Abstract
Digital dermatitis (DD) is an important cause of lameness in dairy cattle worldwide. It has now been reported in beef cattle and also sheep (contagious ovine digital dermatitis [CODD]). Three Treponema phylogroups are consistently isolated from lesions, Treponema medium-like, Treponema phagedenis-like, and Treponema pedis. The gastrointestinal (GI) tract and feces are suggested sites of treponemal infection in dairy cattle; however, isolation of DD-associated treponemes from these areas has previously failed. This study surveyed gingival tissues, rectal tissues, and feces of beef cattle and sheep for the molecular presence (PCR) and isolation of the three cultivable DD-treponeme phylogroups. Of the sheep gingival (n = 40) and rectal (n = 40) tissues, 1/40 gingival tissues was positive for DD-associated treponemes (T. pedis), as were 3/40 rectal tissues (one containing T. medium-like and two containing T. pedis). No DD-associated treponeme DNA was amplified from beef cattle rectal tissues (n = 40); however, 4/40 beef gingival tissues were positive for DD-associated treponemes (all containing T. phagedenis-like). A T. phagedenis-like DD-associated treponeme was isolated from the rectal tissue of a CODD symptomatic sheep. Beef cattle (n = 41) and sheep (n = 79) feces failed to amplify DD-associated Treponema DNA. Twenty-two treponemes were isolated from sheep feces; however, upon phylogenetic analysis, these clustered with the considered nonpathogenic treponemes. This study detected DD-associated treponemes in the GI tract tissues of sheep and beef cattle and successfully isolated a DD-associated treponeme from ruminant rectal tissue. This gives evidence that the GI tract is an important infection reservoir of DD-associated treponemes in multiple DD-infected species.
INTRODUCTION
Digital dermatitis (DD) is an ulcerative lesion of the digital skin (1), causing severe lameness in dairy cattle worldwide. The disease was first reported in dairy cattle in 1974, where it is known as bovine digital dermatitis (BDD) (1, 2). The disease more recently has been reported in sheep (3, 4), where it is known as contagious ovine digital dermatitis (CODD), and now has been confirmed in beef cattle (5, 6).
Over the last few decades, DD has been recognized as an important cause of bovine lameness (7) with serious animal welfare and economic implications, including reduced fertility and milk yield and an increase in the risk of culling (8–10). DD is now a worldwide problem, and controlling the disease on dairy operations has proven difficult due to there being no single effective treatment available. Furthermore, there are no data on treatment of DD in beef cattle, and treatment of CODD is difficult and rarely effective.
The primary causative agents of DD are considered to be the spirochetal bacteria, treponemes, with a polytreponemal etiology (11–13). Cloning bacterial 16S rRNA genes indicated five phylogroups of treponemes in BDD lesions from Germany (14). Three Treponema phylogroups have been consistently isolated from dairy cattle lesions in the United Kingdom and United States (15, 16). The phylogroups are described as Treponema medium-like, Treponema phagedenis-like, and Treponema denticola-like BDD spirochetes (16), with the latter now recognized as a new species, Treponema pedis (17). These DD treponeme phylogroups now have been detected and isolated in beef cattle DD lesions (5, 6) and CODD lesions (18, 19). Additionally, these specific Treponema phylogroups also have been reported in manifestations of DD in goats in the United Kingdom (20) and North American Elk (Cervus elaphus) from Washington State (21). Due to the promiscuous nature of these treponemes and their growing host range, it is important to identify possible infection reservoirs of these bacteria and then routes and means of transmission.
Although the disease has been reported for many years, little is known about the routes of transmission or infection reservoirs of DD. There has been much debate about the gastrointestinal (GI) tract's role as a reservoir of infection of DD treponemes, with the bovine gingival and rectal tissues, rumen fluid, and feces identified as potential infection reservoirs. A previous study detected DD treponemes in the oral cavity and the rectum of dairy cattle (22), and more recent work detected DD treponeme phylogroups in rumen fluid, fecal samples, and slurry (23–25).
To date, isolations of DD treponemes from the bovine GI tract have failed. The sheep GI tract has yet to be investigated as a reservoir of infection, and the tendency of beef cattle to be different breeds, fed different diets, and subjected to different housing regimes than dairy cattle gives reason for further investigations into both of these animals' GI tracts. The current study aimed to further our understanding of the infection reservoirs of DD by surveying a number of beef cattle and sheep gingival tissues, rectal tissues, and feces from both DD symptomatic and asymptomatic animals for the detection and isolation of DD-associated treponemes.
MATERIALS AND METHODS
Animal tissues and fecal collection.
Gingival and rectal tissues were collected using sterile scalpels to extract 3-cm2 biopsy specimens postmortem. The gingival tissue was removed from the mouth of the animal (sheep/beef cattle) where the gum meets the first or second molar. To obtain the rectal tissue samples, the recto-anal junction was removed from the animal (sheep/beef cattle), and an approximately 3-cm2 piece of rectal tissue then was removed from this using scalpel blades, inclusive of the intestinal mucosa. Tissue biopsy samples then were divided in two with half transferred into transport medium and placed on ice for subsequent Treponema culture. Transport medium consisted of oral treponeme enrichment broth (OTEB; Anaerobe Systems, Morgan Hill, CA) with the addition of the antibiotics rifampin (5 μg/ml) and enrofloxacin (5 μg/ml). The remaining half of tissues (for PCR analysis) were transported on ice and stored at −20°C.
