the colon tissue was cut open, and the mesenteric lymph node was released from adipose tissue. Both samples were cut up separately, and approximately 1 g of the respective tissue material was put in a stomacher bag with 7 mL of 0.9% (w/v) Hexadecylpyridinium Chloride solution (0.9% HPC) and was homogenized for 6 min in the stomacher. The homogenized tissue was put in a 50 mL sterile tube (Sarstedt, Nümbrecht, Germany) and shaken at room temperature, 200 U/min for 5–10 min. After that, tubes were place in vertical position in the dark for 24 h at room temperature. After decontamination, the tubes were centrifuged at 1880 ×g, at 20°C for 20 min. The supernatant was discarded, and the sediment was resuspended in PBS-Buffer pH 7.2 and vortexted. Finally, two HEYM slants, supplemented with mycobactin J (Prepared Culture Media, Becton Dickinson, Heidelberg, Germany) were inoculated with 300 μL of the decontaminated pellet. As done with fecal samples, slants were incubated at 37°C for maximum 20 weeks and checked at 1-2-week intervals for mycobacterial growth or contamination. Contamination rate was estimated in 8.3% (3/36) for the fecal and the slurry pooled samples, and 3.7% (1/27) for the individual fecal samples (including fecal cultures from ELISA-C-positive animals of herd 2) and the tissue samples. In all contaminated samples, only one slant of the duplicate was affected. In case of mycobacterial growth, Map was confirmed by the real-time PCR method described as follows.
2.5. Polymerase Chain Reaction (PCR)
PCR was carried out only on individual fecal and slurry samples that were part of positive fecal and slurry pooled samples by culture, and to fecal samples of four positive ELISA-C animals of herd 2 () (Figure 1). DNA isolation from fecal and slurry samples was carried out using a commercial DNA preparation kit (High Pure PCR Template Preparation Kit, Roche, Mannheim, Germany). Briefly, 1.5 g of bovine feces was put in a 15 mL sterile, nonpyrogenic centrifuge tube (Sarstedt, Nümbrecht, Germany). Five mL of a buffer for stabilization (Stool Transport and Recovery-S.T.A.R. buffer, Roche, Mannheim, Germany) was added to fecal sample and homogenized. This suspension was subsequently centrifuged for 1 min by 1000 ×g and 1 mL of the supernatant was put in a 2 mL conical sample tubes (Biozym Scientific, Hess. Oldendorf, Germany) containing ceramic beads, size range 1.4–1.6 mm, Genotype ZY (Zirkonoxid-Beads, Yttrium stabilized) (Sigmund Lidner, Warmensteinach, Germany). A mechanical cell disruption step was carried out in an automated biological sample lyser (Precellys 24, Bertin technologies, Montigny-le-Bretonneux, France) to achieve efficient cell lysis. The mixture was subsequently incubated at 95°C for 10 m and centrifuged 5 min by 5000 ×g. Two hundred microliters of the supernatant was added to a 1.5 mL reaction tube containing 5 μL of lysozyme (Merck, Darmstadt, Germany) solution. Further processing was done according to kit's protocol for isolation of nucleic acids from bacteria and yeast. DNA isolation was always carried out in duplicate.
DNA isolation from bacteria for Map confirmation and molecular characterization was carried out using a commercial preparation kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany). This preparation included overnight lysis buffer incubation at 37°C, proteinase K/AL-buffer incubation for 90 min at 56°C, and final incubation for 15 min at 95°C, as a modification of the protocol of the commercial kit. DNA from fecal samples and from bacterial culture was tested in duplicate for Map with the real-time PCR method targeted to F57 and ISMav2 described by Schönenbrücher et al. [13]. Samples were also tested in duplicate with the nested-PCR targeted to IS900 described by Bull et al. [14]. Additional to the samples, a positive and a negative preparation control, as well as a blank control were included. In the PCR system, a positive Map control (DNA of a positive Map strain), a non-Map negative control (DNA of a non-Map mycobacteria), and a master-mix blank control were also included. The real-time PCR method also included an internal amplification control (IAC) to avoid the misinterpretation of false negative results [13].
2.6. Molecular Characterization
For molecular characterization of the Map strains isolated, a combination of two different genotyping methods based on PCR amplification of repetitive elements of Map genome was applied. The Multilocus Short Sequence Repeat (MLSSR) analysis was carried out by amplification of the short sequence repeats (SSRs) found in locus 1, 2, 8, and 9 according to primers and PCR conditions reported by Amonsin et al. [15]. The final PCR reaction volume (30 μL) contained GeneAmp 10x PCR Puffer (Applied Biosystems, Darmstadt, Germany), dNTP-Mix (10 μM each) (Roche, Mannheim, Germany), 0.2 μM of each primer (Eurofins MWG, Martinsried, Germany), 10% Dimethil Sulfoxide (DMSO) (Roth, Karlsruhe, Germany), 1 U of AmpliTaq Gold Polymerase 5 U/μL (Applied Biosystems, Langen, Germany), and 3 μL of DNA. A master mixture blank (without DNA) was included as control in every PCR reaction. Seven microliters of every PCR product were mixed with 2 μL of loading buffer, and electrophoresed in a 1.5% agarose gel. All amplicons in every SSR locus were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and sequenced by an independent laboratory (Sequence Laboratories, Göttingen, Germany). MLSSR genotypes were expressed as the combination of the number of repeats found in the four loci amplified by PCR.
The Mycobacterial Interspersed Repetitive Units (MIRU) loci MIRU-1, MIRU-2, MIRU-3, and MIRU-4 were selected to analyze the Map isolates according to Bull et al. [16]. The Variable Number of Tandem Repeats (VNTRs) loci VNTR-292, VNTR-1658 (alias X3), VNTR-25, VNTR-47, VNTR-3, VNTR-7, VNTR-10, VNTR-32, and VNTR-259 were selected to analyze the Map isolates according to Overduin et al. [17], Thibault et al. [18], and Castellanos et al. [19]. For all loci, primers used were those suggested by the authors mentioned above. Except for the PCR conditions of MIRU-1, VNTR-7, and VNTR-10 carried out according to Möbius et al. [20], and of VNTR-25 and VNTR-47 according to Castellanos et al. [19]. The final reaction volume of PCR (30 μL) for MIRU-VNTR was the same as described for MLSSR. However, for the PCR amplification of VNTR-32, 5 μL of Betain (Sigma-Aldrich, Schenelldorf, Germany) was additionally added to the mix as suggested by Thibault et al. [18].
Calculation of the number of repeats per locus for MIRU-VNTR was initially performed according to the size of the amplicon determined by electrophoresis in 1.5% agarose gel. Additionally, the amplicons of representative alleles in every locus were purified using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and sequenced by an independent laboratory (Sequence Laboratories, Göttingen, Germany). MIRU and VNTR genotypes were confirmed by the number of repeat units and the number of tandem repeats in selected sequences. MIRU-VNTR genotypes were expressed as the combination of the number of repeats found in every locus in the order MIRU-1, MIRU-2, MIRU-3, MIRU-4, VNTR-292, VNTR-1658 (alias X3), VNTR-25, VNTR-47, VNTR-3, VNTR-7, VNTR-10, VNTR-32, and VNTR-259. The INRA Nouzilly MIRU-VNTR (INMV) nomenclature as defined by Thibault et al. [18] was taken into account for ease comparison with previous studies. For this purpose only the results of loci VNTR-292, VNTR-1658, VNTR-25, VNTR-47, VNTR-3, VNTR-7, VNTR-10, and VNTR-32 were considered.
