Detection of Ureaplasma spp. in semen samples from sheep in Brazil.

A study was conducted to verify the presence of mycoplasmas and ureaplasmas DNA in sheep semen samples from the State of Pernambuco. The PCR assay was conducted of according with standard protocols with generic primers. Mollicutes DNA was detected in 26.0% and Ureaplasma spp. in 12.0% of semen samples.

Mycoplasmas are members of the class of Mollicutes, which are the smallest known prokaryotes with the capacity to self-replicate (Razin and Tully, 1996; Blanchard and Browning, 2005). These mollicutes are found in nature as parasites, endosymbionts, commensals and saprophytes in animals, plants and insects (Whitcomb and Bové, 1983; Garnier ). In ruminants, mycoplasmas can colonize different sites, including the urogenital tract, the respiratory system, the mammary glands, the joints, the ocular mucous, the placenta and the ear canals (Rodríguez ; Razin ). The main mycoplasma species commonly found in goats and sheep are: Mycoplasma agalactiae, M. conjunctivae, M. putrefaciens and several taxa from the Mycoplasma mycoides cluster (MMC), such as Mycoplasma mycoides subspecies capri (Mmc), Mycoplasma capricolum subspecies capripneumoniae (Mccp), Mycoplasma capricolum subspecies capricolum (Mcc) (Manso-Silvan ). In small ruminants mycoplasmosis is associated with various symptoms including mastitis, arthritis, keratoconjunctivitis, pneumonia and septicemia (Thiaucourt and Bolske, 1996). In Brazil, M. agalactiae and Mmc have been reported from contagious agalactia episodes (Nascimento ; Nascimento ; Azevedo ) and M. conjunctivae from outbreaks of keratoconjuntivitis in sheep or in asymptomatic animals (Neto ). Mycoplasmas and ureaplasmas are recognized as some of the most important bacterial agents causing reproductive disorders (Junqueira and Alfieri, 2006). In cattle, goats and sheep, the main related reproductive problems are granular vulvovaginitis, endometritis, being repeatedly on heat, premature birth and miscarriage. In males, the main problems are infertility, a reduction in the frequency of intercourse, balanoposthitis, epididymitis, seminal vesiculitis, orchitis, and pathologies responsible for morphological and functional changes in spermatozoids (Singh ; Jones 1983; Kirkbride 1987; DaMassa ; Trichard ; Cardoso ; Gil ; Junqueira and Alfieri, 2006; Oliveira 2008). The mycoplasmas species involved in these cases in sheep and goats are M. agalactiae, M. putrefaciens and Ureaplasma spp. (Jones 1983; DaMassa ; Azevedo ; Corrales ; Oliveira 2008; De La Fe ), in addition to some species belonging to the Mycoplasma mycoides cluster (MMC) (Nascimento ; Neto ). Mycoplasma spp. and Ureaplasma spp. are also frequently found in animals showing no reproductive disorders (Mccaughey and Ball, 1981; De La Fe ; Rizzo ), which may facilitate the dissemination of these agents among free herds, especially through the use of contaminated semen and the transfer of embryos (De La Fe ; Rizzo ). Studies of mycoplasmas that affect the reproductive system of sheep and goats in Brazil are rare and the true epidemiological situation of this disease and the risk factors associated with these reproductive disorders are unknown, especially in the Northeast region of Brazil, which is the main sheep and goat producing area. The aim of this study is thus to detect the presence of mycoplasmas and ureaplasmas DNA in samples of semen from male sheep using PCR.

