mycoides subsp. mycoides SC identification by PCR in sperm of seminal vesiculitis-affected bulls Giuseppe Stradaioli, Lakamy Sylla, Francesco Mazzarelli, Riccardo Zelli, Georges Rawadi, Maurizio Monaci

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Giuseppe Stradaioli, Lakamy Sylla, Francesco Mazzarelli, Riccardo Zelli, Georges Rawadi, et al.. Mycoplasma mycoides subsp. mycoides SC identification by PCR in sperm of seminal vesiculitis- affected bulls. Veterinary Research, BioMed Central, 1999, 30 (5), pp.457-466. ￿hal-00902586￿

HAL Id: hal-00902586 https://hal.archives-ouvertes.fr/hal-00902586 Submitted on 1 Jan 1999

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Mycoplasma mycoides subsp. mycoides SC identification by PCR in sperm of seminal vesiculitis-affected bulls

Giuseppe Stradaioli Lakamy Syllab Francesco Mazzarellib Riccardo Zellib Georges Rawadic Maurizio Monacib

a Facoltà Medicina Veterinaria, Dipartimento di Scienze della Produzione Animale, Universita di Udine, v. delle Scienze 208, 33100 Udine, Italy b Facoltà Medicina Veterinaria, lstituto di Ostetricia e Ginecologia, Universita di Perugia, v. S. Costanzo 4, 06126 Perugia, Italy ! Département de bactériologie et de mycologie, Institut Pasteur, 25, rue Docteur-Roux, 75724 Paris cedex 15, France

(Received 1 March 1999; accepted 5 July 1999)

Abstract - In this study, by using the polymerase chain reaction (PCR) diagnosis for the detection and identification of Mycoplasma, we investigated contaminating the semen of year- ling bulls affected by seminal vesiculitis. The bulls presented neither subclinical nor clinical conta- gious bovine pleuropneumonia signs and the complement fixation test for specific antibodies was neg- ative. Furthermore, we have investigated mycoplasmas isolated from semen of healthy breeding bulls of several breeds and origins, which routinely underwent breeding soundness examinations and presented no clinical signs of seminal vesiculitis. We were able to demonstrate mycoplasma infection in all tested samples by i) growth on mycoplasma-specific media and ii) a PCR-based method using a mycoplasma-specific MGSO/GPO1 primer set to amplify the 16S fragment rDNA. In addition, the identification of Mycoplasma species was made by PCR using the MSCI/MSC2 primer set that specifically amplifies M. mycoides subsp. mycoides SC or the MM450/MM451 primer set followed by AsnI digestion analysis in order to identify M. mycoides subsp. mycoides LC. The data presented herein clearly show that M. mycoides subsp. mycoides SC infection was associated with sem- inal vesiculitis while M. mycoides subsp. mycoides LC was only found in bull semen from healthy con- trol animals. Our findings confirm that the M. mycoides subsp. mycoides SC is shed in the sperm mak- ing the ejaculate a valuable biological sample for the isolation of these from serologically negative animals. Although the pathogenic role of M. bovigenitalium in bull seminal vesiculitis has been established, our clinical findings, semen characteristics, microbiological and bacterial genomic analysis strongly suggest that M. mycoides subsp. mycoide.s SC may contribute to induce vesicular adenitis in the bull. © Inra/EIsevier, Paris. bovine / sperm / seminal vesiculitis / Mycoplasma / PCR

