Mycoplasma bovis infection in Spanish cattle herds and evaluation of new control strategies

Abstract

The overall goal of this thesis is to contribute to a better understanding of Mycoplasma bovis infection and the biology of the pathogen, as well as to the establishment of new control measures.The specific objectives and methods were: 1) To assess the presence and circulation of M. bovis in Spanish cattle herds using a large collection of samples collected from beef and dairy cattle and from different sources; 2) To investigate the circulation of different subtypes by single-locus sequencing of the polC gene, studying differences in antimicrobial susceptibility between subtypes and the presence of genetic mutations conferring resistance; 3) To address the role of M. bovis in clinical respiratory disease unresponsive to antimicrobials in feedlot calves in Spain through bacteriology, histopathology and immunohistochemistry, and to determine the minimum inhibitory concentration values of the isolates recovered against the specific set of antimicrobials used for the therapy in vivo; 4) To investigate whether mycoplasma chromosomal transfer occurs in the field, analyzing the occurrence of mosaic genomes among M. bovis isolates by whole-genome sequencing; 5) To assess the viability of M. bovis in diluted semen after the addition of an antimicrobial (enrofloxacin or doxycycline), or a Lactobacillus spp.- based probiotic at different concentrations under in vitro conditions; 6) To assess the viability of M. bovis in cervical mucus after the addition of a Lactobacillus spp.-based probiotic at different concentrations under in vitro conditions. The obtained results allowed us to conclude that: 1) M. bovis is widely extended in Spanish beef cattle herds and it is also implicated in mastitis cases affecting dairy herds; 2) Spanish M. bovis isolates analyzed are divided into two groups, ST2 and ST3, both being resistant to macrolides, lincosamides and tetracyclines. Most ST3 isolates are resistant to fluoroquinolones, a situation that illustrates the remarkable capacity of ST3 to accumulate mutations in the quinolone resistance-determining regions and the selective pressure imposed by the indiscriminate use of these antimicrobials. Valnemulin has been shown to be effective against both subtypes in vitro; 3) M. bovis plays a significant role in cases of clinical respiratory disease affecting feedlot calves in Spain. Caseonecrotic bronchopneumonia is the morphological pattern most frequently observed in animals infected with M. bovis, and patterns indicative of other bacteria species and viruses can be concurrently detected; 4) M. bovis isolates recovered from animals with clinical respiratory disease are resistant in vitro to most of the antimicrobials specifically used for therapy in vivo; 5) The co-existence of M. bovis lineages at the herd level and even at the animal level enables events of horizontal gene transfer like mycoplasma chromosomal transfer. These events contribute to wide-genome diversity and may have a negative impact on diagnosis and disease control; 6) Under the conditions analyzed, the addition of Lactobacillus spp., enrofloxacin or doxycycline negatively affects M. bovis viability in diluted semen, which could be useful to treat seminal doses and reduce the risk of transmission of M. bovis during artificial insemination. Dilution with the semen extender, Tris-citrate-fructose also negatively affects the viability of the agent, although to a lesser extent than the addition of the probiotic and antimicrobials; 7) Under the conditions analyzed, the addition of Lactobacillus spp. negatively affects M. bovis viability in cervical mucus, which could be useful to control M. bovis proliferation in the cervico-vaginal tract of cows.