- Email: [email protected]
Prevalence and persistence of Taylorella asinigenitalis in male donkeys
Veterinary Microbiology 160 (2012) 435–442
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Prevalence and persistence of Taylorella asinigenitalis in male donkeys James M. Donahue a, Peter J. Timoney b,*, Carla L. Carleton c, Judy V. Marteniuk c, Stephen F. Sells a, Barry J. Meade b a
University of Kentucky Veterinary Diagnostic Laboratory, P.O. Box 14125, Lexington, KY 40512, USA Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA c Department of Large Animal Clinical Science, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA‘ b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 March 2012 Received in revised form 13 June 2012 Accepted 15 June 2012
This study was undertaken to investigate the prevalence of Taylorella asinigenitalis in a subset of the donkey population of Michigan and in other equids on farms on which the organism was identified. Other aims were to further characterize the carrier state in terms of persistence and preferred sites of colonization of T. asinigenitalis in the male donkey as well as determine the genotype of any isolates of the organism. Initial testing of 43 donkeys and 1 mule turned up 4 (9.3%) donkeys culture positive for T. asinigenitalis. The 4 culture-positive donkeys resided on 2 farms accommodating a collective total of 89 equids, of which 23 (25.8%) were confirmed positive for T. asinigenitalis. The positive equid population on the 2 farms comprised 14 (67%) of 21 gelded donkeys, 8 (36.4%) of 22 intact male donkeys, and 1 (25%) of 4 gelded horses. T. asinigenitalis was not isolated from 27 female donkeys, 11 female horses, 2 female mules, 1 male horse, or 1 male mule resident on these premises. Isolations of the bacterium were obtained from a number of male donkeys whenever they were sampled over a span of 33 months; preferential sites of isolation were the urethral fossa (fossa glandis), dorsal diverticulum of the urethral sinus, and terminal urethra. Isolates of T. asinigenitalis from the 23 culture-positive equids comprised 2 genotypes, one identical to the type strain isolated in California in 1997, and the other identical to 2 strains isolated from donkey jacks in Kentucky in 1998. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Taylorella asinigenitalis Donkeys Normal flora CEM
1. Introduction In late October 1997, a Taylorella equigenitalis-like bacterium was recovered from a Mammoth donkey jack in California (Anonymous, 1998) and in early January 1998, a similar bacterium was recovered from 2 male donkeys, 7 nurse mares and 1 stallion in Kentucky (Jang et al., 2001; Meade et al., 2010). Based on DNA–DNA hybridization studies, these 3 isolates were not T. equigenitalis; they were
* Corresponding author at: Maxwell H. Gluck Research Center, University of Kentucky, Lexington, KY 40546, USA. Tel.: +1 859 218 1094; fax: +1 859 257 8542. E-mail address: [email protected] (P.J. Timoney). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.06.016
a new species in the genus Taylorella named Taylorella asinigenitalis (Jang et al., 2001). Since originally reported, T. asinigenitalis has been recovered from equids in Europe and the USA. The organism was isolated from a 3-year-old stallion in Sweden (Ba˚verud et al., 2006), that previously shared paddock space with a donkey jack in Belgium. In a second report (Franco et al., 2009) T. asinigenitalis was recovered from 2 donkey jacks in Apulia, Italy. They were at the same facility for over 3 years and were previously culture negative for Taylorella species. Breuil et al. (2011) published on the phenotypic and 16S ribosomal RNA gene diversity of 43 strains of T. asinigenitalis isolated from 22 donkey jacks, 2 stallions and 1 mare in France between 1995 and 2009. Based on sequence
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
436
analysis, strains fell into 2 clusters which seemed to correlate with their respective geographic origins. Recent reports from the US confirmed isolation of T. asinigenitalis from a mare in Oklahoma (Reilly et al., 2012) and a miniature donkey in Kentucky (E.S. Ford, personal communication). The source of T. asinigenitalis has not been identified in any of the published reports (Anonymous, 2000; Jang et al., 2001; Katz et al., 2000; Meade et al., 2010), and very little is known about the epidemiology of T. asinigenitalis, particularly its persistence in equid populations. Accordingly, the following study was undertaken to determine: (1) the prevalence of T. asinigenitalis in a subset of the donkey population in Michigan presented for castration, (2) the prevalence of T. asinigenitalis in all equids on farms with a known culture-positive donkey jack, (3) the duration of persistence of T. asinigenitalis in equids, principally donkey jacks, (4) preferential sites of persistence of T. asinigenitalis, and (5) whether multiple genotypes of T. asinigenitalis are present and co-circulate on the same premises.
2. Materials and methods 2.1. Sample population The animals tested consisted of two different populations. The first population consisted of 43 donkeys and 1 male mule that were presented to the College of Veterinary Medicine, Michigan State University (MSU) for castration. Samples were taken from 3 of these 44 animals, even after it was discovered that 3 donkeys had been castrated previously when they arrived at MSU. These 44 equids were part of a joint study with investigators at MSU and the University of Kentucky, Veterinary Diagnostic Laboratory (UKVDL) and the Maxwell H. Gluck Equine Research Center to determine the normal microaerophilic bacterial flora on the external genitalia and in the testes and epididymides of male donkeys (Carleton et al., unpublished data). This population of donkeys was sampled between April 2006 and June 2007. The approximate ages of the 43 donkeys and 1 male mule at the time of initial sampling were 27, 1-year-old or younger, 4, 3-year-olds, and 12, 5–11-year-olds; the mule was 2-years old. Animals submitted for castration represented 12 different owners with premises located throughout Michigan. The castration, blood sampling and swabbing of the external genitalia of the equids included in this study were carried
out in accordance with the guidelines of the College of Veterinary Medicine, Michigan State University Institutional Animal Care and Use Committee. The second population of equids included in this study consisted of donkeys, mules, and horses on each of 2 premises the property of different owners that routinely presented donkeys for castration to the MSU Vet College, some of which subsequently tested culture positive for T. asinigenitalis. The first owner managed a total of 64 animals comprising two groups on closely adjacent premises in southern Michigan and referred to as Farm 1. The equid population consisted of 20 male donkeys, 5 gelded donkeys, 24 female donkeys, 1 male mule, 2 female mules, 1 male horse, and 11 female horses. Samples from the equids located on this premises were collected on July 9 or 10 and November 19, 2007. The second owner maintained a premises (Farm 2) in northern Michigan which accommodated 25 equids including 2 male donkeys, 16 gelded donkeys, 3 female donkeys, and 4 gelded horses. All animals on Farm 2 were sampled on August 13 and November 14, 2007. Additional samples were obtained from some of the culture-positive animals on Farm 1 in November 2008; and from animals on Farm 2 in November 2007, August and November 2008, and March 2010. Histories of the culture-positive equids on these two farms are summarized in Tables 1 and 2. 2.2. Sampling procedures Swabs for the culture of Taylorella species and blood for the detection of antibodies against Taylorella species were taken from all equids. Swabs (one per site) were taken from the urethral fossa (fossa glandis), dorsal diverticulum of the urethral sinus, terminal urethra, and external surface of the penis/prepuce of the entire or gelded male animals and from the clitoral fossa and clitoral sinuses of females; a swab of the cervix was also obtained from three (2 donkeys and 1 horse) animals on Farm 1. Swabs were placed in individual tubes of Amies transport medium with charcoal (CultureSwab Plus; Becton, Dickinson and Company, Sparks, MD, USA). Testes with attached epididymides from each of the males presented for castration were also submitted for culture. Approximately 20 ml of whole blood was collected by jugular venipuncture from each animal. Each blood sample was allowed to clot at room temperature, the serum drawn off and transferred to a chemically clean screw-capped tube, and stored at 20 8C until tested.
Table 1 History of donkeys on Farm 1 that were culture positive for Taylorella asinigenitalis. Animal
Approx. age July 1, 2007
Origin
Duration on farm July 1, 2007
Intact male
Date castrated
1 2 3 4 5 6 7 8 9
10 months 10 months 1 year 4 years 5 years 8 years 2 years 2 years Unknown
Farm 1 Farm 1 Michigan Farm 1 Farm 1 Farm 1 Farm 1 Michigan Unknown
10 months 10 months 1 year 4 years 5 years 8 years 2 years 1 year Not applicableb
Yes/Noa Yes/No Yes Yes Yes Yes Yes No Yes/No
05/15/07 05/15/07 Not applicable Not applicable Not applicable Not applicable Not applicable Unknown 11/20/2007
a b
Yes/No, animal was cultured then castrated. Donkey 9 arrived on Farm 1 in November, 2007 after he was cultured and then castrated at MSU.
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
437
Table 2 History of equids on Farm 2 that were culture positive for Taylorella asinigenitalis. Animal 10 11 12 13 14 15 16 17 18 19 20 21 22 23 a b
Approx. age July 1, 2007 11 years 11 years 11 years 11 years 30 years 5 years 12 years 5 years 6 years 9 years 9 years 19 years 9 years 18 years
Origin a
BLM , NV BLM, AZ BLM, NV BLM, NV Unknown Northeast IN Northeast IN Northeast IN Northeast IN Northern MI Northern MI BLM, AZ Southwest MI Northeast IN
Duration on farm July 1, 2007 1 year 6 months 6 months 6 months 8 years 4.5 years 12 years 4.5 years 2 years 2 years 2 years 2 years 2 years 5 years
Intact male Yes/No Yes/No No No No No No No No No No No No No
b
Date castrated 06/14/07 06/14/07 Unknown Unknown Before July July 2003 Before July July 2003 July 2005 Before July Before July Before July Before July July 2002
1999 2001
2004 2005 2005 2005
BLM, Bureau of Land Management. Yes/No, animal was cultured then castrated.
