Pathogen isolation and antibiogram analysis in dairy cows with clinical mastitis in Adana region, Turkey

The goal of this study was to investigate pathogen types found in milk samples from cows with clinical mastitis, as well as the sensitivity of isolated bacteria to different antibiotics. The study was carried out on 103 milk samples collected from dairy cows with clinical mastitis. Microbiological isolation and identification were performed to identify gram negative and gram positive bacteria, Mycoplasma bovis and fungi. Then, bacteria isolated from infected milks were subjected to antibiogram, using 27 antimicrobials agents. As a result, a total of 146 isolates were obtained from 103 milk samples. The predominant pathogen isolates recovered were fungi (21.9%), Escherichia coli (19.9%) and Staphylococcus aureus (13.7%), followed by Mycoplasma bovis (8.2%) and Streptococcus uberis (6.8%). The mixed predominant pathogen was fungi, (75%) the majority of which mixed with gram positive bacteria. Ampicillin, Imipenem, Chloramphenicol and Streptomycin were the least effective antimicrobial agents, while the most effective agents were Florfenicol, followed by Amikacin and Kanamycin / Cefalexin. In conclusion, the types of microorganisms that cause mastitis and the antibiogram results of the bacteria isolated varied. Above all, fungal mastitis is a severe problem that should always be considered prior to starting antibiotic treatment.


Introduction
Mastitis, which is the inflammation of the mammary gland against infectious and non-infectious factors (Bradley 2002), is known as a multifactorial problem of dairy cows that affects milk quality and quantity (Guimarães et al. 2017;He et al. 2020). Moreover, mastitis is the most common and the most costly production disease in the dairy herds (Abebe et al. 2016;Dalanezi et al. 2020) and it is an account for 38% of total economic losses caused by production diseases (Bradley 2002). Economic losses caused by mastitis are related to the decrease in milk production, treatment and labour cost, milk disposal, decrease in the sale price of milk and the culling of animals (Demir et al. 2012;Guimarães et al. 2017; Ayvazoğlu and Eşki 2019). As a result of all these, mastitis causes an about the annual loss of $159 per cow (Dalanezi et al. 2020). Mastitis occurs in clinical and subclinical forms and their average incidence was reported as 14.2% and 30%, respectively (Sundrum 2015). Furthermore, it has a serious zoonotic risk due to the presence of bacteria and their toxins in milk (Abebe et al. 2016). As understood, mastitis is a global problem that must be tackled effectively due to its high incidence and prevalence (Sundrum 2015;Guimarães et al. 2017) Mastitis has various etiologies (Bradley 2002;Dalanezi et al. 2020) and is most commonly caused by bacteria (Dalanezi et al. 2020). Epidemiologically, its the etiology has been categorized as environmental (Coliform) and contagious pathogens (Smith and Hogan 1993;Abebe et al. 2016). The major reservoir of contagious mastitis pathogens is cows, they are spread among animals (Fox and Gay 1993), and tend to cause chronic subclinical infections after exacerbation of clinical events (Abebe et al. 2016). Fox and Gay (1993) (Smith and Hogan 1993). On the other hand, it is known that viruses, fungi and algae are among the important infectious agents that cause mastitis (Dalanezi at al. 2020), and fungal mastitis (mycotic mastitis) have become an increasing problem in recent years (Jasm and Yassein 2020). The commonly encountered causes of mycotic mastitis are Candida spp., Trichosporon spp., Cryptococcus spp., Saccharomyces spp., Aspergillus spp. (Dubie et al. 2015). Because use of antibiotics may also stimulate the multiplication of fungi, increases in fungal mastitis cases may be associated with antibiotic treatment (Wawron et al. 2010), so misdiagnosed fungal mastitis can be more exacerbated. Considering the differences in pathogen types, it is understood that the fight against mastitis is a difficult and complex situation (Taponen et al. 2017;Rahman et al. 2018). Moreover, the antimicrobial resistance of bacterial agents makes the fight against mastitis more critical (Pitkala et al. 2004;Rajala-Schultz et al. 2004;Tenhagen et al. 2006;Etifu and Tilahun 2019). Consequently, agent isolation and antibiogram test have become indispensable for a successful treatment and struggle.
Antibiotic treatments were reported to be often unsuccessful in mastitis cases in the Adana region. On this situation, we hypothesized that fungal mastitis or antibiotic resistance would be common. This study was planned considering that it would be of economic importance. The aim of the present study was to investigate the pathogen types in milk samples of cows with clinical mastitis and the sensitivity of the isolated bacteria to various antibacterial drugs, in Adana region, Turkey.

