Isolation of Major Pathogens from Cattle with Subclinical Mastitis and Determination of Antibiotic Susceptibilities

Bacterial factors of bovine mastitis have two groups according to origin and transmission routes. The primary contagious agents are S. aureus, Streptococcus sp. and E. coli. These factors are spread widely, especially with handling and milking units. The scope of this study was to determine the isolation and antibiotic susceptibility of S. aureus, Streptococcus sp. and E. coli, which are very important for milk production and frequent agents of subclinical mastitis. In our study, 138 samples of dairy cows were taken aseptically from Aydin province with subclinical mastitis case. In conclusion, 102 (18.5%) S. aureus, 21 (3.8%) Streptococcus sp., and 15 (2.7%) E. coli were isolated from the 552 milk samples examined. After PCR with specific primers in the genotypic identification, all isolates were confirmed about bacterial identification. S. aureus, Streptococcus sp., and E. coli isolates were susceptible to antibiotics of amoxicillin-clavulanic acid, oxytetracycline, cefoperazone and cephalexin-kanamycin.


Introduction
Mastitis, commonly known as inflammation of the mammary glands, is usually associated with microbial infections [1] and is defined as mammary gland inflammation, not as an Intra Mammary (IMI) Infection. Mastitis, an endemic disease in dairy cattle farms around the world, has influence on the loss of milk production. Moreover, mastitis has a direct impact on milk quality, both technically and hygienically. Mastitis treatment is therefore to improve milk quality and the productivity of milk production, there by making milk production more sustainable [2]. Mastitis continues to be the most expensive disease in the world, in clinical or sub clinical form, found in dairy farms [3].
In the spontaneous diseases, the mastitis agents reach the mammary tissue in many ways. These infections emerge by fragile milk ducts, nipple wounds, and infections in the upper respiratory organs, which come into contact with the mammary tissue via blood. There is a significant decrease in milk production both in clinical and sub clinical mastitis. Even after mastitis has been cured in loss of milk production, the yield is not as good as that of the animal; even if the animal recovers [2]. The most common bacterial agents of mastitis are Streptococci (5.43%-20.35%), Coagulase Negative Staphylococci (CNS) (2.86-58.15%) and members of Enterobacteriaceae family (8.47%). Staphylococcus aureus alone accounts for 19.97-65.0% of mastitis cases. Coagulase Negative Staphylococci, which are considered to be the causative pathogens in the development of mastitis now adays, are of great interest in veterinary medicine. CNS is not as pathogenic as other mastitis agents. However, it can cause permanent infections, although it often occurs sub clinically. The bacterial disease leads to an increase in SCC and a drop in milk quality [1].
In recent years, pathogens that produce antibiotic-resistant mastitis have become an increasing concern worldwide. To administer management practices to prevent and treat mastitis, it is necessary to know not only which pathogens cause mastitis, but also which antibiotics are susceptible to these pathogens. The presence of various bacteria in mastitis has been identified in studies conducted, and the distribution of specific infectious agents has been influenced by cattle breeding, lactation stage, milk yield and previous lactation count. Dairy cattle have been shown to be susceptible to mastitis pathogens more than beef cattle.
Today, information about mastitis and optimal technical management and treatment are available. The prevalence of mastitis is lowered with the development of protective measures at the farm level and the correct antibiotic treatment. Antibiotic use is also reduced using prudent antibiotic administration methods.
In appropriate practices can be defined as the reduction of misuse and, where appropriate, the increased use of additional treatments such as alternative medicines. However, producers are still applying inappropriate antibiotics [4].
In this study, isolation of S. aureus, Streptococcus sp. and Escherichia coli strains, determination of antibiotic susceptibility were aimed in milk samples taken from 138 animals showing sub clinical mastitis problem in Aydın province. It is predicted that the results of the study may be useful in the diagnosis and treatment of mastitis cases that may occur in our future. However, there is information in our study results regarding comparing the characteristics of mastitis-affecting organisms of different regions of Turkey and presenting regional data for future studies.

Specimen collection
In this study, 552 milk samples were taken from 138 dairy cows with subclinical mastitis from a dairy cattle enterprise in the Incirliova district, Aydin province within three months period (2017 May-August). The California Mastitis Test (CMT) was used to detect subclinical mastitis cow milk. All of the Holstein breed cows in the study were in lactation but did not receive antibiotic therapy within the last month. The age scale of the cows was between 3 and 11.