Swabs, testes with epididymides were cooled to 4 8C, placed on frozen freezer packs in a Styrofoam container and shipped by overnight delivery to the UKVDL. Because of inclement weather, one set of specimens (collected in early November, 2007) could not be shipped out the day of collection. The specimens were frozen and shipped frozen for overnight delivery on November 19, 2007. If sera had been processed and frozen, they also were included in the same shipment as the swabs. Otherwise, the frozen sera were sent in a separate container on ice packs for overnight delivery. 2.3. Isolation and identification of Taylorella Immediately upon receipt of the swabs at the UKVDL, each swab was plated on a Eugon agar plate (Becton, Dickinson and Company) supplemented with 10% (v/v) laked horse blood as well as on a chocolated Eugon agar plate selective medium (Timoney et al., 1982). If testes and epididymes were received, swabs from each tissue were also plated onto the same media. All plates were incubated in 8% CO2 for 7–10 days at 37 8C. Colonies composed of small gram-negative coccobacilli or bacilli with morphologic and growth characteristics typical or highly suggestive of Taylorella species were further evaluated. Isolates that were microaerophilic, non-motile, and catalase and oxidase positive were tested by a latex agglutination test specific for Taylorella (Bionor Laboratories AS, Skien, Norway). Identification of all isolates as T. asinigenitalis was confirmed using a Taylorella species-specific polymerase chain reaction (PCR) assay (Arata et al., 2001). Cultures of strains isolated from each animal were also sent to the National Veterinary Services Laboratories, Ames, IA, USA for final verification as T. asinigenitalis. Colony counts on culture plates were scored as follows: rare, 1–10 colonies/plate; few, 11–50 colonies/plate; moderate, 51–100 colonies/plate; and numerous, 101 or more colonies/plate. 2.4. Complement fixation test Performance of the complement fixation (CF) test was as previously described (Bryans et al., 1979; Donahue et al., 1978). Sera were screened at a starting dilution of 1:4 and
CF titers against Taylorella were expressed as the reciprocal of the highest 2-fold dilution that completely fixed complement. Since it had been demonstrated that male donkeys can developed detectable CF antibody titers against T. asinigenitalis (Jang et al., 2001; Meade et al., 2010), sera from all equids were tested. 2.5. Sequencing and analysis of 16S rRNA genes The gene encoding the 16S rRNA of each isolate was amplified, purified, and sequenced (at least 1400 base pairs). Genomic DNA was extracted from confirmed T. asinigenitalis isolates by removing growth from a chocolated Eugon agar plate and processed using an UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories Inc, Carlsbad, CA, USA). The gene was amplified and sequenced using the MicroSeq 16S rDNA Gene Kit (Applied Biosystems, Foster City, CA, USA). Reactions were purified, and sequencing gels were run by DNA Analysis, Cincinnati, OH, USA. Genomic data were assembled and edited using Sequencher 4.10.1 Software (Gene Codes Corp., Ann Arbor, MI, USA). 2.6. Data analysis The Fisher exact test was used for univariate analysis to assess the relationship between animal level characteristics and culture positive status for T. asinigenitalis. For the variables gender and species, totals were collapsed into two categories for analysis due to the sparseness of data in some cells such that for the variable gender, males and geldings were combined into one category and, for the variable species, donkeys and mules were grouped together. Odds ratios, with associated significance levels and 95% confidence intervals, were calculated after adjusting for potential confounding using exact conditional logistic regression. Variables were retained for inclusion in the regression model as confounders if the p value for individual variables was 0.25 in the univariate analysis. Statistical analysis was done using Statistical Analysis Software, version 9.2 (SAS Institute, Cary, NC, USA), and Epi Info version 5.0 (CDC, Atlanta GA, USA) (Derr, 2000). All exact p values were two-sided and considered statistically significant if the 2-tailed p value was <0.05.
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
438
Table 3 Culture results by date for Taylorella asinigenitalis culture-positive donkeys on Farm 1. Culture results
Animal
1 2 3 4 5 6 7 8 9
May 2007
July 2007
November 2007
November 2008
D, D, C, Ca C, C, B, A Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled
D, C, C, B Not sampled C, D, C, A B, B, A, C B, A, , , B, A, C, , A, B A, , B, B Not sampled
Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled B, C, C,
Not sampled Not sampled , , , A, , B, B, B, C, C, B C, B, C, Not sampled Not sampled Not sampled
a Culture results of four sites [urethral fossa (fossa glandis), dorsal diverticulum of the urethral sinus, distal urethra, prepuce] from each equid; = negative, A = rare, B = few, C = moderate, and D = numerous colonies of T. asinigenitalis were isolated.
3. Results 3.1. Culture and serological results of initial donkey survey Of the 43 donkeys and 1 mule presented to MSU for routine castration, 4 donkeys (9.3%) were culture positive for T. asinigenitalis. Two of the positive donkeys (animals 1 and 2, Table 1) belonged to the same owner (Farm 1 in southern Michigan); they were presented for castration May 15, 2007. The other 2 positive donkeys (animals 12 and 13, Table 2) were presented by a different owner (Farm 2 in northern Michigan) on June 14, 2007. The latter 2 donkeys plus a third culture-negative donkey submitted at the same time comprised the 3 males that had been castrated previously; nonetheless, they were included in the survey. Sera from 33 (32 donkeys and 1 mule) of the 44 equids were negative for CF antibodies to Taylorella spp.; Sera from an additional 11 donkeys (8 intact and 3 gelded donkeys) were anti-complementary (AC). The sera from the 4 T. asinigenitalis culture-positive donkeys were AC. 3.2. Culture and serological results of equid populations on Farms 1 and 2 The overall culture positivity rate of T. asinigenitalis for the 89 donkeys, mules and horses representing the
combined equid populations on the 2 farms was 25.8%. The rate varied considerably between Farm 1 (14.1%) and Farm 2 (56%). The majority of equids on Farm 1 were swabbed and had blood collected on July 9 or July 10, 2007. Seven (donkeys 1 and 3–8, Tables 1 and 3) or 11.9% of the 59 equids on this farm that were sampled in July 2007 were culture positive for T. asinigenitalis. Five (33%) of the 15 male donkeys and 2 (40%) of the 5 gelded donkeys were culture positive. One of the gelded donkeys (donkey 1) was previously culture positive from samples collected on May 15, 2007 immediately before he was castrated. An additional donkey (donkey 2) which was cultured positive and castrated on May 15, 2007 as a part of the initial survey, died about a week after castration, and therefore was unavailable for subsequent sampling. An additional 4 donkey jacks were sampled and castrated November 19, 2007, of which one (donkey 9) was cultured positive. Including donkey 2 that was castrated May 15 and donkey 9 that was castrated November 19, 2007, the total number of culture positive donkeys on Farm 1 was 9 out of a total equid population of 64. The 24 female donkeys, 11 female horses, 2 female mules, 1 male horse, and 1 male mule were culture negative for T. asinigenitalis. Of the 9 culture-positive donkeys, 2 had CF antibody titers to Taylorella spp. (donkey 4 = 1:8 and
Table 4 Culture results by date for Taylorella asinigenitalis culture-positive equids on Farm 2. Animal
Culture results June 2007
Aug. 2007
November 2007
August 2008
November 2008
March 2010
10 11 12 13 14 15 16 17 18 19 20 21 22 23
, , , a , , , C, D, C, C, C, C, Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled
C, C, C, C B, A, A, O C, D, C, C B, B, B, C, C, B, D, D, , D C, D, C, B D, D, C, C, C, B, C, C, B, B B, C, B, A C, C, B, A B, C, C, B C, C, C,
C, C, B, D B, A, , D D, D, D, D B, C, B, A , , , D, D, , C, C, C, C, C, C, B D, D, C, C B, C, C, C Not sampled D, D, D, D C, C, C, B Not sampled
C, C, C, D Not sampled D, D, C, O C, C, C, A , , , C, D, C, C C, C, C, C, D, C, D, D, C, Not sampled Not sampled Not sampled C, C, C, B C, C, C, C
Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled D, D, D, D C, C, D, Not sampled Not sampled Not sampled
C, C, C, B Not sampled Not sampled B, D, D, A , , , C, D, C, C C, D, C, D, D, D, D Not sampled Not sampled Not sampled C, D, C, C C, C, D, B C, B, B, B
a Culture results of four sites [urethral fossa (fossa glandis), dorsal diverticulum of the terminal urethral sinus, terminal urethra, prepuce] from each equid; = negative, O = the site was overgrown by other bacteria, A = rare, B = few, C = moderate, and D = numerous colonies of T. asinigenitalis were isolated.
