Occurrence of Mycoplasma bovigenitalium and Ureaplasma diversum in dairy cattle from to Pernambuco state , Brazil

The objective of this study was to conduct an investigation of Mycoplasma bovigenitalium and Ureaplasma diversum infections in cattle in the microregion of the Ipanema Valley, state of Pernambuco, Brazil. Vaginal swabs were collected from 355 breeding cows in reproductive age and were analyzed by multiplex PCR (mPCR) and culture. An epidemiological investigation of risk factors was performed for Mollicutes. mPCR analysis showed that, 9.29% (33/355) of the cows were positive for M. bovigenitalium and 21.69% (77/355) for U. diversum; coinfection was observed in 2.81% (10/355) of the cows. The microbiological isolation showed, 81.81% (27/33) of Mycoplasma spp. and 24.67% (19/77) of Ureaplasma spp.. The risk factors related to Mollicutes infection identified were semi-intensive breeding system (OR= 4.6), pasture rent (OR= 3.6), non-isolation of animals with reproductive disorders (OR= 3.2), and natural mounting and artificial insemination (OR= 3.5). There was a significant association between Mollicutes infection and abortions in the first gestational third (P= 0.001). This is the first record of M. bovigenitalium and U. diversum infection in cows in the semiarid region of the state of Pernambuco, Brazil. Preventive measures directed to the identified risk factors can decrease the occurrence of Mollicutes in these herds.

Genital infections caused by Mycoplasma bovigenitalium in females is characterized by granular vulvovaginitis presenting with mucopurulent vaginal discharge, which may cause infertility, and in some cases, necrotizing endometritis (Cardoso and Vasconcellos, 2004).On the other hand, Ureaplasma diversum the infection is associated with abortion, recurrence of estrus, granular vulvovaginitis and infertility in infected herds (Miller et al., 1994).
The risk factors associated with Mollicutes infection in cattle herds are related to dietary stress, such as high milk production, excessive movement of animals (exhibition and trade cattle) (Aebi et al., 2015), and heifers and cows with lower pregnancy rate (León et al., 1995).
Considering that Mollicute infection in cattle was already diagnosed in herds from to Brazil and that there is commercial transport of these animals between Brazilian states, this study aimed to conduct an epidemiological investigation of Mycoplasma bovigenitalium and Ureaplasma diversum infections in cattle in the microregion of the Ipanema Valley, state of Pernambuco.

MATERIALS AND METHODS
The present study analyzed cattle from 18 properties distributed among the six districts of the Ipanema Valley microregion in the Pernambuco semiarid region: Águas Belas (n= 3), Buíque (n= 3),Venturosa (n= 3), Pedra (n= 3), Itaiba (n= 3) and Tupanatinga (n= 3) (Figure 1).A total of 336.221 cattle (IBGE, 2007) and an expected prevalence of 65.6% were considered (Santos et al., 2013).At a significance level of P< 0.05, these parameters provided a minimum sample size (n) of 355 cows (Thrusfield, 2013).The number of animals evaluated in each herd was calculated using the formula of Astudillo (1979), based on the afore mentioned values.The properties were chosen by convenience, and the inclusion criteria was females of reproductive age group.
Vaginal mucous samples were collected after vulvar cleaning and asepsis with alcohol (70 o GL), by using sterile cotton swab, and transported in 3mL of phosphate buffered saline (pH 7.2) (Nascimento et al., 2005).The samples were placed in an isothermal box containing recyclable ice and sent to the laboratory for processing.
An aliquot of 600μL was used for DNA extraction with a commercial kit (Promega ® Wizard Genomic DNA Purification Kit Catalog A1125).Aliquots for microbiological isolation were stored in 2:1 ratio glycerol in microtubes, vortexed and stored in a freezer at -16°C until use.The extracted DNA was analyzed by multiplex polymerase chain reaction (mPCR) technique for Mycoplasma bovigenitalium and Ureaplasma diversum species identification according to the thermal profile and oligonucleotides as described by Tramuta et al. (2011).The following primers were used for mPCR: Mycoplasma bovigenitalium (16S rRNA), 5ʹGTT TGA TCC TGG CTC AGG AT 3ʹ and 5ʹAAG GTA CAT TCA ATA TAG TGG 3ʹ, with amplicon 476bp; Ureaplasma diversum (16S rRNA), 5ʹGTT TGA TCC TGG CTC AGG AT 3ʹ and 5ʹCTC ATA AGC GAG CC GAC ATT 3ʹ, with amplicon 831bp.The reaction mixtures comprised a total of 25μL containing 5μL DNA template, 30pmol of each oligonucleotide and 6.25μL GoTaq ® Green Master Mix (Promega ® Corporation, Madison, WI, USA, Ref. M7122), and ultrapure water.The Ureaplasma diversum strain (GM U132 VUP6, ATCC) and Mycoplasma bovigenitalium strain (PG11, ATCC) were used as positive control.For the negative control, the DNA template was replaced by water.For electrophoresis, 1.5% agarose gel (100V/60min), 9μLPCR reaction mixtures plus 0.3μL Blue Green Loading Dye I (LGC ® Biotecnologia, Cotia, SP, Brazil) and 5μLof molecular size marker (100bp DNA Ladder ® , LGC ® Biotecnologia, Cotia, SP, Brazil) were used.To confirm the species studied, sequencing of PCR positive samples was performed.

