Prevalence and Antibiotic Resistance of Staphylococcus aureus and Risk Factors for Bovine Subclinical Mastitis in District Kasur, Punjab, Pakistan

Abdul Maalik1, Shahzad Ali1,*, Anam Iftikhar1, Muhammad Rizwan1, Haroon Ahmad2 and Iahtasham Khan1

1University of Veterinary and Animal Sciences, Lahore

2Department of Biosciences, COMSATS Institute of Information Technology, Park Road, Chak Shahzad, Islamabad

ABSTRACT

The present study was designed to determine the prevalence and possible risk factors of subclinical mastitis along with antibiotic resistance of one of the causative agent of bovine mastitis, Staphylococcus aureus. Ninety milk samples were collected from cows and buffaloes kept at different localities in district Kasur, Punjab, Pakistan. The possible risk factors inquired from farmers were animal type, breeds, urbanicity, age (years), teat washing, bedding area, lactating stage and previous exposure of mastitis. Initially surf field mastitis test (SFMT) was applied for screening subclinical mastitis followed by bacteriological techniques on positive milk samples for confirmatory isolation of Staph. aureus as a bovine mastitis causing agent. These Staph. aureus isolates were further tested for antibiotic susceptibility. Epidemiological data was analyzed by chi-square methods at 95% confidence interval. The overall prevalence of subclinical mastitis as a result of surf field mastitis test was 42.2%. The prevalence of disease was higher in cows (54.5%) than in buffalo (22.9%). Based on chi-square and P-value the prevalence of mastitis was significantly (P˂0.05) associated with animal type, urbanicity, age of animal and previous exposure of mastitis. There was no association of the prevalence of bovine subclinical mastitis with breed, teat washing, bedding area and lactating stage. Out of 38 positive milk samples, 13 (34.2%) were positive for Staph. aureus. All isolates of Staph. aureus were resistant for ten different antibiotics. It was concluded that multi-drug resistant strains of Staph. aureus were major causative agent of bovine subclinical mastitis and there were multiple risk factors associated with incidence of mastitis.

Article Information

Received 18 September 2018

Revised 20 October 2018

Accepted 25 October 2018

Available online 18 April 2019

Authors’ Contribution

AM, SA, MR and IK conceived and designed the study. AM, HA and IK performed the study and analyzed the data. AM, SA and AI wrote the article. AM, SA, AI, MR, HA and IK proofread the manuscript and approved final version.

Key words

Subclinical mastitis, Risk factors for mastitis, Antibiotic resistance, Bovine, Staphylococcus aureus, multi-drug resistant strain.

DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.3.1123.1130

* Corresponding author: shahzad.ali@uvas.edu.pk

0030-9923/2019/0003-1123 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan

Introduction

Urban and peri-urban dairy production plays an important role in fulfilling the increasing demand of milk and milk products in developing countries. Milk is the essential diet of human being because it has all essential components (Javaid et al., 2009; Ajmal et al., 2015). Recently, due to increase in world population, liquid milk demand has been increased tremendously (FAO, 2018). Apart from other problems, dairy animals are facing heavy economic losses due to mastitis. Mastitis is known as inflammation of mammary tissue and dairy cattle industry is being affected globally by this ailment (Ali et al., 2018; Ashraf and Imran, 2018). The main consequences of mastitis in dairy cattle are altered milk composition, increased number of somatic cells in milk and ultimately low milk production which has now become a serious cause of heavy economic losses (Gianneechini et al., 2002; Hogeveen et al., 2011). Mainly by changing milk color and inflammation of udder, subclinical mastitis has struck in different countries including Pakistan, Ethiopia and Poland (Mungube et al., 2005; Islam et al., 2010; Sztachanska et al., 2016). A number of risk factors like teat or udder lesions play a significant role in occurrence of mastitis among livestock animals (Hussain et al., 2012; Tiwari et al., 2013; Iraguha et al., 2015). Similarly, a number of microorganisms are responsible for mastitis but among all these agents Staphylococcus aureus is a major pathogen which can play vital and significant role in mastitis (Lundberg et al., 2014). Moreover, development of antimicrobial resistance in the case of bovine mastitis caused by Staph. aureus is increasing day by day (Barkema et al., 2006; Gao et al., 2012). Keeping in view the economic importance of bovine mastitis in Pakistan, the present study was designed to determine prevalence, associated risk factors and antibiotic resistance of Staph. aureus from bovine subclinical mastitis in district Kasur, Pakistan.