A total of 40 sheep and 40 beef animals were sampled for gingival and rectal tissues. Twelve of the 40 sheep which had rectal and gingival tissues investigated were CODD symptomatic, and the remaining 28 were asymptomatic. Twelve of the 40 beef animals which had rectal and gingival tissues investigated were BDD symptomatic, and the remaining 28 were asymptomatic. Beef cattle were defined as BDD symptomatic if one or more feet had a clear lesion consistent with the clinical signs of DD (1, 2). Briefly, lesions presented as circular areas of brown/gray moist exudate (30- to 60-mm diameter) in the region of the caudal interdigital cleft at the junction of the skin and horn of the heel with an underlying raw proliferative area. Sheep were defined CODD symptomatic if one or more feet had a clear lesion consistent with CODD clinical signs (26). These signs, although varied, include an ulcerative/granulomatous lesion at the coronary band which may extend under the hoof wall and in some cases lead to avulsion of the entire hoof capsule.
Sheep gingival (n = 12) and rectal tissues (n = 12) (from 12 sheep) were collected from four farms in Wales (United Kingdom), farms 1 to 4, and the remaining 28 rectal and gingival were tissues collected from a fallen stock center which receives animals from farms within Lancashire, Cheshire, and South Cumbria (England, United Kingdom). Prior to tissue biopsy specimen removal, all of these animals had been euthanized for additional reasons. Eleven beef rectal and gingival samples were obtained from two farms, one located in Gloucestershire, England (farm 5), and one located in Wales (farm 6). These beef rectal (n = 11) and gingival (n = 11) samples were collected using a swabbing technique rather than as tissue biopsy specimens, as they were taken from live animals. This was done by running a plain sterile cotton swab over the tissue several times and then processing the sample according to biopsy samples. The remaining 29/40 beef rectal and 29/40 gingival tissues were obtained from the fallen stock center as described above. All GI tissue/swab samples were collected between January 2013 and September 2014.
Fecal samples were collected from sheep (n = 79) and beef cattle (n = 41). All fecal samples were fresh, taken immediately after defecation. Samples were collected individually (∼10 g) into sterile containers (for later PCR analysis), while a small portion (∼1 g) was transferred into transport medium and placed on ice for subsequent Treponema culture. Samples for PCR analysis were transported on ice (without transport medium) and subsequently stored at −20°C. Sheep feces samples were collected from two farms, n = 55 from farm 1 (sampled in this study for GI tissues) and n = 25 from a farm in Wales (farm 7). When fecal samples were collected, DD status was subsequently determined. Of a total of 79 sheep fecal samples, 29 were obtained from CODD symptomatic sheep, and the remaining 50 were from CODD asymptomatic animals. Beef feces were collected similarly to sheep feces from two other farms located in Wales, United Kingdom, namely, farm 6 (also investigated for sheep GI tissues) and another farm located in Wales, United Kingdom (farm 8), where 15/41 fecal samples were from DD symptomatic beef animals and 26/41 were from DD asymptomatic beef animals. Fecal samples were collected from January 2013 to July 2014.
All farms from which sheep rectal, gingival, and fecal samples were obtained were farms where CODD was endemic with the exception of farm 3, which was CODD negative. All farms from which beef cattle rectal, gingival, and fecal samples were obtained were farms where DD is endemic.
Culture of spirochetes.
Bacterial isolation, specifically for treponemes, was attempted on all samples: rectal tissue samples (n = 40), gingival tissue samples (n = 40), and feces (n = 41), from beef cattle and sheep (n = 79), respectively. A (1- to 1.5-mm) piece of tissue/swab was transferred from the transport medium into an anaerobic cabinet (85% N2, 10% H2, and 5% CO2, 36°C) and inoculated into OTEB with 10% fetal calf serum (FCS) containing the antibiotics rifampin (5 μg/ml) and enrofloxacin (5 μg/ml). The same was performed for feces samples, but a concentration of 25 μg/ml for rifampin was used. Bacteria subsequently were subcultured (around 2 to 5 days later) on fastidious anaerobe agar (FAA) plates (LabM, Bury, United Kingdom) with 5% defibrinated sheep blood, 10% FCS, and antibiotics as described above. After 1 to 2 weeks, single colonies were inoculated into growth media and checked for pure culture by phase-contrast microscopy. DNA was extracted from cultures and the isolated organisms identified using a 16S rRNA gene PCR as described previously (16).
DNA extraction from tissue, swab, and feces samples.
For PCR analysis, tissues/swabs were thawed and DNA extracted using a DNeasy blood and tissue kit (Qiagen, United Kingdom) according to the manufacturer's instructions. Fecal samples were thawed and DNA extracted using a QIAamp DNA stool minikit (Qiagen, United Kingdom) according to the manufacturer's instructions. All genomic DNA was stored at −20°C.
Genus and phylogroup-specific treponeme PCR assays.
All samples (n = 280) were subjected to the Treponema genus PCR assay, which detects all Treponema species, both pathogenic and commensal (27). All samples also were subjected to nested PCR assays specific for the three DD-associated treponeme groups, T. medium-like, T. phagedenis-like, and T. pedis, as described previously (13, 16). Resulting PCR products encompassed 300 to 500 bp of the 16S rRNA gene. In each assay, water was used as a negative control, and positive controls included genomic DNA from each of the three treponeme phylogroups.
Phylogenetic analysis of spirochete isolates.
