Abstract
The objective of this study was to evaluate whether cows that were low shedders of Mycobacterium avium subsp. paratuberculosis were passively shedding or truly infected with M. avium subsp. paratuberculosis. We also investigated whether it is possible that these M. avium subsp. paratuberculosis-infected animals could have been infected as adults by contemporary high-shedding animals (supershedders). The M. avium subsp. paratuberculosis isolates were obtained from a longitudinal study of three dairy herds in the northeastern United States. Isolates were selected from fecal samples and tissues at slaughter from all animals that were culture positive at the same time that supershedders were present in the herds. Shedding levels (CFU of M. avium subsp. paratuberculosis/g of feces) for the animals at each culture-positive occasion were determined. Using a multilocus short-sequence-repeat technique, we found 15 different strains of M. avium subsp. paratuberculosis from a total of 142 isolates analyzed. Results indicated herd-specific infection patterns; there was a clonal infection in herd C, with 89% of isolates from animals sharing the same strain, whereas herds A and B showed several different strains infecting the animals at the same time. Tissues from 80% of cows with at least one positive fecal culture (other than supershedders) were culture positive, indicating a true M. avium subsp. paratuberculosis infection. The results of M. avium subsp. paratuberculosis strain typing and observed shedding levels showed that at least 50% of low shedders have the same strain as that of a contemporary supershedder. Results of this study suggest that in a dairy herd, more of the low-shedding cows are truly infected with M. avium subsp. paratuberculosis than are passively shedding M. avium subsp. paratuberculosis. The sharing of strains between low shedders and the contemporary supershedders suggests that low shedders may have been infected by environmental exposure of M. avium subsp. paratuberculosis.
INTRODUCTION
Mycobacterium avium subsp. paratuberculosis is the causative agent of paratuberculosis (or Johne's disease), a debilitating chronic granulomatous enteritis in ruminants (13, 24, 25, 28). Johne's disease is characterized by a very long incubation period of several years (2, 5). This disease is recognized to be of serious economic and animal health consequences in domesticated ruminants (including dairy and beef cattle, sheep, goats, and farmed deer) throughout the world (14, 15, 29). Johne's disease has the greatest economic impact in dairy cattle, where decreased weight gain and milk production loss, premature culling, and reduced carcass value have an estimated cost of $250 million annually in the United States (3, 11, 16, 17). Interest in the pathogenesis and epidemiology of M. avium subsp. paratuberculosis has increased due to the worldwide distribution of Johne's disease, economic losses attributed to the disease (14, 16, 25), and the presence of viable bacteria in products ready for human consumption (4, 6, 8), which have been considered a potential hazard for human inflammatory bowel disease (6, 16, 21).
Cows may shed M. avium subsp. paratuberculosis without showing clinical signs (3, 32). The spread of M. avium subsp. paratuberculosis infection between and within herds is caused by movement of subclinically infected animals between premises and infection in early calfhood (14, 29). Daughters from M. avium subsp. paratuberculosis-infected dams have a high risk of becoming infected (2, 13). Calves from noninfected dams born shortly after the calving of an infected dam and calves growing up with future high shedders are at increased risk of infection (2). Current management practices to control M. avium subsp. paratuberculosis infection include culling cows that test M. avium subsp. paratuberculosis positive or exhibit clinical signs of Johne's disease. Clinical signs of the disease include diarrhea, loss of body weight despite a good appetite, decreased milk production, and mortality (1, 10, 11). Unfortunately, animals on many farms remain infected with M. avium subsp. paratuberculosis even after the farms participate in eradication programs that focus on culling of animals known to be shedding the pathogen (9, 20). In the period preceding the onset of clinical symptoms, infected animals may intermittently shed large quantities of M. avium subsp. paratuberculosis (2, 26). This subclinical shedding is generally thought to be the main driver in keeping herds infected and in maintaining M. avium subsp. paratuberculosis infection in dairy farms (2).
Traditionally, cattle were categorized as either positive or negative for M. avium subsp. paratuberculosis infection based on growth of M. avium subsp. paratuberculosis in fecal culture. However, it is now recognized that differences in M. avium subsp. paratuberculosis shedding levels exist among culture-positive animals. Depending on the amount of bacteria detected in the feces, cattle are classified as low (<10 CFU/tube), moderate (10 to 50 CFU/tube), or high (>50 CFU/tube) fecal shedders (3, 32) (tubes used were 15-ml solid-medium culture slants). The test procedures and the classification of infected cattle by the amount of M. avium subsp. paratuberculosis in their feces have not been standardized (3, 32). Current laboratory practice standards in the United States do not include enumeration of M. avium subsp. paratuberculosis CFU counts beyond 50 visible CFU/tube on solid media, such as Herrold's egg yolk medium (HEYM) (30, 32). Culture tubes with counts exceeding this level are designated too numerous to count (TNTC), and the animal is reported as being a high or heavy shedder (30). Recently, by using serial dilutions to determine the level of shedding in animals that shed more M. avium subsp. paratuberculosis than the TNTC cutoff amount, supershedders were identified and defined as cattle shedding 10,000 to 10 million CFU of M. avium subsp. paratuberculosis/g of fecal material (31).
If a supershedder cow is productive and has no clinical signs of Johne's disease, the animal is likely to stay in the herd even though it is shedding extremely high numbers of M. avium subsp. paratuberculosis into the environment. Based on the concept of supershedders, it has previously been suggested that a significant proportion of the low shedders in a herd are likely to represent passive shedding in many herds (30, 31). Passive shedding is defined as shedding by an animal that has a positive fecal test because of the ingestion of M. avium subsp. paratuberculosis but is not truly infected with M. avium subsp. paratuberculosis (so called “pass-through” of M. avium subsp. paratuberculosis in the intestinal tract). Active shedding occurs due to more extensive infection of the host intestinal tissues with M. avium subsp. paratuberculosis originating within the host's tissues. Although a passive-shedding phenomenon has been demonstrated for M. avium subsp. paratuberculosis organisms in the intestinal tract (26, 27), and some investigators believe that passive shedding may be responsible for a high percentage of low shedders in some heavily infected farms, fecal culture generally is considered to be 100% specific (3). Distinguishing unambiguously among individual animals with regard to the infection status is important in order to better understand the epidemiology of Johne's disease. In this paper, we define a “passive shedder” as an animal that has a positive fecal test and culture-negative tissues and a “truly infected” animal as one that has a positive fecal test and culture-positive tissues (11, 12, 20).