 

Materials and methods

Study area

The present study was conducted in District Kasur, Punjab Province, Pakistan. Kasur is a district of province Punjab, Pakistan. It is the 20th most populated city of Pakistan, bordered to the north by Lahore and to the south and east by India.

Sample collection

Total 90 milk samples were collected from cows (n = 56) and buffaloes (n = 34) from various peri-urban and rural areas of district Kasur from September 2016 to February 2017. Before collection of milk samples each teat was thoroughly washed with water, wiped dry with tissue paper and finally disinfected with 70% alcohol. Milk samples were then collected in sterile sampling falcon tubes (15mL) after discarding first two streams of milk from each teat. These samples were transported in ice box (at 4°C) to the Epidemiology and Microbiology Laboratory of University of Veterinary and Animal Sciences, Lahore, Pakistan.

Confirmation of bovine mastitis

The confirmation of bovine subclinical mastitis was done using 3% Surf-solution (Muhammad et al., 1995). Briefly, one ml of milk was mixed with equal amount of the surf solution in petri-plate and rotated the mixture for 1 min. The mixture was examined for thickening or any other possible change. The positive milk samples for mastitis showed agglutination or color change and vice-versa.

Isolation of Staph. aureus

The positive milk samples (100µL) were inoculated on Mannitol Salt agar (Lab M Limited, UK) for isolation of Staph. aureus and these samples were incubated at 37°C for 24 h. A colony of smooth shaped, golden yellow color was marked as Staph. aureus, which was further confirmed by using gram staining technique: a colony of purple, round and clump shaped finally confirmed the presence of Staph. aureus. Further confirmation was done by using biochemical tests (Pumipuntu et al., 2017).

Antibiotic sensitivity

Disc diffusion method was used to check the susceptibility of Staph. aureus isolates according to the instructions of Clinical and Laboratory Standards Institute (2009). Eighteen antibiotics (Bioanalyse, Ankara, Turkey) including Augmentin (30mcg), Ampicillin (10mcg), Cefoxitin (30mcg), Clindamycin (2mcg), Chloramphenicol (30mcg), Ciprofloxacin (5mcg), Erythromycin (15mcg), Fosfomycin (200mcg), Gentamycin (10mcg), Kanamycin (30mcg), Oxacillin (1mcg), Ofloxacin (5mcg), Penicillin (10mcg), Rifampicin (5mcg), Tetracycline (30mcg), Teicoplanin (30mcg), Trimethoprim (5mcg), and Vancomycin (30mcg) were used.

Statistical analysis

The data were analyzed by using SPSS software version 22.0. The respective prevalence of bovine mastitis was summarized by taking percentage of positive samples and correlation of possible risk factors with bovine mastitis was calculated by chi-square test on the basis of P-value of each possible risk factor. For the test, P < 0.05 was considered statistically significant (Hosseinzadeh and Sei, 2014).

 

Table I.- Risk factors associated with bovine mastitis in peri-urban and rural areas district Kasur.

Para- meters		Sample			Chi-square value (ᵡ2)	P- Value
		Collected	Positive	%age
Animal Type
	Buffalo	35	8	22.9	4.444	0.035
	Cow	55	30	54.5
Breeds
	Local	47	10	21.3	0.178	0.673
	Exotic	43	28	65.1
Urbanicity
	Rural	65	17	26.2	17.778	0
	Urban	25	21	84.0
Age (years)
	≤ 4	24	19	79.1	15.667	0.004
	4.1 to 6	28	4	14.3
	6.1 to 8	19	7	36.8
	8.1 to 10	10	6	60.0
	10.1 to 12	9	2	22.2
Teat washing
	Yes	38	10	26.3	2.178	0.14
	No	52	28	53.8
Bedding area
	Barn	44	26	59.1	0.044	0.833
	Brick	46	12	26.1
Lactating stage
	Early	32	14	43.8	0.267	0.875
	Middle	30	16	53.3
	Late	28	8	28.6
Previous exposure of mastitis
	Yes	37	24	64.9	2.844	0.092
	No	53	14	26.9

 