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All the samples were screened for mycoplasma and ureaplasma by mPCR.Only mPCR confirmed isolates were further tested for recovery of pathogens using microbial culture method.Samples were cultured for Mycoplasma spp.isolation in modified Hayflick medium broth and plate, as described by Whitford et al. (1994), and for Ureaplasma spp. in UB specific medium broth and plate.The samples were observed at stereoscopic microscope and the isolation was considered positive for Mycoplasma spp.when the samples resembling "fried-egg-shaped" colonies, and the small, dark brown granular colonies, for Ureaplasma spp.All samples were monitored for 21 days.
Epidemiological questionnaires were designed and applied to obtain information about the type of farming and management practices and identify possible risk factors of each herd.The questionnaires were applied by a single trained investigator, and answed by people who could provide more about the animals.The variables investigated and the respective categories were: consortium creation (yes/no); (herds of Milk / milk and meat); Wetlands on the property (yes/no); Flooded areas on surrounding properties (yes/no); Presence of hematophagous insects (yes/no); Insect control (yes/no); Herd type (open/closed); Performs quarantine (yes/no); Reproductive management (natural breeding/artificial insemination/both).
Univariate analysis of infection-associated risk factor was performed, by using the Pearson chisquare test ( 2 ) or Fisher's exact test, followed by a logistic regression analysis, which considered the "Gold standard" the mPCR result for Mollicutes as the dependent variable.The independent or explanatories variables considered in the model were those with a statistical significance of <0.20.This probability was stipulated so that possible event risk factors were not excluded from the analysis (Hosmer and Lemeshow, 1989).The EPIINFO TM 3.5.1 program was used to perform the statistical calculations, considering as a risk factor in the final model the variables that presented statistical significance P< 0.05.
The present study was approved by the Ethics Committee on the Use of Animals (CEUA) of the Federal Rural University of Pernambuco, under license N o 042/2015.All authors gave their consent for inclusion in this study.

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An occurrence of 9.3% for Mycoplasma bovigenitalium infection was observed in the present study.These values are simmilar to those found in Ghanem et al. (2013) study, in Japan, which found 7.4% occurrence of Mycoplasma bovigenitalium in cervical vaginal mucus samples from post-partum period of Holstein cows.The low occurrence of Mollicutes infection may be related to the low amount of DNA present in the biological material.Husted (2003) reported that the low amount of DNA in the analyzed samples may result in a low frequency of positive samples.Furthermore, the animals did not present suggestive clinical signs during the gathering, unlike the studies conducted by Santos et al. (2013;2015) which investigated the occurrence of Mollicutes in herds with reproductive disorders.
Historically, M. bovigenitalium and Ureaplasma diversum stand out as agents involved in outbreaks of reprductive diseases in cattle, the main species being diagnosed in mycoplasmosis clinical.M. bovigenitalium have been isolated from the vaginal secretions of cows, this agent has been associated with natural cases of granular vulvovaginitis (Afshar, 1967;Lysnyansky et al., 2009).
In the present study, Ureaplasma diversum was detected in 21.7% (77/355) of the samples.The higher occurrence of Ureaplasma diversum infection compared to Mycoplasma bovigenitalium may be due to the agent colonization characteristic, for the reason that Ureaplasma diversum remains longer in the organism of asymptomatic animals depending on the urea concentrations in the urogenital tract excreta, which is an important nutrient for these bacteria (Sanderson et al., 2000).Although, the silent disease can manifest itself with decreases in the reproductive performance of the cattle (Argue et al., 2013;Santos et al., 2015).
In the seven properties ("A," "B," "C," "D," "H," "I," and "J"), the occurrence of positive animals was high for the agents in question.In these properties, the milk production was higher, which corroborates with Gábor et al. (2008) that reported the Mollicutes occurrence in a herd with higher milk production.Moreover, the use of natural breeding and/or artificial insemination as reproductive management may have contributed for these agents spread between the herd.According to (Nicholas and Ayling, 2003), contaminated semen is an important transmission route in bovine specie.
It was observed that 81.8% (27/33) of the mPCR-positive samples grew Mycoplasma spp. in the specific medium.This percentage is higher than that reported by other authors in Brazil (Nascimento et al., 2005;Buzinhani et al., 2007) who also isolated only Mycoplasma spp.The results of the present study were higher than what was reported in previous studies that isolated Mycoplasma spp.species in cattle in other countries (Petit et al., 2008;Lysnyansky et al., 2009).In addition, the isolation of the genus is more practical than that of different Mycoplasma spp.species.About this, Nagatomo et al. (2001) inoculated several species of Mycoplasma spp. in liquid medium and found different isolation rates compared to the culture media and a sensitivity to temperature variations.
The isolation rate of Ureaplasma spp. was reported as 24.67% (19/77).In previous studies, higher results have been described (Buzinhani et al., 2007;Petit et al., 2008;Marques et al., 2013); however, in the afore mentioned research, the isolation was performed from herds with clinical signs of mycoplasmosis.
The isolation rate of Ureaplasma spp. was lower than that for Mycoplasma spp.It is possible that the presence of dead cells (Ureaplasma spp.) in the samples accounted for the lower rate of growth in the specific medium.
Natural breeding and artificial insemination were identified as risk factors associated with Mollicutes infection in the present study.Some authors confirm the presence of Ureaplasma diversum (Hobson et al., 2013) and Mycoplasma (Cardoso and Vasconcellos, 2004) in semen and indicate the importance of this transmission route for the agent's spread in cattle (Petit et al., 2008).
Animals submitted to the semi-intensive system were 4.6 times more likely to be infected by Mollicutes than those submitted to the extensive system.Santos et al. (2013) detected Mollicutes (65.6% -21/32) and Ureaplasma diversum (15.6% -5/32) in a reproductive disease outbreak where animals were reared in a semi-intensive system.In this system, the transmission of the agent by direct contact is favored (Gambarini et al., 2009) mainly at the time of milking, because the cows have the habit of smelling and licking the external genitalia of each other, which facilitates the agent's dissemination (Rocha, 2009).In properties with pasture rental practice, the animals are more likely to become infected by Mollicutes (OR= 3.6), which is also true for properties where animals with reproductive problems are not isolated (OR= 3.2).Thus, both the pasture rental practice and not isolating animals with reproductive disorders can lead to the contact of cattle with secretions and excretions from infected animals (Nicholas and Ayling, 2003).

