Mycoplasma bovis in cattle : Studies on the transmission and control of infection

Mycoplasma bovis is an important pathogen of cattle. It causes a wide variety of clinical diseases, including mastitis, respiratory disease, arthritis, and otitis media. M. bovis has evolved several mechanisms to avoid the host’s immune system, no effective commercial vaccines are available, and antibiotic resistance is increasingly seen in contemporary strains. The most common route of transmission between herds is the purchase of a latent carrier animal. M. bovis can spread silently within a herd, and once established in a dairy herd it is difficult to eradicate. Hence, it is of utmost importance to try to control and prevent the spread of M. bovis. Sensitive and cost-effective diagnostic methods to detect latent carrier animals, as well as methods that could be used in herd certification are needed. The possible transmission of M. bovis through assisted reproduction needs to be investigated more closely. This thesis examines the course of M. bovis infection in Finnish dairy farms, presents a new route of entry of M. bovis into naive dairy herds, and describes methods that could be utilized in the control of M. bovis infections. In study I, the course of M. bovis infection was followed over two years on 19 recently infected dairy farms. The aim was to identify diagnostic methods that could be used to assess whether the herd had reached a low-risk infection status. In 17 herds, a few cases of clinical mastitis were detected, and these mostly occurred within two months after the index case. On two farms, M. bovis only caused respiratory disease in young stock, and no clinical mastitis was detected. The prevalence of M. bovis in nasal (NS) and deep nasopharyngeal (NP) swabs taken from young calves varied from zero to 75% per herd among the studied calves. An in-house MilA ELISA detected more positive serum samples than the commercial BioX ELISA. The proportion of MilA-positive young stock followed the patterns seen in NS and NP of calves, but no such association was seen in BioX ELISA results. In cows, despite the infection appearing to have resolved in some herds, the proportion of MilA antibody-positive cows remained high for at least one and half year after the detection of the index case. According to the results, M. bovis can be present in calves alone without causing mastitis in cows. Several methods need to be applied to verify the herd infection status. These include regular monitoring for M. bovis in clinical mastitis and calf pneumonia cases, combined with regular PCR testing of nasal swabs from calves and sera for the detection of antibodies against M. bovis using the MilA ELISA. The transmission of M. bovis via semen has been speculated. When epidemiological data to assess the infection source were collected in study I, suspicion arose that contaminated commercial artificial insemination (AI) semen could have been the source in two closed dairy herds. National health care and farm registers were used to collect farm data and farmers were interviewed. Whole genome sequencing was used to compare the genomes of isolated strains. Epidemiological analysis did not reveal any other source than contaminated semen from one bull used for artificial insemination in the cows that were the first ones to develop clinical mastitis in both herds. Core genome multilocus sequence type analysis supported this. The bull had secreted M. bovis intermittently and for only a short time into semen during an approximately seven-week period. On both farms, the incubation period between insemination and clinical mastitis was 32 days. To our knowledge, this is the first study to describe the transmission of M. bovis via AI. Even though this appears to be rare, commercial AI semen needs to be taken into account as a possible transmission route. Following the detection of M. bovis in Finland, a voluntary control program was established. One part of the program is NS taken from young calves and tested for M. bovis to indicate the infection status of the herd. In study