To understand the relationship of the isolated spirochetes with other treponemes and, in particular, those previously isolated from DD lesions and ruminant GI tracts (considered commensals), a phylogenetic tree was produced. The phylogenetic tree compared the aligned and trimmed nearly entire 16S rRNA gene PCR product sequences from the isolates obtained in this study with relevant bacterial sequences available in GenBank and identified using BLAST (28). Sequences were assembled using Chromas Pro 1.41 (Technelysium Pty Ltd.) into a double-stranded consensus sequence. Consensus sequences were aligned by ClustalW (29) in Mega 5.2 (30). For phylogenetic tree analysis, the most appropriate evolution model was predicted using “model test” in the Topali program (31). The final model for nucleotide substitutions chosen was the TrN model (32), which was used to infer a bootstrapped maximum likelihood tree (bootstrapping was performed 10,000 times).
Statistical analysis.
Fisher exact tests were used to investigate associations between the presence of DD-associated Treponema phylogroups in the GI tract of beef cattle and sheep and the DD status of the animal. Additionally, a chi-square test (with Yates correction) was used to identify associations between the DD status of animals and the isolation of Treponema spp. from fecal samples. The statistical test used was determined by the software used based on values for statistical analyses. This was carried out using GraphPad InStat software, version 3.10 (GraphPad Software). In all analyses, an associated probability (P value) of <0.05 was considered significant.
DD treponeme detection rates in beef cattle, sheep, and dairy cattle gastrointestinal tract tissues.
A comparison of PCR detection rates of Treponema DD phylogroups in the GI tissues of dairy cattle, beef cattle, and sheep was carried out using the data produced in this study and previously collected dairy cattle data (22). The data were compared by the number of animals in which a DD treponeme phylogroup (or multiple phylogroups) was detected in GI tract tissue.
Nucleotide sequence accession numbers.
The 16S rRNA gene GenBank accession numbers determined as part of this study are KR052445 to KR052467.
RESULTS
Treponema genus- and phylogroup-specific PCR survey of gastrointestinal tissues.
The results of the specific DD Treponema phylogroup and Treponema genus PCR assays of sheep rectal and gingival tissues and of beef rectal and gingival tissues are shown in Tables 1 and 2, respectively.
TABLE 1.
PCR detection and isolation of treponemes in sheep rectal and gingival tissues
| Animal | Collection date (mo/yr) | Locationa | DD status | Detection result |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Rectal tissue |
Gingival tissue |
||||||||||||
| Specific PCR for groupb: |
Treponema PCR | Isolationc | Specific PCR for groupb: |
Treponema PCR | Isolationc | ||||||||
| 1 | 2 | 3 | 1 | 2 | 3 | ||||||||
| 1 | 06/13 | F1 | + | − | − | − | + | IF | − | − | − | + | IF |
| 2 | 06/13 | F1 | + | − | − | − | + | IF | − | − | − | − | IF |
| 3 | 06/13 | F1 | + | − | − | + | + | IF | − | − | + | + | IF |
| 4 | 06/13 | F1 | + | − | − | + | + | IF | − | − | − | − | IF |
| 5 | 08/13 | F2 | + | − | + | − | + | SR5R | − | − | − | + | IF |
| 6 | 09/13 | F3 | − | − | − | − | + | IF | − | − | − | − | IF |
| 7 | 09/13 | F3 | − | − | − | − | + | IF | − | − | − | − | IF |
| 8 | 09/13 | F3 | − | − | − | − | + | IF | − | − | − | − | IF |
| 9 | 09/13 | F3 | − | − | − | − | + | IF | − | − | − | − | IF |
| 10 | 12/13 | F4 | + | − | − | − | + | IF | − | − | − | − | IF |
| 11 | 12/13 | F4 | + | − | − | − | + | IF | − | − | − | − | IF |
| 12 | 12/13 | F4 | + | − | − | − | + | IF | − | − | − | − | IF |
| 13 | 01/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 14 | 01/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 15 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 16 | 03/14 | FSC | − | − | − | − | − | IF | − | − | − | + | IF |
| 17 | 03/14 | FSC | − | − | − | − | − | IF | − | − | − | + | IF |
| 18 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 19 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 20 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 21 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 22 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 23 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 24 | 03/14 | FSC | + | − | − | − | + | IF | − | − | − | + | IF |
| 25 | 03/14 | FSC | + | − | − | − | + | IF | − | − | − | − | IF |
| 26 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 27 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 28 | 03/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 29 | 04/14 | FSC | − | − | − | − | − | IF | − | − | − | + | IF |
| 30 | 04/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 31 | 04/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 32 | 04/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 33 | 04/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 34 | 04/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 35 | 04/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 36 | 04/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 37 | 04/14 | FSC | + | − | − | − | − | IF | − | − | − | + | IF |
| 38 | 04/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 39 | 05/14 | FSC | + | − | − | − | + | IF | − | − | − | + | IF |
| 40 | 05/14 | FSC | + | − | − | − | + | IF | − | − | − | + | IF |
F, farm with corresponding number; FSC, fallen stock center.
Groups 1, 2, and 3 are T. medium-like, T. phagedenis-like, and T. pedis spirochetes, respectively, which are routinely found in bovine DD lesions.
All isolations are shown for comparison to PCR results. IF, isolation failed. Successful isolations have strains listed.
TABLE 2.