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
donkey 8 = 1:16). Sera from the other 7 culture-positive animals were negative or AC. Of the culture-negative equids, sera from 2 female donkeys were CF positive (1:64 and 1:32), and serum from 1 gelded donkey was positive (1:16). The remaining sera were either CF negative or AC. All equids on Farm 2 were cultured and had blood drawn on August 13 and November 14, 2007. A total of 13 (donkeys 10–13 and 15–23, Tables 2 and 4) or 81.3% of the 16 gelded donkeys and 1 (horse 14, Tables 2 and 4) or 25% of the 4 gelded horses were culture positive for T. asinigenitalis. Two (donkeys 10 and 11) of these 13 donkeys were also cultured on May 14, 2007 before their castration at MSU, and they were culture negative for T. asinigenitalis. The 3 female donkeys on this farm were culture negative. Sera from all 19 donkeys were AC and from the 4 gelded horses negative in the CF test. 3.3. Duration of colonization of equids with T. asinigenitalis Swabs for culture were obtained on more than one occasion from 18 of the 22 culture-positive donkeys and from the one culture-positive horse (Tables 3 and 4). Five donkeys on Farm 1 were cultured on two different occasions. One (donkey 1) was positive on both occasions on swabs taken 2 months apart; three (donkeys 4, 5, and 6) were positive on swabs taken 16 months apart; and one (donkey 3) was culture-negative 16 months after the original positive culture (Table 3). All 14 T. asinigenitalis culture-positive equids on Farm 2 were cultured on 2–5 occasions (Table 4). Animal 14, the only horse that was culture positive, was culture negative 3, 12, and 20 months after T. asinigenitalis was isolated from this horse. All 13 donkeys were culture positive every time they were sampled after the original positive culture. Seven donkeys (animals 10, 15–17, and 21–23) were positive for 31 months and in the case of donkey 13, 33 months after their first set of positive swabs. These 8 individuals were unavailable for further sampling.
439
3.4. Preferred sites of T. asinigenitalis colonization of equids A total of 59 sets of swabs with at least one site culture positive for T. asinigenitalis were obtained from the 23 culture positive equids (Tables 3 and 4). The number (%) of positive cultures for the four sites were 58 (98%) for the urethral fossa (fossa glandis), 56 (95%) for the dorsal diverticulum of the urethral sinus, 55 (93%) for the terminal urethra, and 39 (66%) for the external surface of the penis/prepuce. Plates inoculated with two (3%) of the preputial swabs were overgrown by rapid-growing saprophytic bacteria which made the detection of T. asinigenitalis, if present, impossible. T. asinigenitalis was not cultured from the testes or epididymides of the 3 donkeys (animals 1, 2, and 9) that were positive when cultured immediately before they were castrated, nor from 2 donkeys (animals 10 and 11) that were culture negative when castrated but positive when cultured about 2 months after they were castrated. 3.5. Genotypes of T. asinigenitalis The results obtained from sequencing the 16S rRNA gene divided the isolates from the 23 culture-positive equids into two genotypes of T. asinigenitalis. Isolates from the 9 donkeys on Farm 1 and isolates from 8 equids (animals 13–17, 20, 22, and 23) from Farm 2 were identical to each other and to the type strain of T. asinigenitalis which was isolated in 1997 from a donkey jack in California. Isolates from the remaining 6 equids (animals 10–12, 18, 19, and 21) on Farm 2 were identical to each other and to the two strains recovered from donkey jacks in 1998 in Kentucky. 3.6. Analytical findings Results for the univariate and multivariate analyses are shown in Tables 5 and 6, respectively. A statistical association was demonstrated between positive culture
Table 5 Univariate analysis for animal-based risk factors associated with a culture positive genital swab for T. asinigenitalis among equids residing on two premises in Michigan. Variable
Categorical value
Odds ratio
Farm
1 2 2 years of age >2 years of age Female Male or gelding Horse Donkey or mule No Yes Purchased Raised 1 year >1 year
Ref 7.55 Ref 1.63 Ref 1 Ref 6.38 Ref 6.30 Ref 1.25 Ref 5.56
Age class Genderb Speciesb Offspring Source Length of time on premisesc
Lower 95% CIa
Upper 95% CI
Fisher exact p-value
2.39
25.65
<0.001
0.39
9.83
0.753
2.89
1
<0.001
0.87
284.21
0.060
1.35
60.03
0.008
0.37
4.96
0.786
0.73
72.03
0.099
Ref, reference category. a Confidence intervals. b Cell totals were collapsed into two categories for analysis as the Cochran conditions were not satisfied due to cells with zero counts and more than 80% of the cells with counts less than 5. c Analysis restricted to premises number 2.
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
440
Table 6 Multivariate exact logistic regression analysis of animal-based risk factors associated with a culture positive genital swab for T. asinigenitalis among equids residing on two premises in Michigan. Variable
Categorical value
Odds ratio
Farm
1 2 Male or gelding Female Donkey or mule Horse No Yes
Ref 6.31 Ref 0.091b Ref 0.132 Ref 5.01
Gender Species Offspring
Lower 95% CIa 1.53
Upper 95% CI 30.73
Fisher exact p-value 0.007
0
0.521
0.0172
0.002
1.78
0.174
0.556
246.70
0.229
Ref, reference category. a Confidence intervals. b Indicates a median unbiased estimate.
status and farm location (exact odds ratio, 7.55; 95% CI 2.39, 25.65); an association that was still present after adjusting for other covariates in the multivariate analysis. The effect of prior breeding (exact odds ratio 6.30; 95% CI 1.35, 60.03) identified in the univariate analysis was not present after adjusting for confounders in the multivariate model. The univariate analysis found no significant differences for being culture positive for T. asinigenitalis and the age category of the animal (2 years of age versus >2 years of age). For Farm 2, no differences were observed for the culture status of animals that were purchased compared to those raised on the premises. Overall, the analysis suggests that an animal located on Farm 2 was at a six times greater risk of being culture positive for T. asinigenitalis (exact odds ratio, 6.31; 95% CI 1.53, 36.07) compared to an animal located on Farm 1 and that females are at a eleven times lower risk (exact odds ratio 0.091; 95% CI 0, 0.521) for being culture positive compared to males (stallions or geldings). 4. Discussion A survey of 43 donkey jacks and 1 male mule from 12 premises in Michigan revealed that 9.3% of the donkeys were culture positive for T. asinigenitalis at time of castration (Carleton et al., unpublished data). Based on the findings of this study and those of Jang et al. (2001), Meade et al. (2010), Franco et al. (2009), and Breuil et al. (2011), the donkey is probably the natural host species of T. asinigenitalis, unlike the horse which is considered the reservoir species of T. equigenitalis. The current study is the first to survey a population of donkey jacks for evidence of T. asinigenitalis. As previously reported (Jang et al., 2001; Meade et al., 2010), T. asinigenitalis would appear more widely distributed than suggested initially and may be endemic in the US donkey population. This may well be the situation in other countries reporting isolations of this bacterium (Ba˚verud et al., 2006; Franco et al., 2009; Breuil et al., 2011). Transmission to horses appears to be infrequent and tangential to the primary circulation of this bacterium in the donkey population (Ba˚verud et al., 2006; Meade et al., 2010; Breuil et al., 2011; Reilly et al., 2012). There was no evidence of T. asinigenitalis in the testes or epididymides in 3 of the 4 donkeys cultured positive in the survey. Failure to culture T. asinigenitalis from the proximal
reproductive tract would support the contention that T. asinigenitalis is a preferential inhabitant of the distal reproductive tract, where it can co-exist with the normal bacterial flora on the external genitalia of the intact or gelded male, especially the donkey jack. Of the collective equid populations on the two farms in the second phase of the study, 25.8% were positive for T. asinigenitalis. The culture positivity rate varied widely between the two premises, with 14.1% on Farm 1 and 56% on Farm 2. Of 23 culture positive animals on Farm 2, 22 were donkey jacks, 53.5% of which had been castrated previously. This is supportive evidence of the role of the donkey jack as the primary reservoir of T. asinigenitalis. Detailed investigation failed to turn up any evidence of sharing of donkeys or horses betweens Farm 1 and 2. As reported previously (Anonymous, 1998; Jang et al., 2001; Meade et al., 2010; Katz et al., 2000), neither the source nor when exposure to T. asinigenitalis occurred was determined either for the 4 donkey jacks detected in the initial survey or the 18 donkey jacks and 1 horse gelding subsequently identified on Farms 1 and 2. Many of the donkeys on Farm 2 were of diverse backgrounds. They were either rescue donkeys or donkeys originating from Western or other Mid-Western states. They included 5 relatively recently introduced Bureau of Land Management donkeys, 3 of which originated in Nevada and 2 in Arizona. Failure to detect T. asinigenitalis in mares or female donkeys in this study is similar to the finding of Breuil et al. (2011) but in contrast to the report of Reilly et al. (2012). It is intriguing why this is the case considering that donkeys and horses were allowed to co-mingle at pasture on Farms 1 and 2. An explanation may be the fact that the clitoral fossa and sinuses were the sites sampled in donkey jennies or mares. Based on previous data (Meade et al., 2010), T. asinigenitalis was more often cultured from the proximal (uterus, cervix) rather than the distal reproductive tract (clitoral fossa and sinuses) in a small number of carrier mares. In contrast, most T. equigenitalis chronically infected mares are clitoral carriers. Unfortunately, with the exception of 2 donkey jennies and 1 mare on Farm 1, it was not possible to obtain swabs from both proximal and distal reproductive tract sites in female animals. Alternatively, carriage of T. asinigenitalis may occur less frequently and for a shorter term in the donkey jenny or the mare than in the intact or gelded male, donkey or stallion; This would be consistent with the