III, the suitability of this method was assessed and compared with NP sampling. Furthermore, NS and NP sampling of pneumonic calves to detect M. bovis was compared with bronchoalveolar lavage sampling. Altogether, 1037 NS were taken from calves in 30 recently infected herds, and NP samples were also taken from 284 calves. The overall prevalence in NS was 29.6% and the highest prevalence of 43% was seen in 31- to 60-day-old calves. Thereafter, the shedding rate decreased. At the calf level, NP sampling detected M. bovis in 47% and NS in 33% of studied calves. At the herd level, NS sampling was more sensitive, as it classified 51 out 54 herd visits with a positive infection status as infected, whereas using NP sampling, the respective figure was 43 out of 54 visits (p = 0.061). The reason for the difference seen at the calf and herd levels is the sampling protocol. We took only five NP samples, but number of NS swabs varied from six to 28, depending on the herd size. We conclude that NS swabs taken from calves under six months of age and analyzed with real-time PCR represent a cost-efficient method to be used in a control program. If calves suffering from acute respiratory disease need to be examined, NP samples are a practical and sensitive method to detect M. bovis. The effect of two concentrations of a gentamycin-tylosin-linco/spectinomycin (GTLS) antibiotic combination and a fluoroquinole antibiotic, ofloxacin, on the viability of M. bovis in commercial-scale AI semen production using modern semen extender with plant-derived protein was investigated. A reference strain and a wild-type strains isolated from semen in study II were used in spiking. Three different protocols to extract M. bovis DNA from semen were also compared. At a high spiking concentration of 106 CFU/mL, none of the studied antibiotics had a bactericidal effect. At a low spiking concentration of 103 CFU/mL, the growth of the wild-type strain was inhibited by all other antibiotic protocols except for the low GTLS concentration, which is stated in EU regulation and the OIE Terrestrial Code. Instead, the high GTLS protocol was the only one that inhibited the growth of the reference strain. At a low M. bovis contamination level, GTLS used at a high concentration, according to the Certified Semen Services protocol, is more efficient than GTLS used according to the OIE Terrestrial Code. The Instagene™ matrix was the most efficient method to extract M. bovis DNA from semen.Mycoplasma bovis (M. bovis) on maailmanlaajuisesti merkittävä patogeeni bakteeri naudoilla. Tartunta siirtyy karjasta toiseen yleensä oireettoman kantajaeläimen välityksellä. Jotta bakteerin leviämistä voidaan estää, täytyy tuntea tartuntareitit ja tarvitaan herkkiä ja kustannustehokkaita menetelmiä, joilla voidaan löytää kantajaeläimet sekä käyttää karjan M. bovis -vapauden osoittamiseen. Työssä tutkittiin M. bovis infektion kulkua suomalaisissa lypsykarjoissa, esitetään uusi tartuntareitti karjaan sekä menetelmiä, joilla tartunnan leviämistä voidaan kontrolloida. Infektion kulkua seurattiin kahden vuoden ajan hiljattain tartunnan saaneissa lypsykarjoissa mm. käyttäen kahta erilaista ELISA-testiä vasta-aineiden toteamiseen sekä nuorkarjan sierainlimatutkimuksia. Pääsääntöisesti tartunta ilmeni ensin utaretulehduksina, mutta kahdella tilalla pelkästään nuorkarjan hengitystieinfektioina. M. bovista esiintyi sierainlimassa 0–75 %:lla tutkituista vasikoista tiloittain. Kokeellinen MilA ELISA osoittautui erittäin herkäksi testiksi verrattuna kaupalliseen BioX K260 -testiin. Vasikoissa MilA-vasta-ainepositiivisten eläinten osuus oli verrannollinen M. boviksen esiintymiseen sierainlimanäytteissä, mutta K260 -testillä yhteyttä ei havaittu. Lehmillä MilA-vasta-aineet säilyivät ainakin 1,5 vuotta niissäkin karjoissa, joissa tartunta näytti olevan poistunut. Lypsykarjan M. bovis statuksen arviointi vaatii useiden menetelmien käyttöä: utaretulehdus- ja vasikoiden sierainlimanäytteiden tutkimista PCR-menetelmällä sekä vasta-ainetutkimusta verinäytteistä MilA-ELISAlla. M. boviksen leviämistä sperman välityksellä on