The Addition of Lactobacillus spp., Enrofloxacin or Doxycycline Negatively Affects the Viability of Mycoplasma bovis in Diluted Bovine Semen

Abstract

Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis in cattle. Different transmission routes have been described, including those related to reproduction. The presence of mycoplasma in semen has led to its appearance in infection-free areas through artificial insemination (AI). Semen was recently reported to be the initial source of two M. bovis mastitis outbreaks in two closed dairy herds in Finland. This questions the effectiveness of the antimicrobials currently used in semen extenders to control the pathogens in contaminated semen. They should be re-evaluated, or alternative measures to antimicrobials should be tested to obtain M. bovis-free semen. This in vitro study aimed to assess different strategies to reduce the risk of transmission of M. bovis through AI technologies. The viability of M. bovis (PG45, NCTC 10131) in bull semen diluted (DS) in a Tris-citrate-fructose solution was tested, after the addition of enrofloxacin, doxycycline or a Lactobacillus spp.-based probiotic. The data show the susceptibility of the pathogen to the addition of 0.125 μg/mL of enrofloxacin or 0.0625 μg/mL of doxycycline and to the addition of the probiotic at a concentration of 3.24 × 106 colony forming units (CFU)/mL or 3.24 × 108 CFU/mL in DS. The Tris-citrate-fructose medium negatively affected the viability of M. bovis, although this effect was lower than that observed after the addition of the probiotic and antimicrobials (p < 0.05). Our results may support new strategies for reducing the risk of M. bovis transmission through AI.

The addition of Lactobacillus spp. negatively affects Mycoplasma bovis viability in bovine cervical mucus

“…For instance, the acidification to pH 4 during 1 h was enough to eliminate viable M. bovis organisms in milk [23]. We also found out that the viability of M. bovis was negatively affected by the addition of L2 in diluted bull semen which coincided with a significant decrease of pH after 15 h in incubation [25]. Other authors observed a harmful effect on Mycoplasma agalactiae and Mycoplasma mycoides subsp.…”

“…An inoculum of M. bovis (1 × 10 9 CFU/mL) and two inoculums of Lactobacillus spp., L1 (3.24 × 10 6 CFU/mL) and L2 (3.24 × 10 8 CFU/mL) were prepared in a modified broth SP4 as previously described [25]. The reference strain PG45, and the lyophilized commercial probiotic, NS Femibiotic®, based on a mix of Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus brevis, were employed for preparing the inoculums of M. bovis and L1 and L2 respectively.…”

“…Seven in vitro conditions were experimentally prepared in cervical mucus and/or modified SP4 following an experimental protocol previously described [25]. Table 1 shows the volumes of cervical mucus, SP4, and M. bovis and Lactobacillus spp.…”

“…Counts (CFU/mL) of M. bovis and LAB were conducted on SP4 and MRS agar following a protocol previously described [25]. The counts of M. bovis were only conducted in conditions contaminated with the mycoplasma (Table 1, n = 5) while the counts of LAB were determined in the seven conditions.…”

“…Mycoplasmas lack a cell wall and therefore they are very sensitive to external changes such as pH variations [ 22 ]. In this sense, several authors have observed the effectiveness of the medium acidification to control the growth of mycoplasmas [ 23 – 25 ]. In cows, the in vivo pH of the cervical mucus is generally about 6.8 and rises to about 7.6 to 8.1 during estrus [ 5 , 26 , 27 ].…”