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
findings of Platt et al. (1978) and Timoney et al. (1978) confirming short-term persistence of T. equigenitalis in most experimentally infected mares. Since bacteriological culture is still considered the ‘‘gold standard’’ by the World Organisation for Animal Health, it was selected over real-time PCR (Wakeley et al., 2006) as a proven means of detection of T. asinigenitalis. Various PCR assays have been shown to be highly specific, rapid and at least of equivalent sensitivity to culture. With few exceptions, problems of bacterial overgrowth of plates were not encountered. The preferential sites for isolation of T. asinigenitalis were similar to those reported for T. equigenitalis in the stallion (Timoney, 1996). With one exception (donkey 14), animals cultured positive on the first set of swabs were also positive on subsequent swabbings, regardless of the sampling interval that ranged from 2 to 19 months. A total of 6 donkeys on Farm 2 were culture positive 31 months after initial sampling, and one additional donkey was positive for 33 months, at which point the study had to be terminated. It is highly improbable that some or all of these donkey jacks cleared themselves of T. asinigenitalis and became reexposed to the organism at a later point in the study period. It should be emphasized that with the exception of donkey 14, each individual was culture positive on 2–5 consecutive sets of swabs. On a point of comparison, T. equigenitalis can persist in the stallion for at least 8 years. It should not be surprising therefore that long-term carriage of T. asinigenitalis may also extend over a period of years. This study provides the first documented evidence that this can occur and also, that T. asinigenitalis can persist in the gelded male. In light of the presence of T. asinigenitalis in close to onethird of the gelded donkey jacks in the current study, it is plausible to conjecture that persistence of T. equigenitalis may also occur in horse geldings. If so, it could have implications with regard to the international movement not only of stallions and mares but also geldings as potential carriers of T. equigenitalis, the cause of contagious equine metritis (CEM). The finding of a limited number of donkeys in the current study that gave positive reactions in the CF test using antigen prepared from T. equigenitalis was unexpected. Of a total of 5 CF positive donkeys, 2 were culturepositive donkey jacks and 3 were culture negative (2 jennies and one gelded donkey jack). Detection of CF antibody activity in male donkeys runs counter to the reported absence of a detectable serological response in stallions following natural or experimental exposure to T. equigenitalis (Bryans et al., 1979; Powell, 1981; Platt and Taylor, 1982; Timoney and Powell, 1988). It remains to be determined whether the positive CF reactions were specific for Taylorella spp. or not. Whereas Bryans et al. (1979) found no evidence of non-specific CF antibody activity in the sera of mares naturally infected with T. equigenitalis, Timoney et al. (1985), in an experimental study of T. equigenitalis infection in donkey jennies, concluded that the CF test was of limited diagnostic value due to presence of low levels of non-specific reactivity and anti-complementary activity. Genotyping of isolates of T. asinigenitalis from the 23 culture-positive equids, revealed they belonged to 2
441
clusters. Some 17 were identical to the type strain isolated in California in 1997 (Anonymous, 1998) and 6 to the strains recovered in Kentucky in 1998 (Meade et al., 2010). Whereas all 9 isolates from Farm 1 and 8 of the Farm 2 isolates belonged to the first cluster, the remaining 6 isolates from Farm 2 belonged to the second cluster. This is the first time different genotypes of T. asinigenitalis have been found in circulation on the same premises. Breuil et al. (2011) also found that French isolates of T. asinigenitalis belonged to 2 clusters. While these findings would suggest a lack of genomic diversity among strains recovered in the US and elsewhere, very recently, Reilly et al. (2012) reported a third genotype of the bacterium that was isolated from a mare in the US during routine preexport screening. Based on its relatedness to T. equigenitalis, it is reasonable to assume that most if not all of the transmission of T. asinigenitalis to 22 donkey jacks and 1 horse gelding had to have taken place by direct or indirect venereal contact. As already indicated, there was no evidence of infection in donkey jennies and mares. Even if donkey jacks and the gelding were exposed to T. asinigenitalis by the venereal route, that fails to explain how 2, 10-month-old sexually immature donkey jacks acquired the organism. The only credible explanation would be that they were the offspring of culture positive dams and acquired T. asinigenitalis in utero or at time of parturition. Timoney and Powell (1982) presented a similar argument to account for recovery of T. equigenitalis from a number of sexually immature colts and fillies. 5. Conclusions The study findings provide additional evidence supporting endemicity of T. asinigenitalis in donkeys and their role as the natural host species of the organism. Extended carriage of the bacterium in gelded donkey jacks might suggest that long-term persistence of T. equigenitalis similarly may occur in gelded colts or stallions. Genotypic analysis revealed limited evidence of genomic diversity among of isolates of T. asinigenitalis. Acknowledgements This report was published (12-14-028) by permission of the Dean and Director of the Kentucky Agricultural Experiment Station and the College of Agriculture, University of Kentucky, Lexington. We thank Dr. Dave Dargatz, Center for Epidemiology and Animal Health, USDAS, APHIS, VS for advice and guidance with data analysis, and the serology section, UKVDL for performance of the CF test. The authors acknowledge with gratitude the financial support provided by the University of Kentucky Veterinary Diagnostic Laboratory in carrying out this study. References Anonymous, 1998. CEM in a mammoth jack donkey. J. Equine Vet. Sci. 18, 100–101. Arata, A.B., Cooke, C.L., Jang, S.S., Hirsh, D.C., 2001. Multiplex polymerase chain reaction for distinguishing Taylorella equigenitalis from Taylorella equigenitalis-like organisms. J. Vet. Diagn. Invest. 13, 263–264.
442
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
Ba˚verud, V., Nystro¨m, C., Johansson, K.E., 2006. Isolation and identification of Taylorella asinigenitalis from the genital tract of a stallion, first case of a natural infection. Vet. Microbiol. 116, 294–300. Breuil, M.F., Duquesne, F., Laugier, C., Petry, S., 2011. Phenotypic and 16S ribosomal RNA gene diversity of Taylorella asinigenitalis strains isolated between 1995 and 2008. Vet. Microbiol. 148, 260–266. Bryans, J.T., Darlington, R.W., Smith, B., Brooks, R.R., 1979. Development of a complement fixation test and its application to diagnosis of contagious equine metritis. J. Equine Med. Surg. 3, 467–472. Derr, R.E., 2000. Performing exact logistic regression with the SAS System. In: Proceedings of the Twenty-fifth Annual SAS Users Group International Conference. SAS Institute, Inc., Cary, NC. Donahue, J.M., Swerczek, T.W., Smith, B.J., 1978. Laboratory diagnosis of contagious equine metritis. In: Proceedings of the 21st Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. pp. 497–505. Franco, A., Donati, V., Troiano, P., Lorenzetti, R., Zini, M., Autorino, G.L., Petrella, A., Maggi, A., Battisti, A., 2009. Detection of Taylorella asinigenitalis in donkey jacks in Italy. Vet. Rec. 165, 540–541. Jang, S.S., Donahue, J.M., Arata, A.B., Goris, J., Hansen, L.M., Earley, D.L., Vandamme, P.A.R., Timoney, P.J., Hirsh, D.C., 2001. Taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (Equus asinus). Int. J. Syst. Evol. Microbiol. 51, 971–976. Katz, J.B., Evans, L.E., Hutto, D.L., Schroeder-Tucker, L.C., Carew, A.M., Donahue, J.M., Hirsh, D.C., 2000. Clinical, bacteriologic, serologic, and pathologic features of infections with atypical Taylorella equigenitalis in mares. J. Am. Vet. Med. Assoc. 216, 1945–1948. Meade, B.J., Timoney, P.J., Donahue, J.M., Branscum, A.J., Ford, R., Rowe, R., 2010. Initial occurrence of Taylorella asinigenitalis and its detection in nurse mares, a stallion and donkeys in Kentucky. Prev. Vet. Med. 95, 292–296.