Results

The prevalence of bovine sub-subclinical mastitis is given in Table I. Thirty eight (42.2%) out of 90milk samples were found positive for sub-clinical mastitis using surf field mastitis test. Prevalence of sub-clinical mastitis was significantly (P = 0.035) higher in cows (54.5%) than in buffalo (22.9%). The prevalence of sub-clinical mastitis in animals breeds was not statistically significant (P<0.673). This study revealed that mastitis is much more prevalent at urban site (84%) as compared to rural site (26.2%). Interestingly, the maximum prevalence i.e. 79.1%was reported in animals with age ≤4 years and lowest in the animals with age 4.1 to 6 years.

The prevalence of mastitis in those animals whose teats were being washed before milking was 26.3% while in those whose teats were not being washed before milking was 53.8%. The prevalence measure based on the bedding area was statistically insignificant. The infection was more in those animals which were raised on barn surface (59.1%) than on bricks (26.1%). Lactating stage plays a dynamic and significant role in inflammation of udder. The mastitis was calculated by different lactation stages i.e. early, middle and late with prevalence recorded as 43.8%, 53.3% and 28.6%, respectively. Prominent finding from the results was higher prevalence of sub-clinical mastitis (64.9%) in previously exposure animals of mastitis as compared to not exposed (26.9%).

Prevalence of Staph. aureus

Out of 38 positive milk samples, 13 (34.2%) milk samples were found to be positive in bacteriological examination. Only two (25%) out of 8 SFMT positive milk samples of Nili-Ravi buffalo were contaminated with Staph. aureus. Out of 30 milk samples of cattle, 11 (36.7%) milk samples were positive for Staph. aureus. Out of 10 SFMT positive milk samples of local breeds, 4 (40%) were positive for Staph. aureus. Furthermore, from 28 SFMT positive milk samples of exotic breeds, 9 (32.1%) were positive for Staph. aureus.

 

Table II.- Prevalence of Staph. aureus in milk of cows and buffaloes.

Parameters		Sample tested	Positive culture	Percentage
Animal species
	Buffalo	8	02	25.0
	Cow	30	11	36.7
Animal breed
	Local	10	04	40.0
	Exotic	28	09	32.1

 

Table III.- Breakpoint values of each antimicrobial agent and phenotypic antimicrobial susceptibility profiles of 13 tested Staph. aureus isolates used according to CLSI and EUCAST.

Antimicrobial class	Antimicrobial agents	Reference	Conc. (mcg)	Zone diameter (mm)			S. aureus (n = 13)
				S	I	R	S	I	R
Beta-Lactam	Augmentin	1	30	≥20	-	≤ 19	1	0	12
Beta-Lactam	Ampicillin	2	10	≥15	12-14	≤11	2	0	11
Cephalosporins	Cefoxitin	3	30	≥22	16–20	≤ 20	1	0	12
Lincosamide	Clindamycin	2	2	≥21	15–20	≤14	0	1	12
Phenicol	Chloramphenicol	2	30	≥18	13–17	≤ 12	1	1	11
Fluoroquinolone	Ciprofloxacin	2	5	≥21	16-20	≤ 15	1	2	10
Macrolide	Erythromycin	2	15	≥23	14–22	≤ 13	1	1	11
Fosfomycins	Fosfomycin	2	200/50	≥34	-	≤ 33	0	0	13
Aminoglycoside	Gentamycin	2	10	≥15	13–14	≤ 12	1	0	12
Aminoglycoside	Kanamycin	2	30	≥18	14–17	≤13	0	0	13
Beta-Lactam	Oxacillin	2	1	≥22	-	≤ 21	0	0	13
Fluoroquinolone	Ofloxacin	2	5	≥18	15–17	≤14	2	0	11
Beta-Lactam	Penicillin	2	10	≥29	-	≤ 28	0	0	13
Ansamycins	Rifampicin	2	5	≥20	17–19	≤16	2	0	11
Tetracycline	Tetracycline	2	30	≥19	15-18	≤14	2	0	11
Glycopeptides	Teicoplanin	2	30	≥14	11–13	≤ 10	1	2	10
Folate-Pathway Inhibitors	Trimethoprim	2	5	≥16	11–15	≤ 10	0	0	13
Glycopeptides	Vancomycin	4	30	≥15	-	-	2	0	11

S, sensitive; I, intermediate; R, resistance.