2.7. Data Analysis
The descriptive analysis of age, the estimation of standard deviation (SD), and the determination of confidence intervals 95% (95% CI) were carried out using the program packages BMPD release 8.1 (Berkeley, USA) and BIAS release 8.2 (Hochheim-Darmstadt, Germany). The estimation of the testing agreement between ELISA and culture (Cohen's kappa () coefficient) was done with the program Win-Episcope 1.0 (Zaragoza, Spain). True prevalence was estimated based on the apparent prevalence obtained by ELISA-C using sensitivity (42%), and specificity (99%) values determined previously on asymptomatic infected animals [21]. The relation age versus ELISA results was analyzed descriptively, according to age classes defined arbitrarily.
3. Results
ELISA-C produced positive results in 1.8% (6/329) (95% C.I.; 0.7–3.9%), negative results in 97.5% (321/329), and doubtful results in 0.6% (2/329) of the serum samples examined, as well as positive results in 40% (2/5) of the herds. Of the six positive ELISA-C samples detected, two were detected in herd 1 and four were detected in herd 2 (Tables 2 and 3). The true Map-prevalence based on ELISA-C-apparent prevalence, sensitivity (42%) and specificity (99%) was 2.2%.
The age of the animals sampled ranged between 2.2 and 14 years (mean 5.9, SD 2.8). Analysis of age of animals versus ELISA-C result (positive, negative, doubtful) revealed that the group of >11 years of age, was the group in which the highest proportion (6.3%, 1 out of 16) of ELISA-positive samples were produced (Table 4). However, it was in the group of 5.1–8 years of age in which the highest absolute number of ELISA-positive animals () of the whole study was found. In the group of the youngest cows (>2.2–2.9 years of age), no positive result by ELISA was produced. In the group of 3–5, 5.1–8, and 8.1–10.9 years, 0.8%, 2.9%, and 1.9% of the samples produced positive results, respectively (Table 4).
3.2. Bacteriological Culture and Polymerase Chain Reaction
The strategic pooling procedure for fecal samples from the five herds resulted in 36 pools, including the slurry pool prepared from herd 1, which had a slurry pit collecting liquid manure and wastewater from the herd's milking parlor. Two pools from herd 1 out of 36 pools analyzed produced positive results by culture after 5-6 weeks of incubation with >50 Colony Forming Units (CFU)/tube. The slurry pool produced positive results by culture after 17 weeks of incubation with <10 CFU/tube. Isolates obtained from pooled samples were confirmed as Map by the real-time PCR method described above. Remaining pools of herds 2, 3, 4, and 5 did not show mycobacterial growth by culture in 20 weeks of incubation.
Fecal samples that were a part of the two positive pools of herd 1 () produced four positive results by individual culture (Table 3). All isolates showed no pigmentation and were confirmed as Map by real-time PCR. Two cows (both ELISA-C-negative, PCR-negative, asymptomatic, 7.1 years old) were low shedders (<10 CFU/tube), one cow (ELISA-positive, PCR-positive, symptomatic, 6 years old) was a heavy shedder, and one cow (ELISA-negative, PCR-positive, asymptomatic, 9.5 years old) was a heavy shedder (>50 CFU/tube) (Table 3). The cow 3 from pool 1 (herd 1) also produced positive results by mesenteric lymph node and colon tissue culture (Tables 3 and 5). On HEYM-slants inoculated with mesenteric lymph node tissues, visible colonies grew before 16 weeks of incubation, while in those inoculated with colon tissues no visible Map colonies grew in this period of time. Four fecal samples from positive ELISA-C animals of herd 2 produced negative results by culture and PCR; these samples were all from different pooled fecal samples (Table 3). Surprisingly, although the pooled slurry sample produced positive results by culture and PCR, their individual samples () were negative by PCR, and by culture after 20 weeks of incubation (Table 3).
ELISA-C results were confirmed by culture in only one symptomatic animal (cow 3) of herd 1. Thus, ELISA-C, culture, and PCR only agreed on one single animal out of four animals that delivered positive culture results. According to these results, calculated agreement between ELISA-C and culture was poor (, 95% C.I. 0.09–0.29). ELISA-C did not detect Map antibodies in three serum samples from asymptomatic animals of herd 1 (Cow 1 and Cow 2 from pool 1, and Cow 4 from pool 2) that produced positive results by fecal culture. In fecal samples from two of these three animals, Map was also not detected by PCR. In one case, a cow (cow 4 of pool 1 from herd 1) produced negative results by ELISA-C, but positive results by PCR and culture (Table 3).
3.3. Comparison of Results 2007–2009
Based on results of a previous study [10], it was determined that some animals () tested in 2007 were tested again by ELISA, PCR and culture in this study (2009). None of these animals have presented symptoms of paratuberculosis before 2007 or between the two studies. Results can be classified in five different categories, in which only positive findings are described, meaning that other tests produced negative results. In the category 1, one single animal produced positive results by ELISA-A and real-time PCR (molecular target F57) in 2007 and ELISA-C-positive results in 2009. In the category 2, one single animal produced positive results in ELISA A, PCR and real-time PCR (molecular target ISMav2) in 2007. In the category 3, two animals produced ELISA-A and ELISA-B positive results in 2007. In the category 4, one single animal produced positive results by ELISA-A and PCR in 2007. Finally, in the category 5, six animals produced positive results by ELISA-A (Table 6).
3.4. Molecular Characterization
In total eight Map isolates were recovered. Four isolates were from fecal samples, one from mesenteric lymph node, one from colon tissue sample, and two from pooled slurry samples (Table 5). All isolates were confirmed as Map by the real-time PCR method described above. All isolates were obtained from samples from herd 1. All isolates grew within 6–16 weeks of incubation, except from samples of slurry and colon tissue, which grew after the 16th week.
The four isolates obtained from fecal samples, the isolate obtained from colon tissue, and one of the two isolates obtained from slurry sample were of the MLSSR genotype 7g-10g-4ggt-5tgc (hereafter MLSSR-genotype A). The isolates obtained from mesenteric lymph node and the remaining isolate obtained from slurry sample were of the MLSSR genotype 7g-10g-5ggt-4tgc (hereafter MLSSR-genotype B). Similarly, the combination of MIRU-VNTR showed two different MIRU-VNTR genotypes, genotype 3951-42332228-2 (hereafter MIRU-VNTR genotype 1) and 3751-32332228-2 (hereafter MIRU-VNTR genotype 2). Interestingly, strain types A-1 and B-2 were both identified in cow 4, representing a case of double strain infection.
