From six to zero per cent Mycobacterium avium subsp. paratuberculosis milk ELISA positivity in three years - probably induced by Mycobacterium vaccae.

This research communication reports the results of a study aimed at investigating the effects of introducing Mycobacterium vaccae on paratuberculosis carriage in a dairy herd. M. vaccae is a non-pathogenic member of the Mycobacteriaceae, with immunomodulatory and immunotherapeutic capabilities, acting by stimulating the cellular immune system, important in protection against paratuberculosis. Starting in 2014 we administered, by gavage, 1010 live M. vaccae bacteria to all new-born heifers on a dairy farm, first within 24 h of birth and again 2 weeks later. Paratuberculosis carriage was monitored yearly by milk ELISA. Faecal samples of 50% of cows, aged 3 years, born 1, 2 or 3 years before the experiment's onset, were tested by qPCR for MAP shedding and compared to 100% treated cows of the same age. Within 3 years, milk ELISA positivity was reduced from 6 to 0% and remained unchanged for the subsequent 2 years. One qPCR positive control cow was found each year for a total of 3 animals (2.46%). One positive cow (1%) was found among the treated cows. Two of the 3 positive control animals, still present on the farm at the end of 2019, tested negative whereas the positive test cow continued shedding MAP. M. vaccae shedding heifers mixing with adult cows were the probable means of the microorganism's propagation. The results of this investigation indicate that the introduction of live M. vaccae may be an inexpensive and fast alternative to current paratuberculosis control practices, justifying further exploration of the topic.

Genomics-based epidemiology of bovine Mycoplasma bovis strains in Israel.

BACKGROUND: Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis affecting cattle production and animal welfare. In the past in Israel, M. bovis has been most frequently associated with bovine respiratory disease (BRD) and was rarely isolated from mastitis. This situation changed in 2008 when M. bovis-associated mastitis emerged in Israel. The aim of this study was to utilize whole genome sequencing to evaluate the molecular epidemiology and genomic diversity of M. bovis mastitis-associated strains and their genetic relatedness to M. bovis strains isolated from BRD in local feedlot calves and those imported to Israel from different European countries and Australia. RESULTS: Phylogeny based on total single nucleotide polymorphism (SNP) analysis of 225 M. bovis genomes clearly showed clustering of isolates on the basis of geographical origin: strains isolated from European countries clustered together and separately from Australian and Chinese isolates, while Israeli isolates were found in the both groups. The dominant genotype was identified among local mastitis-associated M. bovis isolates. This genotype showed a close genomic relatedness to M. bovis strains isolated from calves imported to Israel from Australia, to original Australian M. bovis strains, as well as to strains isolated in China. CONCLUSIONS: This study represents the first comprehensive high-resolution genome-based epidemiological analysis of M. bovis in Israel and illustrates the possible dissemination of the pathogen across the globe by cattle trade.

An overview of Mycoplasma bovis mastitis in Israel (2004-2014).

The prevalence of Mycoplasma bovis in milk samples submitted to the Israeli National Service for Udder Health and Milk Quality was determined during the period 2004-2014 and the genetic pattern of the obtained isolates was assessed by multilocus sequence typing (MLST). Mycoplasma spp. were identified in 66 herds including M. bovis (n = 60), M. cottewii (n = 3), M. bovigenitalium (n = 2), M. alkalescens (n = 2) and M. yeatsii (n = 1). The proportion of M. bovis infected herds was relatively low (0-0.68%) in 2004-2007, increased to 3.77% during the 2008 outbreak, and ranged from 0.77 to 2.77% during the 2009-2014 period. Since 2008, about eight M. bovis positive dairy herds have been identified in Israel annually, with six of which on average being newly infected. MLST of 57 M. bovis isolates revealed that sequence type 10 was the dominant genotype identified in 60% of the herds. In conclusion, these data show that M. bovis is the main mycoplasmal mastitic pathogen in Israel.

Acquired resistance to the 16-membered macrolides tylosin and tilmicosin by Mycoplasma bovis.

The molecular mechanism of acquired resistance to the 16-membered macrolides tylosin (Ty) and tilmicosin (Tm) was investigated in Mycoplasma bovis field isolates. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 54 M. bovis isolates showing different minimal inhibitory concentrations (MIC). The presence of any one of the point mutations G748A, C752T, A2058G, A2059G or A2059C (Escherichia coli numbering) in one or both alleles of the 23S rRNAs was correlated with decreased susceptibility to Ty (8-1024 µg/ml) and to Tm (32 to >256 µg/ml) in 27/27 and 27/31 M. bovis isolates, respectively. Although a single mutation in domain II or V could be sufficient to cause decreased susceptibility to Ty, our data imply that a combination of mutations in two domains is necessary to achieve higher MICs (≥ 128 µg/ml). The influence of a combination of mutations in two domains II and V on enhancement of resistance to Tm was less clear. In addition, the amino acid (aa) substitution L22-Q90H was found in 24/32 representative M. bovis isolates with different MICs, but no correlation with decreased susceptibility to Ty or Tm was identified. Multiple aa substitutions were also identified in the L4 protein, including at positions 185-186 (positions 64 and 65 in E. coli) which are adjacent to the macrolide-binding site. This is the first description of the molecular mechanism of acquired resistance to the 16-membered macrolides in M. bovis.

