Mycoplasma 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 To cite this version: 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 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Original article 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 mycoplasmas 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 bacteria 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 ruminants: 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