This study used 50 male sheep of differing breeds and ages from properties located in the State of Pernambuco, in the Northeast region of Brazil. The animals underwent a clinical examination and semen samples were collected using the artificial vagina method. The sperm was tested for macro and microscopic characteristics, in accordance with the guidelines of the Brazilian College of Animal Reproduction (CBRA) (1998). A quantity of 1.5 mL of semen was used for DNA extraction using a commercially available kita, according to the manufacturer’s instructions. PCR for mollicutes was carried out in a mix with a final volume of 25 μL containing: 5 μL of buffer [10 mM of Tris-HCl, pH 8.3), 30 pmol of each “primer”, 25 mM of MgCl2, 50 μM of each dinucleotide, 2.5 units (U) of Taq DNA polymeraseb, Mili-Q ultrapure water and 5 μL of DNA mold. Amplification reactions were carried out in a thermocyclerc and the thermal profile was based on the protocol described in the literature, with primers based on conserved regions of V6 and V7 of the 16SrRNA gene (Van Kuppeveld ; Van Kuppeveld ). The PCR for Ureaplasma spp. was carried out on a final volume of 25 μL containing: 5 μL of DNA; the already described primers by Lauerman (1998) (UGP-F′ and UGP-R′) at 30 pmol; Mili-Q ultrapure water and 6.25 μL of TopTaq Mastermixd in accordance with the manufacturer’s instructions. The thermal profile of the reactions was obtained using the standard protocol (Lauerman 1998). Mycoplasma mycoides mycoides (strain GM12) and Ureaplasma diversum (strain GMU132) were used as positive controls during PCR reactions for Mycoplasma and Ureaplasma detection, respectively. The amplified DNA was visualized by electrophoresis in 1.5% agarose gel with 100 bp molecular weight markere, colored with Bluegreenf, viewed under ultraviolet light and photodocumented. The semen samples that were positive by PCR were cultured in medium specific to Mycoplasma and Ureaplasma (modified Hayflicks and the “U” medium), respectively (Razin and Tully, 1996; Whitford ). The plates and broth were incubated at 37 °C under microaerophilic conditions for up to 21 days and examined daily for colonies under a stereoscopic microscope (40X). In this study, none of the animals examined showed any clinical signs of reproductive disease and 100% exhibited no alteration in sperm and were considered suitable for reproduction. Of the 50 semen samples submitted to PCR, 26.0% (13/50) tested positive for Mollicutes and 12.0% (6/50) for Ureaplasma spp. The amplicons obtained were 280 and 640 base pair, respectively.

It was not possible to identify the two agents in isolation, because, despite the selectivity of the media used for Mycoplasma and Ureaplasma, the samples were contaminated by fungi, making it impossible to confirm the viability of the agents. However, the presence of Ureaplasma DNA, as detected by PCR in the semen samples from the sheep under investigation shows that these agents can be carried among animals from infected and disease-free flocks. It should be noted that the sheep used showed no symptoms of reproductive disease, which could contribute to the dissemination of the two agents, since, in these cases, the rams passed the andrological examination and were used for artificial insemination. The presence of asymptomatic animals has been reported as a risk factor for outbreaks mycoplasmosis, such as those caused by Mycoplasma agalactiae in the Southeast and Northeast of Brazil (Nascimento ; Nascimento ; Gregory ; Neto ; Azevedo ; Oliveira 2008) and in some regions in Spain (Corrales ; De La Fe ). In these cases M. agalactiae was found in goats without reproductive disorders. The frequency of Mollicutes and Ureaplasma detected in this study is similar to that reported in other regions of Brazil in the semen of sheep and goats (Oliveira 2008; Rizzo ). In the present study, even though the frequency of the detection of Ureaplasma was low, this agent should be considered a pathogen that is important for the reproduction of sheep in the Northeast region of Brazil, since it is known that both Mycoplasma and Ureaplasma are sexually transmitted and it has been shown that this is through the semen (Mccaughey and Ball, 1981; Livingston Jr and Gauer, 1982; Corrales ; De La Fe ; Rizzo ). Mycoplasmosis in sheep and goats have still not been studied extensively in Brazil and, like reproductive disease, are often overlooked. The elimination of Ureaplasma from the semen of animals without symptoms should draw attention to the epidemiological role of these animals, which can act as a reservoir of the agents, which may also hinder programs to control diseases, such as Contagious Agalactia of Sheep and Goats, which is endemic in the region. Furthermore, the constant use of biotechnology in reproduction to improve flocks of sheep and goats genetically may contribute to the dissemination of these agents. In order to identify the Mycoplasma and Ureaplasma species present in the semen samples, and document their clinical significance, specific PCR assays are being currently performed in the laboratory.