PCR detection and isolation of treponemes in beef cattle rectal and gingival tissues
| Animal | Collection date (mo/yr) | Locationa | DD status | Detection result |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Rectal tissue |
Gingival tissue |
||||||||||||
| Specific PCR for groupb: |
Treponema PCR | Isolationc | Specific PCR for groupb: |
Treponema PCR | Isolationc | ||||||||
| 1 | 2 | 3 | 1 | 2 | 3 | ||||||||
| 1 | 01/13 | F1 | + | − | − | − | + | IF | − | − | − | − | IF |
| 2 | 01/13 | F1 | + | − | − | − | + | IF | − | − | − | − | IF |
| 3 | 01/13 | F1 | + | − | − | − | + | IF | − | − | − | − | IF |
| 4 | 04/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 5 | 04/14 | FSC | + | − | − | − | − | IF | − | − | − | + | IF |
| 6 | 04/14 | FSC | + | − | − | − | − | IF | − | − | − | − | IF |
| 7 | 05/14 | FSC | − | − | − | − | − | IF | − | − | − | − | IF |
| 8 | 05/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 9 | 05/14 | FSC | − | − | − | − | − | IF | − | − | − | − | IF |
| 10 | 05/14 | FSC | − | − | − | − | − | IF | − | − | − | − | IF |
| 11 | 05/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 12 | 05/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 13 | 05/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 14 | 05/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 15 | 06/14 | FSC | − | − | − | − | − | IF | − | − | − | − | IF |
| 16 | 06/14 | FSC | + | − | − | − | + | IF | − | − | − | − | IF |
| 17 | 06/14 | FSC | + | − | − | − | − | IF | − | − | − | + | IF |
| 18 | 06/14 | FSC | − | − | − | − | − | IF | − | − | − | + | IF |
| 19 | 06/14 | FSC | − | − | − | − | − | IF | − | − | − | − | IF |
| 20 | 06/14 | FSC | − | − | − | − | − | IF | − | + | − | + | IF |
| 21 | 06/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 22 | 06/14 | FSC | − | − | − | − | + | IF | − | − | − | − | IF |
| 23 | 07/14 | F6 | + | − | − | − | + | IF | − | − | − | + | IF |
| 24 | 07/14 | F6 | + | − | − | − | + | IF | − | − | − | − | IF |
| 25 | 07/14 | F6 | − | − | − | − | + | IF | − | − | − | + | IF |
| 26 | 07/14 | F6 | + | − | − | − | + | IF | − | − | − | − | IF |
| 27 | 07/14 | F6 | + | − | − | − | + | IF | − | − | − | + | IF |
| 28 | 07/14 | F6 | − | − | − | − | + | IF | − | − | − | + | IF |
| 29 | 07/14 | F6 | + | − | − | − | + | IF | − | − | − | − | IF |
| 30 | 07/14 | F6 | + | − | − | − | + | IF | − | − | − | − | IF |
| 31 | 08/14 | FSC | − | − | − | − | − | IF | − | − | − | + | IF |
| 32 | 08/14 | FSC | − | − | − | − | − | IF | − | − | − | − | IF |
| 33 | 08/14 | FSC | − | − | − | − | − | IF | − | − | − | − | IF |
| 34 | 08/14 | FSC | − | − | − | − | + | IF | − | + | − | + | IF |
| 35 | 08/14 | FSC | − | − | − | − | + | IF | − | − | − | + | IF |
| 36 | 08/14 | FSC | − | − | − | − | − | IF | − | − | − | + | IF |
| 37 | 08/14 | FSC | − | − | − | − | − | IF | − | − | − | − | IF |
| 38 | 08/14 | FSC | − | − | − | − | + | IF | − | + | − | + | IF |
| 39 | 08/14 | FSC | + | − | − | − | + | IF | − | + | − | + | IF |
| 40 | 09/14 | FSC | + | − | − | − | + | IF | − | − | − | − | IF |
F, farm with corresponding number; FSC, fallen stock center.
Groups 1, 2, and 3 are T. medium-like, T. phagedenis-like, and T. pedis spirochetes, respectively, which are routinely found in bovine DD lesions.
All isolations are shown for comparison to PCR results. IF, isolation failed. Successful isolations have strains listed.
Treponema DNA (as determined using the Treponema genus PCR assay) was present in 36/40 (90%) and 20/40 (50%) of sheep rectal samples and gingival samples, respectively. Phylogroup-specific PCR assays for T. medium-like, T. phagedenis-like, and T. pedis DD spirochetes showed that no T. medium-like DNA was present in any sheep rectal (n = 40) or gingival tissues (n = 40); however, 1/40 sheep rectal tissues were positive for T. phagedenis-like DD spirochetes, and 2/40 sheep rectal tissues were positive for T. pedis DD spirochetes. All three positive rectal tissues were obtained from CODD symptomatic sheep (animals 3, 4, and 5). Neither T. medium-like nor T. phagedenis-like DD spirochetes were detected in any of the sheep gingival tissues; however, T. pedis DD spirochetes were present in 1/40 of the sheep gingival tissues. This T. pedis-infected gingival tissue was obtained from a CODD symptomatic sheep which also had T. pedis DD spirochete DNA present in it is rectal tissue (animal 3).
Treponema DNA (identified using the Treponema genus PCR assay) was present in 25/40 (63%) and 17/40 (43%) of beef cattle rectal samples and gingival samples, respectively. The phylogroup-specific PCRs for T. medium-like, T. phagedenis-like, and T. pedis DD spirochetes amplified no DNA from beef cattle rectal tissues. However, 4/40 gingival tissues were positive for T. phagedenis-like DD spirochetes (animals 28, 34, 38, and 39). No T. medium-like or T. pedis DD spirochete DNA was amplified in beef cattle gingival tissues.
The presence of one or more DD Treponema phylogroups in the GI tract of sheep and beef cattle was analyzed according to the season from which the sample was collected (Table 3). Seasons were defined as winter (December-February), spring (March-May), summer (June-August), and autumn (September-November). As shown in Table 5, only GI tract tissues collected in summer were positive for DD Treponema phylogroup DNA. This was true for both sheep and beef cattle GI tract tissues.
TABLE 3.