spekuloitu. Osatyössä kaksi tutkittiin, voiko kaupallinen sperma olla mahdollinen tartunnan lähde kahdessa suljetussa lypsykarjassa. Tiloista kerättiin tietoa eri rekistereistä sekä omistajien haastattelulla. Kokogenomisekvensointia käytettiin M. bovis -kantojen analysoinnissa. Epidemiologinen selvitys osoitti todennäköiseksi tartuntalähteeksi keinosiemennyksessä käytetyn sonnin sperman. Ydingenomin multilokussekvenssianalyysi tuki tätä. Sonnin spermalla oli siemennetty ne lehmät, joilla ensimmäisenä ilmeni M. bovis -utaretulehdus kummassakin karjassa. Vaikka kaupallisen sperman välityksellä tapahtuva tartunta on todennäköisesti harvinainen, niin tämä reitti on jatkossa otettava huomioon tartunnasta vapailla alueilla. Osatyössä III tutkittiin vasikoiden sierainlima- ja nielun syväsivelynäytteiden soveltuvuutta tilan M. bovis -statuksen osoittamiseen. Eritys sierainlimaan oli suurinta toisella ikäkuukaudella, tämän jälkeen eritys väheni. Vasikkatasolla herkemmäksi menetelmäksi M. boviksen toteamiseen osoittautui syväsively-, mutta karjatasolla taas sierainlimamenetelmä. Tämä selittyi suurimmaksi osaksi menetelmien kustannuseroista johtuneista näytemäärien eroista. Tutkimuksen perusteella alle 6 kk ikäisten vasikoiden sierainlimanäytteiden tutkiminen PCR:lla on kustannustehokas menetelmä käytettäväksi vastustusohjelmassa. Osatyössä neljä tutkittiin kahden eri antibioottivalmisteen, GTLS ja ofloksasiini, tehoa M. bovista vastaan keinosiemennyssperman tuotannossa käyttäen kahta eri antibioottien ja bakteerin pitoisuutta; lisäksi mukana oli M. boviksen villityyppi ja vertailukanta. Kumpikaan tutkituista antibiooteista ei tehonnut suureen M. bovis -pitoisuuteen kummastakaan kannasta. Molemmat ofloksasiinipitoisuudet ja suuri GTLS-pitoisuus estivät villityypin bakteerin kasvun käytettäessä alhaista bakteeripitoisuutta. Vertailukannan kasvu taas estyi vain käyttämällä suurta GTLS-pitoisuutta. Tulosten perusteella keinosiemennyssperma tulisi käsitellä suurella GTLS-pitoisuudella M. boviksen kasvun estämiseksi

Mycoplasma bovis in cattle: Studies on the transmission and control of infection

Mycoplasma bovis is an important pathogen of cattle. It causes a wide variety of clinical diseases, including mastitis, respiratory disease, arthritis, and otitis media. M. bovis has evolved several mechanisms to avoid the host’s immune system, no effective commercial vaccines are available, and antibiotic resistance is increasingly seen in contemporary strains. The most common route of transmission between herds is the purchase of a latent carrier animal. M. bovis can spread silently within a herd, and once established in a dairy herd it is difficult to eradicate. Hence, it is of utmost importance to try to control and prevent the spread of M. bovis. Sensitive and cost-effective diagnostic methods to detect latent carrier animals, as well as methods that could be used in herd certification are needed. The possible transmission of M. bovis through assisted reproduction needs to be investigated more closely. This thesis examines the course of M. bovis infection in Finnish dairy farms, presents a new route of entry of M. bovis into naive dairy herds, and describes methods that could be utilized in the control of M. bovis infections. In study I, the course of M. bovis infection was followed over two years on 19 recently infected dairy farms. The aim was to identify diagnostic methods that could be used to assess whether the herd had reached a low-risk infection status. In 17 herds, a few cases of clinical mastitis were detected, and these mostly occurred within two months after the index case. On two farms, M. bovis only caused respiratory disease in young stock, and no clinical mastitis was detected. The prevalence of M. bovis in nasal (NS) and deep nasopharyngeal (NP) swabs taken from young calves varied from zero to 75% per herd among the studied calves. An in-house MilA ELISA detected more positive serum samples than the commercial BioX ELISA. The proportion of MilA-positive young stock followed the patterns seen in NS and NP of calves, but no such association