Isolation and Detection of Mycoplasma agalactiae from Semen Samples of Goats

Document Type : Original Articles

Authors

1 Mycoplasma Reference Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran

2 Master

3 Assistant Professor

4 Bachelor

Abstract

Contagious agalactia (CA) is a highly infectious disease of goats and sheep, and is a form of Mycoplasmosis,
which is usually enzootic. Since Mycoplasma agalactiae (M. agalactiae) is the main cause of this disease in
goats, the aim of this study was to isolate and detect M. agalactiae from semen of goat bucks. Thirty-nine semen
samples were collected from goat bulks, and all samples were cultured in PPLO broth medium supplemented for
M. agalaciae isolation. The bacteria DNAs were extracted from clinical samples and the PCR assay was applied
to detect Mycoplasma genus and M. agalactiae species using specific primers, which amplified a 163bp
fragment in 16SrRNA gene and a 375bp fragment in lipoprotein gene. The PCR evaluations were performed for
both the clinical samples and the cultures. Out of the 39 samples, 29 (74.3%) of the cultures were shown positive
and typical Mycoplasma colonies grew on PPLO agar, which could be considered as the diagnostic method. In
addition, 38 (97.4%) samples had positive PCR results for Mycoplasma genus and six (15.3%) of the samples
were shown to be positive using PCR for M. agalactiae as the diagnostic method. In the present study, M.
agalactiae was detected in semen of goat bulks for the first time in Iran. Therefore, it is recommended to
concern semen as one of the significant sources for this pathogen and the possibility for transmission to the
female goats through semen is highlighted. Moreover, presence of this microorganism in semen could be
involved in infertility of goat population.

Keywords

Main Subjects


Article Title [French]

L'isolement et l'identification du Mycoplasma agalactiae à partir de sperme de bouc

Abstract [French]

La maladie agalactie est l'une des maladies les plus infectieuses chez les moutons et les boucs. Cette maladie est dans la catégorie des infections à mycoplasme et est habituellement endémique. Mycoplasma agalactiae est la principale cause de cette maladie chez les boucs. Le but de cette étude était l'isolement et l'identification du Mycoplasma agalactiae à partir du sperme de bouc. Au total, 39 échantillons de sperme de boucs ont été prélevés. Une culture cellulaire a été effectuée sur tous les échantillons dans un milieu de culture adapté et spécifique (PPLO Broth) au Mycoplasma agalactiae. Après extraction de l'ADN des bactéries, des tests PCR ont été menés sur tous les échantillons en utilisant des amorces spécifiques à une séquences du gène 16S rRNA de 163 pb de longueur pour l’identification du genre Mycoplasma et une deuxième de 375 pb composant une partie du gène d’une lipoprotéine spécifique à l’espèces Mycoplasma agalactiae. Sur un total de 39 échantillons analysés, 29 échantillon (74.3%) étaient positifs dans le mileu de culture PPLO. Nos analyses par PCR spécifique au genre Mycoplasma ont révélé que 38 des 39 échantillons (97.4%) étaient positifs parmi lesquels 6 (15.3%) étaient également positifs au test PCR spécifique à l’espèce Mycoplasma agalactiae. Ce travail représente la première étude sur l'isolement et l'identification du Mycoplasma agalactiae présent dans le sperme des boucs en Iran. Les résultats de cette étude ont montré que le sperme pourrait être un endroit approprié pour la présence de cet agent pathogène et sa transmission à la femelle. Par conséquent, ce facteur peut être considéré comme l'une des causes potentielles d'infertilité chez les chèvres.

Keywords [French]