Comparison of PCR detection rates of Treponema DD phylogroups in the GI tissues of beef cattle and sheep in different seasonsa
| Season | No. (%) of GI tract tissues positive for infectionb |
|
|---|---|---|
| Sheep | Beef cattle | |
| Winter (December-February) | 0/5 (0) | 0/3 (0) |
| Spring (March-May) | 0/28 (0) | 0/11 (0) |
| Summer (June-August) | 3/5 (60) | 4/25 (16) |
| Autumn (September-November) | 0/4 (0) | 0/1 (0) |
Gingival and rectal tissue results have been combined to give a value and percentage of GI tract tissues positive for each species at the different times of year.
GI tract tissues refers to rectal and oral cavity tissues combined.
TABLE 5.
PCR detection and isolation of treponemes in beef cattle fecal samples
| Sample(s) | Collection date (mo/yr) | Location sample obtaineda | DD status | Treponeme isolatedb | Result |
|||
|---|---|---|---|---|---|---|---|---|
| Specific PCR for groupc: |
Treponema PCR | |||||||
| 1 | 2 | 3 | ||||||
| 1–2 | 03/13 | F6 | + | IF | − | − | − | + |
| 3–7 | 03/13 | F6 | − | IF | − | − | − | + |
| 8 | 03/13 | F6 | + | IF | − | − | − | + |
| 9–13 | 07/14 | F8 | − | IF | − | − | − | + |
| 14, 15 | 07/14 | F8 | + | IF | − | − | − | + |
| 16 | 07/14 | F8 | − | IF | − | − | − | − |
| 17, 18 | 07/14 | F8 | − | IF | − | − | − | + |
| 19 | 07/14 | F8 | + | IF | − | − | − | + |
| 20 | 07/14 | F8 | − | IF | − | − | − | + |
| 21, 22 | 07/14 | F8 | + | IF | − | − | − | + |
| 23–25 | 07/14 | F8 | − | IF | − | − | − | + |
| 26 | 07/14 | F8 | − | IF | − | − | − | − |
| 27–29 | 07/14 | F8 | − | IF | − | − | − | + |
| 30, 31 | 07/14 | F8 | + | IF | − | − | − | + |
| 32, 33 | 07/14 | F8 | − | IF | − | − | − | + |
| 34 | 07/14 | F8 | + | IF | − | − | − | + |
| 35–37 | 07/14 | F8 | − | IF | − | − | − | + |
| 38–41 | 07/14 | F8 | + | IF | − | − | − | + |
F, farm with corresponding number.
All isolations are shown for comparison to PCR results. IF, isolations failed. If isolation was successful, the isolated strains are listed.
Groups 1, 2, and 3 are T. medium-like, T. phagedenis-like, and T. pedis spirochetes, respectively.
Treponema genus- and phylogroup-specific PCR survey of fecal samples.
PCR assay (and isolation) results of sheep and beef fecal samples are shown in Tables 4 and 5, respectively.
TABLE 4.
PCR detection and isolation of treponemes in sheep fecal samples
| Sample(s) | Collection date (mo/yr) | Location sample obtaineda | DD status | Treponeme(s) isolatedb | Result |
|||
|---|---|---|---|---|---|---|---|---|
| Specific PCR for groupc: |
Treponema PCR | |||||||
| 1 | 2 | 3 | ||||||
| 1–4 | 01/13 | F1 | − | IF | − | − | − | + |
| 5 | 01/13 | F1 | + | IF | − | − | − | + |
| 6–10 | 01/13 | F1 | − | IF | − | − | − | + |
| 11 | 01/13 | F1 | − | IF | − | − | − | − |
| 12–19 | 01/13 | F1 | − | IF | − | − | − | + |
| 20 | 01/13 | F1 | + | SF20 | − | − | − | + |
| 21 | 01/13 | F1 | + | SF21a, SF21b | − | − | − | + |
| 22, 23 | 01/13 | F1 | − | IF | − | − | − | + |
| 24 | 01/13 | F1 | + | SF24a, SF24b | − | − | − | + |
| 25 | 01/13 | F1 | − | IF | − | − | − | + |
| 26 | 01/13 | F1 | + | SF26a, SF26b | − | − | − | + |
| 27, 28 | 01/13 | F1 | − | IF | − | − | − | − |
| 29 | 01/13 | F1 | + | SF29 | − | − | − | + |
| 30 | 01/13 | F1 | − | IF | − | − | − | − |
| 31 | 01/13 | F1 | − | IF | − | − | − | + |
| 32 | 01/13 | F1 | + | SF32a, SF32b | − | − | − | + |
| 33 | 01/13 | F1 | + | IF | − | − | − | + |
| 34 | 01/13 | F1 | + | IF | − | − | − | − |
| 35–37 | 01/14 | F1 | − | IF | − | − | − | + |
| 38 | 01/14 | F1 | − | IF | − | − | − | − |
| 39, 40 | 01/14 | F1 | − | IF | − | − | − | + |
| 41 | 01/14 | F1 | + | IF | − | − | − | + |
| 42, 43 | 01/14 | F1 | − | IF | − | − | − | + |
| 44 | 01/13 | F1 | + | IF | − | − | − | + |
| 45, 46 | 01/13 | F7 | − | IF | − | − | − | + |
| 47 | 01/13 | F7 | − | SF47 | − | − | − | + |
| 48- 50 | 01/13 | F7 | − | IF | − | − | − | + |
| 51 | 01/13 | F7 | + | SF51 | − | − | − | + |
| 52 | 01/13 | F7 | + | IF | − | − | − | + |
| 53 | 01/13 | F7 | + | SF53 | − | − | − | + |
| 54, 55 | 01/13 | F7 | + | IF | − | − | − | + |
| 56 | 01/13 | F7 | + | SF56 | − | − | − | + |
| 57 | 01/13 | F7 | + | IF | − | − | − | + |
| 58 | 01/13 | F7 | − | IF | − | − | − | + |
| 59 | 01/13 | F7 | + | SF59 | − | − | − | + |
| 60 | 02/13 | F7 | + | SF60 | − | − | − | + |
| 61 | 02/13 | F7 | − | IF | − | − | − | + |
| 62 | 02/13 | F7 | − | SF62 | − | − | − | + |
| 63 | 02/13 | F7 | − | IF | − | − | − | + |
| 64 | 02/13 | F7 | − | SF64 | − | − | − | + |
| 65 | 02/13 | F7 | + | IF | − | − | − | + |
| 66 | 02/13 | F7 | − | SF66 | − | − | − | + |
| 67 | 02/13 | F7 | + | IF | − | − | − | + |
| 68 | 02/13 | F7 | + | SF68 | − | − | − | + |
| 69 | 02/13 | F7 | + | SF69 | − | − | − | + |
| 70, 71 | 03/13 | F1 | + | IF | − | − | − | + |
| 72 | 03/13 | F1 | − | IF | − | − | − | + |
| 73 | 03/13 | F1 | + | IF | − | − | − | + |
| 74 | 03/13 | F1 | − | SF74 | − | − | − | + |
| 75 | 03/13 | F1 | − | IF | − | − | − | + |
| 76 | 03/13 | F1 | + | IF | − | − | − | + |
| 77–79 | 03/13 | F1 | − | IF | − | − | − | + |
F, farm with corresponding number.