was seen in BioX ELISA results. In cows, despite the infection appearing to have resolved in some herds, the proportion of MilA antibody-positive cows remained high for at least one and half year after the detection of the index case. According to the results, M. bovis can be present in calves alone without causing mastitis in cows. Several methods need to be applied to verify the herd infection status. These include regular monitoring for M. bovis in clinical mastitis and calf pneumonia cases, combined with regular PCR testing of nasal swabs from calves and sera for the detection of antibodies against M. bovis using the MilA ELISA. The transmission of M. bovis via semen has been speculated. When epidemiological data to assess the infection source were collected in study I, suspicion arose that contaminated commercial artificial insemination (AI) semen could have been the source in two closed dairy herds. National health care and farm registers were used to collect farm data and farmers were interviewed. Whole genome sequencing was used to compare the genomes of isolated strains. Epidemiological analysis did not reveal any other source than contaminated semen from one bull used for artificial insemination in the cows that were the first ones to develop clinical mastitis in both herds. Core genome multilocus sequence type analysis supported this. The bull had secreted M. bovis intermittently and for only a short time into semen during an approximately seven-week period. On both farms, the incubation period between insemination and clinical mastitis was 32 days. To our knowledge, this is the first study to describe the transmission of M. bovis via AI. Even though this appears to be rare, commercial AI semen needs to be taken into account as a possible transmission route. Following the detection of M. bovis in Finland, a voluntary control program was established. One part of the program is NS taken from young calves and tested for M. bovis to indicate the infection status of the herd. In study III, the suitability of this method was assessed and compared with NP sampling. Furthermore, NS and NP sampling of pneumonic calves to detect M. bovis was compared with bronchoalveolar lavage sampling. Altogether, 1037 NS were taken from calves in 30 recently infected herds, and NP samples were also taken from 284 calves. The overall prevalence in NS was 29.6% and the highest prevalence of 43% was seen in 31- to 60-day-old calves. Thereafter, the shedding rate decreased. At the calf level, NP sampling detected M. bovis in 47% and NS in 33% of studied calves. At the herd level, NS sampling was more sensitive, as it classified 51 out 54 herd visits with a positive infection status as infected, whereas using NP sampling, the respective figure was 43 out of 54 visits (p = 0.061). The reason for the difference seen at the calf and herd levels is the sampling protocol. We took only five NP samples, but number of NS swabs varied from six to 28, depending on the herd size. We conclude that NS swabs taken from calves under six months of age and analyzed with real-time PCR represent a cost-efficient method to be used in a control program. If calves suffering from acute respiratory disease need to be examined, NP samples are a practical and sensitive method to detect M. bovis. The effect of two concentrations of a gentamycin-tylosin-linco/spectinomycin (GTLS) antibiotic combination and a fluoroquinole antibiotic, ofloxacin, on the viability of M. bovis in commercial-scale AI semen production using modern semen extender with plant-derived protein was investigated. A reference strain and a wild-type strains isolated from semen in study II were used in spiking. Three different protocols to extract M. bovis DNA from semen were also compared. At a high spiking concentration of 106 CFU/mL, none of the studied antibiotics had a bactericidal effect. At a low spiking concentration of 103 CFU/mL, the growth of the wild-type strain was inhibited by all other antibiotic protocols except for the low GTLS concentration, which is stated in EU regulation and the OIE