Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among New York State dairy farms

Abstract

Mycoplasma mastitis is a contagious and costly disease of dairy cattle that significantly affects animal health and milk productivity. Mycoplasma bovis is the most prevalent and invasive agent of mycoplasma mastitis in dairy cattle, and early detection is critical. Other mycoplasma have been isolated from milk; however, the role and prevalence of these species as mastitis pathogens are poorly understood. Routine screening of milk for mycoplasma by bacteriological culture is an important component of a farm control strategy to minimize a herd mycoplasma outbreak, but phenotypic methods have limited ability to speciate mycoplasma, affecting how farms and practitioners can understand the role and effect of species other than M. bovis in herd health. Fastidious mycoplasma culture can be lengthy and inconclusive, resulting in delayed or false negative reports. We developed and validated a multitarget PCR assay that can in the same day confirm or reject a presumptive positive mycoplasma culture found upon bacteriological testing of clinical specimens, further discriminate between Acholeplasma and Mycoplasma, and identify M. bovis. Coupled with sequence analysis isolates can be further identified as bovine mycoplasma Mycoplasma arginini, Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovirhinis, Mycoplasma bovigenitalium, Mycoplasma californicum, Acholeplasma laidlawii, and Acholeplasma oculi. Assay validation included analysis of 845 mycoplasma representing these species and 30 additional bacterial species obtained from routine milk submissions to the Quality Milk Production Services from New York State farms and veterinary clinics between January 2012 and December 2015. Among 95 herds, we found 8 different Mycoplasma species and 3 different Acholeplasma species, with an overall prevalence of M. bovirhinis of 1%, A. oculi of 2%, M. arginini of 2%, M. californicum of 3%, M. canadense of 10%, M. bovigenitalium of 10%, A. laidlawii of 11%, M. alkalescens of 17%, and M. bovis of 78%. More than one mycoplasma was found in 14% of the herds tested, and both M. bovis and Acholeplasma were found in 6% of the farms. Incorporation of the validated molecular diagnostic assay into routine bacteriological screening as a supportive confirmation and identification tool will lead to an improved assessment of Mycoplasma and Acholeplasma prevalence data, which will facilitate increased knowledge about the role of these mycoplasma in mastitis.

Antimicrobial Resistance in Members of the Bacterial Bovine Respiratory Disease Complex Isolated from Lung Tissue of Cattle Mortalities Managed with or without the Use of Antimicrobials

“…Primers and annealing conditions for MH, PM, and HS were as described by Klima et al [2]. For MB, the three targets included uvrC, 16S rDNA, and Mycoplasma 16S to 23S rDNA intergenic transcribed spacer region with primers and annealing conditions as described by Gioia et al [19]. Only PCR-confirmed MH, PM, HS, or MB isolates were included in subsequent analyses.…”

Gap analysis ofMycoplasma bovisdisease, diagnosis and control: An aid to identify future development requirements

“…In general, molecular methods can be divided into those allowing specific detection of M. bovis (using either conventional or real‐time PCRs; Cai, Bell‐Rogers, Parker, & Prescott, ; Ghadersohi, ; Hotzel, Sachse, & Pfützner, ; Rossetti, Frey, & Pilo, ; Sachse et al., ; Subramaniam et al., ) and those allowing simultaneous detection of M. bovis with other mycoplasma species or Mollicutes . The latter includes multiplex PCRs (Boonyayatra et al., ; Cornelissen et al., ; Gioia, Werner, Nydam, & Moroni, ; Justice‐Allen, Trujillo, Goodell, & Wilson, ; Parker, House, Hazelton, Bosward, & Sheehy, ; Righter, Rurangirwa, Call, & McElwain, ), PCR with denaturing gradient gel electrophoresis (DGGE); (McAuliffe, Ellis, Lawes, Ayling, & Nicholas, ) and a DNA microarray based on oligonucleotide probes derived from the 23S rRNA gene and species‐specific probes from the tuf gene target (Bottinelli et al., ; Schnee et al., ). It is important to note that although multiplex detection may help in the diagnosis of mixed mollicute infections, it may introduce additional complexity into the determination of disease aetiology; multiple mycoplasma species may be found, for example, in the upper respiratory or genital tracts that are apparent commensals with questionable significance in disease (Brown et al., ).…”

Virulence, antimicrobial resistance and phylogenetic analysis of zoonotic walking pneumonia Mycoplasma arginini in the one-humped camel (Camelus dromedarius)

“…We also followed the steps of Gioia et al (2016) Compared to other bacterial pathogens, the current knowledge of the molecular basis of pathogenicity of mycoplasmas is limited, and disruption at the molecular and cellular level remain to be elucidated. Several studies in the past years have shown that pathogenic mycoplasmas are equipped with sophisticated genetic systems, which allow these agents to spontaneously change their surface antigenic make-up (Jeqchlingeret al, 2004;Chopra-Dewasthaly et al, 2017).…”