All isolations are shown for comparison to PCR results. IF, isolations failed. If isolation was successful, the isolated strains are listed.
Groups 1, 2, and 3 are T. medium-like, T. phagedenis-like, and T. pedis spirochetes, respectively.
Using the Treponema genus PCR assay, Treponema DNA was identified as present in 73/79 (92%) and 39/41 (95%) of sheep and beef cattle feces samples, respectively.
All sheep (n = 79) and beef cattle feces (n = 41) were negative for T. medium-like, T. phagedenis-like, and T. pedis DD spirochete DNA as determined using the respective PCR assays.
Isolation and phylogenetic analysis of spirochetes.
The isolation of spirochetes was attempted from all rectal, gingival, and fecal samples. All isolation attempts were unsuccessful from beef cattle rectal and gingival tissues. Isolation attempts from sheep gingival tissues also were unsuccessful. However, a spirochete was isolated from 1 out of 40 sheep rectal tissues (animal 5). This isolate, SR5R (GenBank accession number KR052467), isolated from animal 5, was identified as belonging to the T. phagedenis-like DD spirochete group, sharing 100% 16S rRNA gene sequence identity with the T. phagedenis-like DD spirochete strain T320A (GenBank accession number EF061261), previously isolated from a United Kingdom dairy cow BDD lesion (16).
All treponeme isolation attempts from beef cattle fecal samples were unsuccessful. However, 22 spirochetes were successfully isolated from 18/79 (23%) sheep fecal samples. Multiple spirochetes were isolated from some fecal samples. All 22 isolates shared over 99% 16S rRNA gene sequence identity with Treponema sp. strain CHPA (GenBank accession number GU566699), previously isolated from the GI tract contents of a DD-positive dairy cow (22). Four of the 22 isolates shared 100% 16S rRNA sequence identity with Treponema sp. strain CHPA (GenBank accession number GU566699). Of the Treponema sp. strain CHPA isolates obtained from sheep feces, 17/22 (77%) were isolated from the feces of CODD symptomatic sheep. Sequence analysis revealed that the 22 isolates could be separated into four groups based on 16S rRNA gene sequences. Within each group, isolates shared between 99.7% and 100% 16S rRNA gene sequence identity with each other. These groups appeared to relate to the farm from which the animal which had produced the feces had originated (either farm 1 or farm 7). One of the groups of isolates consisted of eight treponemes, all of which came from animals from farm 1. The second group of isolates consisted of 10 treponemes, of which eight originated from farm 7 (the remaining two were from farm 1), and a third group of two isolates came from animals from farm 7. The last group consisted of two treponemes, one of which was from the feces of a sheep from farm 1 and the other from the feces of a sheep from farm 7.
There was a marked difference in phylogenetic location of the rectal tissue and fecal isolates, with rectal tissue isolate SR5R clustering with the T. phagedenis-like DD spirochete group, as would be expected, within the larger group of the DD pathogenic treponemes (Fig. 1). The 22 isolates obtained from sheep feces samples grouped with the commensal treponemes (lower half of Fig. 1), in particular Treponema sp. strain CHPA (GenBank accession number GU566699).
FIG 1.
Maximum-likelihood tree based on 16S rRNA gene sequence comparisons of ∼1,000 aligned bases. The tree shows the relationship between the strains isolated here (shown in boldface) from ruminant feces and GI tissue and other DD-associated and commensal treponeme 16S rRNA gene sequences. Bootstrapping was performed 10,000 times, and for clarity only bootstrap values above 70% are shown. An asterisk indicates previously reported 16S rRNA gene sequences from BDD lesions.
Statistical analysis.
From the results of the statistical analyses, no significant association was found between the presence of DD-associated Treponema spp. in the GI tract and the DD status of the animal for sheep (P = 0.58) or beef cattle (P > 0.99). However, a statistically significant association between DD status and the isolation of Treponema sp. strain CHPA was identified (P = 0.041).
DD treponeme detection in beef cattle, sheep, and dairy cattle GI tract tissues.