4. Discussion
To our knowledge, this is the first report of isolation and molecular characterization of Colombian Map-strains from dairy herds. For achievement of this goal, herds with history of Johne's disease (report of cases and/or positive diagnosis) were selected to increase the likelihood of detection and isolation of Map. Despite the importance of the cattle production in Colombia, paratuberculosis has remained relatively uninvestigated, and very limited epidemiological information and data on molecular characterization of Map were available. For about 60 years, some studies have tried to widen the clinical and epidemiological information of paratuberculosis in the country through research on diagnosis, treatment, epidemiology, and molecular biology. Now, a consistent study including a significant cattle population, using different diagnostic tests, and including the molecular characterization of the circulating causal agent is presented. As previously suggested in some studies, ELISA, PCR, and culture were used to increase sensitivity of Map detection, in order to confirm whether herds with history of Johne's disease or Map diagnosis were truly infected [22].
The lower proportion of the current ELISA-C positive results (1.8%) compared to the previous ELISA-A positive results (10.1%) in four of the five herds examined was surprising at first sight, but it is explained by the characteristics of the ELISA tests used in both studies. ELISA-C is an absorbed test using purified Map extract, IgG-conjugate and preincubation with M. phlei, which are characteristics that have been considered of critical influence on the increment of specificity for the serological diagnosis of paratuberculosis [23–26]. Therefore, the use of an absorbed test (ELISA-C) has produced negative or a lower proportion of positive results in herds with previous Map diagnosis (ELISA-A and PCR), or even with previous history of clinical cases of paratuberculosis (e.g., herd 1), compared to the previous study of 2007. In this previous study, herds of a dairy region mostly without previous diagnosis of paratuberculosis were tested using an unabsorbed test that used LAM as Map antigen (ELISA-A), which produced a higher proportion of seropositives confirmed only in two animals by an absorbed ELISA (ELISA-B). Interestingly, the results of both absorbed tests, ELISA-B in 2007 and ELISA-C in 2009, produced closer results (5.1% versus 1.8%) than those obtained with the unabsorbed ELISA-A (10.1%) in 2007. This suggests that the characteristics of the tests used were determinant in the different proportions of seropositives obtained in both studies.
Furthermore, the absence of reliable preliminary epidemiological information on the disease makes also plausible that dairy herds in the region of study were of a very low prevalence or even negative for Map, or at least undetectable with the current diagnostic tests, if only cross-sectional studies instead of a longitudinal study or serial testing is carried out. In any case, these studies are the first step of the systematic epidemiological study of paratuberculosis in Colombia, and therefore further studies have to be conducted to elucidate the situation of the disease in the country.
The case of herd number 2, in which clinical paratuberculosis has never been reported, but some animals were positive by ELISA-A and PCR in 2007, and again in 2009 (four positive ELISA-C, but negative results by culture) is striking and difficult to explain. In this case, it could be possible that other mycobacteria could influence the positive results of the unabsorbed ELISA-A in 2007 and of the ELISA-C positive in 2009, making the proportion of positive animals higher than it really is [27]. In the study of 2007, atypical mycobacteria (Mycobacterium engbaekii) were isolated [10]. However, the lack of testing of the half of the adult cattle population of this herd for ELISA-C limits any definitive conclusion about the current negative PCR and culture results obtained from seropositive animals, taking into account the high quality of the tests employed. Additionally, the four fecal samples from the positive ELISA-C animals were cultured individually, which could have been reduced the chances of a probable concentration of low quantities of Map, if these animals were really shedders. Likewise, it is possible that the positive ELISA results, not only in herd 2, but all herds of both studies have been produced due to the interference with tuberculin from intradermal tests (caudal fold tuberculin test) [28] applied occasionally in order to be certified as free herd from bovine tuberculosis in frame of the national program for eradication of tuberculosis. In conclusion, some of these results of ELISA-A and even ELISA-C could have been simply false positives in 2007 and in 2009, respectively.
The low apparent prevalence and the true prevalence obtained (1.8 versus 2.2%) was probably related to the high specificity of test used (99%), as previously reported for studies with these characteristics [29]. Although our study was biased for prevalence determination, due to analysis of herds with previous history or diagnosis of paratuberculosis, animal level apparent prevalence calculated appeared to be lower compared to prevalences obtained in European countries [30]. However, no similar studies were found aiming at the determination of prevalence of Map infection by using the ELISA-C, which made impossible a better comparison of results.
In this study, only 0.8% of ELISA-C-positive cows were detected in the group of 3–5 years and the only symptomatic animal found was a 6- year- old cow. This results slightly disagree with a report of a higher probability of testing positive by ELISA between 2.5 and 5.5 years in infected animals [31] and with the knowledge that most clinical cases of paratuberculosis occur between 3–5 years [32]. Cattle in Colombia tend to be kept longer in production and to be culled later compared to North American or European countries. In this manner, cows can live long enough to be tested and detected by ELISA or fecal culture, or to show symptoms of paratuberculosis out of the age limits reported for other countries.
As reported before, culture of pooled fecal samples of 8–12 animals per pool permitted the examination of a high number of fecal samples by culture at low cost and with acceptable sensitivity [6, 33, 34]. The option of pooling 5 fecal samples instead of 10 was not considered due to economic reasons. Furthermore, precise information about Map within-herd prevalence was absence to take a better decision of the best pool size according to a previous modeling study [35]. This option was also discarded because of the reported insignificant difference in sensitivity between pooling 5 or pooling 10 cows in a previous study in a comparable South American cattle production system, in which it was concluded that the sensitivity of the pool is related more to the prevalence of the herd and to the infection status of the cows as with the size of the pool [6]. In the same way, a study in the United States reported acceptable sensitivity with 10 samples per pool (35%), compared to pooled samples of 5 animals (44%), leading to the conclusion that in herds with at least one high fecal shedder, pools of more than five samples might also detect Map [36]. Although some studies have concluded a better sensitivity of pooling five animals instead of 10 or more, these studies have been based on the detection of Map using radiometric fecal cultures to reliably detect low-shedders, which could be not comparable to classical bacteriological methods [37]. Other studies refer more to a theoretical calculation than to a sensitivity estimation, difficult to extrapolate to South American field conditions [35], or reported the use of five samples per pool focusing on the determination of the sensitivity of culture of pooled fecal samples compared with culture of individual fecal samples, with special attention to the number of pooled fecal samples per herd, rather than to the number of animals to be included in the pooled fecal samples [36].
The detection of a positive pooled slurry sample by culture from a positive ELISA-C herd (herd 1) agrees with the knowledge of the correlation of this finding with seropositive results, and with the higher probability of isolation from lagoon samples compared to other environmental samples [7]. The result of the single slurry samples producing negative results by individual culture and PCR has been reported and has been attributed to uneven distribution of Map in the fecal sample [6], to the lack of homogeneity in the fecal sample or to different sensitivities of individual fecal culture procedure between laboratories [34], to the presence of Map in the feces of at least one animal within the pooled fecal sample, although this animal was not detected by bacteriological culture of individual fecal samples [38], or to unclear reasons [36]. In any case, Kalis et al. properly concluded that there is an element of chance apart from the element of dilution related to the detection of Map in feces, particularly when samples contain low numbers of the organism, and the bacteria are not uniformly distributed in the fecal samples [33]. Thus, although a complete homogenization of the pooled slurry sample was achieved and the PCR systems used are very reliable, it is possible that the 3 g or 1.5 g of slurry samples taken to test by individual culture and by PCR, respectively, lacked enough Map cells to be detected by bacteriological culture in a 20-week period of incubation, and in two PCR systems (F57-IsMav2-real-time and IS900-nested-PCR) carried out in duplicate. The PCR systems used, specially the real-time PCR, are strict tested for specificity, included an IAC and use multiple reaction controls, which avoid the misinterpretations of results due to disturbed contamination or very improbable false positive results [13].