Platt, H., Atherton, J.G., Simpson, D.J., 1978. The experimental infection of ponies with contagious equine metritis. Equine Vet. J. 10, 153–159. Platt, H., Taylor, C.E., 1982. Contagious equine metritis. Med. Microbiol. 1, 49–96. Powell, D.G., 1981. Contagious equine metritis. Adv. Vet. Sci. Comp. Med. 25, 161–184. Reilly, J.R., Calcutt, M.J., Ganjam, I.K., Erdman, M.M., Aalsburg, A.M., Blyden, J.F., Fales, W.H., 2012. Isolation and identification of Taylorella asinigenitalis from a mare in Oklahoma, USA. Clin. Theriogenol. 4, 163–167. Timoney, P.J., 1996. Contagious equine metritis. Comp. Immunol. Microbiol. Infect. Dis. 19, 199–204. Timoney, P.J., McArdle, J.F., O’Reilly, P.J., Ward, J., 1978. Infection patterns in pony mares challenged with the agent of contagious equine metritis 1977. Equine Vet. J. 10, 148–152. Timoney, P.J., Powell, D.G., 1982. Isolation of the contagious equine metritis organism from colts and fillies in the United Kingdom and Ireland. Vet. Rec. 111, 478–482. Timoney, P.J., Powell, D.G., 1988. Contagious equine metritis-epidemiology and control. J. Equine Vet. Sci. 8, 42–46. Timoney, P.J., O’Reilly, P.J., McArdle, J.F., Ward, J., Harrington, A.M., 1985. Contagious equine metritis: experimental infection in the donkey. Vet. Microbiol. 10, 259–268. Timoney, P.J., Shin, S.J., Jacobson, R.H., 1982. Improved selective medium for isolation of the contagious equine metritis organism. Vet. Rec. 111, 107–108. Wakeley, P.R., Errington, J., Hannon, S., Roest, H.I.J., Carson, T., Hunt, B., Heath, P., 2006. Development of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from T. asinigenitalis. Vet. Microbiol. 118, 247–254.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Prevalence and persistence of Taylorella asinigenitalis in male donkeys James M. Donahue a, Peter J. Timoney b,*, Carla L. Carleton c, Judy V. Marteniuk c, Stephen F. Sells a, Barry J. Meade b a
University of Kentucky Veterinary Diagnostic Laboratory, P.O. Box 14125, Lexington, KY 40512, USA Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA c Department of Large Animal Clinical Science, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA‘ b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 March 2012 Received in revised form 13 June 2012 Accepted 15 June 2012
This study was undertaken to investigate the prevalence of Taylorella asinigenitalis in a subset of the donkey population of Michigan and in other equids on farms on which the organism was identified. Other aims were to further characterize the carrier state in terms of persistence and preferred sites of colonization of T. asinigenitalis in the male donkey as well as determine the genotype of any isolates of the organism. Initial testing of 43 donkeys and 1 mule turned up 4 (9.3%) donkeys culture positive for T. asinigenitalis. The 4 culture-positive donkeys resided on 2 farms accommodating a collective total of 89 equids, of which 23 (25.8%) were confirmed positive for T. asinigenitalis. The positive equid population on the 2 farms comprised 14 (67%) of 21 gelded donkeys, 8 (36.4%) of 22 intact male donkeys, and 1 (25%) of 4 gelded horses. T. asinigenitalis was not isolated from 27 female donkeys, 11 female horses, 2 female mules, 1 male horse, or 1 male mule resident on these premises. Isolations of the bacterium were obtained from a number of male donkeys whenever they were sampled over a span of 33 months; preferential sites of isolation were the urethral fossa (fossa glandis), dorsal diverticulum of the urethral sinus, and terminal urethra. Isolates of T. asinigenitalis from the 23 culture-positive equids comprised 2 genotypes, one identical to the type strain isolated in California in 1997, and the other identical to 2 strains isolated from donkey jacks in Kentucky in 1998. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Taylorella asinigenitalis Donkeys Normal flora CEM
1. Introduction In late October 1997, a Taylorella equigenitalis-like bacterium was recovered from a Mammoth donkey jack in California (Anonymous, 1998) and in early January 1998, a similar bacterium was recovered from 2 male donkeys, 7 nurse mares and 1 stallion in Kentucky (Jang et al., 2001; Meade et al., 2010). Based on DNA–DNA hybridization studies, these 3 isolates were not T. equigenitalis; they were
* Corresponding author at: Maxwell H. Gluck Research Center, University of Kentucky, Lexington, KY 40546, USA. Tel.: +1 859 218 1094; fax: +1 859 257 8542. E-mail address: [email protected] (P.J. Timoney). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.06.016
a new species in the genus Taylorella named Taylorella asinigenitalis (Jang et al., 2001). Since originally reported, T. asinigenitalis has been recovered from equids in Europe and the USA. The organism was isolated from a 3-year-old stallion in Sweden (Ba˚verud et al., 2006), that previously shared paddock space with a donkey jack in Belgium. In a second report (Franco et al., 2009) T. asinigenitalis was recovered from 2 donkey jacks in Apulia, Italy. They were at the same facility for over 3 years and were previously culture negative for Taylorella species. Breuil et al. (2011) published on the phenotypic and 16S ribosomal RNA gene diversity of 43 strains of T. asinigenitalis isolated from 22 donkey jacks, 2 stallions and 1 mare in France between 1995 and 2009. Based on sequence
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
436
analysis, strains fell into 2 clusters which seemed to correlate with their respective geographic origins. Recent reports from the US confirmed isolation of T. asinigenitalis from a mare in Oklahoma (Reilly et al., 2012) and a miniature donkey in Kentucky (E.S. Ford, personal communication). The source of T. asinigenitalis has not been identified in any of the published reports (Anonymous, 2000; Jang et al., 2001; Katz et al., 2000; Meade et al., 2010), and very little is known about the epidemiology of T. asinigenitalis, particularly its persistence in equid populations. Accordingly, the following study was undertaken to determine: (1) the prevalence of T. asinigenitalis in a subset of the donkey population in Michigan presented for castration, (2) the prevalence of T. asinigenitalis in all equids on farms with a known culture-positive donkey jack, (3) the duration of persistence of T. asinigenitalis in equids, principally donkey jacks, (4) preferential sites of persistence of T. asinigenitalis, and (5) whether multiple genotypes of T. asinigenitalis are present and co-circulate on the same premises.
2. Materials and methods 2.1. Sample population The animals tested consisted of two different populations. The first population consisted of 43 donkeys and 1 male mule that were presented to the College of Veterinary Medicine, Michigan State University (MSU) for castration. Samples were taken from 3 of these 44 animals, even after it was discovered that 3 donkeys had been castrated previously when they arrived at MSU. These 44 equids were part of a joint study with investigators at MSU and the University of Kentucky, Veterinary Diagnostic Laboratory (UKVDL) and the Maxwell H. Gluck Equine Research Center to determine the normal microaerophilic bacterial flora on the external genitalia and in the testes and epididymides of male donkeys (Carleton et al., unpublished data). This population of donkeys was sampled between April 2006 and June 2007. The approximate ages of the 43 donkeys and 1 male mule at the time of initial sampling were 27, 1-year-old or younger, 4, 3-year-olds, and 12, 5–11-year-olds; the mule was 2-years old. Animals submitted for castration represented 12 different owners with premises located throughout Michigan. The castration, blood sampling and swabbing of the external genitalia of the equids included in this study were carried
out in accordance with the guidelines of the College of Veterinary Medicine, Michigan State University Institutional Animal Care and Use Committee. The second population of equids included in this study consisted of donkeys, mules, and horses on each of 2 premises the property of different owners that routinely presented donkeys for castration to the MSU Vet College, some of which subsequently tested culture positive for T. asinigenitalis. The first owner managed a total of 64 animals comprising two groups on closely adjacent premises in southern Michigan and referred to as Farm 1. The equid population consisted of 20 male donkeys, 5 gelded donkeys, 24 female donkeys, 1 male mule, 2 female mules, 1 male horse, and 11 female horses. Samples from the equids located on this premises were collected on July 9 or 10 and November 19, 2007. The second owner maintained a premises (Farm 2) in northern Michigan which accommodated 25 equids including 2 male donkeys, 16 gelded donkeys, 3 female donkeys, and 4 gelded horses. All animals on Farm 2 were sampled on August 13 and November 14, 2007. Additional samples were obtained from some of the culture-positive animals on Farm 1 in November 2008; and from animals on Farm 2 in November 2007, August and November 2008, and March 2010. Histories of the culture-positive equids on these two farms are summarized in Tables 1 and 2. 2.2. Sampling procedures Swabs for the culture of Taylorella species and blood for the detection of antibodies against Taylorella species were taken from all equids. Swabs (one per site) were taken from the urethral fossa (fossa glandis), dorsal diverticulum of the urethral sinus, terminal urethra, and external surface of the penis/prepuce of the entire or gelded male animals and from the clitoral fossa and clitoral sinuses of females; a swab of the cervix was also obtained from three (2 donkeys and 1 horse) animals on Farm 1. Swabs were placed in individual tubes of Amies transport medium with charcoal (CultureSwab Plus; Becton, Dickinson and Company, Sparks, MD, USA). Testes with attached epididymides from each of the males presented for castration were also submitted for culture. Approximately 20 ml of whole blood was collected by jugular venipuncture from each animal. Each blood sample was allowed to clot at room temperature, the serum drawn off and transferred to a chemically clean screw-capped tube, and stored at 20 8C until tested.
Table 1 History of donkeys on Farm 1 that were culture positive for Taylorella asinigenitalis. Animal
Approx. age July 1, 2007
Origin
Duration on farm July 1, 2007
Intact male
Date castrated
1 2 3 4 5 6 7 8 9
10 months 10 months 1 year 4 years 5 years 8 years 2 years 2 years Unknown
Farm 1 Farm 1 Michigan Farm 1 Farm 1 Farm 1 Farm 1 Michigan Unknown
10 months 10 months 1 year 4 years 5 years 8 years 2 years 1 year Not applicableb
Yes/Noa Yes/No Yes Yes Yes Yes Yes No Yes/No
05/15/07 05/15/07 Not applicable Not applicable Not applicable Not applicable Not applicable Unknown 11/20/2007
a b
Yes/No, animal was cultured then castrated. Donkey 9 arrived on Farm 1 in November, 2007 after he was cultured and then castrated at MSU.
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
437
Table 2 History of equids on Farm 2 that were culture positive for Taylorella asinigenitalis. Animal 10 11 12 13 14 15 16 17 18 19 20 21 22 23 a b
Approx. age July 1, 2007 11 years 11 years 11 years 11 years 30 years 5 years 12 years 5 years 6 years 9 years 9 years 19 years 9 years 18 years
Origin a
BLM , NV BLM, AZ BLM, NV BLM, NV Unknown Northeast IN Northeast IN Northeast IN Northeast IN Northern MI Northern MI BLM, AZ Southwest MI Northeast IN
Duration on farm July 1, 2007 1 year 6 months 6 months 6 months 8 years 4.5 years 12 years 4.5 years 2 years 2 years 2 years 2 years 2 years 5 years
Intact male Yes/No Yes/No No No No No No No No No No No No No
b
Date castrated 06/14/07 06/14/07 Unknown Unknown Before July July 2003 Before July July 2003 July 2005 Before July Before July Before July Before July July 2002
1999 2001
2004 2005 2005 2005
BLM, Bureau of Land Management. Yes/No, animal was cultured then castrated.
