Bacteriology, Antimicrobials Resistance and Susceptibility Profiles of Bacteria from Mastitic Cattle and Buffaloes of Khyber Pakhtunkhwa

Bovine mastitis (inflammation of mammary gland) is one of the most prevailing and economically destructive diseases affecting dairy animals across the globe (De Vliegher et al., 2012; Mbindyo et al., 2020), including Pakistan (Bachaya et al., 2011; Bhat et al., 2017; Ali et al., 2021). Bovine mastitis imparts huge financial losses, adversely affecting animal’s health and production, and welfare; besides, it deteriorates the milk quality and quantity capacities (De Vliegher et al., 2012). Bacterial pathogens are the major causing agents of mastitis. In addition, traumatic and mechanical injuries to the udder are also minor causes of mastitis (Bradley et al., 2002). Approximately 135 different bacterial species have been isolated from mastitic animals; however, about 20 types of bacterial pathogens are reported to be mostly associated with mastitis in dairy cattle (Bradley et al., 2002; Gao et al., 2017; Ali et al., 2021). Amongst the bacterial mastitis causing pathogens, coagulase-positive and –negative staphylococci, streptococci spp. Escherichia coli, Klebsiela spp. and Salmonella spp. are the main bacteria associated with mastitis (Ali et al., 2016; Yang et al., 2020;Aslam et al., 2021).

Different antimicrobials are commercially used to treat bacterial infections in livestock including intramammary bacterial infections. It has been reported that reproductive and mammary gland diseases are the main reasons to use antimicrobial agents in dairy animals for prolonged and frequent uses (Nobrega et al., 2017). These conditions paved the way to produce multi-drug resistant (MDR) bacterial pathogens (Ali et al., 2016; 2017). There is always a need of robust antimicrobial resistance surveillance systems of pathogenic bacteria from various sources (WHO, 2014). However, because of the economic constraints, national surveillance programs including monitoring systems of mastitis, the associated bacteria and their resistance profiles, are not present in Pakistan. Although, some regional studies were conducted on these aspects to fill the gaps (Mustafa et al., 2011; Ali et al., 2014; 2021). Thus, the current study was conducted with the aims to investigate the current prevalence of mastitis in lactating cattle, and buffaloes (Bubalus bubalis) in Khyber Pakhtunkhwa during 2019-2020, to detect bacterial pathogens associated with mastitis, and to evaluate their antimicrobial resistance.

To conduct this study, ethical and technical guidance was obtained from the ethical and technical committee of the Veterinary Research Institute (VRI) Peshawar, Pakistan. Milk samples were obtained from cattle and buffaloes as per the guidelines of the National Mastitis Council (NMC) with the prior consent of the owners and without any harm to the animals.

A total of 1013 milk samples were either collected directly from the animals or brought by the owners/farm managers to detect mastitis. Samples were processed at the Center of Microbiology and Biotechnology (CMB), VRI Peshawar, Pakistan during July, 2019 to June, 2020. Aseptic milk samples were collected from individual small farmers with 01 to 03 cattle or buffaloes. Dairy farmers were guided for the collection of aseptic samples from their animals as per NMC protocols. A total of 1013 milk samples from 726 cattle and 287 buffaloes of different areas of Khyber Pakhtunkhwa (KPK) were brought to our Mastitis Laboratory for detection of clinical mastitis (CM) and subclinical mastitis (SCM) as shown in Table 1 and Table 2. CM was detected with the help of visual observations as well as from the abnormal changes in milk (Ali et al., 2014). While, California mastitis test (CMT) kit (ImmuCell, Portland, ME) was used to diagnose SCM.

A total of 541 samples from mastitic animals were further cultured to identify the bacterial pathogens. The identification of bacterial isolates was carried out up to the level of genus as described earlier (Ali et al., 2021). In addition, antimicrobial resistance and susceptibility profiling of the isolated bacteria from mastitic cattle and buffaloes was carried out on Muller-Hinton agar (OxoidTM, Thermo Scientific Inc. USA). Antimicrobial susceptibility testing of the bacterial isolates from mastitic milk samples was performed on Mueller-Hinton agar (OxoidTM) by the Kirby-Bauer disc diffusion assay according to the recommendations of the Clinical Laboratory Standard Institute (CLSI, 2014). Fourteen different antimicrobial agents were tested, including ampicillin, amoxicillin, augmentin, oxytetracycline, gentamicin, streptomycin, kanamycin, norfloxacin, enrofloxacin, sulphamethoxazole, doxycycline, erythromycin, florfenicol and flumequine.