* Correspondence and reprints Tel.: (39) 075 585 4420; fax: (39) 075 585 4424; e-mail: [email protected] Résumé - Identification de Mycoplasma mycoides subsp. mycoides SC dans le sperme de taurillons atteints de vésiculite séminale avec la technique d’amplification en chaîne (PCR). Dans la présente étude, nous avons appliqué la technique d’amplif ïcation en chaîne par polymérase (PCR) pour la détection et l’identification des mycoplasmes dans les prélèvements de sperme de taurillons atteints de vésiculite séminale. Les prélèvements ont été obtenus de taurillons lors de l’épidémie de pleu- ropneumonie bovine contagieuse survenue en Italie en 1994, mais ne présentant aucun signe clinique ou subclinique de pleuropneumonie. Les taurillons ont été montrés négatifs pour l’agent de pleu- ropneumonie bovine contagieuse par le test de fixation de complément. Nous avons inclus dans notre étude la recherche de Mycoplaslllil spp. isolés du sperme de taurillons témoins, non atteints de vésiculite. Nous avons pu mettre en évidence la présence de mycoplasmes dans tous les prélève- ments d’abord par culture sur milieu spécifique ensuite par amplification spécifique du gène d’ARN 16S ribosomal de mycoplasmes en utilisant la paire d’amorces GPOI/MGSO. L’identification des mycoplasmes isolés a été rendue possible par PCR utilisant la paire d’amorces MSCI/MSC2 pour l’identification de M. n?vcoides subsp. mycoides SC ou utilisant la paire d’amorces MM450/MM4511 suivi de digestion enzymatique par A.snf pour la détection de M. mycoides subsp. mycoicles LC. Les résultats montrent clairement l’identification de M. mvcoides subsp. mycoides SC dans les prélève- ments de sperme de taurillons atteints de vésiculite séminale, alors que M. on’f’M!M subsp. mycoides LC a été identifié dans les prélèvements du groupe témoin. Nos résultats confirment que le sperme est susceptible de véhiculer M. mvcoicfo.s subsp. ttnt!«:/M Se. et ainsi constitue une source intéres- sante pour la détection de ce pathogène chez les animaux séronégatifs. Bien que le rôle de M. boui- genitalium ait été clairement établi dans la vésiculite séminale, les résultats présentés ici suggèrent que M. mycoide.s subsp. rnycnides SC pourrait jouer un rôle dans l’induction d’adénite vésiculaire chez les taureaux. © InraElsevier, Paris. bovin / sperme / vésiculite séminale / Mycoplasma / PCR

