Identification of Mycobacterium avium subspecies paratuberculosis strains isolated from dairy goats and dairy sheep in Ontario, Canada

Abstract

The main objective of this study was to identify the circulating strains of Mycobacterium avium subspecies paratuberculosis (Map) in fecal isolates obtained from dairy goat (N = 29 farms) and dairy sheep (N = 21 farms) populations in Ontario, Canada. Further subtyping was performed to determine if there was adequate diversity between strains that could be used to establish Map transmission patterns. Type C was the dominant strain of Map isolates (95.2%) identified in dairy goats (n = 21). Sub-typing of the Type C strains, based on variable number tandem repeats (VNTR) and mycobacterial interspersed repetitive units, identified 3 VNTR types: INMV 1 (n = 10), INMV 2 (n = 10), and a type not previously identified (n = 1). Only 2 sheep isolates could be identified; both were Type S, sub-type III. Current typing methods demonstrate little Map diversity in the dairy goat population and are therefore of limited use to investigate infection patterns.

Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of paratuberculosis, a chronic, incurable wasting disease of domestic ruminants. Early paratuberculosis research recognized that Map was more difficult to culture from suspect cases of paratuberculosis in sheep than from cattle and it was suspected that 2 strains of the bacteria existed, one more commonly infecting cattle and one more commonly infecting sheep (1). It was not until genotyping was developed as a diagnostic tool that this was confirmed (2). Initially, the strains were labeled Type C/Type II/cattle type and Type S/Type I/sheep type. However, Type C had also been isolated from goats, sheep, and other ruminant and non-ruminant animals, indicating the strain had a broad host range and was not limited to one species (2). An intermediate strain was also subsequently identified from a Canadian sheep that demonstrated a DNA hybridization pattern that was a composite of both Type C and Type S (2). However, whole genome sequencing has now re-classified this strain as a subtype of Type S and it is now referred to as Type S/subtype III (1).

The Map genome of Type C (K-10) was the first strain to be completely sequenced (3–5). This genome consists of a single, circular sequence of 4 829 781 base pairs (4 Mb) divided into genes (4587) and intergenic spaces. Type S (both subtypes I and III) has now also been sequenced (6). Comparisons of the genomes of different mycobacteria and different strains of Map allow for the identification of genomic regions possessing different nucleotide compositions that are unique to each; these can be subsequently used as diagnostic test targets and to better understand the molecular evolution of the bacteria and disease (7). These genomic variations or mutations are either intergenic (between genes) or intragenic (within genes) with large sequence polymorphisms, insertion sequences (IS), repeated sequences, and single nucleotide polymorphisms.

To date, genomic-based Map typing has been primarily used to differentiate Map from other M. avium subspecies. Mycobacterial culture mediums are not selective for Map; therefore, laboratories rely on polymerase chain reaction (PCR) confirmation tests based on the hspX gene (8) or insertion sequences such as IS900 (7) to reduce the number of false positive test results. The further discovery of genomic variations in the insertion sequence IS1311 has allowed PCR-restriction enzyme analysis (PCR-REA) based testing to be used to differentiate Type C and Type S strains of Map (9), while variations in the gene MAP1506 and the use of denaturing gradient gel electrophoresis have provided the ability to not only differentiate Type C and Type S, but also subtypes I and III (10). To further identify intra-strain variability, methods based on short-sequence repeats (SSR) (11), variable number tandem repeats (VNTR), and mycobacterial interspersed repetitive units (MIRU), have been developed (12). These tools aim to identify related and unrelated isolates to ultimately provide insight into the routes and origins of infection (e.g., mixing of animal species), and to identify husbandry and animal movement practices that may increase infection risk (e.g., pasturing, post-natal management).

While a Map Type S/subtype III isolate has been previously collected from a sheep in Canada (2), there has been no other published literature regarding the type or subtypes of Map commonly circulating in the small ruminant, and specifically, the dairy small ruminant populations. Therefore, the 2 main objectives of this study were to: i) identify the circulating strains of Map that were cultured from the feces of randomly sampled populations of Ontario dairy goats and dairy sheep; and ii) explore the subtype diversity of these isolates to determine if they were unique to the small ruminant populations and could therefore be used to investigate transmission patterns.

Fecal samples were obtained as part of a larger study of the Ontario dairy small ruminant industries, between October 2010 and August 2011, with the purpose to determine the herd- and flock-level prevalence of Map infection and to evaluate the accuracy of 7 individual-animal diagnostic tests for paratuberculosis. This study received approval from the University of Guelph Research Ethics Board (10JN004) for human participants and the University of Guelph Animal Care Committee (10R057) for use of animals. The sampling methodology was previously described (13,14). In brief, dairy goat herds (N = 29) and dairy sheep flocks (N = 21) were randomly and conveniently selected, respectively. A complete random sample of the dairy sheep population was not possible, as these producers are not required to be licensed under the Ontario Milk Act; therefore, a complete list of farms was not available. Twenty lactating animals (n = 20) 2 y and older that appeared to be healthy were randomly selected from each goat and sheep farm. Fecal samples were obtained per-rectum using individual single-use polyethylene gloves and samples were stored between 4°C and 8°C until submission for processing.

