Mastitis (inflammation of the mammary gland) is one of the most prevalent diseases of dairy animals (1). It is the most costly and devastating disease to the dairy industry because it imparts vast economic losses, compromising the health and welfare of animals, and due to its adverse effects on the quality and quantity of milk (1). Cattle and buffaloes (Bubalus bubalis) are the major dairy animals in Pakistan. The disease is associated with considerable alterations in milk chemical composition with a significant decrease in milk synthesis and changes in cell permeability (2). Bacterial pathogens are majorly (70%) involved in the etiology of mastitis. Besides, minor causes (30%) include non-infectious agents such as physical trauma, mechanical injuries to the gland, etc. (3). More than 135 types of bacterial species have been reported to be associated with bovine mastitis, but 20 different pathogenic bacteria are most commonly involved in mastitis of dairy animals (3, 4). The most common mastitis-causing bacterial pathogens are Staphylococcus aureus, Escherichia coli, coagulase-negative staphylococci (CNS), Streptococcus dysgalactiae, Streptococcus uberis, and Streptococcus agalactiae (4–6).

Generally, antimicrobial agents are used to treating bacterial infections including intramammary infections (IMI) in livestock. In dairy animals, mastitis and reproductive disorders are some of the most important reasons for frequent and prolonged use of antimicrobials (7). Quite usual, the treatment of mastitis takes a longer duration due to relapse and failure to flush out those bacteria which are resistant to a wide range of antimicrobials. The therapy of mastitis should be as efficient and quick as possible; however, there is a rapid increase in the prevalence of multidrug-resistant bacteria globally (5). According to the World Health Organization (WHO), an efficient monitoring system for the antimicrobial resistance of various bacterial pathogens is urgently required at national levels to fulfill the requirements of international standards (8). Data regarding the prevalence of bovine mastitis and the knowledge of pathogens causing mastitis is important to chalk out prevention and control strategies and to adopt appropriate therapeutic protocols (4).

In Pakistan, national surveillance programs for monitoring the prevalence of bovine mastitis and antimicrobial resistance in bacterial pathogens are lacking. Only some discrete studies were conducted in different areas of Pakistan (9, 10). Thus, there is always a dire need to monitor the prevalence of mastitis at local levels, as well as its etiology, and to evaluate antimicrobial susceptibility of bacterial isolates. Therefore, this study was carried out with the objectives to investigate: (i) the prevalence of bovine mastitis in cattle and buffaloes located in the northwest of Pakistan; (ii) detection of bacterial etiology of mastitis; and (iii) antimicrobial susceptibility of the bacterial isolates.

Mastitis is one of the most prevalent diseases of dairy animals all over the world (1), including Pakistan (11, 14, 15). In the current study, 20 and 53% prevalence of clinical and subclinical mastitis was noted in cattle, whereas it was 11 and 66% in buffaloes belonging to different areas of Khyber Pakhtunkhwa. This prevalence of subclinical mastitis was quite higher than the previous reports of 35% subclinical mastitis in cattle of district Muzaffar Gharr (14), 30% in cattle of district Lahore (9), and 44% in Punjab (11), and our previously reported 42% subclinical mastitis in buffaloes of district D. I. Khan (10). This considerable increase in the prevalence of bovine mastitis is alarming, which might be because dairy farming is rapidly growing in Khyber Pakhtunkhwa, and it is reported that mastitis is significantly increasing with an increase in the number of dairy animals (10). The other reasons might be that the number of milk samples processed and areas under study in the present study were considerably more than the other studies. The prevalence of clinical mastitis was in line with previous studies which reported 11.5% clinical mastitis in cattle of the Jammu and Kashmir region (15) and 11% in Nili Ravi buffaloes (10). However, Mustafa et al. (9) and Sharif et al. (16) reported a higher prevalence of clinical mastitis in cattle and buffaloes. They reported a higher prevalence of 40% and 61% of clinical mastitis in buffaloes and cattle. The highest prevalence of clinical mastitis was observed in cattle followed by buffaloes, which is also in line with a previous study (9). This might be attributed to the decreased immunity of exotic and crossbred cattle in Pakistan. However, as buffaloes are local to the environment, they are more resistant to various diseases including mastitis. In addition, 4.7 and 4.0% blood-mixed milk samples were observed in cows and buffaloes, which is in agreement with other studies conducted in this region (9, 16). The prevalence of bovine mastitis in cattle was also in partial agreement with several global studies conducted in India (17), China (4), Ethiopia (18), and Poland (19). Additionally, the highest prevalence of mastitis was noted in the hot and humid months of summer in both species. Similar findings were also observed by Sinha et al. (17); they reported the highest incidence of mastitis during the monsoon season. The prevalence of mastitis varied with different regions, the highest prevalence being noted in the cattle (81%) and buffaloes (82%) of district Peshawar. Previous studies also reported that the prevalence of mastitis varied with geography (10, 11).