1. INTRODUCTION induced genital infections in cattle [ 18, 24, 39]. The seminal vesiculitis syndrome affects The mycoides cluster includes six bulls of all ages. The pathogenesis has not Mycopla.sma species or subspecies that are been clearly established; however, several all significant pathogens in small and large pathogenic organisms such as Brucella : M. rnycaides subsp. mycoide,s abortus [5], Pseudomonas tiertigiiios!i and with two biotypes, small-colony (SC) and .4(’!/w!ac///M.’) actinoides [ 17!, Corynebac- large-colony (LC); M. mycoides subsp. terium pyo!;ene.s ! 12!, Proteu.s [28],1, capr-i; M. cnpricnlum subsp. capricolum: Haemophilus A’!/!MM.s’ [ 16], Mycoplasma M. capricolum subsp. (-apripneuiiioiiiae spp. [ 1 ] and Uplasma rea diversum [ 4 1 have (type F38) and M. sp. group 7 of Leach (type been associated with seminal vesiculitis. PG50) [8, 25]. Mycoplasma mycoides subsp. mycoides SC is responsible for contagious Mycoplasma organisms are widely dis- bovine pleuropneumonia (CBPP), a spo- tributed in animals and human beings and radic infection in Europe [26]. The com- have been observed in different tracts of the pleiiieiit fixation test for the detection of this reproductive system in cattle 13, 23 A pathogen is highly specific in approximately high incidence of Mycoplasma and Ure- 70 % of infected animals, while it is poorly aplasma contamination in fresh and frozen responsive in asymptomatic animals in the bull semen has been reported [2, 29, 351 by early stages of infection and in chronically the Artificial Inscmination Industry. Some infected ones [31 In affected animals, M. species of mycoplasma have been associ- /HVt’!;’<’/6’.s’ subsp. mycoides SC and its anti- ated with both natural and experimentally gens have been found in the lungs, thoracic lymph nodes, blood and extrathoracic organs grcssive motile spermatozoa were evaluated in such as the kidney and liver [27, 36, 37]. diluted semen according to the World Health M. mycoides subsp. mycoides SC has Organization [42J. The concentration (l0y sper- matozoa/mL) was measured by means of the recently been identified in bull semen, Burker haemocytometer. The spermatozoa mor- clinical associated with although findings phology was evaluated with the eosin-nigrosin genital infections were not observed [14]. stain [6] and defective cells were grouped into and secondary abnormalities [7]. Addi- In the present we bac- primary study, investigated tional smears were also stained with Giemsa for teria isolated from semen of seminal vesi- leukocyte identification. culitis-affected yearling bulls, derived from an outbreak of CBPP in Italy in 1994 and from healthy control bulls presenting no 2.2. Microbiological analysis clinical signs of seminal vesiculitis. The data reported herein, using specific Seminal vesicle secretions and semen sam- PCR assays for the identification of bovine ples from affected animals were diluted with Mycoplasma species, has clearly shown the PBS solution pH 7.2 and were centrifuged at 1 500 30 min. The (0.5 mL) infection of seminal vesiculitis bull semen g for supernatant was plated onto agar, blood agar and Gassner with M. mycoides subsp. mycoides SC and media and then incubated at 37 °C for 24 h. For the semen from control bulls with M. mycoplasma isolation, 0.5 mL of the supernatant mycoides subsp. mycoides LC. was added to 6 mL PPLO broth (Difco, Milano, Italy) supplemented with 0.5 % yeast extract (Difco, Milano, Italy), 20 °l° heat-inactivated horse serum Milano, 10 2. MATERIALS AND METHODS (Difco, Italy), g of tryp- tose (Difco, Milano, Italy), I % glucose, 500 000 IU of Penicillin (Carlo Erba, Milano, Italy), thal- lium acetate (Carlo Erba, Milano, Italy) and dis- 2.1. Animals and sample collections tilled 7.6-7.8. Four 10-fold dilutions ( I (r ! towater I 0 )pH were titrated from this in the same broth and incubated at 37 °C for 3 The IW4 The study was carried out on four yearling days. dilution of the PPLO broth was sub- fresian bulls (from 1 l to l3 months of age) routinely cultured on the PPLO agar. were affected by seminal vesiculitis derived from an Agar plates outbreak of contagious bovine pleuropneumo- incubated in an aerobic and anaerobic environ- and observed for on the nia in 1994 in (bulls A, B, C, D). The bulls ment colony appearance Italy 3rd and 6th of in vitro culture. presented neither subclinical nor clinical CBPP days Mycoplasma colonies were isolated into culture medium and the fixation test [15J for liquid signs complement and maintained at―80 °C until specific antibodies proved to be negative. genomic analysis. In order to identify Mvcoplasma spp. by bio- Furthermore, we included mycoplasma- chemical tests, glucose fermentation [33], argi- infected semen samples from healthy breeding nine hydrolysis [3], film and spot formation, bulls of several breeds and origin, which rou- casein and serum and tetrazolium salt tinely underwent breeding soundness examina- digestion reduction were performed [I I].1. tions, presenting no clinical signs of seminal vesiculitis in our investigation (bulls E, F, G, H). The clinical evaluation of the genital tract, including transrectal ultrasound examination of 2.3. DNA preparation the accessory sexual glands (Medison, SONACE from mycoplasma colonies 88P, 5 MHz linear array transducer), were carried out. Seminal vesicle secretions were harvested DNA was prepared from mycoplasma sam- using a sterile catheter via the urethra by tran- ples as described by Kellog and Kwok [211.] . srectal massage, as described by Parsonson et Briefly, mycoplasmas were centrifuged at al. [30]. 17 000 g for 30 min at 4 °C, and the cell pellet was in 500 mL of K In addition, two consecutive ejaculates were resuspended proteinase collected by the artificial vagina method. The (Gibco BRL, France) solution (10 mM Tris-HCI, 8.3, 50 mM KCI, 2.5 mM 0.5 5 % volume of each ejaculate was determined. Pro- pH MgCl2, Tween 20, 0.5 % Triton X-100, and 60 mg/mL 373 software. Sequencing was performed with proteinase K) and incubated at 37 °C for 2 h. MGSO or GPO I primers. Heating at 95 °C for 10 min denatured proteinase K, and the samples were cooled in ice prior to use in PCR amplification. At this stage the sam- ples could be stored at-20 °C for several months. 2.6. PCR assay for mycoplasma species identification