Close analysis of the individual results obtained in herd 1 revealed that one single symptomatic animal producing positive results by ELISA-C and PCR, confirmed that regardless of the ELISA or PCR type used sensitivity is higher for detection of symptomatic animals and fecal high shedders [21, 39]. The results of three asymptomatic ELISA-C-negative cows that produced positive results by culture could be: two cases (Map low-shedders and PCR-negative) of the known "passing through" phenomenon previously described [40], and one case (Map high-shedder and PCR-positive) of a positive animal with undetectable antibodies. On the other hand, the results of one ELISA-C positive-animal of herd 1, that produced negative results by culture, does not necessarily mean that the animal was not really infected, but that the shedding phase has probably not yet started (infected animal in a noninfectious phase) or was absent at the moment of fecal sampling (intermittency). Another possibility is that in this animal Map-antibodies have been detected prior to the start of bacterial shedding, which could begin later and could be then detected by PCR or culture [41]. Map is shed in feces of infected animals at all stages but at different levels and sporadically, which demands repeated testing to detect animals shedding very low number of Map, which could anyway go undetected [5].
One ELISA-C-negative animal was positive by PCR, real-time PCR, and culture. On the contrary, one ELISA-C-positive animal in the same herd showed negative results by PCR, real-time PCR, and culture. In herd 2 four ELISA-C-positive animals produced negative results by fecal PCR and fecal real-time PCR, as well as negative results by individual fecal culture. Muskens et al. found a low percentage of ELISA-positive cattle testing fecal culture-positive for all age groups included. Among their arguments, they stated a possible limited sensitivity of the fecal culture and/or false-positive ELISA test results and a nonhomogeneous distribution of Map in feces especially for low shedders [42]. In general, explanations for the poor concordance of diagnostic tests could be attributed to false-positive ELISA results, to nonhomogeneous distribution of Map in feces (especially for low shedders), to relatively low prevalence of Map infection, and very low positive predictive value of ELISAs applied. In addition, it has to be taken into account that not only the combination of different tests, but repeated sampling is necessary to achieve the identification of individual animals [5].
Although many animals sampled in a previous study (2007) were no longer in the herds at the time of the second sampling (2009), it was an interesting finding to compare the diagnostic results of animals sampled in 2007 with those results obtained from the same animals in 2009, simulating a longitudinal study or repeating testing for these animals. Changes in our diagnostic test results between 2007 and 2009 agree with studies that report fluctuations of serum ELISA, PCR, and culture results overtime [43–45]. Many test factors (sensitivity, specificity, within-herd prevalence of herd) in every diagnostic procedure influence the variability of results, when the same animals are tested more than once overtime. Particularly for ELISA, fluctuations in test results have been attributable to false-positive results on the first or on the second test, fluctuation in antibody production by the cow, application of tests to low prevalence herds, in which the positive predictive value of tests is lower, or to analytic error. Analytic error occurs when samples were not tested in duplicate as suggested by manufacturers, and repeat analysis gives negative results [43]. Nevertheless multiple testing over time increases the chance of detection of an infected animal, this would also increase the chances of a false-positive result [43]. Therefore, ELISA results have to be analyzed carefully when this test is applied for individual animal diagnosis [21, 46]. However, this is not an uniform process because, as it has been reported, cows with negative results are less likely to change ELISA status than cows with positive results, regardless of within-herd prevalence [44].
Phenotypic characteristics of fast growth, mycobactin dependency, and no pigmentation of Colombian Map isolates coincide with the description of type II (or cattle type) strains described in previous studies [47]. The combination of MIRU-VNTR and MLSSR, as done previously [48] made possible the reliable differentiation for the first time of two Map genotypes among eight different Map isolates of one herd in Colombia. These methods were applied combined to increase the minimum discriminatory ability needed and not reached if one single method had been used, as reported before by Stevenson et al. 2009 [49]. According to MLSSR, the types isolated in our study are commonly found in cattle and other species in different countries [48, 50–52]. Interestingly, a bovine isolate from Colombia's neighbor country Venezuela has shown a different genotype (11g-10g-5ggt-5ggt), suggesting strain diversity in the northern part of the subcontinent [48].
Although comparison with other studies is very difficult because of the use of different loci for analysis, genotype 1 (INMV1) and genotype 2 (INMV 2) were previously reported as the most common genotypes found in isolates from Argentina and Venezuela [18], and in European isolates [49, 53]. Cases of double strain infection has been also reported at herd level in the United States [54], Germany [20], and the Netherlands [55], while cases of double strain infection at animal level have been reported in Germany [51].
The finding of two strain types among eight isolates recovered from herd 1, including isolates from four cows all born in the herd, but unrelated each other, and isolates of slurry samples of the slurry pit collecting liquid manure and wastewater from the herd's milking parlor, suggests the circulation of Map from and to the environment, and among different animals in the herd. In the same way, the isolation of two different types in one single animal, types that were also isolated in slurry samples, supports the idea of a highly Map contaminated environment, which leads to the infection with more than one different strain genotype in the herd. In herd 1, animal feces are used as fertilizer on the pastures and no paratuberculosis control program is carried out. It has been presenting sporadic cases of animals with symptoms of paratuberculosis confirmed by histopathology (unpublished data), and Map has been detected by PCR and serology [10, 11]. Shedding cows were relatively old cows (≥6 years) at the time of sampling suggesting that these animals have been contaminating the environment with Map until they are removed from the herd, contributing to the perpetuation of Map and the presentation of new infections, if no control program is established.
Regarding technical considerations of the genotyping methods, we agree that MLSSR could be less accessible and more expensive than MIRU-VNTR due to the sequencing step required [48]. This aspect could represent a limitation in some developing countries (e.g., Colombia) in which sometimes sequencing has to be carried out abroad incrementing even more the costs of application of MLSSR method. However, we agree that MLSSR analysis is an excellent Map molecular characterization method in terms of in vitro stability and discriminatory index [54], which could justify the cost of the sequencing step needed.
The results of this study confirm the presence and suggest the circulation and transmission of different Map strains types between individuals of the infected herd. In addition, the study confirmed the limitations of current tests for individual diagnosis of subclinical Map infections in cattle, and the usefulness of pooled fecal samples and environmental sampling to screen herds for Map.
The authors acknowledge the ALECOL Program (DAAD, Universidad de Antioquia, Colciencias-MEN-Icetex) for financial support; M. Fischer, C. Walter, K. Simon, Ö. Akineden, and K. Failing of the Universität Giessen; J. E. Pérez, C. Correa, O. Arroyave, M. Olivera, M. Palacio, M. Zapata, R. Ramírez, J. G. Maldonado, and collaborating students of the Universidad de Antioquia; M. Suárez of the Universidad Nacional de Colombia, for technical and logistical support; Departamento de Formación Académica de Haciendas of the Universidad de Antioquia; participating farmers for their cooperation; and IDVET innovative diagnostics for the donation of the ELISA-C test kit.
