Mycoplasmas are common inhabitants of the respiratory mucosa, the urogenital tract, conjunctival surfaces, the gastrointestinal tract and the mammary gland; they can be commensals, opportunistic pathogens or primary pathogens associated with respiratory disease, otitis, mastitis, arthritis, reproductive disease, meningitis and conjunctivitis in cattle ^5,11,13. Some Mycoplasma species are highly contagious, spread rapidly within a herd, and respond poorly to antibiotic therapy, which often determines the need of segregating or culling affected animals13. Hence, a rapid and accurate diagnosis is of outmost importance both for the control and for prevention of disease outbreaks. Early stud-ies using biochemical methods detected the presence of M. bovis associated with mastitis in dairy cattle in Argentina3. In the last few years, Mycoplasma infections in cattle have received increasing attention, being the organisms identi-fied either by biochemical and molecular methods^12,14,15 or by immunohistochemistry10.

Several conventional PCR methods have been developed in different countries to identify Mycoplasma species either from isolates grown through classical culture or directly from milk samples13. However, so far only few Mycoplasma species have been isolated from dairy cattle in Argentina and there is scarce information about their frequency and the epidemiological and clinical features associated with their presence in local dairy herds. Hence, there is a need to generate methods for rapid identification at the species level to be used as a diagnostic tool both during outbreaks and epidemiological surveys. We have recently reported the iso-lation of M. leachii associated with arthritis in dairy calves and the development of a multiplex PCR for detecting both this organism and M. californicum12. Since other species have been reported to be associated with diseases in dairy cattle in Argentina^10,14,15, the objectives of this study were to develop a multiplex PCR for identifying other Mycoplasma species (M. bovis and M. canadense) and to describe the frequency of Mycoplasma species isolated from clinical samples submitted to a diagnostic laboratory.

A total of 1548 samples were submitted to the Microbiology Laboratory of the Rafaela Experiment Station of INTA from January 2009 to July 2016 by veterinarians who suspected the presence of Mycoplasma as etiologic agent of diseases in dairy cattle. One hundred and seven of these samples were from bulk tank milk, 1412 from composite milk from individual cows and 29 from various organs and biological fluids of dairy calves. The samples came from dairy farms located in the central dairy area of Argentina (Santa Fe and Córdoba provinces) and in the Mar y Sierras dairy area (Buenos Aires province). In a first stage, biologi-cal specimens were processed by the classical methodology using modified Hayflick medium at 37 °C in a 10% CO2 atmosphere incubated for 7-10 days and Mycoplasmas were presumptively identified on the basis of their colony mor-phology under a microscope at 15-25x magnification as previously described12. DNAfrom bacterial growth in culture medium from colonies suspected to be Mycoplasma spp. was extracted using the procedure previously described12 and subjected to a PCR test to confirm that isolates belonged to this genus2. For further identification of M. leachii and M. californicum a multiplex PCR to amplify specific DNA frag-ments from the conserved spacer region between the 16S and 23S ribosomal RNA gene was carried out as previously described12.

For designing a multiplex PCR for amplifying specific DNA fragments of M. bovis and M. canadense two steps were followed. First, the conserved spacer regions between the 16S and 23S ribosomal RNA gene1 of M. bovis ATCC 25025 and of six unidentified isolates previously characte-rized as belonging to genus Mycoplasma by a genus-specific PCR2 were amplified and sequenced (ABI3130xl, Applied Biosystems). The sequences obtained were aligned against the NCBI database using BLASTN and then compared with the reported hits in GenBank using the ClustalX multiple sequence alignment program. The 16S-23S rRNA gene sequence obtained from the unidentified strain showed 99% identity with M. canadense strain 275C (DQ847417) while the other was confirmed as M. bovis. The anal-ysis of the 16S-23S rRNA region allowed the design of species-specific primers, but with very similar amplicon size. Then, specific primers only for M. canadense were designed using FastPCR (https://primerdigital.com/tools/pcr.html) for the 16S-23S rRNA region. A multiplex-reaction was set up using those specific primers for M. canadense while primers for M. bovis were those previously described to amplify a conserved segment of the uvrC gene suitable for specific diagnosis17. Primers for M. bovis were UVRF (5'-TTACGCAAGAGAATGCTTCA-3') and UVRR (5'-TAGGAAAGCACCCTATTGAT-3') and for M. canadense were CANDF (5'-GCGGAACATTAGTTAGTTGGTA-3') and CANDR (5'-CGTTAGCTGCGTCAGTGAATT-3'). Expected DNA amplificaron fragments were 1626 bp for M. bovis and 623 bp for M. canadense, respectively. The amplification program was: 94 °C 2 min, 30 cycles of 94 °C 30 s, 54 °C 30 s, 72 °C 60 s, 72 °C 5 min. Primers designed for amplifying specific fragments of the 16S-23S rRNA region for differentiation of M. canadense as well as those previously described12

Table 1: Frequency and origin of Mycoplasma isolated from biological samples obtained from dairy cattle. 