Swabs, testes with epididymides were cooled to 4 8C, placed on frozen freezer packs in a Styrofoam container and shipped by overnight delivery to the UKVDL. Because of inclement weather, one set of specimens (collected in early November, 2007) could not be shipped out the day of collection. The specimens were frozen and shipped frozen for overnight delivery on November 19, 2007. If sera had been processed and frozen, they also were included in the same shipment as the swabs. Otherwise, the frozen sera were sent in a separate container on ice packs for overnight delivery. 2.3. Isolation and identification of Taylorella Immediately upon receipt of the swabs at the UKVDL, each swab was plated on a Eugon agar plate (Becton, Dickinson and Company) supplemented with 10% (v/v) laked horse blood as well as on a chocolated Eugon agar plate selective medium (Timoney et al., 1982). If testes and epididymes were received, swabs from each tissue were also plated onto the same media. All plates were incubated in 8% CO2 for 7–10 days at 37 8C. Colonies composed of small gram-negative coccobacilli or bacilli with morphologic and growth characteristics typical or highly suggestive of Taylorella species were further evaluated. Isolates that were microaerophilic, non-motile, and catalase and oxidase positive were tested by a latex agglutination test specific for Taylorella (Bionor Laboratories AS, Skien, Norway). Identification of all isolates as T. asinigenitalis was confirmed using a Taylorella species-specific polymerase chain reaction (PCR) assay (Arata et al., 2001). Cultures of strains isolated from each animal were also sent to the National Veterinary Services Laboratories, Ames, IA, USA for final verification as T. asinigenitalis. Colony counts on culture plates were scored as follows: rare, 1–10 colonies/plate; few, 11–50 colonies/plate; moderate, 51–100 colonies/plate; and numerous, 101 or more colonies/plate. 2.4. Complement fixation test Performance of the complement fixation (CF) test was as previously described (Bryans et al., 1979; Donahue et al., 1978). Sera were screened at a starting dilution of 1:4 and
CF titers against Taylorella were expressed as the reciprocal of the highest 2-fold dilution that completely fixed complement. Since it had been demonstrated that male donkeys can developed detectable CF antibody titers against T. asinigenitalis (Jang et al., 2001; Meade et al., 2010), sera from all equids were tested. 2.5. Sequencing and analysis of 16S rRNA genes The gene encoding the 16S rRNA of each isolate was amplified, purified, and sequenced (at least 1400 base pairs). Genomic DNA was extracted from confirmed T. asinigenitalis isolates by removing growth from a chocolated Eugon agar plate and processed using an UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories Inc, Carlsbad, CA, USA). The gene was amplified and sequenced using the MicroSeq 16S rDNA Gene Kit (Applied Biosystems, Foster City, CA, USA). Reactions were purified, and sequencing gels were run by DNA Analysis, Cincinnati, OH, USA. Genomic data were assembled and edited using Sequencher 4.10.1 Software (Gene Codes Corp., Ann Arbor, MI, USA). 2.6. Data analysis The Fisher exact test was used for univariate analysis to assess the relationship between animal level characteristics and culture positive status for T. asinigenitalis. For the variables gender and species, totals were collapsed into two categories for analysis due to the sparseness of data in some cells such that for the variable gender, males and geldings were combined into one category and, for the variable species, donkeys and mules were grouped together. Odds ratios, with associated significance levels and 95% confidence intervals, were calculated after adjusting for potential confounding using exact conditional logistic regression. Variables were retained for inclusion in the regression model as confounders if the p value for individual variables was 0.25 in the univariate analysis. Statistical analysis was done using Statistical Analysis Software, version 9.2 (SAS Institute, Cary, NC, USA), and Epi Info version 5.0 (CDC, Atlanta GA, USA) (Derr, 2000). All exact p values were two-sided and considered statistically significant if the 2-tailed p value was <0.05.
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
438
Table 3 Culture results by date for Taylorella asinigenitalis culture-positive donkeys on Farm 1. Culture results
Animal
1 2 3 4 5 6 7 8 9
May 2007
July 2007
November 2007
November 2008
D, D, C, Ca C, C, B, A Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled
D, C, C, B Not sampled C, D, C, A B, B, A, C B, A, , , B, A, C, , A, B A, , B, B Not sampled
Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled B, C, C,
Not sampled Not sampled , , , A, , B, B, B, C, C, B C, B, C, Not sampled Not sampled Not sampled
a Culture results of four sites [urethral fossa (fossa glandis), dorsal diverticulum of the urethral sinus, distal urethra, prepuce] from each equid; = negative, A = rare, B = few, C = moderate, and D = numerous colonies of T. asinigenitalis were isolated.
3. Results 3.1. Culture and serological results of initial donkey survey Of the 43 donkeys and 1 mule presented to MSU for routine castration, 4 donkeys (9.3%) were culture positive for T. asinigenitalis. Two of the positive donkeys (animals 1 and 2, Table 1) belonged to the same owner (Farm 1 in southern Michigan); they were presented for castration May 15, 2007. The other 2 positive donkeys (animals 12 and 13, Table 2) were presented by a different owner (Farm 2 in northern Michigan) on June 14, 2007. The latter 2 donkeys plus a third culture-negative donkey submitted at the same time comprised the 3 males that had been castrated previously; nonetheless, they were included in the survey. Sera from 33 (32 donkeys and 1 mule) of the 44 equids were negative for CF antibodies to Taylorella spp.; Sera from an additional 11 donkeys (8 intact and 3 gelded donkeys) were anti-complementary (AC). The sera from the 4 T. asinigenitalis culture-positive donkeys were AC. 3.2. Culture and serological results of equid populations on Farms 1 and 2 The overall culture positivity rate of T. asinigenitalis for the 89 donkeys, mules and horses representing the
combined equid populations on the 2 farms was 25.8%. The rate varied considerably between Farm 1 (14.1%) and Farm 2 (56%). The majority of equids on Farm 1 were swabbed and had blood collected on July 9 or July 10, 2007. Seven (donkeys 1 and 3–8, Tables 1 and 3) or 11.9% of the 59 equids on this farm that were sampled in July 2007 were culture positive for T. asinigenitalis. Five (33%) of the 15 male donkeys and 2 (40%) of the 5 gelded donkeys were culture positive. One of the gelded donkeys (donkey 1) was previously culture positive from samples collected on May 15, 2007 immediately before he was castrated. An additional donkey (donkey 2) which was cultured positive and castrated on May 15, 2007 as a part of the initial survey, died about a week after castration, and therefore was unavailable for subsequent sampling. An additional 4 donkey jacks were sampled and castrated November 19, 2007, of which one (donkey 9) was cultured positive. Including donkey 2 that was castrated May 15 and donkey 9 that was castrated November 19, 2007, the total number of culture positive donkeys on Farm 1 was 9 out of a total equid population of 64. The 24 female donkeys, 11 female horses, 2 female mules, 1 male horse, and 1 male mule were culture negative for T. asinigenitalis. Of the 9 culture-positive donkeys, 2 had CF antibody titers to Taylorella spp. (donkey 4 = 1:8 and
Table 4 Culture results by date for Taylorella asinigenitalis culture-positive equids on Farm 2. Animal
Culture results June 2007
Aug. 2007
November 2007
August 2008
November 2008
March 2010
10 11 12 13 14 15 16 17 18 19 20 21 22 23
, , , a , , , C, D, C, C, C, C, Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled
C, C, C, C B, A, A, O C, D, C, C B, B, B, C, C, B, D, D, , D C, D, C, B D, D, C, C, C, B, C, C, B, B B, C, B, A C, C, B, A B, C, C, B C, C, C,
C, C, B, D B, A, , D D, D, D, D B, C, B, A , , , D, D, , C, C, C, C, C, C, B D, D, C, C B, C, C, C Not sampled D, D, D, D C, C, C, B Not sampled
C, C, C, D Not sampled D, D, C, O C, C, C, A , , , C, D, C, C C, C, C, C, D, C, D, D, C, Not sampled Not sampled Not sampled C, C, C, B C, C, C, C
Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled Not sampled D, D, D, D C, C, D, Not sampled Not sampled Not sampled
C, C, C, B Not sampled Not sampled B, D, D, A , , , C, D, C, C C, D, C, D, D, D, D Not sampled Not sampled Not sampled C, D, C, C C, C, D, B C, B, B, B
a Culture results of four sites [urethral fossa (fossa glandis), dorsal diverticulum of the terminal urethral sinus, terminal urethra, prepuce] from each equid; = negative, O = the site was overgrown by other bacteria, A = rare, B = few, C = moderate, and D = numerous colonies of T. asinigenitalis were isolated.