Multiple locus variable number tandem repeat analysis of Mycoplasma bovis isolated from local and imported cattle.

Mycoplasma bovis is an important and emerging pathogen of cattle. In this study, multiple locus variable number tandem repeat (VNTR) analysis was used to differentiate M. bovis type strain PG45 and 68 M. bovis field isolates, including 34 isolates from calves imported to Israel from Australia, Lithuania and Hungary in the period 2006-2011, 32 isolates from mastitic dairy cows in Israel in the period 2000-2011, one isolate from the pneumonic lungs of a calf in Israel in 2010 and one isolate from frozen bull semen in Israel in 2008. A total of 35 VNTR types were distinguished, including three, eight and 10 different VNTR types among isolates from calves imported from Australia, Hungary and Lithuania, respectively, and 17 VNTR types among isolates from dairy cows in Israel. The VNTR types in isolates from Lithuanian calves were not identified among isolates from Israeli dairy cows. VNTR type XX, present in the Hungarian group, was identified in one Israeli mastitis-associated isolate. A cluster of 16 M. bovis isolates from Israeli dairy cows possessed the same VNTR type III as three Australian isolates from a single shipment of calves in 2006. The other cluster of isolates contained M. bovis strain 883, isolated from a mastitic cow, strain 72236, isolated from a calf with pneumonia, two isolates from calves imported from Australia to the same farm 3 months previously and four isolates from calves in quarantine imported to Israel from Australia in 2009-2010. Multiple locus VNTR analysis is a useful tool for understanding the movement and spread of strains of M. bovis within and across international boundaries.

Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry.

The macrolide class of antibiotics, including tylosin and tilmicosin, is widely used in the veterinary field for prophylaxis and treatment of mycoplasmosis. In vitro susceptibility testing of 50 strains of M. gallisepticum isolated in Israel during the period 1997-2010 revealed that acquired resistance to tylosin as well as to tilmicosin was present in 50% of them. Moreover, 72% (13/18) of the strains isolated from clinical samples since 2006 showed acquired resistance to enrofloxacin, tylosin and tilmicosin. Molecular typing of the field isolates, performed by gene-target sequencing (GTS), detected 13 molecular types (I-XIII). Type II was the predominant type prior to 2006 whereas type X, first detected in 2008, is currently prevalent. All ten type X strains were resistant to both fluoroquinolones and macrolides, suggesting selective pressure leading to clonal dissemination of resistance. However, this was not a unique event since resistant strains with other GTS molecular types were also found. Concurrently, the molecular basis for macrolide resistance in M. gallisepticum was identified. Our results revealed a clear-cut correlation between single point mutations A2058G or A2059G in domain V of the gene encoding 23S rRNA (rrnA, MGA_01) and acquired macrolide resistance in M. gallisepticum. Indeed, all isolates with MIC ≥ 0.63 µg/mL to tylosin and with MIC ≥ 1.25 µg/mL to tilmicosin possess one of these mutations, suggesting an essential role in decreased susceptibility of M. gallisepticum to 16-membered macrolides.

In vitro antimicrobial susceptibility of Mycoplasma bovis isolated in Israel from local and imported cattle.

Monitoring of susceptibility to antibiotics in field isolates of pathogenic bovine mycoplasmas is important for appropriate choice of treatment. Our study compared in vitro susceptibility profiles of Mycoplasma bovis clinical strains, isolated during 2005-2007 from Israeli and imported calves. Minimal inhibitory concentration (MIC) values were determined for macrolides by the microbroth dilution test, for aminoglycosides by commercial Etest, and for fluoroquinolones and tetracyclines by both methods. Notably, although correlation between the methods was generally good, it was not possible to determine the MIC endpoint for enrofloxacin-resistant strains (MIC > or =2.5 microg/ml in the microtest) by Etest. Comparison of antibiotic susceptibility profiles between local and imported M. bovis strains revealed that local strains were significantly more resistant to macrolides than most isolates from imported animals, with MIC(50) of 128 microg/ml vs. 2 microg/ml for tilmicosin and 8 microg/ml vs. 1 microg/ml for tylosin, respectively. However, local strains were more susceptible than most imported strains to fluoroquinolones and spectinomycin. Difference in susceptibility to tetracycline, doxycycline and oxytetracycline between local and imported strains was expressed in MIC(90) values for imported strains in the susceptible range compared to intermediate susceptibility for local strains. The marked difference in susceptibility profiles of M. bovis strains isolated from different geographical regions seen in this study emphasizes the necessity for performing of the antimicrobial susceptibility testing periodically and on a regional basis.