The results of this study showed 21.2% and 62.3% of overall prevalence of CM and SCM in cattle and 17.8% and 68.9% in buffaloes, respectively (Table 1). Thus, CM was highest in cattle (21.2%) as compared to buffaloes (17.8%); however, prevalence of SCM

Table 1: Species-wise prevalence of mastitis in cattle and buffaloes of Khyber Pakhtunkhwa.

Table 2: Area-wise prevalence of subclinical mastitis (SCM) and clinical mastitis (CM).

Table 3: Bacteriology of mastitic milk from cattle and buffaloes.

was highest in buffaloes (68.9%) in comparison to cattle (62.3%). Additionally, 12.7% of milk samples of cattle were mixed with blood, and 5.3% blood-mixed milk was observed in buffaloes. Table 2 shows the area-wise prevalence of CM and SCM in cattle and buffaloes of different areas of KPK. In districts Peshawar and Charsada, the topmost prevalence of CM and SCM was noted in cattle and buffaloes.

Isolation of different bacterial pathogens from mastitic dairy animals is presented in Table 3. Amongst the bacterial isolates from mastitic milk, Staphylococci spp. (24.6%) were the main prevailing bacteria. Besides, the other bacterial isolates included E. coli (23.7%), Streptococci spp. (8%), Klebsiella spp. (5.9%), Candida spp. (4.1%), and Proteus spp. (1.5%). Additionally, no bacterial growth was observed in 29.8% of samples from mastitic cattle and buffaloes.

Table 4 reveals the antimicrobial resistance and susceptibility profiles of different bacterial pathogens against fourteen antimicrobial agents. The isolated bacteria were highly resistant to ampicillin (96.5%), sulphamethoxazole (96.4%), streptomycin (95.1%), oxytetracycline (85%), and amoxicillin (78.1%). In addition, these isolates were mainly sensitive to enrofloxacin (86.2%), gentamicin (83.5%), and florfenicol (82.6%).

This study reported an overall prevalence of 21.2% and 62.3% of clinical mastitis (CM) and subclinical mastitis (SCM) in cattle; however, it was 17.8% and 68.9% in buffaloes of different areas of KPK. Thus, CM was highest in cows in comparison to buffaloes however SCM was highest in buffaloes. Mustafa et al. (2011) and our recent study (Ali et al., 2021) also reported higher prevalence of CM in cows than in buffaloes. It has been reported that buffaloes are generally more resilient to different diseases such as bovine mastitis due to their strong immune system (Pal and Chakravarty, 2020). Besides, the other contributing factors of high prevalence of mastitis in cattle might be the harsh environmental conditions of the province, and diminished immunity of exotic and crossbred cows. Also, this study reported higher prevalence of SCM in cows than reported by other studies such as Bachaya et al. (2011) reported 35% prevalence of SCM in cows of district Muzafar-Gharr, 30% in cattle belonging to Lahore (Mustafa et al., 2011), and 44% in Punjab province (Ali et al., 2011), and 42% in buffaloes belonging to district Dera Ismail Khan (Ali et al., 2014). This increase in the occurrence of bovine mastitis is worrying, which might be due to the reason that the dairy sector is swiftly growing in the KPK, and large numbers of commercial dairy farms are now developed in this area. Our previous study also reported that with the increase in herd size there is significant increase in the incidence of mastitis in buffaloes (Ali et al., 2014). This study is in line with other studies (Bhat et al., 2017; Ali et al., 2014) that reported a prevalence of 11.5% CM in cattle belonging to Azad Jamu Kashmir, and 11% in buffaloes of district Dera Ismail Khan. In contrast, the studies of Sharif et al. (2009) and Mustafa et al. (2011) observed the highest prevalence of CM in cows (61%) and buffaloes (61%). Additionally, this study was in line with some other studies (Mustafa et al., 2011: Sharif et al., 2009), we reported that 4.7% milk samples from cattle and 4.0% from buffaloes were mixed with blood. The findings of this study were partially according to other studies across the world, which reported the prevalence of bovine mastitis in India (Sinha et al., 2016), in China (Gao et al., 2017), in Ethiopia (Birhanu et al., 2017), in Poland (Sztachańska et al., 2016), and in Kenya (Mbindyo et al., 2020). Geo-climatic variations in different regions also affect the prevalence of bovine mastitis as investigated in this study and are also in line with other published work (Ali et al., 2011).