Bacteriology of mastitic milk samples from cattle and buffaloes showed that Staphylococci spp. (34%) and E. coli (19%) were the most frequently isolated bacterial pathogen from cattle and buffaloes suffering mastitis. This was followed by streptococci spp. (9%), Klebsiella spp. (8%), Salmonella spp. (2%), and Proteus spp. (1%). This was in agreement with the other studies conducted in various regions of Pakistan (9, 11, 15, 20, 21), which also predominantly isolated Staphylococci spp. and E. coli from cases of bovine mastitis. We identified E. coli isolates (n = 186) by molecular assay using PCR amplification of uidA gene. This was in agreement with our previous work (5) and the study of Tantawiwat et al. (12). In a recent large-scale Chinese study, Song et al. (22) also reported that staphylococci spp. were the main pathogens associated with mastitis in cattle. Similarly, Bhat et al. (15) reported that Staphylococcus aureus (61%) was the most prevalent bacteria isolated from the mastitic cattle of Jammu and Kashmir, followed by E. coli (13%), coagulase-negative staphylococci (13.04%), Streptococcus uberis (4.35%), and Streptococcus dysgalactiae (8.69%). Gao et al. (4) and Ali et al. (5) also reported these pathogens from Chinese cattle. However, in another large-scale Chinese study by Gao et al. (4), they concluded that E. coli (14%) were the most prevalent bacteria isolated cases of bovine clinical mastitis followed by Klebsiella spp. (13%), coagulase-negative staphylococci (10%), Streptococcus dysgalactiae (11%), Staphylococcus aureus (10%), Streptococcus agalactiae (3%), and Streptococcus uberis (2%). This might be because the bacteria causing mastitis are changing with topographical and management conditions.

In the current study, gentamicin (92%), enrofloxacin (88%), and ciprofloxacin (79%) showed the highest susceptibility to various bacterial isolates from milk of cows and buffaloes suffering mastitis. However, the highest resistance of bacterial isolates was observed against sulphamethoxazole (99%), lincomycin (98%), oxytetracycline (89%), ampicillin (86%), and doxycycline (85%). This was in partial agreement with the study of Iqbal et al. (20); they also reported that gentamicin, enrofloxacin, norfloxacin, and kanamycin were the most effective antimicrobial drugs against the isolated bacteria from bovine mastitis (20). Similarly, several other studies also reported the highest susceptibility of bacterial isolates toward aminoglycosides including gentamicin, fluoroquinolone like enrofloxacin, and ciprofloxacin (11, 16). Notably, gentamicin and enrofloxacin antibiotics are listed in the approved list of antimicrobial agents approved for veterinary medicine by the World Health Organization and World Organization for Animal Health (23). This study, describes for the first time the prevalence of clinical and subclinical mastitis in a large number of dairy cattle and buffaloes belonging to different areas of Khyber Pakhtunkhwa, and identifies different bacterial pathogens associated with mastitis and their antimicrobial susceptibility profiles. Thus, this study would be beneficial for scientists, researchers, and clinicians working with the dairy sector to sketch prevention and control strategies and to adopt appropriate therapeutic protocols for bovine mastitis.

This study concludes the high prevalence of clinical and subclinical mastitis in cattle and buffaloes belonging to the northwest of Pakistan. Staphylococci spp. was the major bacterial pathogen associated with mastitis. This indicates unhygienic and poor management practices at the local farms. In addition, based on susceptible antimicrobial profiling of the isolated bacterial pathogens from cases of bovine mastitis, gentamicin and enrofloxacin might be appropriate antimicrobial agents in the treatment of bovine mastitis.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by Ethical Committee of Veterinary Research Institute Peshawar, Khyber Pakhtunkhwa. Written informed consent was obtained from the owners for the participation of their animals in this study.

TA, AR, MA, and GL designed and conceived the study. TA, Kamran, IW, and RU performed the experiments. TA, PS, and BH analyzed the data and wrote the article. BH and GL critically reviewed and revised the manuscript. All authors read and approved the final version.

This study was carried out with the financial support of the Annual Development Program (ADP) of the Government of Khyber Pakhtunkhwa under ADP scheme No. 25/150090, with the title Establishment of Livestock Research and Development Station at Swabi.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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