The summary of results for DD treponeme detection rates in beef cattle, sheep, and dairy cattle GI tract tissues is shown in Table 6. Of the beef cattle, dairy cattle (22), and sheep, 10%, 7.1%, and 2.5% had DD treponeme phylogroup DNA present in their gingival tissues, respectively. In terms of rectal tissues, 0%, 11.1%, and 7.5% were positive for DD treponeme phylogroup DNA in beef cattle, dairy cattle, and sheep, respectively.
TABLE 6.
Comparison of PCR detection rates of Treponema DD phylogroups in the GI tissues of dairy cattle, beef cattle, and sheep
| Animala (source or reference) | No. (%) of positive tissues |
|
|---|---|---|
| Rectal-anal junctionb | Gingival tissuec | |
| Dairy cattle (22) | 3/27 (11.1) | 1/14 (7.1) |
| Beef cattle (this study) | 0/40 (0) | 4/40 (10) |
| Sheep (this study) | 3/40 (7.5) | 1/40 (2.5) |
Animal GI tissues originated with reference to the corresponding study.
Only rectal-anal junction tissue positives have been used for the comparison and not rectal wall results.
GI tissue results from Evans et al. (22) have been corrected to give a figure for detection rate per tissue per animal in cases. Where tissues from the same animal were sampled multiple times, this has only been counted as 1 positive.
DISCUSSION
Previous studies of BDD in dairy cattle and beef cattle, and more recently CODD in sheep, show that in all DD lesions, one or more of the three cultivable DD treponeme phylogroups are present (6, 12, 13, 19, 23). However, although these phylogroups are consistently detected and isolated from cattle and sheep lesions, studies have failed to isolate these pathogenic treponemes from potential transmission vectors and infection reservoirs. The only evidence of DD treponemes surviving outside the foot itself was a recent study that isolated a T. phagedenis-like DD spirochete from a piece of trimming equipment used to trim the hoof of a BDD-positive cow foot (33).
Recently, work has focused on the GI tract as a possible reservoir of DD treponemes in dairy cows. Previously, DD treponemes were identified in two GI tract regions of dairy cattle, the oral cavity and rectal tissue, suggesting these areas as treponeme reservoirs (22). However, the study failed to isolate live DD treponemes from these tissues. Various other studies now have detected one or more of the commonly associated DD treponeme phylogroups in dairy cow GI tract-associated areas. One study detected at least one of the three cultivatable DD treponeme phylogroups in 60% of rumen fluid samples tested (24), and using a metagenomic approach, another identified DD treponemes in rumen fluid and fecal samples (25). Additionally, DD-associated treponemes have been identified in environmental samples such as manure slurry from dairy farms using high-throughput sequencing (23). The present study has, however, isolated one of the DD Treponema phylogroups from a ruminant rectal tissue sample. Importantly, this DD treponeme was isolated from a CODD-positive sheep, a host species of which the GI tract was not previously investigated as a reservoir for DD treponemes. The isolation of a DD treponeme from a deep-tissue sample from the rectal tissue of a DD-infected animal is evidence of live and, as a result, transmissible DD treponemes within host tissues other than the foot.
Although DD treponemes are detectable in ruminant GI tract tissues, they appear to be present in only a small percentage of animals. Three sheep had DD Treponema phylogroup DNA (7.5%) in their rectal tissue, one of which had T. pedis DD treponeme phylogroup DNA in both its gingival and rectal tissue. Although all beef cattle rectal tissues were negative for DD Treponema phylogroup DNA, four (10%) of the beef animals had T. phagedenis-like DD spirochete DNA present in their gingival tissue. The number of animals with DD treponeme DNA in their oral and rectal tissue is similar to what has been found in dairy cattle GI tract tissues (22). In terms of gingival tissues, the percentages of dairy cattle and beef cattle which contained Treponema DD phylogroup DNA were extremely similar, 7.1% and 10%, respectively. However, the percentage of sheep which contained Treponema DD phylogroup DNA in their gingival tissue was lower, 2.5% of animals tested. Conversely, the percentages of dairy cattle and sheep which contained Treponema DD phylogroup DNA in their rectal tissue were similar, 11.1% and 7.5%, respectively. In contrast, no Treponema DD phylogroup DNA was detected in beef rectal tissues. What is clear is that the percentage of animals with DD treponemes present in these GI tract tissues appears small; however, it is possible that only a small proportion of animals carry (in the GI tract) and shed these bacteria into feces, and this could be all that was needed to effectively spread these bacteria in the farm environment. This is particularly true for the closely related spirochetal bacterium Brachyspira hyodysenteriae, the causative agent of swine dysentery in pigs, in which there is often only a small number of carrier animals which are able to sustain infection in an affected farm (34, 35).
Interestingly, animals which were found to contain DD treponeme phylogroup DNA in either their oral cavity tissue or rectal tissue were not necessarily DD-positive animals. Only one of the four beef animals which contained DD treponeme phylogroup DNA in their gingival tissue was DD symptomatic. It might be predicted that it would be cows with DD lesions which had DD treponeme DNA in their GI tract (and possibly capable of shedding the bacteria), but it appears from these results that this is not necessarily the case, and statistical analysis confirmed this. Similarly, Evans et al. (22) showed that although gingival tissues were positive only for DD treponeme carriage in cattle that were BDD positive, rectal tissue DD treponeme carriage showed no indication of an association with symptomatic BDD. Additionally, the sheep which, upon PCR analysis, was found to contain DD treponeme phylogroup DNA (T. pedis) in its gingival tissue and its rectal tissue also had the same DD-associated Treponema phylogroup, T. pedis, present in its active CODD lesion (19). This has been observed in a previous study where a DD symptomatic dairy cow contained T. phagedenis-like DNA in it is gingival, rumen, and rectal tissues as well as in an active DD lesion (22).