Of the total samples processed, 38 isolates were phe-notypically identified as Mycoplasma spp. This preliminary identification was confirmed by the genus-specific PCR tech-nique. Identification of the isolates at species level was achieved using both multiplex PCR described. Table 1 sum-marizes the frequency of isolated Mycoplasma species and their origin.

Since convenience samples were used, the results of this study are not indicative of the prevalence of the dif-ferent isolated species. The low number of isolates in relation to the total of processed samples indicates that the presence of this genus detected by classical culture is not a frequent finding. No systematic studies have been performed in Argentina to determine prevalence of the different Mycoplasma species associated with defined syn-dromes in dairy cattle; rather, disease outbreaks and clinical cases have been reported so far. Three isolates (7.9%) were identified as M. bovis. Within the Mycoplasma genus, M. bovis is currently considered the most frequently isolated species associated with disease in cattle worldwide11. Since the first report causing mastitis in a dairy herd, M. bovis has been isolated from cases of respiratory disease, mastitis and arthritis in cattle, being considered an emergent pathogen in several countries5,11. In Argentina its presence was first reported in a mastitis outbreak in a dairy farm located in Buenos Aires province where 11.8% of cows presented clinical mastitis that in various cases affected the four mammary quarters, caused a marked decrease in milk production and did not respond to antibiotic therapy. The organism was iso-lated from composite milk samples from cows with clinical and subclinical mastitis, bulk tank milk and a preputial swab from a bull by classical culture and characterized as M. bovis by biochemical tests and indirect immunofluorescence of the isolated colonies3. More recently, M. bovis was detected in milk samples both from cows with clinical and subclinical mastitis and bulk tank milk from dairy herds from Buenos Aires and Córdoba provinces and characterized by ampli-fying a fragment of the uvrC gene15; however, no further information was provided about the cases from which the processed samples came. In addition, M. bovis was identified by immunohistochemistry in cases of bronchopneumonia unresponsive to antibiotic treatment both in beef and dairy calves from Córdoba and Santa Fe provinces10. These results, together with those obtained in the present study confirm the importance of M. bovis as a causative agent of mastitis, bronchopneumonia and arthritis as has been pointed out in different countries5,11 and indicate the need to include diag-nostic methodology in routine laboratories to identify this pathogen.

M. canadense was isolated from a single case of intra-mammary (IMI) infection from a dairy herd in Buenos Aires province. Diseases and clinical cases associated with this organism are not as well characterized as those associated with M. bovis. This species has been reported to cause not only IMI5, but also to be associated with granulopapular vul-vovaginitis in dairy cattle in single or mixed infections with Mycoplasma bovigenitalium8 and with arthritis in a calf9. In Argentina, this species was isolated from two bulk tank milk samples from a dairy farm located in Buenos Aires province15.

M. californicum is considered to be the second most fre-quently isolated Mycoplasma species from bovine mastitis cases in USA after M. bovis5, sharing with the latter similar characteristics regarding both the clinical mastitis features and the importance of the presence of asymptomatic car-riers in the dissemination of the organism among cows and dairy herds6. In the present study, this organism was iso-lated from 4 cows during an outbreak of mastitis refractory to antibiotic therapy, as well as from two bulk tank milk sam-ples from the same dairy farm located in Córdoba province. In Argentina, the isolation of M. californicum was reported from bulk tank milk samples15 and clinical mastitis samples from the same dairy herd14 located in Buenos Aires province.

Twenty eight isolates (73.7%) were characterized as M. leachii. These isolates belonged to a single dairy farm where cases of polyarthritis in calves were previously detected12.

In this farm, milk samples were taken from clinical mastitis cases and also from individual cows (composite milk sam-ples) as part of a control program based on the detection of asymptomatic carriers. In addition, bulk tank milk samples were taken for monitoring the presence of the organism in the lactating dairy herd as the control program progressed. M. leachii, previously referred to as Mycoplasma sp. bovine Group 716, was early recognized as a cause of polyarthritis, mastitis and abortion in dairy cattle in Australia7 and recent studies characterized clinical signs and histopathological lesions induced by experimental IMI with this pathogen4. The present study, as well as our previous report12 confirms the ability of this organism to cause diverse clinical syndromes.

In conclusion, the multiplex PCR technique for detecting M. bovis and M. canadense described in this study, along with the one previously described for detecting M. californicum and M. leachii, provide a useful diagnostic tool for characterizing some of the Mycoplasma species that cause disease in cattle in Argentina. The results of this study confirm the presence of several species of Mycoplasma associated with different disease syndromes in dairy cattle. The techniques used in the present study can be useful to broaden knowl-edge about Mycoplasma infections in cattle, since the search for these organisms is not usually included in routine diagnoses.