The identification of 2.4. PCR for Mycoplasma mycoides assay mycoplasma SC was as described detection subsp. mycoides performed by Dedieu et al. [9]. Mycoplasma mycoides subsp. mycoides SC-specific amplification using MGSO and GPO1 PCR primers (table n [32] MSC1 and MSC2 primers (table I) was per- were used to amplify mycoplasma 16S rDNA formed on DNA samples as described above, from samples as described by Roulland-Dussoix except that the annealing temperature was 53 °C. et al. 40 mM of each were [34]. Here, primer The identification of mixed in 50 mM KCI, 10 mM Tris-HCI 8.3, Mycoplasma mycoides pH LC was as described I .5 mM 0.2 mM dNTP and I unit of Taq subsp. mycoides performed MgCI2’ Bashiruddin et al. [4]. Amplification from DNA polymerase (Appligene Oncor, Montreuil, by DNA samples was performed using MM450 and France). DNA sample (2 !tL) was added to 48 ¡.t.L MM451 (table I) as described above of PCR mix, then heated for 15 min at 95 °C and primers that the was 50 °C. was carried out for 35 as except annealing temperature amplification cycles DNA fragments using the follows: 95 °C for 30 s, 58 °C for I min and 30 s, Amplified MM450/MM451 set were extracted with 72 °C for 1 min and 30 s. primer phenol/chloroform and precipitated with ethanol. Precipitated DNA was digested with 10 units of Asnl in a total volume of 20 mL. The digests 2.5. PCR product sequencing were then electrophoresed in 2.5 % Metaphor (FMC, Le Perray en-Yvelines, France) gel. Mycoplasmal 16S rDNA fragments ampli- M. agalactiae identification was performed fied from DNA samples were purified by phe- as described by Subramaniam et al. [38] by spe- nol/chloroform extraction and precipitated with cific amplification of the uvrC gene. MAGAU- ethanol. Purified DNA fragments were sequenced VRC1-L and MAGAUVRC1-R primers were using the automated ABI sequencer employing used to carry out M. agalactiae identification in the DNA samples. PCR amplification was carried dilated, presenting an irregularly echogenic out as indicated above except that the annealingg content due to abnormal fluid accumulation temperature was 50 °C. (figure 1).

The ejaculates were stringy, creamy-yel- 2.7. Analysis of amplified PCR low and contained clots of pus. Semen char- products acteristics, compared to breeding bulls, are reported in table II. No significant differ- Each amplified DNA (20 mL) was subjected ences were observed in semen characteris- to electrophoresis with 1.5 % agarose (Sigma, tics, although total defective sperm cells St. Quentin Fallavier, France) Asnl DNA gel. were more than 6 % higher in the seminal was carried out for M. digestion mycoides subsp. vesiculitis-affected bulls. Clusters of mycoides identification and the digestion products agglu- tinated cells were observed. Further- were analysed by electrophoresis on 2.5 % sperm Metaphor agarose (FMC, Le Perray en-Yvelines, France). DNA was detected by UV fluorescence after ethidium bromide staining. 0X 174 digested with NaeIII (Boerhinger Mannheim GmbH, Ger- many) was used as a marker to estimate the molecular weight of the amplified bands.

3. RESULTS

3.1. Clinical findings

Neither congenital nor acquired abnor- malities of the external genitalia were observed while internal genital examination was characterized by moderate enlargement, loss of lobulation and fluctuation areas of the seminal vesicles. The animals presented a monolateral enlargement of the glands, except bull D in which both seminal glands were affected. Upon transrectal ultrasound examination, the glandular lumen appeared more, the Giemsa-stained smears presented Biochemical data showed that isolated polymorphonuclear leukocytes and epithe- mycoplasmas fermented glucose, reduced lial cells. tetrazolium salts, were negative for arginine and did not digest serum and casein.