- R. W. Sweeney, "Transmission of paratuberculosis," The Veterinary clinics of North America. Food Animal Practice, vol. 12, no. 2, pp. 305–312, 1996. View at Google Scholar · View at Scopus
- "Possible links between Crohn's disease and paratuberculosis.," Report of the Scientific Comitee on Animal Health and Welfare, pp. 2–76, European Comission, Brussels, Belgium, 2000.
- D. L. Clark Jr., J. J. Koziczkowski, R. P. Radcliff, R. A. Carlson, and J. L. E. Ellingson, "Detection of Mycobacterium avium subspecies paratuberculosis: comparing fecal culture versus serum enzyme-linked immunosorbent assay and direct fecal polymerase chain reaction," Journal of Dairy Science, vol. 91, no. 7, pp. 2620–2627, 2008. View at Publisher · View at Google Scholar · View at Scopus
- S. S. Nielsen and N. Toft, "Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-γ assay and faecal culture techniques," Veterinary Microbiology, vol. 129, no. 3-4, pp. 217–235, 2008. View at Publisher · View at Google Scholar · View at Scopus
- K. Stevenson, "Diagnosis of Johne's disease: current limitations and perspectives," Cattle Practice, vol. 18, no. 2, pp. 104–109, 2010. View at Google Scholar
- G. van Schaik, M. Pradenas, A. Mella, and J. Kruze, "Diagnostic validity and costs of pooled fecal samples and individual blood or fecal samples to determine the cow- and herd-status for Mycobacterium avium subsp. paratuberculosis," Preventive Veterinary Medicine, vol. 82, no. 1-2, pp. 159–165, 2007. View at Publisher · View at Google Scholar · View at Scopus
- R. D. Berghaus, T. B. Farver, R. J. Anderson, C. C. Jaravata, and I. A. Gardner, "Environmental sampling for detection of Mycobacterium avium ssp. paratuberculosis on large California dairies," Journal of Dairy Science, vol. 89, no. 3, pp. 963–970, 2006. View at Google Scholar · View at Scopus
- M. T. Collins, I. A. Gardner, F. B. Garry, A. J. Roussel, and S. J. Wells, "Consensus recommendations on diagnostic testing for the detection of paratuberculosis in cattle in the United States," Journal of the American Veterinary Medical Association, vol. 229, no. 12, pp. 1912–1919, 2006. View at Publisher · View at Google Scholar · View at Scopus
- A. S. Motiwala, L. Li, V. Kapur, and S. Sreevatsan, "Current understanding of the genetic diversity of Mycobacterium avium subsp. paratuberculosis," Microbes and Infection, vol. 8, no. 5, pp. 1406–1418, 2006. View at Publisher · View at Google Scholar · View at Scopus
- J. A. Fernández-Silva, A. Abdulmawjood, O. Akineden, and M. Bülte, "Serological and molecular detection of Mycobacterium avium subsp. paratuberculosis in cattle of dairy herds in Colombia," Tropical Animal Health and Production. In press. View at Publisher · View at Google Scholar
- M. M. Zapata, O. Arroyave, R. Ramírez, C. Piedrahita, J. D. Rodas, and J. G. Maldonado, "Identification of Mycobacterium avium subspecies paratuberculosis by PCR techniques and establishment of control programs for bovine paratuberculosis in dairy herds," Revista Colombiana de Ciencias Pecuarias, vol. 23, pp. 17–27, 2010. View at Google Scholar
- "Mycobacterium avium subsp. paratuberculosis," Working Group on diagnosis of infections in veterinary medicine (AVID) of the German Veterinary Society (DVG), 2007, http://www.dvg.net/fileadmin/Bilder/PDF_AVID_Alt/methoden/AB_AVID-Methode_Paratb.pdf.
- H. Schönenbrücher, A. Abdulmawjood, K. Failing, and M. Bülte, "New triplex real-time PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces," Applied and Environmental Microbiology, vol. 74, no. 9, pp. 2751–2758, 2008. View at Publisher · View at Google Scholar · View at Scopus
- T. J. Bull, E. J. McMinn, K. Sidi-Boumedine et al., "Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease," Journal of Clinical Microbiology, vol. 41, no. 7, pp. 2915–2923, 2003. View at Publisher · View at Google Scholar · View at Scopus
- A. Amonsin, L. L. Li, Q. Zhang et al., "Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis Strains," Journal of Clinical Microbiology, vol. 42, no. 4, pp. 1694–1702, 2004. View at Publisher · View at Google Scholar · View at Scopus
- T. J. Bull, K. Sidi-Boumedine, E. J. McMinn, K. Stevenson, R. Pickup, and J. Hermon-Taylor, "Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex," Molecular and Cellular Probes, vol. 17, no. 4, pp. 157–164, 2003. View at Publisher · View at Google Scholar · View at Scopus
- P. Overduin, L. Schouls, P. Roholl et al., "Use of multilocus variable-number tandem-repeat analysis for typing Mycobacterium avium subsp. paratuberculosis," Journal of Clinical Microbiology, vol. 42, no. 11, pp. 5022–5028, 2004. View at Publisher · View at Google Scholar · View at Scopus
- V. C. Thibault, M. Grayon, M. L. Boschiroli et al., "New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing," Journal of Clinical Microbiology, vol. 45, no. 8, pp. 2404–2410, 2007. View at Publisher · View at Google Scholar · View at Scopus
- E. Castellanos, B. Romero, S. Rodríguez et al., "Molecular characterization of Mycobacterium avium subspecies paratuberculosis types II and III isolates by a combination of MIRU-VNTR loci," Veterinary Microbiology, vol. 144, no. 1-2, pp. 118–126, 2010. View at Publisher · View at Google Scholar · View at Scopus
- P. Möbius, G. Luyven, H. Hotzel, and H. Köhler, "High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing," Journal of Clinical Microbiology, vol. 46, no. 3, pp. 972–981, 2008. View at Publisher · View at Google Scholar · View at Scopus
- H. Köhler, B. Burkert, I. Pavlik et al., "Evaluation of five ELISA test kits for the measurement of antibodies against Mycobacterium avium subspecies paratuberculosis in bovine serum," Berliner und Munchener Tierarztliche Wochenschrift, vol. 121, no. 5-6, pp. 203–210, 2008. View at Publisher · View at Google Scholar · View at Scopus
- P. J. Pinedo, D. O. Rae, J. E. Williams, G. A. Donovan, P. Melendez, and C. D. Buergelt, "Association among results of serum ELISA, faecal culture and nested PCR on milk, blood and faeces for the detection of paratuberculosis in dairy cows," Transboundary and Emerging Diseases, vol. 55, no. 2, pp. 125–133, 2008. View at Publisher · View at Google Scholar · View at Scopus
- C. H. J. Kalis, H. W. Barkema, J. W. Hesselink, C. Van Maanen, and M. T. Collins, "Evaluation of two absorbed enzyme-linked immunosorbent assays and a complement fixation test as replacements for fecal culture in the detection of cows shedding Mycobacterium avium subspecies paratuberculosis," Journal of Veterinary Diagnostic Investigation, vol. 14, no. 3, pp. 219–224, 2002. View at Google Scholar · View at Scopus