Animal and management
This study was approved by the Local Ethics Committee of Ceyhan Veterinary Faculty, Cukurova University, Adana, Turkey (approval number 8/1 and 23.09.2020). The presented study was carried out on 103 milk samples collected from dairy cows with clinical mastitis in around 40 medium-scale commercial farms, Adana region, Turkey. The dairy cows had a similar lactation period, milk yield, age, parity, milking system and body condition score, and they were managed in free-stall barns under the same conditions, had free access to water. And cows did not receive any treatment before the study.

Udder examination and milk sample collection
Palpation and inspection methods were performed to examine typical signs of clinical mastitis by a veterinarian. The examination included abnormal milk, quarter asymmetry, blindness and inflammation such as redness, hotness, swelling and painful sensation. Milk samples (20-25 ml) were collected from individual quarters with clinical mastitis to sterile falcon tubes under asepsis and antisepsis conditions. Briefly, milk sampling procedure was performed according to Laboratory Handbook on Bovine Mastitis of the National Mastitis Council (Hogan et al. 1999). After the milk samples were collected, they were immediately transported to the laboratory under the cold chain (4°C).

California mastitis test (CMT)
California mastitis test (CMT) was performed for confirmation of the examination applied by inspection and palpation. The test process and the interpretation of CMT results were made according to the Baştan et al. (2008)'s instructions.

Microbiological examination
Microbiological identification and isolation were performed according to National Mastitis Council's instructions (Hogan et al. 1999

Bacterioscopy
The milk samples collected from infected quarters were centrifuged and preparations were prepared from the sedimentation. Then, they were stained with gram staining methods for bacterioscopy examination.

Culture and Biochemical Identification
The milk samples were individually inoculated into blood agar (Oxoid, CM0055), MacConkey agar (Oxoid, CM0007) and Sabouraud Dextrose agar (Merck, 105438) using a quadrant streaking method, and they were incubated at 37°C for 24-48 h. While blood agar and MacConkey agar were used for bacteriological isolation, Sabouraud Dextrose agar was used for fungal isolation. Morphologic characteristics of isolated microorganisms were observed on these primary cultures. And Gram staining was applied to define the gram reaction and shape of the cultures.