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
donkey 8 = 1:16). Sera from the other 7 culture-positive animals were negative or AC. Of the culture-negative equids, sera from 2 female donkeys were CF positive (1:64 and 1:32), and serum from 1 gelded donkey was positive (1:16). The remaining sera were either CF negative or AC. All equids on Farm 2 were cultured and had blood drawn on August 13 and November 14, 2007. A total of 13 (donkeys 10–13 and 15–23, Tables 2 and 4) or 81.3% of the 16 gelded donkeys and 1 (horse 14, Tables 2 and 4) or 25% of the 4 gelded horses were culture positive for T. asinigenitalis. Two (donkeys 10 and 11) of these 13 donkeys were also cultured on May 14, 2007 before their castration at MSU, and they were culture negative for T. asinigenitalis. The 3 female donkeys on this farm were culture negative. Sera from all 19 donkeys were AC and from the 4 gelded horses negative in the CF test. 3.3. Duration of colonization of equids with T. asinigenitalis Swabs for culture were obtained on more than one occasion from 18 of the 22 culture-positive donkeys and from the one culture-positive horse (Tables 3 and 4). Five donkeys on Farm 1 were cultured on two different occasions. One (donkey 1) was positive on both occasions on swabs taken 2 months apart; three (donkeys 4, 5, and 6) were positive on swabs taken 16 months apart; and one (donkey 3) was culture-negative 16 months after the original positive culture (Table 3). All 14 T. asinigenitalis culture-positive equids on Farm 2 were cultured on 2–5 occasions (Table 4). Animal 14, the only horse that was culture positive, was culture negative 3, 12, and 20 months after T. asinigenitalis was isolated from this horse. All 13 donkeys were culture positive every time they were sampled after the original positive culture. Seven donkeys (animals 10, 15–17, and 21–23) were positive for 31 months and in the case of donkey 13, 33 months after their first set of positive swabs. These 8 individuals were unavailable for further sampling.
439
3.4. Preferred sites of T. asinigenitalis colonization of equids A total of 59 sets of swabs with at least one site culture positive for T. asinigenitalis were obtained from the 23 culture positive equids (Tables 3 and 4). The number (%) of positive cultures for the four sites were 58 (98%) for the urethral fossa (fossa glandis), 56 (95%) for the dorsal diverticulum of the urethral sinus, 55 (93%) for the terminal urethra, and 39 (66%) for the external surface of the penis/prepuce. Plates inoculated with two (3%) of the preputial swabs were overgrown by rapid-growing saprophytic bacteria which made the detection of T. asinigenitalis, if present, impossible. T. asinigenitalis was not cultured from the testes or epididymides of the 3 donkeys (animals 1, 2, and 9) that were positive when cultured immediately before they were castrated, nor from 2 donkeys (animals 10 and 11) that were culture negative when castrated but positive when cultured about 2 months after they were castrated. 3.5. Genotypes of T. asinigenitalis The results obtained from sequencing the 16S rRNA gene divided the isolates from the 23 culture-positive equids into two genotypes of T. asinigenitalis. Isolates from the 9 donkeys on Farm 1 and isolates from 8 equids (animals 13–17, 20, 22, and 23) from Farm 2 were identical to each other and to the type strain of T. asinigenitalis which was isolated in 1997 from a donkey jack in California. Isolates from the remaining 6 equids (animals 10–12, 18, 19, and 21) on Farm 2 were identical to each other and to the two strains recovered from donkey jacks in 1998 in Kentucky. 3.6. Analytical findings Results for the univariate and multivariate analyses are shown in Tables 5 and 6, respectively. A statistical association was demonstrated between positive culture
Table 5 Univariate analysis for animal-based risk factors associated with a culture positive genital swab for T. asinigenitalis among equids residing on two premises in Michigan. Variable
Categorical value
Odds ratio
Farm
1 2 2 years of age >2 years of age Female Male or gelding Horse Donkey or mule No Yes Purchased Raised 1 year >1 year
Ref 7.55 Ref 1.63 Ref 1 Ref 6.38 Ref 6.30 Ref 1.25 Ref 5.56
Age class Genderb Speciesb Offspring Source Length of time on premisesc
Lower 95% CIa
Upper 95% CI
Fisher exact p-value
2.39
25.65
<0.001
0.39
9.83
0.753
2.89
1
<0.001
0.87
284.21
0.060
1.35
60.03
0.008
0.37
4.96
0.786
0.73
72.03
0.099
Ref, reference category. a Confidence intervals. b Cell totals were collapsed into two categories for analysis as the Cochran conditions were not satisfied due to cells with zero counts and more than 80% of the cells with counts less than 5. c Analysis restricted to premises number 2.
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
440
Table 6 Multivariate exact logistic regression analysis of animal-based risk factors associated with a culture positive genital swab for T. asinigenitalis among equids residing on two premises in Michigan. Variable
Categorical value
Odds ratio
Farm
1 2 Male or gelding Female Donkey or mule Horse No Yes
Ref 6.31 Ref 0.091b Ref 0.132 Ref 5.01
Gender Species Offspring
Lower 95% CIa 1.53
Upper 95% CI 30.73
Fisher exact p-value 0.007
0
0.521
0.0172
0.002
1.78
0.174
0.556
246.70
0.229
Ref, reference category. a Confidence intervals. b Indicates a median unbiased estimate.
status and farm location (exact odds ratio, 7.55; 95% CI 2.39, 25.65); an association that was still present after adjusting for other covariates in the multivariate analysis. The effect of prior breeding (exact odds ratio 6.30; 95% CI 1.35, 60.03) identified in the univariate analysis was not present after adjusting for confounders in the multivariate model. The univariate analysis found no significant differences for being culture positive for T. asinigenitalis and the age category of the animal (2 years of age versus >2 years of age). For Farm 2, no differences were observed for the culture status of animals that were purchased compared to those raised on the premises. Overall, the analysis suggests that an animal located on Farm 2 was at a six times greater risk of being culture positive for T. asinigenitalis (exact odds ratio, 6.31; 95% CI 1.53, 36.07) compared to an animal located on Farm 1 and that females are at a eleven times lower risk (exact odds ratio 0.091; 95% CI 0, 0.521) for being culture positive compared to males (stallions or geldings). 4. Discussion A survey of 43 donkey jacks and 1 male mule from 12 premises in Michigan revealed that 9.3% of the donkeys were culture positive for T. asinigenitalis at time of castration (Carleton et al., unpublished data). Based on the findings of this study and those of Jang et al. (2001), Meade et al. (2010), Franco et al. (2009), and Breuil et al. (2011), the donkey is probably the natural host species of T. asinigenitalis, unlike the horse which is considered the reservoir species of T. equigenitalis. The current study is the first to survey a population of donkey jacks for evidence of T. asinigenitalis. As previously reported (Jang et al., 2001; Meade et al., 2010), T. asinigenitalis would appear more widely distributed than suggested initially and may be endemic in the US donkey population. This may well be the situation in other countries reporting isolations of this bacterium (Ba˚verud et al., 2006; Franco et al., 2009; Breuil et al., 2011). Transmission to horses appears to be infrequent and tangential to the primary circulation of this bacterium in the donkey population (Ba˚verud et al., 2006; Meade et al., 2010; Breuil et al., 2011; Reilly et al., 2012). There was no evidence of T. asinigenitalis in the testes or epididymides in 3 of the 4 donkeys cultured positive in the survey. Failure to culture T. asinigenitalis from the proximal
reproductive tract would support the contention that T. asinigenitalis is a preferential inhabitant of the distal reproductive tract, where it can co-exist with the normal bacterial flora on the external genitalia of the intact or gelded male, especially the donkey jack. Of the collective equid populations on the two farms in the second phase of the study, 25.8% were positive for T. asinigenitalis. The culture positivity rate varied widely between the two premises, with 14.1% on Farm 1 and 56% on Farm 2. Of 23 culture positive animals on Farm 2, 22 were donkey jacks, 53.5% of which had been castrated previously. This is supportive evidence of the role of the donkey jack as the primary reservoir of T. asinigenitalis. Detailed investigation failed to turn up any evidence of sharing of donkeys or horses betweens Farm 1 and 2. As reported previously (Anonymous, 1998; Jang et al., 2001; Meade et al., 2010; Katz et al., 2000), neither the source nor when exposure to T. asinigenitalis occurred was determined either for the 4 donkey jacks detected in the initial survey or the 18 donkey jacks and 1 horse gelding subsequently identified on Farms 1 and 2. Many of the donkeys on Farm 2 were of diverse backgrounds. They were either rescue donkeys or donkeys originating from Western or other Mid-Western states. They included 5 relatively recently introduced Bureau of Land Management donkeys, 3 of which originated in Nevada and 2 in Arizona. Failure to detect T. asinigenitalis in mares or female donkeys in this study is similar to the finding of Breuil et al. (2011) but in contrast to the report of Reilly et al. (2012). It is intriguing why this is the case considering that donkeys and horses were allowed to co-mingle at pasture on Farms 1 and 2. An explanation may be the fact that the clitoral fossa and sinuses were the sites sampled in donkey jennies or mares. Based on previous data (Meade et al., 2010), T. asinigenitalis was more often cultured from the proximal (uterus, cervix) rather than the distal reproductive tract (clitoral fossa and sinuses) in a small number of carrier mares. In contrast, most T. equigenitalis chronically infected mares are clitoral carriers. Unfortunately, with the exception of 2 donkey jennies and 1 mare on Farm 1, it was not possible to obtain swabs from both proximal and distal reproductive tract sites in female animals. Alternatively, carriage of T. asinigenitalis may occur less frequently and for a shorter term in the donkey jenny or the mare than in the intact or gelded male, donkey or stallion; This would be consistent with the