Methods for differentiation or subtyping of bacterial strains provide important information for molecular epidemiologic analysis (1, 16, 21). Multilocus short-sequence-repeat (MLSSR) sequencing is a highly discriminatory method that has been used for typing M. avium subsp. paratuberculosis isolates, and this analysis may enable molecular epidemiologic investigations that will lead to a better understanding of strain transmission and spread of M. avium subsp. paratuberculosis (1, 7). Also, sequencing of the M. avium subsp. paratuberculosis strain K-10 genome (10) has permitted the identification and application of multiple short-sequence repeats (SSR) to the study of the diversity, strain sharing, and host specialization among M. avium subsp. paratuberculosis isolates (7). While only a limited number of cross-sectional studies (1, 15) have used this method, and with a restricted set of isolates, it has been recognized that the use of well-designed longitudinal studies using several herds in multiple states is essential for applying the MLSSR sequencing technique to understand the epidemiology of M. avium subsp. paratuberculosis (7). Although several investigations have been conducted to address the molecular diversity of M. avium subsp. paratuberculosis isolates from a variety of hosts (1, 7, 14, 15, 21, 25), our understanding of the epidemiology of Johne's disease is limited, specifically with respect to M. avium subsp. paratuberculosis shedding levels and strain transmission in dairy herds.
In this study, we used the MLSSR sequencing technique for discrimination of M. avium subsp. paratuberculosis isolates in combination with the observed M. avium subsp. paratuberculosis shedding levels from a longitudinal study involving three dairy herds in the northeastern United States to evaluate whether low shedders of M. avium subsp. paratuberculosis were passively shedding M. avium subsp. paratuberculosis or whether they were truly infected. Furthermore, we investigated whether it is possible that these M. avium subsp. paratuberculosis-infected animals could have been infected as adults by the supershedders.
MATERIALS AND METHODS
Dairy farms and bacterial isolates.
Isolates used in this study were obtained from three commercial dairy farms in the northeastern United States: farm A in New York State, farm B in Pennsylvania, and farm C in Vermont. All three farms participated in the Regional Dairy Quality Management Alliance (RDQMA) project, which is a multistate research program conducted under a cooperative research agreement between the USDA Agricultural Research Service (ARS) and four Universities, Cornell University, Pennsylvania State University, University of Pennsylvania, and University of Vermont. This project emphasizes longitudinal data collection in areas in which infectious diseases of public and animal health concern in dairy herds are endemic. For a more complete description, including information on farms, samplings, and microbial analyses, see the work by Pradhan et al. (18). Briefly, the milking herds consisted of approximately 330, 100, and 140 cows on farms A, B, and C, respectively. Sampling commenced in February, March, and November 2004 on farms A, B, and C, respectively, and continued for approximately 5 yr. The project design included biannual collection of individual fecal samples from all milking and nonlactating cows. In special-interest situations, sampling frequency was increased and individual fecal samples were collected at shorter individuals from those selected animals. Additionally, culled cows were tracked from the farm to the slaughterhouse, and at the slaughterhouse (Cargill processing plant, Wyalusing, PA), four gastrointestinal (GI) tissues and a fecal sample were collected with the cooperation of USDA Food Safety and Inspection Service (FSIS) personnel. The harvested tissues included two lymph nodes located at the ileocecal (IC) junction and two pieces of ileum, one taken from 20 cm proximal to the IC valve (hereinafter referred to as the ileum sample) and the other taken from very near the IC valve (hereinafter referred to as the IC valve sample).
All collected samples were placed in coolers with ice packs and transported overnight to the Johne's Research Laboratory (New Bolton Center at the University of Pennsylvania) for M. avium subsp. paratuberculosis testing using a solid-medium culture technique as described previously (18, 32). The shedding levels (CFU of M. avium subsp. paratuberculosis/g of feces) for animals at each culture-positive occasion were determined (i) by additional dilutions for TNTC samples (30, 31) and (ii) based on the regular colony count for samples not designated TNTC; these samples were tested by four-tube fecal culture for the presence of viable M. avium subsp. paratuberculosis organisms (18, 32). To determine CFU of M. avium subsp. paratuberculosis/g of feces, the total sum of CFU across four tubes was multiplied by 5.3 (22) to take into account the dilution inherent in the sampling and isolation of M. avium subsp. paratuberculosis as performed at the Johne's Research Laboratory (New Bolton Center at the University of Pennsylvania). Isolates of M. avium subsp. paratuberculosis as distinct colonies on HEYM slants containing mycobactin J were sent from the Johne's Research Laboratory to Quality Milk Production Services (QMPS) at Cornell University for further molecular characterization. Isolates were selected from samples taken from supershedders, from all animals that were culture positive at the same time supershedders were present in the herds, and from all slaughterhouse samples available for these animals.
Criteria for evaluation of infection status.
We used the following criteria to evaluate the infection status of animals with fecal culture-positive test results. (i) To be considered passively shedding, an animal shed only a few times (<3) with low levels of shedding each time (i.e., ≤ 21 CFU M. avium subsp. paratuberculosis/g of feces, which is equivalent to 1 M. avium subsp. paratuberculosis colony per tube in each of four tubes containing the solid culture medium HEYM) and with subsequent fecal samples testing culture negative, shed the same M. avium subsp. paratuberculosis strain as a contemporary supershedder, and subsequently have culture-negative tissues obtained at slaughter. (ii) To be considered infected, an animal shed M. avium subsp. paratuberculosis and was subsequently culture positive for tissues obtained at slaughter. To evaluate whether these animals could have been infected as adults by contemporary supershedders, all M. avium subsp. paratuberculosis isolates were strain typed using MLSSR sequencing.