Discussion and Conclusion
In the present study, firstly the types of microorganisms that cause clinical mastitis were determined. Then, the sensitivity of isolated bacterial pathogens to 27 different antibacterial agents was investigated. Among the microorganisms isolated in this study, fungi, E. Coli and S. aureus were determined as the major cause of mastitis, but the obtained result showed some differences compared to the previous report (Green et al. 2002;Baştan et al. 2008;Rahman et al. 2018).
It has been reported that fungi are usually agents in bovine mastitis and are commonly considered to be an environmental mastitis agent due to poor hygiene (Krukowski et al. 2001). There are different types of fungi that cause mycotic mastitis (Du et al. 2018). However, its most common cause was found the genus Candida (Zaragoza et al. 2011;Eldesouky et al. 2016;Du et al. 2018). They may be also associated with clinical mastitis (Jasm and Yassein 2020). In the present study, because the main aim was to investigate the importance of fungi in mastitis cases, no species distinction was performed. It has been reported that the prevalence of mycotic mastitis has considerably increased in recent years (Zhou et al. 2013) and the rates of fungi isolated from milk with mastitis differed significantly by country. And this rate was noted as 12.1% in Turkey (Jasm and Yassein 2020). However, it was found to be higher (31.06%) in our study. We considered that it may be due to Adana's subtropical climate (Krukowski and Saba 2003). Antibiotics are administered more frequently in routine mastitis treatment and antifungal treatment is not given importance (Pachauri et al. 2013). However, the presence of fungi in mastitis cases reveals the importance of pathogen isolation for treatment and control. Moreover, the differences in bacterial species isolated and their antibiotic resistance make the situation even more critical. The results demonstrated that 48.27% of E. coli isolated developed resistance to all antimicrobial agents used in this study. And the most effective antimicrobial agent against E. coli was Kanamycin / Cefalexin (37.93%). Another study showed that E. coli has a sensitivity of 94.59% against Chloramphenicol in buffaloes (Charaya et al. 2014). Mohanty et al. (2013) reported that E. coli were the most sensitive toward Levofloxacin (96.66%) and Chloramphenicol (90%). Similarly, other studies showed differences in antimicrobial agents to which E. coli was sensitive (Tenhagen et al. 2006;Bhat et al. 2017;Taponen et al. 2017). It also is known that the treatment process of cases of E. coli is easier than cases of S. aureus, another major pathogen (Luoreng et al. 2018). And its rate and spread in the herd is closely related to management (Rainard et al. 2018). In a previous study, all staphylococcal species including S. aureus were determined at a rate of 20.4% (Twomey et al. 2000), which is similar to the ratio of our study. Antibiotic treatment is mostly ineffective for mastitis caused by S. aureus (Luoreng et al. 2018). In the present study, results of antibiogram test for S. aureus showed that Amikacin (40%) and Florfenicol (40%) to be the most effective of all antibiotics. Charaya et al. (2014) also reported that Amikacin (88.57%) the most effective antibiotic for S. aureus isolated in buffaloes. On the other hand, several studies declared that S. aureus is most sensitive to Enrofloxacin and Ciprofloxacin (Ismail 2017), Levofloxacin (88.23%) and Enrofloxacin (88.23%) (Mohanty et al. 2013), Gentamicin (100%) and Erythromycin (100%) (Tenhagen et al. 2006). It was also observed that there was a difference in the rates of isolated S. aureus in these studies. Other important pathogens isolated in the present study were M. bovis (8.2%) and S. uberis (6.8 %). Mastitis caused by mycoplasma is not as common as mastitis caused by other bacteria (Nicholas et al. 2016). However, it generally does not respond to antibiotic therapy and highly contagious (Fujimoto et al. 2020;Liu et al. 2020). It also has been reported that mycoplasma has no cell wall (Parker et al. 2018) and to be resistant to all major classes of antimicrobial drugs. Therefore, the control of mycoplasma mastitis with antibiotic treatment is usually unsuccessful (Nicholas et al. 2016). As understood, protection and prevention come to the fore in the fight against this pathogen. For these reasons, we did not perform an antibiogram test against M. bovis.
It was declared that S. uberis is among the most important factors causing bovine mastitis (Käppeli et al. 2019), has an increasing prevalence worldwide and responsible for 14 to 26% of clinical mastitis (Phuektes et al. 2001). On the other hand, Käppeli et al. (2019) reported that clinical mastitis cases caused by S. uberis have decreased with preventive control studies. Antibiogram results of the study conducted by Phuektes et al. (2001) showed that all isolates of S. uberis sensitive towards Vancomycin and Cephalexin. Other studies revealed that the most effective antimicrobial agents against to Streptococcus species isolated were Chloramphenicol and Tetracycline (100%) (Kurjogi and Kaliwal 2011), Levofloxacin, Enrofloxacin and Ciprofloxacin (Mohanty et al. 2013) However, no similar results were observed by the current study, we found that S. uberis the most sensitive to (80%) Amikacin, followed by Kanamycin / Cefalexin (60%).
Antimicrobial agents effective against minor pathogens isolated in our study and their effectiveness rates showed different distributions and results. Furthermore, some antibiotics had a higher success rate on them compared to major pathogenes. We interpreted this as that these bacteria develop less antibiotic resistance since they are not the common cause of mastitis. We also thought that the differences in isolated microorganism and antibiotic resistance vary according to the regions where the study was conducted, management conditions, hygiene and the antibiotics preferred in previous treatments.
In conclusion, the types of microorganisms that cause mastitis and the antibiogram results of the bacteria isolated varied, and the predominant pathogens were fungi, E. coli and S. aureus. Fungal mastitis is serious problem, should always be considered before routine antibiotic therapy and emphasis should be given on antifungal therapy. Considering all bacterial cases, the most effective antibiotic was Florfenicol, followed by Amikacin and Kanamycin / Cefalexin, respectively. However, a one-sided treatment cannot be successful in mastitis. Therefore, determine the pathogens that cause mastitis and the selection of the appropriate treatment are very important in the prevention, control and treatment process.
Ethic statement: Ethical approval This article has not been published previously and is not under consideration for publication elsewhere. The publication of this article is approved by all authors and explicitly by the responsible authorities where the work was carried out. This study was approved by the Local Ethics Committee of Ceyhan Veterinary Faculty, Cukurova University, Adana, Turkey (approval number 8/1 and 23.09.2020).