Primer pairs
The primer pairs for identification of E. coli, S. aureus, and Streptococcus sp. were shown in (Table 1).

Collection of specimen
Before collecting samples from animals, subclinical mastitis was detected by CMT. All mammary nipples were cleansed with 70% alcohol. After a few milliliters of milk were poured off, 10 ml of milk sample were collected from 4 mammary lobes. The samples were sent to the Routine Diagnostic Laboratory of Department of Microbiology, Department of Veterinary Medicine of Adnan Menderes University under cold chain for isolation.

S. aureus and Streptococcus sp. isolation : Milk samples
were inoculated onto blood agar containing 5% sheep blood and allowed to incubate for 48 hours at 37 °C. Hemolytic colonies on blood agar were then inoculated to Tryptic Soy Agar (TSA) to recover the pure culture, and allowed to incubate at 37°C for 24 hours. Gram staining was applied to determine the Gram(+), and Gram(-) isolates in order to identify the bacterial strains S. aureus, Streptococcus sp. and Escherichia species. Catalase test was performed for differentiation of Staphylococcus and Streptococcus sp. Catalase negative colonies were preliminarily identified as Streptococcus sp. Coagulase positive colonies were regarded as S. aureus. Gram(-) isolates were inoculated to Eosin Methylene Blue (EMB) agar and Mac Conkey agar, then incubated at 37°C for 24 h for identification of E. coli). The isolates with a greenish metallic sheen on EMB agar and lactose positive pink colored isolates were preliminarily identified as E. coli after IMVIC (Indole, Methyl red, Voges-Proskauer, Citrate) reactions.

Genotypic identification
DNA extraction from isolates was performed via DNA Extraction Kit (Fermentas®, Lithuania) according to manufacturer's specifications. The concentrations of DNA obtained by extraction kit were determined with a micro-volume spectrophotometer (ProNano PN-913, Maestrogen®, Taiwan). PCR primers were synthesized by manufacturer highly divergent and species-specific regions of the DNA coding for 16S-23S ISR rRNA, 16S r RNA and Trat gene for the detection of Staphylococcus, Streptococcus sp. and E. coli respectively.
The primers were diluted in TE buffer to a final concentration of 1000 pmol/μl and stored at-80 ˚C as stock. The working concentration of 20pmol/μl was prepared with sterile filtered deionized water, aliquoted and stored at-20 ºC. PCR was performed in a Gene Amp PCR Systems 9700 (Applied Biosystems®) in a final volume of 25μl consisting of 5pmol each of the primer pair, 1 U of Taq DNA polymerase (Fermentas®), 25mM MgCl2, and 100μM each of the four deoxyribonucleotide triphosphates (Fermentas®) in 25mM Tris-HCl, pH 8.3. Initial denaturation was performed at 95 ˚C for 2 min before applying 35 cycles, each cycle with denaturation at 95 ˚C for 45 s, annealing 50 ˚C for 1min, and extension at 72 ˚C for 30sec. After the final cycle, one cycle of extension at 72 ˚C for 5min was carried out to complete the reaction. PCR assay was performed for each pathogen using known standard DNA and with 5pmol each of the specific primer pair described before [5]. Subsequently, PCR was performed with a mixture of standard DNAs with ten-fold serial dilutions of templates to rule out non specific amplification. The PCR products were loaded on agarose electrophoresis gel, and stained with three µl ethidium bromide. The bands were referenced to a Gene Ruler 100-bp DNA ladder (Fermentas®, Canada) and a 1-kb DNA ladder (Fermentas®, Canada) to size the amplicons.
coli isolates and Trat (complement resistance protein) genes were genotypically examined. After PCR with specific primers, S. aureus, Streptococcus sp. and E. coli positive PCR product bands were screened in lengths of 229, 561 and 313bp, respectively ( Figure 1).  Antibiotic susceptibility rates of bacterial isolates as a test result are presented in Table 2.