DNA extraction.
A single bacterial colony from a positive sample was substreaked on HEYM slants. These substreaked bacterial cells from the slants were harvested using a 10-μl loop and suspended in a 2-ml sterile vial (Biospec Products, Inc., Bartlesville, OK) containing 1 ml of sterile distilled water (DNase-free, RNase-free; Invitrogen Corporation, Carlsbad, CA) and 250 mg of 0.1-mm zirconia/silica beads (Biospec). A QIAamp DNA minikit (Qiagen, Inc., Valencia, CA) was used for DNA extraction with few modifications. Briefly, 650 μl of AL lysis buffer (Qiagen) was added to the contents of the 2-ml sterile vial, which were homogenized with a Mini-BeadBeater-8 (BioSpec) for 5 min, followed by incubation at 70°C for 30 min. In the next step, 600 μl of the incubated sample was transferred into a new 2-ml tube containing 600 μl of AL buffer and 60 μl proteinase K; the combination was vortex mixed thoroughly and then incubated at 70°C for 30 min. Following the incubation, 600 μl of 100% ethanol (Sigma-Aldrich, Inc., St. Louis, MO) was added, and the combination was vortex mixed thoroughly. Subsequently, the DNA was bound to spin columns, washed, and eluted in 150 μl of sterile distilled water (Invitrogen) as suggested by the manufacturer. During elution, the spin column was placed in a 1.5-ml microcentrifuge tube, 100 μl sterile water was added, and the mixture was incubated at room temperature for 10 min and then centrifuged at 6000 × g for 3 min. This was repeated with another 50 μl of sterile water to make a final volume of 150 μl, which was placed in storage at −20°C until further analysis. An extraction blank was included in each extraction set and used as a negative control for molecular analyses.
Molecular characterization using the MLSSR sequencing technique.
Amonsin et al. (1) identified 11 loci for use in the MLSSR sequencing of M. avium subsp. paratuberculosis isolates. Subsequently, Harris et al. (7) used four of the 11 SSR loci (locus 1, locus 2, locus 8, and locus 9) to study the diversity of M. avium subsp. paratuberculosis isolates collected from animals throughout the United States. These four loci were selected due to their highest genetic diversity indices and were identified as the most discriminatory, stable, and informative SSR loci (1, 7). We used these four loci for MLSSR sequencing analysis of our M. avium subsp. paratuberculosis isolates. PCR amplification was carried out with extracted DNA for all isolates using the previously published primers for the four loci (1). PCR amplification was unsuccessful for a few isolates for locus 1; however, these samples were successfully amplified with modified primers: 5′-GTG TTC GGC AAA GTC GTT GT-3′ and 5′-GCG GTA CAC CTG CAA G-3′. Information about modified primers for locus 1 was obtained from the Center for Genomics and Veterinary Population Medicine Department, University of Minnesota, St. Paul, MN.
The 25-μl PCR amplification reaction mixture for each SSR comprised 1× GoTaq Green Master Mix (Promega Corporation, Madison, WI), 0.625 μl of 10 μM (each) upstream and downstream primers (Integrated DNA Technologies, Coralville, IA), and 5 μl of genomic DNA. The amplification conditions consisted of an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min, with a final extension step at 72°C for 7 min. A PCR master mixture blank was included as a negative control for each batch. A total of 5 μl of the PCR product was electrophoresed at 104 V for 45 min in 1.5% (wt/vol) agarose gel in 0.5× TBE buffer (0.45 M Tris-Borate, 0.01 M EDTA [pH 8.3]). Gels were stained in an ethidium bromide bath for 2 min followed by destaining in water for approximately 1 h. The gels were visualized for a quality check through UV transillumination with a Molecular Imager Gel Doc XR system and Quantity One software, version 4.4.1 (Bio-Rad, Hercules, CA) and then photographed.
PCR products were purified in 30 μl of elution buffer with a PureLink PCR purification kit (Invitrogen) following the purification procedure suggested by the manufacturer. The amount of DNA in the purified PCR products was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). The PCR amplicons were sequenced using standard dye terminator chemistry and AmpliTaq-FS DNA polymerase. Sequences were analyzed with an automated DNA sequencer (3730 DNA analyzer; Applied Biosystems, Inc., Foster City, CA) at the Cornell University Life Sciences Core Laboratories Center (http://cores.lifesciences.cornell.edu/brcinfo/). All chromatograms were visually inspected, and sequences were analyzed with the EditSeq, SeqMan, and MegAlign programs (DNAStar, Inc., Madison, WI). For each locus, the number of tandem repeats was determined, and allele numbers were assigned to reflect the number of copies represented in the SSR sequence. On the basis of the unique combination of alleles for each locus, MLSSR types were then assigned. The dendrogram depicting genetic relationships among isolates was generated by the unweighted pair group method with arithmetic averages (UPGMA) with the program PAUP (version 4.0; Sinauer Associates, Inc. Sunderland, MA).
RESULTS
Distribution of different genotypes.
PCR-amplified products showing strong bands after gel electrophoresis with a good DNA yield were subsequently sequenced. We identified 15 different strains of M. avium subsp. paratuberculosis (i.e., genotypes or MLSSR types) from three farms with a total of 142 isolates from fecal and tissue samples: 9 types for farm A, 7 types for farm B, and 6 types for farm C (Fig. 1). Seven of the MLSSR types were isolated from more than one farm, but the remaining eight types were farm specific. In a few animals, more than one strain was found, indicating polyclonal infection. A mostly clonal infection was identified for farm C, with 89% of isolates (n = 52) determined to be MLSSR type 2; samples from 95% of cows (n = 21) on farm C were identified as MLSSR type 2. Type 4 was the most predominant type on farm A and represented 59% of the isolates (n = 81) from this farm; also, samples from 59% of cows (n = 27) on this farm were strain typed as type 4. Seven MLSSR types were identified from a limited number of farm B isolates (nine isolates from eight animals).
