Mastitis is the most important problem of dairy production. In cows, mastitis is an infection that occurs in all farms, including modern farms, and causes significant economic loss. It has been shown that economic losses in cows are more frequent in subclinical mastitis than in clinical mastitis. Mastitis is the leading cause of economic loss in milk production when yield, product losses and treatment costs are evaluated together. Mastitis frequency can be observed up to 40%, especially in modern intensive farms. Despite all hygienic practices, the continuation of the mastitis problem has brought medical preventive measures to the agenda and the development of control programs, including vaccines, has become unavoidable. However, in order to be able to effectively combat an infection, definite etiopathogenesis and its associated epidemiology must be known on the basis of region and herd
10.
In cattle, mastitis is widespread all over the world and leads to significant economic losses. Pathogens of mastitis usually exhibit a contagious course during transport, and 2 groups are separated as infectious agents from environmental reservoirs. Many streptococcal species are among those isolated as mammary pathogens. S. uberis is an important pathogen in bovine mastitis, and is associated mostly with subclinical and clinical intramammary infections in both culled and untreated cows. These species are often a problem because they are ubiquitous around the dairy farms. Increasing the hygiene procedures against these opportunist pathogens results in the reduction of consequent mastitis, however does not yield an effective result in environmental contamination 12.
The isolation rates of streptococci from milk samples vary. In a previous research 13, S. dysgalactiae was isolated from 2.3% of the milk samples examined, S. uberis and S. agalactiae was ddetected in the ratio of 0.3% on the farm to have a low isolation rate. Sampimon et al. 14 found the prevalence of S. uberis at 1.1%, S. dysgalactiae at 0.9%, and did not encounter S. agalactiae. Tenhagen et al. 15 have identified S. agalactiae as 0.1%, S. dysgalactiae and S. uberis as 2.3% from cow milk. In a study conducted in UK 16, bacteriological sampling in the milk resulted in the isolation of S. uberis with ratio of 23.5%, whereas S. agalactiae and S. dysgalactiae were not identified.
Bentley et al. 17 reported that only three of the 206 gram-positive cocci isolated from mastitic milk were S. parauberis. Researchers have indicated that it is difficult to distinguish S. parauberis agents from S. uberis and that there is limited information on epidemiology. Pitkala et al. 18 identified two S. uberis isolates as S. parauberis in their study in Finland and found that the antibiotic resistance of S. parauberis strains resembled that of S. uberis. Devriese et al. 19 reported that catalase-negative and esculin-positive Gram-positive cocci isolated from clinical and subclinical mastitis were mostly identified as S. uberis and that they were not correctly identified.
In studies conducted in Turkey, Dakman 20 reported that 12.5% of cow mastitis cases were caused by Streptococcus spp. and that 4.5% of these Streptococci were S. agalactiae. Karahan 21 examined 24.3% of mastitic milk samples from Streptococcus spp. 10.4% of isolates were S. agalactiae and 13.9% of other isolates were identified as other Streptococci.
Şahin et al. 22 found that Streptococci isolation rate from milk was 29.82% and 14.03% of S. agalactiae, 8.77% of S. dysgalactiae and 7.02% of S. uberis were identified.
The average isolation rate obtained in this study was found to be correlated with the findings obtained from previous studies conducted in Turkey. These findings may arise from the lack of application of mastitis control programs in dairy cows when assessed based on the mean Streptococcal isolation rate. The isolation rates obtained in other studies are at the same level.
Jayarao and Oliver 23 identified 11 different enzymes in the RFLP-PCR method to isolate mastitic Streptococci at the species level by single enzyme cuts. McDonald et al. 9 performed identification of Streptococci at the species level by using dual enzyme combinations in a single reaction in the RFLP-PCR method.
Different molecular methods have been used for species-level identification of streptococci. These methods are used especially in the typing of S. uberis 2.
Duarte et al. 24 identified RFLP-PCR as an efficient method for diagnosing in a single reaction with shorter time than conventional methods. Odierno et al. 25 reported that RFLP-PCR was 100% accurate after RFLP-PCR, but that conventional test results did not fully support RFLP-PCR.
Jayarao and Oliver 23 compared the results with classical biochemical results in RAPD-PCR evaluation and distinguished near genotypic factors by conventional method.
Duarte et al. 24 showed that this method could be used in typing microorganisms in the RAPD PCR procedure they applied for group B Streptococci, but the use of additional typing methods in the interpretation of the results increased the reliability.
Chatellier et al. 26 identified 71 profiles in 114 strains of RAPD-PCR for S. agalactiae and found that these profile differences could be due to the geographical areas where the farms were located.