Discussion
In this study, PCR method has overcome the restrictions for detection of multiple-bacterial subclinical mastitis. The test depends on multiplex PCR that can distinguish pathogens. PCR techniques are proficient for the location of pathogens at low fixation. Techniques including ribotyping are valuable for pathogen location. Along these lines, PCR based location of 16S rRNA or 23S rRNA primers have been effectively connected to the distinguishing of numerous microorganisms. The favourable position of PCR is its affectability as a couple of picogram amounts of nucleic acid is sufficient to distinguish the detection of various pathogens, permitting the confirmation of culture. Also, rRNA is available in numerous duplicates, which increases the specifity. Primers depicted here were turned out to be particular since on agarose gel just a single band was screened for each fragment apart from other ones, and no amplification was identified with negative controls.
The procedure was applicable for multiplex PCR and regarded as specific for S. aureus, Streptococcus sp. and E. coli. Subclinical bovine mastitis is a costly and multi factorial disease for breeders due to reduced milk production, economic losses and untreated chronic mortality rates in the dairy industry. Mastitis is often caused by a variety of infectious agents (usually bacteria), along with physical and chemical agents. More than 120 different microorganism species, including bacterial, viral and fungal, have been isolated from mastitis cows' milk. The most common causes of bacterial mastitis are E. coli, S. aureus, Streptococcus sp. and Enterococcus faecalis [6,7].
The scope of this study is to identify isolated strains by phenotypic and molecular methods. Obaidat et al. [8] conducted a research to identify significant anti microbial agents and resistance against E. coli from cattle in Jordan. A total of 520 E. coli isolates from 43 farms were investigated. 12 antimicrobials were tested for resistance to mastitis in 51.2% of the samples and the most commonly used antimicrobials were oxy tetracycline and streptomycin. E. coli isolates showed high resistance to streptomycin (47.5%), tetracycline (45.4%) and ampicillin (34.2%). Less than 10% of the isolates were resistant to kanamycin, gentamicin, ciprofloxacin and ceftriaxone. In general, 64.6% and 37.1% of E. coli isolates were resistant to ≥1 antimicrobial and multidrug resistance (resistance to ≥3 antimicrobial classes), respectively. The isolates exhibited an antimicrobial resistance profile of 107, and E. coli antibiotic resistance was found to be high in cows in Jordan [8,9] collected 489 milk samples from the breast lobes of 273 dairy cows and 393 (80.37%) samples showed bacterial isolation while 55 (11.25%) samples showed no breeding. Also, 8.38% of the specimens were found to have 3 or more bacterial species in the medium, suggesting that the samples were contaminating. Eightynine (20.10%) bacterial culture positive strains were found to be E. coli strains, and the incidence of E. coli from different farms was between 15.7% and 25%.
In the antimicrobial susceptibility tests of E. coli isolates, they were found to have resistance to sulfamethoxazole 100%, sulfamerazine 98.73%, tiamfenicole 93.67% and oxytetracycline 79.75%. Also, they were found to have high resistance to colistin 49.37%, ceftiofur 22.91%, cefazolin 31.65%, azithromycin 31.65%, ampicillin 25.32% and tetracycline 25.32%, and low resistance to doxycycline, chloromycetin, florfenicol, amoxicillin, ciprofloxacin, streptomycin, and amikacyn. Although there are antimicrobial agents that are effective against the isolates ofloxacin, gentamicin, and enrofloxacin, they have been reported to be resistant to these antimicrobial agents.
Milk samples from cows with acute clinical mastitis were collected using the Vet Path program in nine countries, and 934 isolates were examined for subsequent antimicrobial resistance tests between 2009 and 2012. Milk samples were taken from cows with acute clinical mastitis from Belgium, the Czech Republic, Denmark, France, Germany, Italy, the Netherlands, Spain and the United Kingdom. When the samples were taken, 79.1% of the animals were found not to use antibiotics for at least three weeks.
The remaining 20.9% was treated as unknown. Only one sample per year was taken from each run. The samples were sent to the central laboratories for identification by microbiological and biochemical tests according to gender and species level. Here, the MIC values of all samples were determined as twofold dilutions, by CLSI standards, amoxicillin-clavulanic acid, cefquinome, ceftiofur, cephalin, cephapirin, cefalonium, cloxacillin, penicillin G, enrofloxacin, marbofloxacin, erythromycin, tylosin, kanamycin, cephalexin / kanamycin, neomycin, and tetracycline Suleiman et al. [11] surveyed to determine the prevalence of sub clinical mastitis and the antibacterial susceptibility patterns of isolated mastitis pathogens in cows in different farms in Zanzibar, between January and July 2014. To test the susceptibility ratio of common isolated bacteria, disc diffusion technique was used, 1664 mammary lobes were scanned, and 1648 samples were sent to the laboratory for bacterial culture after CMT. According to the results of CMT, the general subclinical mastitis prevalence was determined as 28.6% in mammary lobes, 48.8% in individual level and 64.7% in farm level respectively.
The CMT positive cow rate was found to be 51.28%, while 63% of their milk samples had bacteriological growth. E. coli (28.9%), Candida sp. (24.21%), S. uberis (19.53%), Coagulase Negative Staphylococci (19.53%), S. aureus (3.9%) and Proteus sp. (2.34%) were identified from samples. Also, no statistically significant difference was found between the number of lactations of CMT positive cows on farms. In other words, farm location, age, lactation number/period, localization of mammary lobes did not affect microbiological results. In microbiologically positive samples, microorganisms were found to be sensitive to amoxicilline clavulanic acid and oxytetracycline, while resistant to gentamicin, ceftiofur, enrofloxacin and cefquinom [13].
Rüegsegger et al. [14] evaluated the antimicrobial resistance of 3954 milk samples isolated from samples collected from animals with subclinical and clinical mastitis diagnoses from various regions of Switzerland between 2011 and 2013. Staphylococcus spp, 598 The highest resistance levels were polymyxine (86.0%), oxacillin (64.7%), and lincomycin (53.7%). Thus, resistance to at least one of the antimicrobial agents has been demonstrated. A total of 256 milk samples from 61 animals were collected from 86 straws during the lactation period in the Sarikamis district of Kars. Of these samples, 72(91.1%) were subclinical, and 7(8.9) samples were related to clinical mastitis mammary lobes. When the microbiological examination of the milk was carried out, 67 isolates were detected from samples. As a result of the CMT test, the subclinical mastitis ratio in cows was found to be 22.3%, and the clinical mastitis rate was found to be 1.5%. It has been determined in a study that a significant portion of mastitis cases was subclinical. Of the 47 mastitic milk, Staphylococcus sp. and Micrococcus sp. Streptococcus sp. and 4 were lactose positive and metallic reflections colonies on EMB agar. Staphylococcus sp., isolated and identified from mastitis milk [15], Antibiotics against staphylococci were determined to be amoxicilline-clavulanic acid, cloxacillin, enrofloxacin, and vancomycin, which are the most effective antibiotics against staphylococci.
In our study, 102 (18.5%) S. aureus, 21 (3.8%) Streptococcus sp., and 15 (2.7%) E. coli were isolated from the 552 milk samples examined. After PCR with specific primers in the genotypic identification, all isolates were identified as S. aureus, Streptococcus sp. and E. coli. The obtained S. aureus, Streptococcus sp. and E. coli isolates were susceptible to antibiotics of amoxicillin-clavulanic acid, oxytetracycline, cefoperazone and cephalexin-kanamycin. The resulting data were determined by the studies carried out.