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There was an association between abortion in the first gestational third (P= 0.001) and Mollicutes infection, and history of abortion was observed in 45.6% of the positive cows.Petit et al. (2008) indicated that the detection of Mycoplasma bovigenitalium is greater in animals with a history of abortion.Another study implicated the occurrence of abortion in the first (25%) and third (75%) third of gestation.Given that there are divergences between the studies related to the timing of abortion, it is suggested experimental or observational studies be conducted with the purpose of establishing the time of pregnancy at which the cows have a higher risk of aborting when infected with Mollicutes.
It was observed that 9.41% (8/85) of the positive cows (3/8 for Mycoplasma bovigenitalium and 5/8 for Ureaplasma diversum) showed signs of granular vulvovaginitis during the gathering of samples.Gambarini et al. (2009) and Cardoso et al. (1997) reported that granular vulvovaginitis may be associated with the presence of Ureaplasma diversum.Oliveira-Filho et al. (2005) affirmed that the presence of Ureaplasma diversum is associated with the occurrence of granular vulvovaginitis syndrome, and it is independent of the injury extension observed in the vulvovaginal mucosa.Some animals had granules in the vulva and were negative for Mollicutes, representing 90.5% (77/85).The clinical condition of VVG is not pathognomonic for Mollicutes infection.

CONCLUSION
This is the first record of the occurrence of M. bovigenitlium and U. diversum infection in dairy cattle in the semiarid region of Pernambuco.The semi-intensive breeding system, pasture rent, non-isolation of animals, natural mounting and artificial insemination are a risk for the occurrence of M. bovigenitalium and U. diversum in the cattle studied.Mollicutes are associated with abortions in the in the first gestational third.Preventive measures directed at these identified risk factors may contribute to a decrease in the occurrence of these agents in the herds.
Figure 1.Area of investigation for Mollicutes in reproductive trac of cattle, microregion of the Ipanema Valley -Pernambuco-PE, Brazil.

Table 1 .
Results of PCR for the M. bovigenitalium and U. diversum in dairy cows in the Ipanema Valley microregion, Pernambuco state, Brazil

Table 2 .
Analysis of risk factors associated with Mollicutes infection in dairy cattle from to Ipanema Valley, Pernambuco state, Brazil . Bras.Med.Vet.Zootec., v.70, n.6, p.1798-1806, 2018(A) Fisher's Exact Test; (B)  2 Test; 1 different database (N= 296); N -Total Number of Samples; OR -Odds Ratio; I.C. -Confidence Interval;*Significant association at the P< 0.05level.This is the first record of Mycoplasma bovigenitalium and Ureaplasma diversum infections in dairy cattle in the semiarid region of the state of Pernambuco, Brazil.Mollicutes identification in the reproductive tract of bovine females was reported in Brazil (Nascimento

Table 3 .
Reproductive history associated with Mollicutes (M.bovigenitalium and U. diversum or both) infection in dairy cows in Ipanema Valley, Pernambuco state, Brazil