Terrestrial Code. Instead, the high GTLS protocol was the only one that inhibited the growth of the reference strain. At a low M. bovis contamination level, GTLS used at a high concentration, according to the Certified Semen Services protocol, is more efficient than GTLS used according to the OIE Terrestrial Code. The Instagene™ matrix was the most efficient method to extract M. bovis DNA from semen.Mycoplasma bovis (M. bovis) on maailmanlaajuisesti merkittävä patogeeni bakteeri naudoilla. Tartunta siirtyy karjasta toiseen yleensä oireettoman kantajaeläimen välityksellä. Jotta bakteerin leviämistä voidaan estää, täytyy tuntea tartuntareitit ja tarvitaan herkkiä ja kustannustehokkaita menetelmiä, joilla voidaan löytää kantajaeläimet sekä käyttää karjan M. bovis -vapauden osoittamiseen. Työssä tutkittiin M. bovis infektion kulkua suomalaisissa lypsykarjoissa, esitetään uusi tartuntareitti karjaan sekä menetelmiä, joilla tartunnan leviämistä voidaan kontrolloida. Infektion kulkua seurattiin kahden vuoden ajan hiljattain tartunnan saaneissa lypsykarjoissa mm. käyttäen kahta erilaista ELISA-testiä vasta-aineiden toteamiseen sekä nuorkarjan sierainlimatutkimuksia. Pääsääntöisesti tartunta ilmeni ensin utaretulehduksina, mutta kahdella tilalla pelkästään nuorkarjan hengitystieinfektioina. M. bovista esiintyi sierainlimassa 0–75 %:lla tutkituista vasikoista tiloittain. Kokeellinen MilA ELISA osoittautui erittäin herkäksi testiksi verrattuna kaupalliseen BioX K260 -testiin. Vasikoissa MilA-vasta-ainepositiivisten eläinten osuus oli verrannollinen M. boviksen esiintymiseen sierainlimanäytteissä, mutta K260 -testillä yhteyttä ei havaittu. Lehmillä MilA-vasta-aineet säilyivät ainakin 1,5 vuotta niissäkin karjoissa, joissa tartunta näytti olevan poistunut. Lypsykarjan M. bovis statuksen arviointi vaatii useiden menetelmien käyttöä: utaretulehdus- ja vasikoiden sierainlimanäytteiden tutkimista PCR-menetelmällä sekä vasta-ainetutkimusta verinäytteistä MilA-ELISAlla. M. boviksen leviämistä sperman välityksellä on spekuloitu. Osatyössä kaksi tutkittiin, voiko kaupallinen sperma olla mahdollinen tartunnan lähde kahdessa suljetussa lypsykarjassa. Tiloista kerättiin tietoa eri rekistereistä sekä omistajien haastattelulla. Kokogenomisekvensointia käytettiin M. bovis -kantojen analysoinnissa. Epidemiologinen selvitys osoitti todennäköiseksi tartuntalähteeksi keinosiemennyksessä käytetyn sonnin sperman. Ydingenomin multilokussekvenssianalyysi tuki tätä. Sonnin spermalla oli siemennetty ne lehmät, joilla ensimmäisenä ilmeni M. bovis -utaretulehdus kummassakin karjassa. Vaikka kaupallisen sperman välityksellä tapahtuva tartunta on todennäköisesti harvinainen, niin tämä reitti on jatkossa otettava huomioon tartunnasta vapailla alueilla. Osatyössä III tutkittiin vasikoiden sierainlima- ja nielun syväsivelynäytteiden soveltuvuutta tilan M. bovis -statuksen osoittamiseen. Eritys sierainlimaan oli suurinta toisella ikäkuukaudella, tämän jälkeen eritys väheni. Vasikkatasolla herkemmäksi menetelmäksi M. boviksen toteamiseen osoittautui syväsively-, mutta karjatasolla taas sierainlimamenetelmä. Tämä selittyi suurimmaksi osaksi menetelmien kustannuseroista johtuneista näytemäärien eroista. Tutkimuksen perusteella alle 6 kk ikäisten vasikoiden sierainlimanäytteiden tutkiminen PCR:lla on kustannustehokas menetelmä käytettäväksi vastustusohjelmassa. Osatyössä neljä tutkittiin kahden eri antibioottivalmisteen, GTLS ja ofloksasiini, tehoa M. bovista vastaan keinosiemennyssperman tuotannossa käyttäen kahta eri antibioottien ja bakteerin pitoisuutta; lisäksi mukana oli M. boviksen villityyppi ja vertailukanta. Kumpikaan tutkituista antibiooteista ei tehonnut suureen M. bovis -pitoisuuteen kummastakaan kannasta. Molemmat ofloksasiinipitoisuudet ja suuri GTLS-pitoisuus estivät villityypin bakteerin kasvun käytettäessä alhaista bakteeripitoisuutta. Vertailukannan kasvu taas estyi vain käyttämällä suurta GTLS-pitoisuutta. Tulosten perusteella keinosiemennyssperma tulisi käsitellä suurella GTLS-pitoisuudella M. boviksen kasvun estämiseksi