Submitted fecal samples were decontaminated and cultured according to manufacturer’s instructions at the Animal Health Laboratory (AHL, University of Guelph, Ontario) using the BD BACTEC MGIT 960 Mycobacterial Detection system (Becton Dickinson and Company, Franklin Lakes, New Jersey, USA). The standard incubation length for samples in the system is 49 d; however, this was extended to 240 d to give the opportunity for slower-growing strains of Map to be detected (14). Culture positive samples underwent acid-fast staining and PCR confirmation using the MAP-specific hspX gene (8) (Culture Confirmation Protocol, MAP Extraction System, Tetracore, Rockville, Maryland, USA).

At the beginning of October 2012, uncontaminated mycobacteria growth indicator tubes (MGIT) from culture positive feces were subcultured on modified Middlebrook 7H10 solid media (15) to obtain isolated colonies of Map and purify the cultures. Every 500 mL of media contained: 7H10 (9.5 mg), water (440 mL), glycerol (2.5 mL), oleic acid, albumin, dextrose, and catalase (OADC; 50 mL), Mycobactin J (0.5 mL of 1 mg/mL), and PANTA (10 mL of 2%) (Becton Dickinson and Company, Franklin Lakes, New Jersey, USA). The samples were incubated at 37°C and checked weekly for growth; any visibly contaminated samples were immediately discarded. All samples demonstrating growth by February 1, 2013 were submitted for strain typing, which was performed at the Department of Production Animal Health at the University of Calgary.

Prior to genetic analysis, DNA extraction of subcultured colonies was achieved through a 2-min bead-beating step in lysis buffer, followed by the spin-column DNeasy blood and tissue kit protocol (Qiagen, Mississauga, Ontario). Amplification for Map confirmation (IS900) and strain typing (MAP1506) was performed as described previously (10,11). Amplified DNA was submitted for Sanger sequencing (University Core DNA Services, University of Calgary, Alberta) and analyzed using Geneious software v.6.1 (Biomatters available at www.geneious.com).

In the original project (13,14), 106 dairy goat and 30 dairy sheep fecal samples had tested positive using the abovementioned culture method and confirmatory procedure. Subculturing and strain typing were performed after all fecal samples had been processed in batches and incubated until either growth was observed or 240 d had passed. Therefore, MGIT tubes ranged in age from 8 to 18 mo and a high proportion of contamination had occurred (47.2% in goat samples and 53.3% in sheep samples). Cultures of 56 goat fecal isolates (52.8%) and 14 sheep fecal isolates (46.6%) were uncontaminated and available to be subcultured onto modified Middlebrook 7H10 solid agar medium. After 4 mo of incubation, 26 goat samples (46.4%) and 8 sheep samples (57.1%) demonstrated visible colonies (all non-pigmented) and were submitted for Map PCR confirmation using IS900. The 8 sheep isolates and 25 of the 26 goat isolates were confirmed as Map based on IS900 testing, indicating these were Map. One goat isolate was IS900 negative and was likely a contaminant. Of the colonies submitted for typing, 22 goat (88.0%) and 2 sheep samples (25.0%) were successfully amplified. Based on MAP1506 typing, both samples from sheep were determined to be Type S/subtype III; one goat sample was also Type S/subtype III (4.8%). The remaining goat samples were Type C (n = 21; 95.2%) and no Type S/subtype I were identified. The predomination of the Type C strain in the goat population of Ontario was expected based on similar results generated by studies undertaken in goat populations in Mexico and Spain (16,17) and supports the use of the BACTEC MGIT 960 system for culturing fecal samples from goats suspected to be shedding Map. The Mexican and Spanish studies also reported the sporadic occurrence of Type S/subtype III in their goat populations (16,17). This subtype has also been previously identified in fecal samples of dairy cattle in Canada (12/602; 0.2%) (18). The dairy goat farm in the present study that owned the goat with Type S/subtype III was a relatively new producer who had amalgamated a herd from 2 smaller herds. While there was no concurrent cohabitation with sheep on his farm, it could not be ruled out that the goat had not had prior contact with sheep.

Overall, the sample size of typeable isolates in this study was small due to the high proportion of contamination in the original samples and the poor success when subculturing these bacteria. With only 2 typeable sheep samples available, this is too small a sample to make any generalizations other than they were both Type S/subtype III. While no Type S/subtype I was isolated from sheep or goats in this study, it does not imply that the strain is not present in animals in Ontario. Since Type S/subtype I is the predominant strain in sheep in other countries (19) the lack of isolation of this strain from sheep in this study supports the concern that the laboratory method used to culture the feces in the initial phase of the study likely does not support growth of Type S/subtype I (14,20). Therefore, prevalence results based only on this fecal culture method may under-represent the true number of sheep that were shedding Map in their feces.