- E. A. Sugden, K. Stilwell, and A. Michaelides, "A comparison of lipoarabinomannan with other antigens used in absorbed enzyme immunoassays for the serological detection of cattle infected with Mycobacterium paratuberculosis," Journal of Veterinary Diagnostic Investigation, vol. 9, no. 4, pp. 413–417, 1997. View at Google Scholar · View at Scopus
- Y. Yokomizo, H. Yugi, and R. S. Merkal, "A method for avoiding false-positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine paratuberculosis," Nippon Juigaku Zasshi, vol. 47, no. 1, pp. 111–119, 1985. View at Google Scholar · View at Scopus
- U. Jark, I. Ringena, B. Franz, G. F. Gerlach, M. Beyerbach, and B. Franz, "Development of an ELISA technique for serodiagnosis of bovine paratuberculosis," Veterinary Microbiology, vol. 57, no. 2-3, pp. 189–198, 1997. View at Publisher · View at Google Scholar · View at Scopus
- J. B. Osterstock, G. T. Fosgate, B. Norby, E. J. B. Manning, M. T. Collins, and A. J. Roussel, "Contribution of environmental mycobacteria to false-positive serum ELISA results for paratuberculosis," Journal of the American Veterinary Medical Association, vol. 230, no. 6, pp. 896–901, 2007. View at Publisher · View at Google Scholar · View at Scopus
- R. Varges, C. D. Marassi, W. Oelemann, and W. Lilenbaum, "Interference of intradermal tuberculin tests on the serodiagnosis of paratuberculosis in cattle," Research in Veterinary Science, vol. 86, no. 3, pp. 371–372, 2009. View at Publisher · View at Google Scholar · View at Scopus
- S. W. Martin, "Estimating disease prevalence and the interpretation of screening test results," Preventive Veterinary Medicine, vol. 2, no. 1–4, pp. 463–472, 1984. View at Google Scholar · View at Scopus
- S. S. Nielsen and N. Toft, "A review of prevalences of paratuberculosis in farmed animals in Europe," Preventive Veterinary Medicine, vol. 88, no. 1, pp. 1–14, 2009. View at Publisher · View at Google Scholar · View at Scopus
- S. S. Nielsen and A. K. Ersbøll, "Age at occurrence of Mycobacterium avium subspecies paratuberculosis in naturally infected dairy cows," Journal of Dairy Science, vol. 89, no. 12, pp. 4557–4566, 2006. View at Google Scholar · View at Scopus
- R. J. Chiodini, H. J. Van Kruiningen, and R. S. Merkal, "Ruminant paratuberculosis (Johne's disease): the current status and future prospects," The Cornell Veterinarian, vol. 74, no. 3, pp. 218–262, 1984. View at Google Scholar · View at Scopus
- C. H. J. Kalis, J. W. Hesselink, H. W. Barkema, and M. T. Collins, "Culture of strategically pooled bovine fecal samples as a method to screen herds for paratuberculosis," Journal of Veterinary Diagnostic Investigation, vol. 12, no. 6, pp. 547–551, 2000. View at Google Scholar · View at Scopus
- S. Tavornpanich, I. A. Gardner, R. J. Anderson et al., "Evaluation of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds," American Journal of Veterinary Research, vol. 65, no. 8, pp. 1061–1070, 2004. View at Publisher · View at Google Scholar · View at Scopus
- G. Van Schaik, S. M. Stehman, Y. H. Schukken, C. R. Rossiter, and S. J. Shin, "Pooled fecal culture sampling for Mycobacterium avium subsp. paratuberculosis at different herd sizes and prevalence," Journal of Veterinary Diagnostic Investigation, vol. 15, no. 3, pp. 233–241, 2003. View at Google Scholar · View at Scopus
- S. J. Wells, R. H. Whitlock, C. J. Lindeman, and T. Fyock, "Evaluation of bacteriologic culture of pooled fecal samples for detection of Mycobacterium paratuberculosis," American Journal of Veterinary Research, vol. 63, no. 8, pp. 1207–1211, 2002. View at Google Scholar · View at Scopus
- G. J. Eamens, R. J. Whittington, M. J. Turner, S. L. Austin, S. A. Fell, and I. B. Marsh, "Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle," Veterinary Microbiology, vol. 125, no. 1-2, pp. 22–35, 2007. View at Publisher · View at Google Scholar · View at Scopus
- S. J. Wells, S. M. Godden, C. J. Lindeman, and J. E. Collins, "Evaluation of bacteriologic culture of individual and pooled fecal samples for detection of Mycobacterium paratuberculosis in dairy cattle herds," Journal of the American Veterinary Medical Association, vol. 223, no. 7, pp. 1022–1025, 2003. View at Publisher · View at Google Scholar · View at Scopus
- K. Bögli-Stuber, C. Kohler, G. Seitert et al., "Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by real-time PCR and culture: a comparison of the two assays," Journal of Applied Microbiology, vol. 99, no. 3, pp. 587–597, 2005. View at Publisher · View at Google Scholar · View at Scopus
- R. W. Sweeney, R. H. Whitlock, A. N. Hamir, A. E. Rosenberger, and S. A. Herr, "Isolation of Mycobacterium paratuberculosis after oral inoculation in uninfected cattle," American Journal of Veterinary Research, vol. 53, no. 8, pp. 1312–1314, 1992. View at Google Scholar · View at Scopus
- S. S. Nielsen, "Transitions in diagnostic tests used for detection of Mycobacterium avium subsp. paratuberculosis infections in cattle," Veterinary Microbiology, vol. 132, no. 3-4, pp. 274–282, 2008. View at Publisher · View at Google Scholar · View at Scopus
- J. Muskens, M. H. Mars, A. R. W. Elbers, K. Van Maanen, and D. Bakker, "The results of using faecal culture as confirmation test of paratuberculosis-seropositive dairy cattle," Journal of Veterinary Medicine, Series B, vol. 50, no. 5, pp. 231–234, 2003. View at Publisher · View at Google Scholar
- R. W. Sweeney, R. H. Whitlock, S. McAdams, and T. Fyock, "Longitudinal study of ELISA seroreactivity to Mycobacterium avium subsp. paratuberculosis in infected cattle and culture-negative herd mates," Journal of Veterinary Diagnostic Investigation, vol. 18, no. 1, pp. 2–6, 2006. View at Google Scholar · View at Scopus
- H. L. Hirst, F. B. Garry, and M. D. Salman, "Assessment of test results when using a commercial enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in repeated samples collected from adult dairy cattle," Journal of the American Veterinary Medical Association, vol. 220, no. 11, pp. 1685–1689, 2002. View at Google Scholar · View at Scopus
- E. Geisbauer, J. L. Khol, M. Wassertheurer et al., "Longterm investigation in an Austrian dairy herd with low prevalence of paratuberculosis detection of antibodies in blood and milk," Veterinary Quarterly, vol. 29, no. 4, pp. 138–148, 2007. View at Google Scholar · View at Scopus
- S. L. B. McKenna, H. W. Barkema, G. P. Keefe, and D. C. Sockett, "Agreement between three ELISAs for Mycobacterium avium subsp. paratuberculosis in dairy cattle," Veterinary Microbiology, vol. 114, no. 3-4, pp. 285–291, 2006. View at Publisher · View at Google Scholar · View at Scopus