From the PCR survey of GI tract tissues, it is interesting that there is a possible association between season and the carriage of DD treponemes in the GI tract. No DD-associated treponeme DNA was amplified from GI tract samples, for either sheep or beef, in any other season apart from summer. Reasons for this are unknown and contradict previous similar data on dairy cows (22) where the winter housing season was positively associated with DD treponeme carriage in the GI tract. This finding may be due to different management practices between the different host species. Alternatively, it suggests that there are episodes of shedding on farms, as the majority of positive sheep GI tract tissues were collected from one farm on 1 day. Similarly, the majority of the positive beef GI tract tissues were collected from the fallen stock center on the same day (although unknown, it is possible they originated from the same farm).
Compared to previous work detecting the commonly isolated DD treponeme phylogroups in dairy cattle feces (23, 25), it is interesting that we were unable to detect these same bacteria in either sheep (n = 79) or beef cattle (n = 41) fecal samples. This implies that these bacteria are in too low an abundance in fecal samples for detection using the DNA extraction and PCR assay techniques we have commonly used for detection of these bacteria in DD-affected feet. Alternatively, like the GI tract tissues, there may be only a small number of animals that shed the bacteria into their feces, so only an extremely large number of feces would be sufficient to detect the treponemal bacteria.
There was an extremely high isolation rate of spirochetes from sheep feces which was entirely due to isolation of Treponema sp. strain CHPA, previously isolated from the feces of a dairy cow (22). However, what is striking is the percentage of these isolates that were obtained from CODD symptomatic sheep. A surprising proportion, 17/22 (77%), of these spirochete isolates were isolated from CODD symptomatic sheep, confirmed by statistical analysis which identified a positive association between the DD status of the animal and the isolation of Treponema sp. strain CHPA from its feces. It is interesting to speculate whether these animals are in fact shedding various species of treponemes into the environment (including DD treponeme species), but due to the issues involved in treponeme culture biasing toward the growth of predominant bacterial species (nonpathogenic treponemes), we were only able to isolate this commensal species of treponeme. The commensal association data are based solely on isolation results; thus, given the fastidious nature of the bacteria, they may have unknown biases. Hence, further investigations could investigate this association further by using the development of a specific PCR assay or use of metagenomics with relevant primer sets to specifically target this treponeme species and to quantify Treponema sp. strain CHPA in both CODD symptomatic and asymptomatic sheep feces. The reason for such a high isolation rate of commensal treponemes from sheep feces yet no successful isolations from beef feces is unknown but may be due to differences in diet in beef animals and, as a result, feces constituents. Despite there being a wide diversity of treponemes isolated/identified in the rumen (36), it is interesting that the same phylogroup of rumen treponemes was consistently isolated. Treponema sp. strain CHPA, when previously isolated, was from dairy cow feces in the same geographical area. This suggests that this is not only a prominent, and previously not reported, ruminal commensal species in sheep but also is a commensal shared by ruminants in this geographical region. Further studies are needed to characterize and taxonomically appraise Treponema sp. strain CHPA toward proposition as a new species, given that it shares less than 97% 16S rRNA gene sequence similarity with the taxonomically defined closest relatives, suggesting it is a novel species (37).
Upon further genetic analysis of the treponemes isolated from the sheep fecal samples, it was apparent that there were four groups which were highly similar (sharing >99.9% sequence identity) based on 16S rRNA gene sequence analysis, with said groupings corresponding to the farm the fecal samples were collected from. This apparent evolution of ruminal treponemes on farms is something that has not been reported in the DD treponemes with dairy cattle BDD, beef cattle BDD, and sheep CODD treponemes from different farms/geographical areas showing little diversity (6, 16, 19).
The morphology and structure of treponemes, particularly their flagella, allows them to be highly motile bacteria capable of rotational and translational movement (38). It has previously been demonstrated, by antitreponemal immunohistochemical staining, that treponemes are capable of breaching the skin barrier via hair follicles and causing infection down into deep layers of surrounding tissues (17). The ability of treponemes to access such breaches in skin barriers and bury deep into tissues makes it unsurprising to find them in deep-tissue biopsy specimens of gingival and rectal tissue. However, this finding and the detection of treponemes in GI tract fluids suggest these treponemes are more adaptable bacteria than previously thought. Their role as a pathogen in the feet of ruminants is well known; however, their exact role while in these GI tissues is currently unknown.
The detection of DD-associated treponeme phylogroup DNA in both sheep and beef cattle GI tract tissues, even in low numbers, indicates that the GI tract is an important infection reservoir of DD treponemes in multiple DD-suffering host species. The isolation of live pathogenic treponemes from the rectal tissue of a CODD-positive sheep highlights that these bacteria can be live, and possibly are transmissible if shed, in at least this GI tract tissue. The presence of pathogenic DD-associated treponemes in both the gingival and rectal tissue of one animal suggests that certain animals carry DD treponemes throughout their GI tract. The inability to detect these same bacteria in feces indicates that only a small number of animals are able to shed these bacteria, and this may be key to disease transmission on farms. The role of Treponema sp. strain CHPA needs further investigation to fully understand any specific contribution to the GI tract microbiome and to the clarify the identified association with DD symptomatic animals.
ACKNOWLEDGMENTS
This work was funded by QMS, HCC, and EBLEX, a division of the Agriculture and Horticulture Development Board, United Kingdom.
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