Fig. 1.
Bar diagram showing the percentage of each type (MLSSR type or genotype) from a total of 81, 9, and 52 isolates (fecal and tissue) from farms A, B, and C, respectively.
A dendrogram showing the genetic relationships among all M. avium subsp. paratuberculosis isolates analyzed by MLSSR sequencing is given in Fig. 2. The numbers of isolates for each strain type and the numbers of isolates from supershedders belonging to that particular type are also presented in Fig. 2. On farm A, 48 out of 81 isolates were type 4. Among these, 20 were from four supershedders. Nine of 81 isolates were type 3, and of these, 3 were from two supershedders. Samples from approximately 26% of cows (7 out of 27) on farm A were strain typed as type 3. All isolates from farm B were obtained from animals that were purchased in 2001 from different sources. These animals were born on different farms. On farm C, where we found a clonal infection with 46 of the 52 isolates sharing the same strain (type 2), 18 out of 19 isolates from supershedders (four animals) belonged to this type.
Fig. 2.
Dendrogram showing genetic relationships among all M. avium subsp. paratuberculosis isolates determined by MLSSR sequencing analysis. The dendrogram was generated by the unweighted pair group method with arithmetic averages (UPGMA) with the PAUP program. For each farm, the distribution of genotypes is shown to the right of the dendrogram. The numbers of isolates (n) identified as a particular MLSSR type and (after the semicolon) the numbers of isolates from supershedders (nss) identified as that same MLSSR type (“n” includes “nss”) are shown. For locus 1, the numbers of G mononucleotides present for 14 or more repeats were assigned the value >14 per the nomenclatures in the works by Amonsin et al. (1) and Harris et al. (7).
Infection patterns.
The longitudinal follow-up data for individual animals, showing M. avium subsp. paratuberculosis genotypes and the observed shedding level at each sampling along with genotype information in the available fecal and tissue samples collected at the slaughterhouse, are shown in Tables 1, 2, and 3 for farms A, B, and C, respectively. In the first sampling on farm A, animal 693 was the only animal with supershedder status (based on the criterion of ≥10,000 CFU of M. avium subsp. paratuberculosis/g of feces). At that time, the same strain (type 4) as that found in the supershedder was found in the fecal samples of five other animals. No tissue samples were available for this supershedder.
Table 1.
Longitudinal follow-up of individual animals showing the genotype of isolates and the shedding level at each farm sampling on farm Aa
| Cow IDb | Birth date | MLSSR type (shedding level [CFU of M. avium subsp. paratuberculosis/g of feces]) on indicated sampling date |
Cull date | Result for Fec; LN1, LN2, Il, ICVc | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 2/17/2004 | 10/5/2004 | 4/12/2005 | 10/3/2005 | 5/1/2006 | 10/9/2006 | 4/16/2007 | 10/15/2007 | ||||
| 693S,H | 8/31/1997 | 4 (462,000) | 7/27/2004 | NA | |||||||
| 1085S,H | 10/20/1999 | 3 (159) | 3 (39,060) | 3/22/2005 | NA | ||||||
| 1099S,H | 12/11/1999 | Neg | 3 (71,400) | 3/29/2005 | NA | ||||||
| 1148S,H | 7/15/2000 | Neg | Neg | Neg | 4 (16) | 4 (16) | 4 (223) | 4 (1,778) | 4 (28,000) | 3/4/2008 | 4; NA, NA, 4, 4 |
| 1160S,H | 8/19/2000 | Neg | 4 (620) | 4 (42,000) | 8/16/2005 | NA | |||||
| 1180S,H | 11/17/2000 | 4 (164) | 4 (7,000) | 4 (28,000) | 4 (98,000) | 1/17/2006 | 4; 4, 4, 4, 4 | ||||
| 1078H | 10/7/1999 | Neg | Neg | Neg | 11 (5) | Neg | 9/28/2006 | Neg; 4, 4, Neg, 4 | |||
| 1127H | 1/1/1998 | 4 (11) | 7 (11) | 1/18/2005 | Neg; 4, 4, 3, Neg | ||||||
| 1149H | 7/23/2000 | Neg | 4 (11) | 12/7/2004 | NA | ||||||
| 1161H | 8/28/2000 | 4 (80) | 9/28/2004 | NA | |||||||
| 1167H | 9/17/2000 | 4 (1,039) | 8/24/2004 | 4; 2, 4, 4, 4 | |||||||
| 1171H | 10/12/2000 | Neg | Neg | Neg | 4 (5) | Neg | Neg | 10/19/2006 | Neg; 2, 2, 2, 2 | ||
| 1179H | 11/11/2000 | Neg | 4 (16) | 4 (127) | 4 (313) | 7 (594) | 7/31/2006 | NA; 4, 4, 4, 4 | |||
| 1181H | 11/22/2000 | Neg | 7 (16) | Neg | 7 (276) | 7 (1,590) | 8/29/2006 | 7; 7, 7, 7, 7 | |||
| 1182H | 11/24/2000 | 4 (7,000) | 8/9/2004 | NA | |||||||
| 1194H | 12/6/2000 | Neg | Neg | Neg | 4 (5) | 10/11/2005 | NA | ||||
| 1200H | 12/25/2000 | Neg | Neg | Neg | 4 (11) | Neg | Neg | Neg | 6/9/2007 | NA | |
| 1255H | 3/10/2001 | 3 (5) | Neg | Neg | Neg | Neg | Neg | 1 (5) | 9/18/2007 | NA | |
| 1280H | 5/23/2001 | Neg | Neg | Neg | 2 (11) | 11/15/2005 | NA | ||||
| 1288H | 6/10/2001 | 7 (5) | Neg | Neg | 6/28/2005 | Neg; Neg, 4, 7, 4 | |||||
| 1294H | 7/1/2001 | Neg | Neg | 3 (11) | Neg | Neg | Neg | Neg | Neg | 5/18/2009 | NA |
| 1416H | 12/1/1999 | Neg | Neg | 9 (5) | Neg | 5 (5) | 6/22/2006 | NA | |||
| 1645H | 10/25/2003 | NS | NS | NS | 4 (5) | Neg | Neg | 3/22/2007 | NA | ||
| 2005H | 8/10/1998 | Neg | 6 (5) | Neg | Neg | Neg | 9/19/2006 | NA | |||
| 2044H | 9/12/1998 | Neg | Neg | 6 (11) | 4 (5) | 12/27/2005 | NA | ||||
| 2653H | 12/26/1998 | Neg | Neg | 3 (5) | Neg | Neg | Neg | Neg | 9/18/2007 | NA | |
| 2669H | 2/5/1999 | 3 (196) | 3 (48) | 2/22/2005 | NA | ||||||
Dates are given in month/day/year format. Fec, fecal sample; LN1 and LN2, respective samples from two lymph nodes located at the ileocecal (IC) junction; Il, ileum sample (i.e., from the ileum 20 cm proximal to the IC valve); ICV, IC valve sample, as defined in the text (i.e., from the ileum close to the IC valve); NA, information/results not available; Neg, sample was culture negative; NS, animal was not sampled at this visit—sampling from this animal started at a later date.