- K. Stevenson, V. M. Hughes, L. De Juan, N. F. Inglis, F. Wright, and J. M. Sharp, "Molecular characterization of pigmented and nonpigmented isolates of Mycobacterium avium subsp. paratuberculosis," Journal of Clinical Microbiology, vol. 40, no. 5, pp. 1798–1804, 2002. View at Publisher · View at Google Scholar · View at Scopus
- V. C. Thibault, M. Grayon, M. L. Boschiroli et al., "Combined multilocus short-sequence-repeat and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing of Mycobacterium avium subsp. paratuberculosis isolates," Journal of Clinical Microbiology, vol. 46, no. 12, pp. 4091–4094, 2008. View at Publisher · View at Google Scholar · View at Scopus
- K. Stevenson, J. Alvarez, D. Bakker et al., "Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants," BMC Microbiology, vol. 9, article 212, 2009. View at Publisher · View at Google Scholar
- A. H. Ghadiali, M. Strother, S. A. Naser, E. J. B. Manning, and S. Sreevatsan, "Mycobacterium avium subsp. paratuberculosis strains isolated from Crohn's disease patients and animal species exhibit similar polymorphic locus patterns," Journal of Clinical Microbiology, vol. 42, no. 11, pp. 5345–5348, 2004. View at Publisher · View at Google Scholar · View at Scopus
- A. El-Sayed, A. A. Hassan, S. Natour et al., "Evaluation of three molecular methods of repetitive element loci for differentiation of Mycobacterium avium subsp. paratuberculosis (MAP)," Journal of Microbiology, vol. 47, no. 3, pp. 253–259, 2009. View at Publisher · View at Google Scholar · View at Scopus
- J. L. Corn, E. J. B. Manning, S. Sreevatsan, and J. R. Fischer, "Isolation of Mycobacterium avium subsp. paratuberculosis from free-ranging birds and mammals on livestock premises," Applied and Environmental Microbiology, vol. 71, no. 11, pp. 6963–6967, 2005. View at Publisher · View at Google Scholar · View at Scopus
- P. E. Douarre, W. Cashman, J. Buckley, A. Coffey, and J. O'Mahony, "Molecular characterization of Mycobacterium avium subsp. paratuberculosis using multi-locus short sequence repeat (MLSSR) and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) typing methods," Veterinary Microbiology, vol. 149, no. 3-4, pp. 482–487, 2011. View at Publisher · View at Google Scholar
- N. B. Harris, J. B. Payeur, V. Kapur, and S. Sreevatsan, "Short-sequence-repeat analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium isolates collected from animals throughout the United States reveals both stability of loci and extensive diversity," Journal of Clinical Microbiology, vol. 44, no. 8, pp. 2970–2973, 2006. View at Publisher · View at Google Scholar · View at Scopus
- K. J. E. Van Hulzen, H. C. M. Heuven, M. Nielen, J. Hoeboer, W. J. Santema, and A. P. Koets, "Different Mycobacterium avium subsp. paratuberculosis MIRU-VNTR patterns coexist within cattle herds," Veterinary Microbiology, vol. 148, no. 2–4, pp. 419–424, 2011. View at Publisher · View at Google Scholar


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Daily Reload: Iron Maiden, Linkin Park + More
What's up Loudwire peeps? Time for your Daily Reload. Iron Maiden released a new trailer for their upcoming live DVD / documentary 'En Vivo!' and we celebrate the 30th anniversary of 'Number of the Beast' with a special readers poll; Slash confirms that Guns N' Roses will not be performing at the Rock and Roll Hall of Fame induction ceremony; and Disturbed frontman David Draiman announces the guitarist for his industrial project.
Also, Marilyn Manson unveils the cover artwork and track listing for 'Born Villain'; Linkin Park's Chester Bennington promises to return to their "familiar" sound in their next album; Bret Michaels drafts KISS, Pantera, Van Halen + Aerosmith members for his new album; Black Keys half-apologize to Nickelback after poking fun at the band and much more.
Check out all of today's stories on Loudwire, plus some favorites from around the Web, in the links below:
Today on Loudwire:
Marilyn Manson Unveils AlbumCover Art + Track Listing for 'Born Villain'
Slash: Guns N' Roses Not Performing at Rock and Roll Hall of Fame Induction Ceremony
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Disturbed's David Draiman Recruits Ex-Filter Guitarist Geno Lenardo for Industrial Project
Bret Michaels Drafts KISS, Pantera, Van Halen + Aerosmith Members for New Album
Black Keys Drummer Half-Apologizes to Nickelback: 'There's Much Worse Bands'
Iron Maiden Drop Trailer for 'En Vivo!' Steel Book DVD
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subset: commoncrawl


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- afckredietenAsked on November 13, 2015 at 06:50 AM
I use currency validation in a couple off textfields, but apperently this only stands for use of $, how can i use euro or € instead of dollar?
- JotForm UI DevelopermertAnswered on November 13, 2015 at 09:36 AM
Currently, we have currency validator widget called "Masked Input" for $; but unfortunately, it doesn't support €.
If you set the "Properties" for the "Text" field, you can do something similar. You can set the "Size" of the field, change the "Validation" and add a "Default Value" like the image below:
I hope this one will meet your needs. If it is not, please let us know the updates.
- JotForm UI DevelopermertAnswered on November 13, 2015 at 09:44 AM
I also raised your thread as a feature request, so our developers will be learned about this issue. I can't tell you a exact release date;but they will be on it as soon as possible.
- JotForm UI DevelopermertAnswered on November 13, 2015 at 10:07 AM
In addition, if you are planning to use one of the payment tools, you can set the currency as a € in the wizard. By doing this, its currency field works like a currency validator for €.
- afckredietenAnswered on November 13, 2015 at 10:27 AM
ok thanks, i use the validation with default value
Thanks for you're response.
But i doesn't work.
1. in Europe we put the currency symbol behind the number, so how can i do this.
2. i tried exactly as you discribed in youre response above
and this is what i get
It's a car loan calculator annex application form for that calculted loanamount.
So the cost of a car (Kostprijs vht voertuig) needs to be a free field meaning sometimes people will put 5000€ but also 30.000€ for instance.
No i put 10 in default value, and apparently it will not accept different values?
This is the form itself https://form.jotformeu.com/53163025529351
is there a solution for speed issues? i work on a 200MBPS internetline, but i'm having issues building a 6 pages form. sometimes it takes up to a minute before i can do a next item, or it crashes and i need to restart (closing chrome, edge, firefox) and restart.