All samples identified as Type C underwent further testing to determine VNTR type based on 8 specific VNTR loci: 292, X3, 25, 47, 3, 7, 10, and 32, as previously described (12). VNTR types [INRA Nouzilly MIRU-VNTR (INMV) profile number] were assigned according to the MAC-INMV database (21). Typing of the 21 goat samples (representing 11 farms) identified 3 different VNTR types: INMV 1 (10 isolates; 47.6%), INMV 2 (10 isolates; 47.6%), and 1 type (4.8%) that had not been reported previously and therefore had not been assigned a type number. To gain insight into whether these VNTR types were goat-specific or present in other animal species, results were compared to those obtained from a recent study of Canadian dairy cattle isolates (18). When 527 cattle isolates from across the country were typed, 76% were typed as INMV 2 (n = 398 isolates) and 7% were typed as INMV 1 (n = 36 isolates). Of the 52 Ontario dairy cattle isolates typed in that study, the predominant INMV types were 17, 2, 3, 6 with no VNTR Type 1 identified (18). Sample sizes for both the dairy goat and dairy cattle populations are too small to draw any conclusions that a particular VNTR type is strictly limited to either species.

The 21 VNTR typed isolates represented 11 goat farms, with 6 farms having isolates from multiple goats. Two of these farms had 2 VNTR types circulating (INMV 1 and 2), 1 farm had 2 VNTR types (INMV 1 and 2) circulating in addition to the isolate identified as Type S subtype III, while 3 of the farms only had 1 VNTR type circulating. No information was available on the history of movements between these farms, but they are all a significant distance from one another (> 100 km). A more detailed exploration of an infected farm is needed, wherein the isolates from all infected animals would undergo subtyping, to determine the true diversity within these herds before performing traceback investigations.

The typing method reported here proved very useful in an Australian investigation of the origin of paratuberculosis cases in a previously low prevalence region. The study demonstrated that the subtypes of Map isolated from the infected animals were genotypically different from those previously isolated from the higher prevalence regions of Australia and were thus unlikely to have originated from violations of the livestock travel bans (11). The subtypes of the isolates in Ontario goats do not demonstrate great diversity. The only difference between the predominant subtypes INMV 1 and INMV 2 is the number of copies at the VNTR-MIRU 292 locus (3 versus 4). Therefore, this apparent lack of diversity currently limits the use of this method in this Ontario population to investigate patterns of transmission. While Kasnitz et al (22) concluded that VNTR subtypes are very stable through multiple in vitro cultivations and in vivo passage, they concluded that this method still lacks discriminatory power to be epidemiologically relevant and should be used in conjunction with other testing.

When these isolates were also analyzed using SSR-typing based on the 8-ggt locus (only 16 produced bands), samples typed as INMV 1 had 4 copies of 8-ggt, while INMV 2 had 5 copies. The 3 Type S/subtype III isolates had 3 copies of 8-ggt as did the isolate that had the previously unreported INMV type. Therefore, SSR-typing, using only the 8-ggt locus, did not discriminate the isolates any further when used with VNTR-typing; however, greater diversity may be observed when VNTR is combined with multiple SSR-loci (11).

In summary, this study identified the dominant circulating Map strain in the isolates of this dairy goat population to be Type C, of which half were subtyped as INMV 1 and the other half as INMV 2. These results support interspecies transmission between dairy goats and dairy cattle. The 2 isolates from sheep were identified as Type S/subtype III. In the future, increasing the incubation period of sheep samples and using culture methods that are more supportive of Type S/subtype I Map would help discern if subtype I or III is the dominant strain in dairy sheep in Ontario. This would also help inform determination of the optimum method for culturing future sheep fecal samples. With only 1 goat isolate identified as Type S/subtype III, this type appears to occur at a comparatively low level in the goat population.

The current study demonstrates that understanding the epidemiology of Map is inherently challenged by the difficulties associated with culturing the different strains of the bacteria and the lack of genomic diversity (4) using SSR and VNTR typing. Caution needs to be exercised when using VNTR subtyping in the future. Ahlstrom et al (23) recently raised the concern about the potential to misclassify relatedness when typing is based on VNTR loci alone. When they compared whole genome sequencing to the results from VNTR testing, they observed a large degree of variability between isolates of the same VNTR type and a large degree of similarity between isolates of different VNTR types. Therefore, as the barriers to the routine use of whole genomic sequencing come down, this will likely be the default method for investigating Map transmission dynamics in small ruminant dairy populations in Ontario.