In our study, 35 isolates identified as S. uberis by bacteriological culture and biochemical identification method were all identified as S. uberis in PCR result using 16S rRNA gene specific primer. The fact that the primer used has a high specificity and that it is derived from the 16S rRNA gene, which is more stable than other gene regions, indicates that PCR identification can be performed as a reliable and rapid method in routine diagnostic laboratories. The use of molecular methods those are more specific and more sensitive than those of conventional methods with lower sensitivity and would be more beneficial.
In a study conducted by Hassan et al. 27, positive for cfb gene was detected in S. agalactiae isolates, phenotypically negative for CAMP, by PCR. This suggests that the cfb gene-specific PCR assay can avoid false negative conditions that may arise from the CAMP reaction, which is considered to be one of the key criteria in the biochemical characterization of streptococci.
Ekin et al. 28 reported a positive phenotype in 73.3% of S. agalactiae isolates obtained from cattle milk in terms of CAMP test in our country. In another study 26.3% of cattle-born Streptococcal isolates were positive for CAMP test 29. Although this test is generally considered to be a determinant test for the identification of Streptococci, it is likely that CAMP testing cannot be used effectively for identification from earlier studies. Reinoso et al. 10 found that the cfu gene distribution in their study was 76.9%.
The cfu gene responsible for the CAMP factor in S. uberis was identified in 30 (86%) strains in our study. These results were in parallel with findings in our study. Thus, the result that cfu gene identification can be used for identification in S. uberis quickly and reliably.
The operon in the capsule formation consists of the hasA, hasB and hasC gene loci, which are essential genes for capsule formation 11. HasA, hasB and hasC gene identification rates were 83%, 91% and 86%, respectively, in our study. Reinoso et al. 10 identified the hasA gene as 74.3%, the hasB gene as 66.6% and the hasC as 89.7%. According to the findings obtained, capsule formation in Streptococcal mastitis cases seems to play an important role in the development of infection.
The oppF gene plays an important role in the milk production of S. uberis agents 30. In our study, oppF gene identification rate was 80%. Reinoso et al. 10 identified the oppF gene by 64.1%. Zadoks et al. 4 reported that this gene could not be isolated from all strains. The findings obtained correlate with the data in our study.
Serine protease enzymes that convert plasminogenetic plasmids are essential components for the degradation of extracellular matrix proteins and the colonization of bacterial agents in tissues. In addition, the milk proteins that are responsible for the activation of endogenous plasminogenes in the milk are hydrolyzed, thus forming the molecules necessary for S. uberis 11. The skc gene responsible for the synthesis of these enzymes has been identified in 91% of our studies. Reinoso et al. 10 identified 63% of the skc gene in their study.
The pauA gene 31 that was detected in association with plasminogen activation and intramammary colonization was isolated in 71% of our study. The pauB gene has not been identified in our study. In the study conducted by Smith et al. 30, only one strain of S. uberis was identified as pauB. Expression of pauB genes has not been shown to play a role in the formation of intramammary infections in the direction of the obtained data.
Multiplex PCR-based molecular assays have been found to be reliable for detecting the genotypic relationships of the virulence genes at the genus level of the S. uberis agent, a common effect of subclinical bovine mastitis cases. In addition, in this study, the close proximity of the homozygous for the identification rates as a result of PCR amplification of the hasA, hasB and hasC gene operon used for the molecular identification of S. uberis has shown that this gene region allows rapid and reliable identification of cattle origin S. uberis agents. It has been found that the molecular methods used in this study may be useful for detecting virulent types among strains of S. uberis which are encountered as an important problem in subclinical mastitis cases and may contribute to putting the pathogen of infection in the basis of field strains. Significant control and strategies to be undertaken in the face of these findings are expected to provide a significant reduction in the incidence of S. uberis-induced mastitis. Thus, effective control programs against S. uberis mastitis, which are very difficult to eradicate or control due to various factors that are effective in the host environment and active agent triplet, will provide important advances both in terms of animal health and public health. It is also believed that the identification of additional genotypic methods and methods for detection of new virulence genes of S. uberis strains as well as speeding up detailed genetic analysis studies will be useful in obtaining more specific results and more detailed information on the epidemiological characteristics of these factors. As a result, when the distribution of virulence genes of S. uberis isolates isolated from mastitis cases is investigated, it has been shown that the genes which exert capsular formation in pathogenicity of the agent play an important role and the genes involved in the synthesis of serine protease enzyme are distributed in a remarkable manner. It is also evaluated that the identification of additional genotypic methods and methods for detection of new virulence genes of S. uberis strains as well as speeding up detailed genetic analysis studies will be useful in obtaining more specific results and more detailed information on the epidemiological characteristics of these factors. The S. uberis factors identified in our study were at the same level as the other studies in our country. The presence of the bacterial agent has been demonstrated in the Aydın region and with the help of regular preventive control strategies it will become possible that the incidence of S. uberis-induced mastitis cases can be lowered and therefore economic losses can be minimized.
)
)
)