Species distribution and in vitro antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitic milk

Background: Coagulase-negative staphylococci (CoNS) are the most common bovine mastitis causing bacteria in many countries. It is known that resistance for antimicrobials is in general more common in CoNS than in Staphylococcus aureus but little is known about the antimicrobial resistance of specific CoNS species. In this study, 400 CoNS isolates from bovine mastitic milk samples were identified to species level using ribotyping and MALDI-TOF MS, and their antimicrobial susceptibility was determined using a commercially available microdilution system. The results were interpreted according to the epidemiological cut-off values by the European Committee on Antimicrobial Susceptibility testing. Results: The most common CoNS species were S. simulans, S. epidermidis, S. chromogenes and S. haemolyticus. Penicillin resistance was the most common type of antimicrobial resistance. Staphylococcus epidermidis was the most resistant among the four major species. Almost one-third of our S. epidermidis isolates were resistant to >2 antimicrobials and close to 7 % were multidrug resistant. The majority of S. epidermidis isolates were resistant to benzylpenicillin. On the contrary, only few S. simulans isolates were penicillin-resistant. Phenotypic oxacillin resistance was found in all four main species, and 34 % of the isolates were oxacillin resistant. However, only 21 isolates (5 %) were positive for the mecA gene. Of these, 20 were S. epidermidis and one S. sciuri. mecC positive isolates were not found. Conclusion: Staphylococcus epidermidis differed from the three other major CoNS species as resistance to the tested antimicrobials was common, several isolates were multidrug resistant, and 19 % of the isolates carried the mecA gene encoding methicillin resistance.Peer reviewe

Suitability of Nasal and Deep Nasopharyngeal Swab Sampling of Calves in the Mycoplasma bovis Control Program

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Semen as a source of Mycoplasma bovis mastitis in dairy herds

Mycoplasma bovis infections are responsible for substantial economic losses in the cattle industry, have significant welfare effects and increase antibiotic use. The pathogen is often introduced into naive herds through healthy carrier animals. In countries with a low prevalence of M. bovis, transmission from less common sources can be better explored as the pathogen has limited circulation compared to high prevalence populations. In this study, we describe how M. bovis was introduced into two closed and adequately biosecure dairy herds through the use of contaminated semen during artificial insemination (AI), leading to mastitis outbreak in both herds. Epidemiological analysis did not reveal an infection source other than semen. In both farms the primary clinical cases were M. bovis mastitis in cows inseminated with the semen of the same bull four weeks before the onset of the disease. One semen straw derived from the semen tank on the farm and other semen lots of this bull were positive for M. bovis. In contrast, semen samples were negative from other bulls that had been used for insemination in previous or later oestrus to those cows with M. bovis mastitis. Furthermore, cgMLST of M. bovis isolates supported the epidemiological results. To our knowledge this is the first study describing the introduction of M. bovis infection into a naive dairy herd via processed semen. The antibiotics used in semen extenders should be re-evaluated in order to provide farms with M. bovis-free semen or tested M. bovis-free semen should be available.Peer reviewe

Extraintestinal pathogenic Escherichia coli in poultry meat products on the Finnish retail market