In the present study, contagious mastitis pathogens Staphylococci spp. (24.6%) were the most prevalent bacteria, followed by environmental mastitis pathogens E. coli (23.7%). The other bacterial isolates were Salmonella spp. (5.9%), Klebsiella spp. (5.7%), Candida spp. (4.1%) and Proteus spp. (1.5%). Several studies from Pakistan (Iqbal et al., 2004, Ali et al., 2011; Mustafa et al., 2011; Bhat et al., 2017; Aslam et al., 2021) and other countries (Ali et al., 2017; Birhanu et al., 2017) have also reported these bacterial pathogens from cases of bovine mastitis; although the bacterial prevalence was slightly different in different regions. The findings of Bhat et al. (2017) were in agreement with our findings. They also reported 61% prevalence of S. aureus, 13% E. coli, 13% coagulase-negative streptococci (CNS), 9% Streptococcus dysgalactiae, and 4% Streptococcus uberis in cattle of Jammu and Kashmir. Nevertheless, in contrast, researchers in China investigated 14% prevalence of E. coli, 10% CNS, 10% Staph. aureus, 11% Strep. dysgalactiae, 3% Strep. agalactiae, and 2% Strep. uberis (Gao et al., 2017). The reasons of this variation might be that the prevalence of bacterial pathogens associated with bovine mastitis is varying with topographical conditions and managemental practices.

Table 4: Antimicrobial susceptibility of bacterial isolates from mastitic cattle and buffaloes.

In our most recently published work (Ali et al., 2021), we isolated different bacteria from mastitic cattle and buffaloes, which were highly susceptible to enrofloxacin (86.2%), gentamicin (83.5%), and florfenicol (82.6%). These isolates were resistant to ampicillin (96.5%), sulphamethoxazole (96.4%), streptomycin (95.1%), oxytetracycline (85%), and amoxicillin (78.1%). Similarly, the findings of Iqbal et al. (2004) also suggested that several antimicrobial drugs such as aminoglycoside, enrofloxacin, and norfloxacin were highly effective drugs against bacterial isolates recovered from mastitic animals in Pakistan. Also, gentamicin, enrofloxacin, norfloxacin and ciprofloxacin were also found susceptible against bacterial pathogens in several studies (Sharif et al., 2009; Ali et al., 2011; Aslam et al., 2021). Strikingly, gentamicin and enrofloxacin are also in the approved and listed for veterinary usage by World Organization for Animal Health (OIE) and World Health Organization (WHO, 2016).

Our study revealed that contagious mastitis and environmental mastitis due to Staphylococcus spp. and E. coli were highly prevalent in cows and buffaloes of KPK and the recovered bacteria were mostly susceptible to enrofloxacin, gentamicin, and florfenicol. This study might be helpful to clinicians and researchers for designing and implementing prophylactic and therapeutic plans for bovine mastitis. In addition, this study recommends national level surveillance study on prevalence of bacterial pathogens associated with bovine mastitis and their resistance profiles across the country.

We highly appreciate the assistance of our para-Lab staff at the Center, especially, Mr. M. Rauf, Mr. Raham Dad, Mr. Haseeb Gul and Mr. Qaisar Khan.

This is an important surveillance study on bovine mastitis, the prevalence of different bacteria associated with mastitis and their antimicrobial resistance and susceptibility profiling. Such studies are always needed by the clinicians and researchers associated with dairy industry to design and implement strategies in order to control and prevent mastitis in cattle and buffaloes.

Tariq Ali, Abdur Raziq and Muhammad Ijaz Ali: Designed and perceived this study.

Tariq Ali, Kamran, Anwar Ali and Inam Ullah Wazir: Performed the experimental work.

Muhammad Shuaib Khan, Shakeeb Ullah and Inam Ullah Wazir: Performed data analysis and wrote this article.

Abdur Raziq, Muhammad Ijaz Ali, Shakeeb Ullah and Sher Hayat Khan: Critically reviewed and revised the article.

All authors had read and approved the final draft for submission.

No conflict of interest is declared by the authors.