Cow ID, cow identification number. Superscript letters indicate supershedder animals (S) and homegrown animals (H).
Shown are results for the indicated strain types of isolates from samples collected at the slaughterhouse.
Table 2.
Longitudinal follow-up of individual animals showing the genotype of isolates and the shedding level at each farm sampling on farm Ba
| Cow ID | Birth date | MLSSR type (shedding level [CFU of M. avium subsp. paratuberculosis/g of feces]) on indicated sampling date |
Cull date | Result for Fec; LN1, LN2, Il, ICV | ||||
|---|---|---|---|---|---|---|---|---|
| 3/22/2004 | 9/12/2004 | 12/15/2004 | 6/6/2005 | 12/5/2005 | ||||
| 51S,P | 12/1/1999 | 6 (210,000) | 6 (742,000) | 10/27/2004 | NA | |||
| 52S,P | 1/20/1999 | 15 (546,000) | 3/29/2004 | NA | ||||
| 30P | 11/2/1998 | 13 (5) | Neg | Neg | Neg | Neg | 1/13/2006 | NA |
| 73P | 10/15/1999 | 12 (5) | Neg | Neg | Neg | Neg | 1/9/2006 | Neg; Neg, Neg, Neg, Neg |
| 74P | 11/5/1999 | 3 (186) | Neg | 10/18/2004 | NA | |||
| 87P | 1/15/2000 | 14 (11) | Neg | Neg | 3/25/2005 | NA | ||
| 214P | 8/2/1999 | 8 (5) | Neg | Neg | Neg | Neg | 3/10/2006 | NA |
| TulipP | 9/15/1999 | Neg | 3 (11) | 9/19/2004 | NA | |||
See the footnotes to Table 1. For Cow ID, superscript letters indicate supershedder animals (S) and purchased animals (P).
Table 3.
Longitudinal follow-up of individual animals showing the genotype of isolates and the shedding level at each farm sampling on farm Ca
| Cow ID | Birth date | MLSSR type (shedding level [CFU of M. avium subsp. paratuberculosis/g of feces]) on indicated sampling date |
Cull date | Result for Fec; LN1, LN2, Il, ICV | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 11/15/2004 | 5/9/2005 | 11/14/2005 | 5/15/2006 | 11/13/2006 | 5/21/2007 | 11/12/2007 | ||||
| 66S,H | 6/21/1999 | 2 (32) | 2 (991) | 2 (70,000) | 2 (238,000) | 6/19/2006 | NA; 2, 2, 2, 2 | |||
| 102S,H | 4/27/2000 | Neg | 10 (16) | 2 (3,010,000) | 12/15/2005 | NA | ||||
| 106S,H | 6/22/2000 | 2 (21) | 2 (58) | 2 (98,000) | 2/2/2006 | NA | ||||
| 152S,H | 7/10/2001 | 2 (1,260,000) | 2/9/2005 | 2; 2, 2, 2, 2 | ||||||
| 40H | 10/16/1998 | 2 (5) | 4/14/2005 | Neg; Neg, Neg, Neg, Neg | ||||||
| 107H | 6/27/2000 | Neg | 2 (5) | Neg | Neg | 7/6/2006 | Neg; 2, 1, 1, Neg | |||
| 114H | 7/26/2000 | 2 (5) | 2 (5) | 2 (27) | 2 (461) | 10/10/2006 | 2; 2, 2, 2, 2 | |||
| 124H | 10/11/2000 | 2 (5) | 4 (477) | 8/29/2005 | NA | |||||
| 133H | 1/17/2001 | 2 (11) | Neg | Neg | Neg | 7/6/2006 | 8; Neg, 2, Neg, Neg | |||
| 136H | 2/20/2001 | 2 (5) | 4/14/2005 | Neg; Neg, Neg, Neg, Neg | ||||||
| 164H | 10/1/2001 | 2 (11) | Neg | Neg | Neg | Neg | 3/11/2009 | NA | ||
| 170H | 11/13/2001 | 2 (21) | Neg | Neg | 3/15/2006 | NA | ||||
| 171H | 11/17/2001 | 2 (5) | Neg | Neg | 12/29/2005 | Neg; 2, Neg, Neg, Neg | ||||
| 178H | 1/30/2002 | Neg | 7 (5) | Neg | Neg | Neg | Neg | 4/5/2008 | NA | |
| 184H | 4/9/2002 | 2 (5) | Neg | Neg | Neg | Neg | Neg | Neg | NA | NA |
| 189H | 4/26/2002 | 2 (21) | Neg | Neg | Neg | Neg | Neg | Neg | 11/25/2007 | NA |
| 193H | 5/22/2002 | 2 (16) | Neg | Neg | Neg | Neg | 1/22/2007 | NA | ||
| 208H | 8/13/2002 | NS | Neg | 2 (5) | Neg | Neg | Neg | Still in herd | ||
| 491P | 9/29/2001 | 2 (11) | Neg | 8/8/2005 | Neg; 2, Neg, Neg, Neg | |||||
| 493P | 1/1/2000 | 2 (5) | Neg | 8/15/2005 | NA | |||||
| 495P | 1/1/2000 | 2 (382) | Neg | Neg | Neg | 6/26/2006 | NA | |||
See the footnotes to Table 1. For Cow ID, superscript letters indicate supershedder animals (S), homegrown animals (H), and purchased animals (P).