 

Antibiotics sensitivity of Staph. aureus

Staph. aureus isolates were completely resistant (100%) to Fosfomycin, Kanamycin, Oxacillin, Penicillin and Trimethoprim (Table III). Twelve (92.3%) isolates were resistant to Augmentin, Cefoxitin, Clindamycin and Gentamycin. Ampicillin, Chloramphenicol, Erythromycin, Ofloxacin, Rifampicin and Vancomycin were 84.6% resistant while Ciprofloxacin and Teicoplanin was 76.9 % resistant (Table III).

 

Discussion

Staph. aureus is an economically important pathogen and can survive in the extreme ranges of temperature. It is responsible for intra-mammary infections in dairy animals. In present study, 42.2% animals were positive for subclinical mastitis which is higher than previous studies conducted in Lahore (22.4%) and Faisalabad (27%), Punjab, Pakistan (Khan and Muhammad, 2005; Mustafa et al., 2011).

The prevalence of sub-clinical mastitis was significantly higher (P = 0.035) in cattle (54.5%) than in buffalo (22.9%). In contrast to our findings, Khan and Muhammad (2005) concluded that the overall prevalence of sub-clinical mastitis was 27% in buffaloes while 36% in crossbred cows by using surf field mastitis test. However, a very low prevalence of bovine subclinical mastitis was reported from Egypt (3.9% in cattle and 0% in buffalo) (Osman et al., 2009). The prevalence of subclinical mastitis was higher in exotic breeds as compared to local breeds. A higher prevalence was previously reported for exotic (29.2%) breeds as compared to local (23.8%) breed using surf field mastitis test as screening test in Khyber Pakhtunkhwa, Pakistan (Khan et al., 2015). Similarly slightly higher prevalence was reported in exotic breed (12.7%) as compared to local (4.2%) in Bangladesh (Hossain et al., 2016). Based on urbanicity, the prevalence of bovine subclinical mastitis was higher (84%) in urban areas and lower (26.2%) in rural areas in present study. These findings are in line with Hardenberg (2016), who concluded slightly lower prevalence in rural (36.8%) and higher prevalence in urban (43.9%) area of Bihar India. However, these values are not in line with findings of Bilal et al. (2004), who reported the higher prevalence of mastitis in rural areas (25.1%) and lower (19.7%) in urban areas of Faisalabad District, Punjab province, Pakistan. In our study, the prevalence of mastitis with respect to different age groups showed statistically significant (P<0.05) results. There is perfect agreement between our findings and the findings of studies conducted in Ethiopia; the prevalence of bovine subclinical mastitis was statically significant with respect to age (Zeryehun et al., 2013). However, Hussain et al. (2012) showed no-significant (P>0.05) association of mastitis with age in his study in Pakistan. In case of teat washing, our results were considerably different from the findings of Tesfaye and Abera (2018), who find higher prevalence of mastitis (45.5%) in animals whose udder being washed before milking in Jimma Town Dairy Farms, Western Ethiopia. With respect to bedding area, our study showed the higher prevalence in the animals with barn floor while lower prevalence in the animals with brick floor. Our results considerably differs from the findings of Chishty et al. (2007), who observed that the animals which were used to reside on brick floors showed a higher prevalence (36.53%) in case of cattle and (41.53%) buffaloes, and the prevalence on Kaccha floor were the lowest, (10.34%) for cattle and (15.00%) for buffalo in cattle and buffaloes of tehsil Gojra, Pakistan. Our results are consistent with the results of Rahman et al. (2009) who reported high prevalence of mastitis on brick floor (30.6%) and low in the animals with barn floor (20%) in dairy cows of various Districts of Sirajganj and Pabna, Bangladesh. From our study the stages of lactation indicated that they are statistically insignificant. The current study does not support the previous research in this area; Tekle and Berihe (2016) reported that lactation stage had significant (P<0.05) effect on the prevalence of mastitis in animals in Ethiopia. This could be due to the delayed inflammation of neutrophils to mammary glands. Even though our findings differ from some previously published literature yet the lactation stage was significantly associated with mastitis (Qayyum et al., 2016) in Cholistani cattle of Bahawalpur District, Punjab province, Pakistan. Our findings were in good agreement with Biffa et al. (2015), higher prevalence of mastitis (57%) in previously exposed dairy animals as compared to not previously exposed (22%) in Ethiopia.