Conclusion
Cattle mastitis is a standout amongst the most monetarily critical ailments that influence the dairy industry. Billions of dollars are lost all over the world due to decreased quality and amount of drain delivered. In spite of the fact that the mastitis is of multietiological, the high level of misfortune is because of the three noteworthy bacterial pathogens, Staphylococcus sp., Streptococcus sp., and E. coli. Mastitis, if distinguished at subclinical level can be controlled by antimicrobial treatment. Diagnosis of subclinical mastitis and the causative agent is essential for under taking preventive measures. Customary methodology for identified proof of pathogens of mastitis is worked serious, and the greater part of the conventional tests is not intended to distinguish dominant veterinary pathogens. Location of mastitis at sub-clinical form needs tests that are exceedingly sensitive and PCR based tests can meet this necessity [16].
However, a few of these accessible techniques still need enhancement of the way of culture, which is tedious. Additionally to recognize the pathogen the PCR responses are to be conveyed independently for various pathogens. Molecular identification is an elective strategy, be that as it may, the similarity of the primer sets is essential for the response to work productively with high affectability of identification. Mastitis is an important mammary gland disease caused by microorganisms. Due to mastitis, big economic losses occur every year. The economic losses caused by the disease are not only limited to a reduction in milk yield but also include expenditures such as the treatment of the disease and the removal of diseased animals. Deterioration in the quality of the milk, which is an important food source, also has negative consequences for public health. It is expected that the results obtained in this study past will be beneficial for future studies.