In order to confirm mycoplasma con- 3.2. Mycoplasma detection in semen tamination in these semen samples, DNA samples was extracted from samples and subjected to PCR amplification using the primer set, MGSO/GPO1, that specifically amplifies No significant bacterium was isolated the 16S rRNA gene from the mycoplasma from the LB agar, blood agar and Gassner . All samples were found to be positive media. However, on PPLO agar plates the for mycoplasma based on the amplification typical mycoplasma colonies called ’fried- results obtained with the mycoplasma genus- egg’, were shown in all seminal fluids and specific PCR as indicated in table III. These ejaculate samples. These data clearly indi- data indicate that the specimens were con- cated that the specimens were infected with taminated with mycoplasmas and confirmed mycoplasmas. earlier findings. 3.3. Identification of mycoplasma order to determine whether these samples contamination were contaminated with other Mycoplasma species related to the iii-v(-oides cluster, such We further pursued our investigation to as M. agalactiae or M. bovis, we carried out identify the Mycoplas/l1a species that had a specific PCR for these two species. No been isolated from the semen samples. PCR amplification was obtained from our sam- products obtained from the MGSO/GPOII ples, thus ruling out this possibility (data amplification were purified and sequenced. not shown). The sequences obtained were compared with those of the Genebank database by using a blast and fast program. The sequence com- 4. DISCUSSION parison data showed that the amplified 16S rRNA gene from the semen samples was The aetiology of seminal vesiculitis has related to the mycoides cluster and highly never been completely elucidated. Overfed similar to the M. mvcwicles subsp. rrrycoides and growing yearling bulls appear to be LC and SC (98 % similarity; data not more susceptible to seminal vesiculitis, shown). Yet, given the high similarity (up to although some affected bulls have presented 97 %) between these two subspecies, it was a history of pneumonia or septicaemia. The inaccurate to clearly identify the bulls that were the object of our study had, Mycoplasma species or subspecies based however, no previous history of disease. on a 16S rRNA sequence comparison. Lowered fertility has been related to the seminal vesiculitis From these data, we could predict that syndrome; however, affected bulls overcome the our samples were most likely contaminated young may infection with a mycoides SC or LC subtype; there- spontaneously i0j. fore, we set up and performed specific PCR Although the occurrence of such con- assays for the identification of these sub- taminants as U. diversulll, M. bovis and types. The data are summarized in table III. M. bovigcl1italium has been reported in the When PCR was carried out using the reproductive tract and bull semen, our study MSC1/MSC2 primer set, only samples from demonstrates semen contamination by the affected bulls were found positive, indi- M. Illycoidcs subsp. l11ycoidcs SC in year- cating that the mycoplasma contaminating ling bulls shown to be negative by the CBPP these samples was M. mycoides subsp. complement fixation test, as previously /I1ycoides SC. Sequencing of PCR products observed in Portugal by Gon!:alves [ 14]. confirmed our results not ). (data shown). Within the last few years, M. l11ycoidcs In addition, when PCR was carried out cluster identification has been dramatically using the MM450/MM451 primer set, all improved through the use of species-spe- samples were found to be positive (lable cific DNA probes and PCR. Several PCR III). Digestion of thc PCR products from assays have been previously described for samples from bulls B, C and D with A.Ylil, the identification of M. l11ycoidcs subsp. yielded two bands allowing the identilication l11ycoilcs LC [41 and SC [9]. The of subsp. IIlvcoides SC and thus confirmed MSCI/MSC2 primer set described by the previous identification. Furthermore, the Dedieu et al. !9] allows the specific identi- digestion of PCR products from samples fication of M. l11ycoide.B’ subsp. rnycoidc,v from bulls F and G yielded three bands indi- SC. On the contrary, the MM450/MM4511 cating that the Mvcopla.B/I1(( species con- primer set described by Bashiruddin et al. taminating these samples was M. mvcnicles [4] allows the specific detection of both M. subsp. mvcoides LC. Identification of the l11ycoides subsp. l11ycoides SC and LC. LC subtype was confirmed by sequencing of Digestion of the PCR products using A.snl PCR products (data not shown). 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