On farm A, 55.6% (5 of 9), 12.5% (1 of 8), 20.0% (1 of 5), and 70.0% (7 of 10) of culture-positive animals shed the same strain as that of contemporary supershedders at the first, second, third, and fourth sampling, respectively. Approximately 60% of isolates on this farm were the same (dominant) strain. On farm C, 100% of culture-positive animals shed the same strain as that shedded by contemporary supershedders during the first (16 of 16 animals), third (2 of 2 animals), and fourth (1 of 1 animals) sampling. There were no supershedder animals at the time of the second sampling on farm C, and four distinct MLSSR types were identified at this sampling. Most of the M. avium subsp. paratuberculosis-positive animals on farm B were shedding unique strains, although type 3 was identified for two animals (Table 2). These three farms showed three different patterns of M. avium subsp. paratuberculosis strain infection. Farm C is a clear example of a clonal outbreak, whereas farm B shows no evidence for transmission between cows at all. Farm B is a recently constituted herd that includes animals purchased from multiple sources. Farm A showed a dominant strain type, indicating clonal transmission, but also a number of other strain types.
Cows 40 and 136 on farm C were low shedders (shedding only 5 CFU/g of feces) and were shedding the same strain (type 2) as that shedded by a contemporary supershedder (cow 152). At slaughter, their tissues were culture negative, and they were therefore classified as passive shedders. As indicated above, we defined truly infected animals as animals that were culture positive for M. avium subsp. paratuberculosis in tissues. For example, cows 133, 171, and 491 on farm C and cows 1127 and 1288 on farm A were classified as truly infected animals, because they were low shedders (shedding 5 to 11 CFU/g of feces) and their tissue samples were culture positive. Out of 15 animals (other than supershedders) for which we had tissue culture data, 10 could be classified as truly infected (67%) with the same strain in fecal samples and tissues, 2 could be classified as truly infected (13%) but with different strains in fecal samples and tissues, and 3 could be classified as passive shedders (20%). Because of the unavailability of tissue samples for other animals in the study, it was not possible to classify these animals as passive shedders or truly infected animals. By using all available tissue data, tissues from 80% (12 of 15) of nonsupershedder cows were found to be culture positive for M. avium subsp. paratuberculosis, indicating a true infection.
During the study period, 6, 2, and 4 animals on farms A, B, and C, respectively, were identified as supershedders. For low shedders in the three herds, 64% of cows (21 of 33) for fecal samples and 58% of cows (19 of 33) for fecal samples and available tissue samples shared the same strains as those of the identified supershedders in the herds. For example, on farm C at the first sampling, all 17 animals with culture-positive results were shedding the same strain (type 2). On farm C, cows 491, 493, and 495 were purchased from other farms (unknown sources), and these animals were brought into the herd as older heifers, at the age of approximately 20 to 24 months. Table 3 shows that cow 491 was still infected with the same strain as that infecting the contemporary supershedder.
DISCUSSION
This study describes the use of an MLSSR sequencing technique for subtyping M. avium subsp. paratuberculosis isolates. Using this strain-typing technique in conjunction with fecal M. avium subsp. paratuberculosis shedding data and slaughterhouse tissue culture data, we attempted to improve our understanding of the infection dynamics of M. avium subsp. paratuberculosis in dairy herds. To our knowledge, this is the first report describing molecular epidemiology of M. avium subsp. paratuberculosis isolates collected from individual animals over time in multiple dairy herds. Our results indicated that (i) cows shedding low levels of M. avium subsp. paratuberculosis in the presence of supershedders were more likely truly infected with than passively shedding M. avium subsp. paratuberculosis, (ii) supershedders appear to be spreading M. avium subsp. paratuberculosis among herd mates, which may lead to an increased M. avium subsp. paratuberculosis prevalence on dairy farms, and (iii) many different strains could be found within and between dairy herds, suggesting herd-specific M. avium subsp. paratuberculosis infection patterns.
Cows shedding low levels of M. avium subsp. paratuberculosis in the presence of supershedders were more likely truly infected with than passively shedding M. avium subsp. paratuberculosis.
The principal strength of this study was that the isolates were obtained from samples of a longitudinal study that involves tracking infection and disease dynamics from farm to slaughterhouse (18). Slaughterhouse data are rarely collected in a large-scale observational study, because this process (i) is time and resource intensive and (ii) needs continuous coordination between the herd owner, the buying agents, the slaughter plant, and the research lab performing microbial analyses of M. avium subsp. paratuberculosis detection and isolation. Although it was not possible for all culled cows in our study to be identified in the slaughterhouse, enough care was taken to track and sample as many culled cows as possible.