Background: Extraintestinal pathogenic Escherichia coli bacteria (ExPEC) exist as commensals in the human intestines and can infect extraintestinal sites and cause septicemia. The transfer of ExPEC from poultry to humans and the role of poultry meat as a source of ExPEC in human disease have been discussed previously. The aim of the present study was to provide insight into the properties of ExPEC in poultry meat products on the Finnish retail market with special attention to their prevalence, virulence and phylogenetic profiles. Furthermore, the isolates were screened for possible ESBL producers and their resistance to nalidixic acid and ciprofloxacin was tested. Methods: The presence of ExPEC in 219 marinated and non-marinated raw poultry meat products from retail shops has been analyzed. One E. coli strain per product was analyzed further for phylogenetic groups and possession of ten virulence genes associated with ExPEC bacteria (kpsMT K1, ibeA, astA, iss, irp2, papC, iucD, tsh, vat and cva/cv) using PCR methods. The E. coli strains were also screened phenotypically for the production of extended-spectrum β-lactamase (ESBL) and the susceptibility of 48 potential ExPEC isolates for nalidixic acid and ciprofloxacin was tested. Results: E. coli was isolated from 207 (94.5%) of 219 poultry meat products. The most common phylogenetic groups were D (50.7%), A (37.7%), and B2 (7.7%). Based on virulence factor gene PCR, 23.2% of the strains were classified as ExPEC. Two ExPEC strains (1%) belonged to [O1] B2 svg+ (specific for virulent subgroup) group, which has been implicated in multiple forms of ExPEC disease. None of the ExPEC strains was resistant to ciprofloxacin or cephalosporins. One isolate (2.1%) showed resistance to nalidixic acid. Conclusions: Potential ExPEC bacteria were found in 22% of marinated and non-marinated poultry meat products on the Finnish retail market and 0.9% were contaminated with E. coli [O1] B2 svg+ group. Marinades did not have an effect on the survival of ExPEC as strains from marinated and non-marinated meat products were equally often classified as ExPEC. Poultry meat products on the Finnish retail market may have zoonotic potential

Transmission of Mycoplasma bovis infection in bovine in vitro embryo production

Mycoplasma bovis (M. bovis) causes several costly diseases in cattle and has a negative effect on cattle welfare. There is no effective commercial vaccine, and antimicrobial resistance is common. Maintaining a closed herd is the best method to minimize the risk of introduction of M. bovis. Assisted reproduction is crucial in a closed herd to make genetic improvements. M. bovis has been found in commercial semen, and contaminated semen has been the source of disease in naïve dairy herds. The objective of this study was to evaluate M. bovis transmission in bovine in vitro embryo production (IVP) using several possible exposure routes. We used a wild-type M. bovis strain isolated from semen at a final concentration of 106 CFU/mL to infect cumulus–oocyte complexes, spermatozoa, and 5-day-old embryos. We also used naturally contaminated semen in fertilization. Blastocysts were collected on day 7–8 and zona pellucida (ZP)-intact embryos were either washed 12 times, including trypsin washes as recommended by the International Embryo Technology Society (IETS), or left unwashed. Washed and unwashed embryos, follicular fluids, maturation medium, cumulus cells, fertilization medium, and G1 and G2 culture media, as well as all wash media were analyzed using enrichment culture followed by real-time PCR detection of M. bovis. Altogether, 76 pools containing 363 unwashed embryos and 52 pools containing 261 IETS washed embryos were analyzed after oocytes, spermatozoa, or 5-day-old embryos were infected with M. bovis or naturally contaminated semen was used in fertilization. We could not detect M. bovis in any of the embryo pools. M. bovis was not found in any of 12 wash media from different exposure experiments. M. bovis did not affect the blastocyst rate, except when using experimentally infected semen. Contrary to an earlier study, which used a cell co-culture system, we could not demonstrate M. bovis in embryo wash media or tight adherence of M. bovis to ZP-intact embryos. Naturally infected semen did not transmit M. bovis to embryos. We conclude that by using our IVP system, the risk of M. bovis transmission via IVP embryos to recipient cows is very low