Also, this form once published is verry slow. Is it because im working from in Europe? would transferring data to the data center in Germany be a solution for the speed issue?
this is my speedtest
Ok, i could work with seperate forms, and use the thank you redirect to the seccond form, but because i need plenty of submissions from the first form in the seccond it"'s easier to work with everything in one form so i can use update from another field, instead of prepolulations, and looking for field id's and so one.
The question i'm having is that now the speed is one thing, but once embedded in my wordpress site (i use a verry fast hosting digital ocean, or amazon) would the speed wouldn't be more slower than now? If so that would be a verry important issue. I need to be sure that the form is reasonable fast, (normal openingspeed).
Are there solutions out there to fix this.
- JotForm SupportKiranAnswered on November 13, 2015 at 12:13 PM
If you are not having any decimal points in the input value, you can have the generic validation by using # for numbers and € sign at the end as shown below :
Since # allows only numbers, user is not allowed to enter any other character other than number. In this case, the validation option can be left to None. Please see if that helps for you and let us know if you need any further assistance. We will be happy to help.
The other question related to speed related issue is moved to a separate thread and shall be addressed there shortly.
- JotForm UI DevelopermertAnswered on November 16, 2015 at 03:24 AM
In addition to what have mentioned earlier, you can set the "Masked Input" widget for € currency. On its setting wizard, you need to change following values:
After this changes, the result will be like the following:
- afckredietenAnswered on November 16, 2015 at 04:37 AM
Right, i completly forgot the input mask widget and the suffix, thanks for the reminder
- JotForm UI DevelopermertAnswered on November 16, 2015 at 04:55 AM
Hi again,
You are kindly welcome. In the beginning, I forgot to do that too; but thanks to TitusN who is the one of the widget developers of JotForm, I realized that we can customize the widget "Mask Input" for € currency, so sorry for the earlier confusing methods.
For further assistance, please let us know.


text:
Lumbini Natural Minral Water
Sunday, June 13, 2010
ASI carry out archaeological excavations
It is said that good thing come in big packet after 150 years of Mahuadabar was razed finally with great effort from Mr. Latif ji and help from ASI. ASI has started excavations of Mahuadabar to find its history. The Exavations work started on 13th June 2010.
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Mohd. Latif Ansari Mobile: +91-9451621458 Tel: 05542-281373
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Monday, 26 December 2016 11:09
Few nations obtain the opportunity to lead the world of nearly two hundred nations of diverse cultures, histories, and economies. After the fall of the Soviet Union in 1991, the United States emerged as the sole superpower with formidable military and economic strengths.
As a well-functioning democracy under which governments changed periodically and peacefully and where constitutional rights were being democratized for previously excluded communities, the United States was proving the thesis that liberty and prosperity can coexist within a decent legal system. Many highly gifted individuals all over the world viewed the United States as a miracle nation and wished to live and work here.
Gradually but steadily, arrogance misguided politicians and policy makers in Washington D.C. The presidency, congress, the military, and think-thanks – all dominated mostly by men intoxicated with power — embarked upon juvenile rhetoric and conduct that insulted diverse cultures, ridiculed world religions, and asserted raw power even in diplomatic circles of the United Nations. In 1995, Speaker of the House Newt Gingrich proclaimed with unmitigated hubris: "We're the only country complicated enough, sophisticated enough, big enough to lead the human race."
The 2001 terrorist attacks on the United States bamboozled the power-drivers in Washington D.C. The fall of the iconic twin towers and an assault on the Pentagon inflicted a national trauma anchored in disbelief, shaking the confidence in progressive American values. "The whole world is under the control of the evil" was the new consensus even among the highly educated secular elites, let alone the ordinary people indoctrinated with militaristic patriotism.
An erratic analysis of the "evil world" forced the United States into a self-squandering trajectory of war mongering. When an empire or a nation begins to fight the world through aggressive means, its tragic end is predictable. Recall how the Soviets invaded, occupied, and bullied Hungary (1956), Czechoslovakia (1968), and Afghanistan (1979), paving the way for its own demise. The U.S. invasions of Afghanistan (2001) and Iraq (2003), though vengeful and projections of power, were manifest errors.
Bombing villages in Afghanistan, destroying neighborhoods in Iraq, wholesale violations of human rights, and faith-based torture committed at Bagram, Abu Gharib, and Guantanamo shattered the morality of a superpower that lectured the whole world about the inherent dignity of human beings. Through inhumane invasions, America was losing its status as a beloved country of liberty and economic freedoms. The city on a hill, as President Ronald Reagan called the United States, turned off its lights.
Congress, more haughty and fitful than the presidency, has embarked on its own visionless path of imposing sanctions on various countries inviting ill-will and hatred of their people. Trade and financial sanctions rarely distinguish between friends and foes. Foes including Cuba, Iran, Russia, Venezuela, and Zimbabwe, and friends including Saudi Arabia, Pakistan, Egypt, and Mexico, all have been subject to various types of sanctions, embargos, and financial constraints. These sanctions have also been hurting the U.S. economy.
The rise of China as an enormous economic power is also quickening the self-squandering of the United States. While the United States armed forces are killing and getting killed in Afghanistan, China is investing in Afghanistan's natural resources. While the United States is drifting away from Pakistan, China is reconstructing the historic silk route with billions of dollars. While the United States is engrossed in military conflicts in Syria and Libya, China is weighing its options to rebuild these worn-torn countries. What the United States did to the Soviet Union, China is doing to the United States.
Having squandered its influence abroad, the United States is now turning on its own domestic future. The rise of Trumpism is more petrifying for the country's future than Trump's fatuous twittering. Trumpism as a phenomenon, though controversial, yearns to revive the "good old days" of a pre-1954 America (Brown v. Board of Education)(a case that outlawed apartheid) when non-white immigrants were fewer in number, when there were no mosques, when American-Japanese were interned, when blacks were under the white thumb, when women had little access to higher education and corporate America, and when there was no social pressure to recognize Spanish as a language.
Under Trumpism, diversity is turning into a dirty word. Ignoring contemporary ethnic complexity of the nation, employment hiring is blatantly reverting to dominant communities already disproportionately represented in corporations, universities, and government. Academic freedom is under attack as teachers and professors are harassed and punished for speaking up against hegemony of the National Rifle Association, for the rights of Palestinians, and for the dignity of Muslim women wearing religious hijab. The lands of Native Americans, whatever is left of them after centuries of theft, are being appropriated for laying fetid oil pipelines.
The promise of a complex and diverse America seems to have been torn apart. The Jeffersonian America of inalienable rights for all, once a cherished ideal, is being publicly mocked. Creatures like Carl Paladino and Bill O'Reilly are reinventing a bigoted America. Ugliness is replacing tolerance. Sentiments for secessions on part of various states, including Texas and California, though mere unpleasant rhetoric at this time, challenge the core integrity of the Union.
At this time of weakness both home and abroad, the slogan of Make America Great Again sounds much like the Siren Song in Homer's Odyssey. It is tragic entrapment for a shipwreck. Students of history know how nations squander to lose their God-gifted competitive military, economic, and moral advantage. The fall of the United States, and its possible dismemberment, will be yet another heart-rending story in human history.
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