  • boucs
  • gène lipoprotéine
  • Mycoplasma agalactiae
  • Sperme
  • 16S rRNA
Abo-Elnaga, T.R., Gouda, A.S.A., Kamel, Y.M., Mahmoud, M.A., Osman, W.A., 2012. Application of the Polymerase Chain Reaction for identification of Mycoplasma isolated from aborted she camel. Glob Vet 8, 386-392.
Ak, K., Ak, S., Gurel, M., Hasoksuz, A., Barau, Y., Ozturkeler, I., et al., 1995. Experimental studies on the effects of Mycoplasma agalactiae on the spermatozoa and genital organs of rams. Pendik Vet Mikrobiyol Derg 26, 139-155.
Bergonier, D., Berthelot, X., Pourmarat, F., 1997. Contagious agalactia of small ruminants current of knowledge concerning epidemiology diagnosis and control. Rev Sci Techenol 16, 848-873.
Bermudez, V.M., Miller, R.B., Rosendal, S., Fernando, M.A., Johnson, W.H., O'Brien, P.J., 1992. Measurement of the cytotoxic effects of different strains of Mycoplasma equigenitalium on the equine uterine tube using a calmodulin assay. Can J Vet Res 56, 331-338.
Bielanski, A., Devenish, J., Phipps-Todd, B., 2000. Effect of Mycoplasma bovis Mycoplasma bovigenitalium in semen on fertilization and association with in vitro produced morula and blastocyst stage embryos. Theriogenology 53, 1213-1223.
Breard, A., Poumarat, F., 1988. [Isolation of Mycoplasma capricolum from bull semen]. Rev Elev Med Vet Pays Trop 41, 149-150.
Byrne, J., Brennan, P., McCormack, R., Ball, H.J., 1999. Isolation of <em>Mycoplasma bovis</em> from the abomasal contents of an aborted bovine fetus. Vet Record 144, 211.
Corrales, J.C., Esnal, A., De la Fe, C., Sánchez, A., Assunçao, P., Poveda, J.B., et al., 2007. Contagious agalactia in small ruminants. Small Rumin Res 68, 154-166.
de la Fe, C., Amores, J., Martin, A.G., Sanchez, A., Contreras, A., Corrales, J.C., 2009. Mycoplasma agalactiae detected in the semen of goat bucks. Theriogenology 72, 1278-1281.
de la Fe, C., Gomez Martin, A., Amores, J., Corrales, J.C., Sanchez, A., Poveda, J.B., et al., 2010. Latent infection of male goats with Mycoplasma agalactiae and Mycoplasma mycoides subspecies capri at an artificial insemination centre. Vet J 186, 113-115.
Dedieu, L., Mady, V., Lefevre, P.-C., 1995. Development of two PCR assays for the identification of mycoplasmas causing contagious agalactia. FEMS Microbiology Letters 129, 243-249.
Gil, M.C., Peña, F.J., Hermoso De Mendoza, J., Gomez, L., 2003. Genital Lesions in an Outbreak of Caprine Contagious Agalactia Caused by Mycoplasma agalactiae and Mycoplasma putrefaciens. J Vet Med 50, 484-487.
Givens, M.D., Marley, M.S., 2008. Pathogens that cause infertility of bulls or transmission via semen. Theriogenology 70, 504-507.
Gomez-Martin, A., Corrales, J.C., Amores, J., Sanchez, A., Contreras, A., Paterna, A., et al., 2012. Controlling contagious agalactia in artificial insemination centers for goats and detection of Mycoplasma mycoides subspecies capri in semen. Theriogenology 77, 1252-1256.
Hasso, S.A., Al-Aubaidi, J.M., Al-Darraji, A.M., 1993. Contagious agalactia in goats: It's severity as related to the route of infection and pregnancy. Small Rumin Res 10, 263-275.
Jones, G.E., Rae, A.G., Holmes, R.G., Lister, S.A., Jones, J.M., Grater, G.S., et al., 1983. Isolation of exotic mycoplasmas from sheep in England. Vet Rec 113, 540.
Jurmanova, K., Sterbova, J., 1977. Correlation between impaired spermatozoan motility and mycoplasma findings in bull semen. Vet Rec 100, 157-158.
Kissi, B., Juhasz, S., Stipkovits, L., 1985. Effect of mycoplasma contamination of bull semen on fertilization. Acta Vet Hung 33, 107-117.
Kojima, A., Takahashi, T., Kijima, M., Ogikubo, Y., Nishimura, M., Nishimura, S., et al., 1997. Detection of Mycoplasma in avian live virus vaccines by polymerase chain reaction. Biologicals 25, 365-371.
Kotani, H., Nagatomo, H., Ogata, M., 1980. Isolation and Serological Comparison of Ureaplasmas from Goats and Sheep. Japanese J Vet Sci 42, 31-40.
Lambert, M., 1987. Contagious agalactia of sheep and goats. Rev Sci Technol OIE 6, 699-711.
Le Grand, D., Poumarat, F., Martel, J.L., 1995. [Genital Ureaplasma diversum infection: investigations in cattle in France]. Vet Res 26, 11-20.
Miller, R., Chelmonska-Soyta, A., Smits, B., Foster, R., Rosendal, S., 1994. Ureaplasma Diversum As a Cause of Reproductive Disease in Cattle. Vet Clin North Am: Food Anim Prac 10, 479-490.
Nicholas, R.A.J., Ayling, R.D., 2003. Mycoplasma bovis: disease, diagnosis, and control. Res Vet Sci 74, 105-112.
Nunez-Calonge, R., Caballero, P., Redondo, C., Baquero, F., Martinez-Ferrer, M., Meseguer, M.A., 1998. Ureaplasma urealyticum reduces motility and induces membrane alterations in human spermatozoa. Hum Reprod 13, 2756-2761.
OIE., 2008. Contagious agalactia
Singh, N., Rajya, B.S., Mohanty, G.C., 1974. Granular vulvovaginitis (GVV) in goats associated with Mycoplasma agalactiae. Cornell Vet 64, 435-442.
Spergser, J., Aurich, C., Aurich, J.E., Rosengarten, R., 2002. High prevalence of mycoplasmas in the genital tract of asymptomatic stallions in Austria. Veterinary Microbiology 87, 119-129.
Sylla, L., Stradaioli, G., Manuali, E., Rota, A., Zelli, R., Vincenti, L., et al., 2005. The effect of Mycoplasma mycoides ssp. mycoides LC of bovine origin on in vitro fertilizing ability of bull spermatozoa and embryo development. Anim Reprod Sci 85, 81-93.
Tola, S., Idini, G., Manunta, D., Galleri, G., Angioi, A., Rocchigiani, A.M., et al., 1996. Rapid and specific detection of Mycoplasma agalactiae by polymerase chain reaction. Vet Microbiol 51, 77-84