The infection definition for truly infected cows, i.e., having culture-positive tissues, was based on previously published reports (11, 12, 20, 24, 28). No histological examination was done in this study. In this study, we assumed that the presence of live M. avium subsp. paratuberculosis in mesenteric lymph node, ileum, and/or IC valve samples is associated with a true infection. As most cows had multiple tissues that were M. avium subsp. paratuberculosis culture positive at the time of slaughter, a true infection would be the most likely explanation of these findings. Previous studies (11, 12, 26) found that histological testing was less sensitive than bacteriologic culture of tissues for detecting infected cattle. McKenna et al. (12) analyzed mesenteric lymph nodes and ileum samples from 984 culled dairy cows and found that the overall prevalence of M. avium subsp. paratuberculosis was 16.1% and 0.7% by bacteriologic culture and histologic methods, respectively. Martinson et al. (11) tested ileum and lymph node samples and compared bacterial culture, histopathology, and immunochemistry results for the diagnosis of Johne's disease in culled dairy cows. Histopathology and immunochemistry tests in their study were much less sensitive than bacterial culture, detecting less than 6% of cows that were M. avium subsp. paratuberculosis culture positive.
Passive shedding due to pass-through of M. avium subsp. paratuberculosis organisms in the intestinal tracts of experimentally infected cattle has been previously demonstrated (26, 27). The term “passive shedding” appears to imply that the host is not actively involved in the shedding process and only serves as a pass-through vector. In this paper, we argue that culture-positive or culture-negative tissues provided a better insight into the infection status of individual animals with a low level of M. avium subsp. paratuberculosis shedding (11, 12, 20). The detection of M. avium subsp. paratuberculosis-positive tissues is dependent on the number of tissues collected from multiple sites of an animal and analyzed by bacteriologic culture (26), which indicates that culture of a high number of tissues would more likely identify a M. avium subsp. paratuberculosis-infected animal. Sweeney et al. (26) demonstrated what they defined as passive shedding of M. avium subsp. paratuberculosis in calves experimentally inoculated with high doses of M. avium subsp. paratuberculosis (2 × 1010 CFU) at day 2 or 3 of age. They used the term “passive shedding” to describe the passage of orally ingested M. avium subsp. paratuberculosis organisms through the gastrointestinal (GI) tract of the animal and the presence of these organisms in feces during the first few days following inoculation; cessation of passive shedding occurred within 48 h after inoculation (26). However, extensive tissues collected from multiple sites of these calves at day 42 to day 44 of age showed that all calves were culture positive for M. avium subsp. paratuberculosis in tissues (26). Utilizing the definitions that we put forward in this paper, these calves would be defined as truly infected, as multiple tissues collected postmortem were M. avium subsp. paratuberculosis culture positive. In addition to the culture results, strain information from MLSSR sequencing provided further insight into the potential M. avium subsp. paratuberculosis infection with the same strains as those infecting contemporary supershedders. Based on the observed shedding levels and MLSSR strain types shed by individual animals, our results indicated that more of the low-shedding cows in the observed herds were truly infected than were passively shedding M. avium subsp. paratuberculosis. Tissues from 80% of cows with at least one positive fecal culture (other than supershedders) were culture positive, indicating a true M. avium subsp. paratuberculosis infection status of the animals. Shedding levels for truly infected animals varied within and between farms, which is common in dairy herds (3, 32).
Supershedders appear to spread M. avium subsp. paratuberculosis among herd mates, which may lead to an increased M. avium subsp. paratuberculosis prevalence on dairy farms.
In the presence of supershedders in our study farms A and C, several culture-positive animals were shedding the same strains as the contemporary supershedders at different samplings. MLSSR types 3 and 4 on farm A and type 2 on farm C were shared between the low-shedding animals and contemporary supershedders. In contrast, from a limited number of M. avium subsp. paratuberculosis isolates on farm B, we did not find any culture-positive animal that shed the same strains as contemporary supershedders. However, based on limited differences in repeating elements at some loci, some of the strain types being carried by these animals are closely related. For example, supershedder 51 on farm B was shedding MLSSR type 6 (7-11-5-5), and some other animals shed type 3 (7-10-5-5) and type 8 (7-12-5-5), with a difference of only 1 G repeat for locus 2.
Although animals up to 1 year old are thought to be most susceptible to M. avium subsp. paratuberculosis infection and assumed to have acquired infection in early calfhood (2, 29), it is reasonable to speculate that animals may also acquire M. avium subsp. paratuberculosis infection as adults (13, 19). Adult infection is characterized by an animal that is free of M. avium subsp. paratuberculosis infection in her initial year of life and becomes infected as an adult. Since many M. avium subsp. paratuberculosis-infected cows have been raised with an eventual supershedder, it is difficult to distinguish between the low-shedding M. avium subsp. paratuberculosis-infected cows that have become infected together with the supershedder in early life and those that were infected by the supershedder as an adult. In the two infection scenarios, a similar strain would be present in supershedders and in low-shedding M. avium subsp. paratuberculosis-infected animals. Animals purchased from another herd would be less likely to have experienced exposure during early life to the same M. avium subsp. paratuberculosis strain as that which infected the supershedder. Therefore, the observation that a purchased adult animal (cow 491) on farm C was infected with the same strain as a contemporary supershedder may suggest a case of adult infection following exposure to high doses of M. avium subsp. paratuberculosis. However, details pertaining to the MLSSR strain types of M. avium subsp. paratuberculosis circulating on the farm from which this animal was purchased are not available; thus, it is not possible to rule out infection on the farm of birth.
M. avium subsp. paratuberculosis infection in individual animals in dairy herds may occur through different routes, such as acquiring in utero infection from infected dams (vertical transmission), through contaminated colostrum, or through contaminated environments. Interestingly, for the data set presented in this report, dams of all low shedders born on these three dairy farms were not M. avium subsp. paratuberculosis positive. It appeared that for low shedders on these farms, M. avium subsp. paratuberculosis was spread mainly by the fecal-oral route. Supershedders may contribute substantially to the herd environmental M. avium subsp. paratuberculosis bioburden and the exposure of uninfected animals to M. avium subsp. paratuberculosis organisms. Supershedders can play an important role in the spreading of M. avium subsp. paratuberculosis infection among herd mates, potentially even adult herd mates. We therefore argue that the presence of supershedders may lead to an increased M. avium subsp. paratuberculosis prevalence on dairy farms.