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
findings of Platt et al. (1978) and Timoney et al. (1978) confirming short-term persistence of T. equigenitalis in most experimentally infected mares. Since bacteriological culture is still considered the ‘‘gold standard’’ by the World Organisation for Animal Health, it was selected over real-time PCR (Wakeley et al., 2006) as a proven means of detection of T. asinigenitalis. Various PCR assays have been shown to be highly specific, rapid and at least of equivalent sensitivity to culture. With few exceptions, problems of bacterial overgrowth of plates were not encountered. The preferential sites for isolation of T. asinigenitalis were similar to those reported for T. equigenitalis in the stallion (Timoney, 1996). With one exception (donkey 14), animals cultured positive on the first set of swabs were also positive on subsequent swabbings, regardless of the sampling interval that ranged from 2 to 19 months. A total of 6 donkeys on Farm 2 were culture positive 31 months after initial sampling, and one additional donkey was positive for 33 months, at which point the study had to be terminated. It is highly improbable that some or all of these donkey jacks cleared themselves of T. asinigenitalis and became reexposed to the organism at a later point in the study period. It should be emphasized that with the exception of donkey 14, each individual was culture positive on 2–5 consecutive sets of swabs. On a point of comparison, T. equigenitalis can persist in the stallion for at least 8 years. It should not be surprising therefore that long-term carriage of T. asinigenitalis may also extend over a period of years. This study provides the first documented evidence that this can occur and also, that T. asinigenitalis can persist in the gelded male. In light of the presence of T. asinigenitalis in close to onethird of the gelded donkey jacks in the current study, it is plausible to conjecture that persistence of T. equigenitalis may also occur in horse geldings. If so, it could have implications with regard to the international movement not only of stallions and mares but also geldings as potential carriers of T. equigenitalis, the cause of contagious equine metritis (CEM). The finding of a limited number of donkeys in the current study that gave positive reactions in the CF test using antigen prepared from T. equigenitalis was unexpected. Of a total of 5 CF positive donkeys, 2 were culturepositive donkey jacks and 3 were culture negative (2 jennies and one gelded donkey jack). Detection of CF antibody activity in male donkeys runs counter to the reported absence of a detectable serological response in stallions following natural or experimental exposure to T. equigenitalis (Bryans et al., 1979; Powell, 1981; Platt and Taylor, 1982; Timoney and Powell, 1988). It remains to be determined whether the positive CF reactions were specific for Taylorella spp. or not. Whereas Bryans et al. (1979) found no evidence of non-specific CF antibody activity in the sera of mares naturally infected with T. equigenitalis, Timoney et al. (1985), in an experimental study of T. equigenitalis infection in donkey jennies, concluded that the CF test was of limited diagnostic value due to presence of low levels of non-specific reactivity and anti-complementary activity. Genotyping of isolates of T. asinigenitalis from the 23 culture-positive equids, revealed they belonged to 2
441
clusters. Some 17 were identical to the type strain isolated in California in 1997 (Anonymous, 1998) and 6 to the strains recovered in Kentucky in 1998 (Meade et al., 2010). Whereas all 9 isolates from Farm 1 and 8 of the Farm 2 isolates belonged to the first cluster, the remaining 6 isolates from Farm 2 belonged to the second cluster. This is the first time different genotypes of T. asinigenitalis have been found in circulation on the same premises. Breuil et al. (2011) also found that French isolates of T. asinigenitalis belonged to 2 clusters. While these findings would suggest a lack of genomic diversity among strains recovered in the US and elsewhere, very recently, Reilly et al. (2012) reported a third genotype of the bacterium that was isolated from a mare in the US during routine preexport screening. Based on its relatedness to T. equigenitalis, it is reasonable to assume that most if not all of the transmission of T. asinigenitalis to 22 donkey jacks and 1 horse gelding had to have taken place by direct or indirect venereal contact. As already indicated, there was no evidence of infection in donkey jennies and mares. Even if donkey jacks and the gelding were exposed to T. asinigenitalis by the venereal route, that fails to explain how 2, 10-month-old sexually immature donkey jacks acquired the organism. The only credible explanation would be that they were the offspring of culture positive dams and acquired T. asinigenitalis in utero or at time of parturition. Timoney and Powell (1982) presented a similar argument to account for recovery of T. equigenitalis from a number of sexually immature colts and fillies. 5. Conclusions The study findings provide additional evidence supporting endemicity of T. asinigenitalis in donkeys and their role as the natural host species of the organism. Extended carriage of the bacterium in gelded donkey jacks might suggest that long-term persistence of T. equigenitalis similarly may occur in gelded colts or stallions. Genotypic analysis revealed limited evidence of genomic diversity among of isolates of T. asinigenitalis. Acknowledgements This report was published (12-14-028) by permission of the Dean and Director of the Kentucky Agricultural Experiment Station and the College of Agriculture, University of Kentucky, Lexington. We thank Dr. Dave Dargatz, Center for Epidemiology and Animal Health, USDAS, APHIS, VS for advice and guidance with data analysis, and the serology section, UKVDL for performance of the CF test. The authors acknowledge with gratitude the financial support provided by the University of Kentucky Veterinary Diagnostic Laboratory in carrying out this study. References Anonymous, 1998. CEM in a mammoth jack donkey. J. Equine Vet. Sci. 18, 100–101. Arata, A.B., Cooke, C.L., Jang, S.S., Hirsh, D.C., 2001. Multiplex polymerase chain reaction for distinguishing Taylorella equigenitalis from Taylorella equigenitalis-like organisms. J. Vet. Diagn. Invest. 13, 263–264.
442
J.M. Donahue et al. / Veterinary Microbiology 160 (2012) 435–442
Ba˚verud, V., Nystro¨m, C., Johansson, K.E., 2006. Isolation and identification of Taylorella asinigenitalis from the genital tract of a stallion, first case of a natural infection. Vet. Microbiol. 116, 294–300. Breuil, M.F., Duquesne, F., Laugier, C., Petry, S., 2011. Phenotypic and 16S ribosomal RNA gene diversity of Taylorella asinigenitalis strains isolated between 1995 and 2008. Vet. Microbiol. 148, 260–266. Bryans, J.T., Darlington, R.W., Smith, B., Brooks, R.R., 1979. Development of a complement fixation test and its application to diagnosis of contagious equine metritis. J. Equine Med. Surg. 3, 467–472. Derr, R.E., 2000. Performing exact logistic regression with the SAS System. In: Proceedings of the Twenty-fifth Annual SAS Users Group International Conference. SAS Institute, Inc., Cary, NC. Donahue, J.M., Swerczek, T.W., Smith, B.J., 1978. Laboratory diagnosis of contagious equine metritis. In: Proceedings of the 21st Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. pp. 497–505. Franco, A., Donati, V., Troiano, P., Lorenzetti, R., Zini, M., Autorino, G.L., Petrella, A., Maggi, A., Battisti, A., 2009. Detection of Taylorella asinigenitalis in donkey jacks in Italy. Vet. Rec. 165, 540–541. Jang, S.S., Donahue, J.M., Arata, A.B., Goris, J., Hansen, L.M., Earley, D.L., Vandamme, P.A.R., Timoney, P.J., Hirsh, D.C., 2001. Taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (Equus asinus). Int. J. Syst. Evol. Microbiol. 51, 971–976. Katz, J.B., Evans, L.E., Hutto, D.L., Schroeder-Tucker, L.C., Carew, A.M., Donahue, J.M., Hirsh, D.C., 2000. Clinical, bacteriologic, serologic, and pathologic features of infections with atypical Taylorella equigenitalis in mares. J. Am. Vet. Med. Assoc. 216, 1945–1948. Meade, B.J., Timoney, P.J., Donahue, J.M., Branscum, A.J., Ford, R., Rowe, R., 2010. Initial occurrence of Taylorella asinigenitalis and its detection in nurse mares, a stallion and donkeys in Kentucky. Prev. Vet. Med. 95, 292–296.
Platt, H., Atherton, J.G., Simpson, D.J., 1978. The experimental infection of ponies with contagious equine metritis. Equine Vet. J. 10, 153–159. Platt, H., Taylor, C.E., 1982. Contagious equine metritis. Med. Microbiol. 1, 49–96. Powell, D.G., 1981. Contagious equine metritis. Adv. Vet. Sci. Comp. Med. 25, 161–184. Reilly, J.R., Calcutt, M.J., Ganjam, I.K., Erdman, M.M., Aalsburg, A.M., Blyden, J.F., Fales, W.H., 2012. Isolation and identification of Taylorella asinigenitalis from a mare in Oklahoma, USA. Clin. Theriogenol. 4, 163–167. Timoney, P.J., 1996. Contagious equine metritis. Comp. Immunol. Microbiol. Infect. Dis. 19, 199–204. Timoney, P.J., McArdle, J.F., O’Reilly, P.J., Ward, J., 1978. Infection patterns in pony mares challenged with the agent of contagious equine metritis 1977. Equine Vet. J. 10, 148–152. Timoney, P.J., Powell, D.G., 1982. Isolation of the contagious equine metritis organism from colts and fillies in the United Kingdom and Ireland. Vet. Rec. 111, 478–482. Timoney, P.J., Powell, D.G., 1988. Contagious equine metritis-epidemiology and control. J. Equine Vet. Sci. 8, 42–46. Timoney, P.J., O’Reilly, P.J., McArdle, J.F., Ward, J., Harrington, A.M., 1985. Contagious equine metritis: experimental infection in the donkey. Vet. Microbiol. 10, 259–268. Timoney, P.J., Shin, S.J., Jacobson, R.H., 1982. Improved selective medium for isolation of the contagious equine metritis organism. Vet. Rec. 111, 107–108. Wakeley, P.R., Errington, J., Hannon, S., Roest, H.I.J., Carson, T., Hunt, B., Heath, P., 2006. Development of a real time PCR for the detection of Taylorella equigenitalis directly from genital swabs and discrimination from T. asinigenitalis. Vet. Microbiol. 118, 247–254.