In present study, Staph. aureus is a prominent pathogen constituting 34.2% of SFMT positive milk samples; our results are slightly higher than the findings of Zeryhum et al. (2013), who reported 28.7% isolates of Staph. aureus and their primary role in bovine subclinical mastitis in small holder dairy farms of Addis Ababa, Ethiopia.

Resistance in all isolates of Staph. aureus against Penicillin has been reported from cow milk in Afar, Ethiopia (Beyene, 2016). Resistance against Penicillin was also reported in Staph. aureus isolates from subclinical mastitis in dairy cow from Germany (74.28%) (EI-Behiry et al., 2012). Resistance in Staph. aureus isolates against Oxacillin has been reported from buffalo infected with mastitis in Brazil (50%) (Garcia et al., 2017). Staph. aureus isolated from mastitic milk of cows were also resistant against Trimethoprim in Ethiopia (21%) and Kososvo (11.1%) (Abera et al., 2013; Hamidi et al., 2016). Staph. aureus isolates from cattle were found resistant to Kanamycin in Romania (28%) and China (17.37%) (Brinda et al., 2010; Shi et al., 2010). Similarly, previous work from Brazil on buffaloes affected with subclinical mastitis showed high antimicrobial resistance (97.98%) (Cuhna et al., 2006). However, comparatively low (57%) resistance was recorded against Gentamycin from bovine mastitis milk samples in India (Pati and Mukherjee, 2016). Interestingly, in case of Staph. aureus resistance against Vancomycin, our findings are in line (88.89%) with a study conducted on cattle and buffaloes in India (Sharma et al., 2015). However, comparatively very low resistance (6%) was reported in dairy products in Malaysia (Sasidharan et al., 2011). Our results differ from some previous studies (Szweda et al., 2014; Hamidi et al., 2016), in which comparatively low resistance against Ampicillin was reported from cows infected with mastitis (46.1% from Poland and 22.8% from Kosovo). Surprisingly, the findings of Goa et al. (2012) are in line with our study; which showed very high resistance (98.1%) of Staph. aureus isolates against Tetracycline from bovine mastitis in single herd in China. Tetracycline however revealed moderately low resistance (26%) in cows with sub clinical mastitis in Poland (Malinowski et al., 2002). Our results validate the findings of Leigue et al. (2017) who declared that Staph. aureus have resistance (52%) against Erythromycin in cattle with mastitis in Brazil. The results of our study supports previous research in this area: Tetracycline showed comparatively high resistance (61.9%) against Staph. aureus isolated from bovine mastitis in Algeria (Saidi et al., 2015). In contradiction with earlier findings; El-Behiry et al. (2012) who described resistance of Staph. aureus showed a very low resistance (2.85%) against Chloramphenicol in cows with subclinical mastitis in Germany. This study is in good agreement with the results of Abraha et al. (2018) in which Chloramphenicol was found highly resistance (81.8%) against Staph. aureus isolated from raw cow milk in Ethiopia. In case of Clindamycin, we found much higher resistance than Kumar et al. (2010) who found a low resistance (14.1 %) against Staph. aureus isolates from milk of mastitic crossbred cattle in India. The findings of Younis et al. (2018) showed relatively low resistance (41.25%) as compared to our study against Clindamycin in dairy cows affected with bovine clinical and sub clinical mastitis in of Egypt.

The results of the present study proved that bovine subclinical mastitis is prevalent in cattle and buffalo of Pakistan and Staph. aureus is the major pathogen associated with this disease. Multiples risk factors i.e. species, urbanicity, age and previous exposure of animal with mastitis were associated with bovine subclinical mastitis. High antibiotic resistance was investigated in mastitis causing Staph. aureus isolates. This is an alarming condition regarding presence of multi-drug-resistant Staph. aureus strains in food (i.e. milk). The results of the present study and knowledge of disease causing pathogen and their antibiotic resistance patterns against various drugs will be very helpful in mastitis control, and will play a vital and significant role in the improvement of health status of dairy herds in Pakistan.

 

Statement of conflict of interest

The authors declare no conflict of interest.

 

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