Many different strains could be found within and between dairy herds, suggesting herd-specific M. avium subsp. paratuberculosis infection patterns.
Our results indicated herd-specific infection patterns, with a clonal infection on farm C and several different strains being found on farms A and B. Some strains were found on multiple farms; this could be interpreted as a coincidence, or it may be concluded that these strains were dominant strains in the geographic region. The observed predominant strains in this study, MLSSR type 2 and type 4, may be environmentally stable or more infectious than other strains. It has been suggested that some M. avium subsp. paratuberculosis strains interfere more successfully than others with the ability of macrophages to kill intracellular pathogens, which may make it more important to include strain typing when designing control programs (5).
In a recent study, a total of 61 distinct genotypes were identified among the 211 M. avium subsp. paratuberculosis isolates collected from dairy herds throughout the United States and analyzed by MLSSR sequencing with the same four loci as in our study (7). Most of the strains found in our study are among the strains observed throughout the United States (7), suggesting that these strains are representative of the strains found nationwide. Previous studies also reported a high diversity of strains in M. avium subsp. paratuberculosis isolates collected from a variety of hosts (cows, sheep, goats, deer, etc.) (1, 14, 15, 21, 25). On farms A and C, we identified a few animals that were infected with more than one strain (polyclonal infection), and this observation was similar to that in some recent studies (7, 21). Several distinct SSR genotypes were identified within the herds and the individual animals studied. This may reflect the presence of animals infected with multiple strains of M. avium subsp. paratuberculosis and multiple introductions of infected animals into herds (1, 7). In the presence of multiple strains on a farm, there is a potential for strain competition, with several strains competing for the same niche in the host system (5). Such a strain competition would eventually lead to a dominant strain within a dairy, which is exactly what we observed for two of the three farms in our study. We recognize that there may be technical artifacts associated with the strand slippage during either PCR or sequencing reactions that may result in the erroneous assignment of genotypes. To minimize the occurrence of such slippage errors, where relevant, we increased the amount of sequence coverage by confirming the sequences in both directions (forward and reverse) and by testing samples in duplicate.
Infected animals enter a latent, nonshedding stage that can vary in length, followed by a period of low and intermittent shedding of M. avium subsp. paratuberculosis with no obvious clinical symptoms (23, 32). It has been reported that animals may transiently contaminate the environment by intermittent shedding; thus, the immediate cow environment may serve as a proxy for the SSR genotypes within a herd (7). The transient shedding status may be an important factor in maintaining M. avium subsp. paratuberculosis infection on farms. Given the low apparent prevalence of M. avium subsp. paratuberculosis within a dairy herd, Johne's disease should be expected to fade out in herds when all infected animals are continuously culled. Such herds follow best management practices that include hygienic calf-rearing practices and culling of cows that test M. avium subsp. paratuberculosis positive or exhibit clinical signs of the disease. However, M. avium subsp. paratuberculosis infection continues to be endemic in the bovine populations worldwide. A detailed understanding of the infection process and mechanism for persistence is essential for controlling M. avium subsp. paratuberculosis incidence and prevalence in dairy herds. It is reasonable to speculate that nonshedding (as defined by our current diagnostic programs) or transiently shedding infected animals have an important role in maintaining infections in dairy herds.
An increased understanding of strain-specific dynamics of M. avium subsp. paratuberculosis infection in herds will make it possible to design more effective, targeted, and robust disease control programs (5). The presence of herd-specific infection patterns in bovine populations suggests that different control strategies may be warranted depending on whether a new infection is the result of introducing infected but clinically healthy cattle from another farm or is attributable to exposure within the animal's own farm environment (such as contaminated manure, feed, water, pasture, etc.). In the latter case, it appears that strongly infectious supershedders may play an important role in maintaining infections in dairy herds. Although the sample size is relatively small, the precision and detail of our data allowed us to explore shedding patterns and transmission routes of M. avium subsp. paratuberculosis on dairy farms. Given the small sample size, validation of our results with independently collected data would be prudent.
In conclusion, based on the molecular epidemiology of M. avium subsp. paratuberculosis strains and M. avium subsp. paratuberculosis shedding levels, at least 50% of low shedders in these herds had the same strain as that of contemporary supershedders. Results of this study suggest that in a dairy herd, more of the low-shedding cows are truly infected with M. avium subsp. paratuberculosis than are passively shedding M. avium subsp. paratuberculosis cells. Shedding of indistinguishable strains by the low shedders and their contemporary supershedders suggests that low shedders may have been infected due to environmental exposure of M. avium subsp. paratuberculosis. Successful control strategies for Johne's disease require a good understanding of the epidemiology of the disease. The use of SSR strain typing combined with the observed shedding levels provided a unique opportunity to get a better insight into herd infection and dynamics of M. avium subsp. paratuberculosis.
ACKNOWLEDGMENTS
This project was supported in part by the USDA ARS (agreements 58-1265-3-155, 58-1265-3-156, 58-1265-3-158, 58-1265-4-020, and 58-1265-8-064) for the Regional Dairy Quality Management Alliance (RDQMA), the Johne's Disease Integrated Program (JDIP; USDA contract 45105), and USDA-CSREES-NRI award 2007-35204-18391.
We express our appreciation to the farm owners and personnel that participated in the study both at the farms and in the laboratories. We thank USDA FSIS personnel for their support in collecting slaughterhouse samples for culled cows from our study farms. We also thank Henk C. den Bakker for his help with the phylogenetic analysis.
Footnotes
Published ahead of print on 5 January 2011.
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