JAHP_14_3_1176-1183

INTRODUCTION

Pakistan’s economy is predominantly agrarian, with livestock production constituting a critical component of the agricultural sector and serving as a major source of income, particularly in rural communities. Livestock contributes substantially to the national economy, accounting for approximately 11.8% of the Gross Domestic Product (GDP) and more than half of the overall agricultural value. Among livestock commodities, milk represents the most valuable product, surpassing the combined economic value of staple crops such as wheat and rice. Pakistan ranks among the leading milk-producing nations globally, with buffaloes as the principal source of milk, accounting for a substantial share of total production (Rehman et al., 2017; FAO, 2001). Infectious and non-infectious diseases significantly affect animal health, productivity, and profitability. Among infectious diseases, mastitis remains one of the most prevalent and economically damaging conditions, leading to substantial reductions in milk yield and quality (Hussain et al., 2013; Farooq et al., 2008). Moreover, mastitis induces compositional alterations in milk and compromises udder integrity (Ali et al., 2022). From a public health perspective, mastitis is of particular concern because contaminated milk can harbor pathogenic microorganisms, thereby posing risks to consumers and compromising the quality and safety of dairy products (Schukken et al., 2009; Bilal et al., 2004). Methicillin-resistant Staphylococcus aureus (MRSA) is among the most frequently isolated etiological agents, attributable to its array of virulence factors, including toxins and enzymes that facilitate colonization and tissue damage in the mammary gland (Neelam et al., 2022). Subclinical mastitis, characterized by the absence of overt clinical symptoms, further complicates disease detection and control while facilitating bacterial transmission through milk (Rola et al., 2015; Persson et al., 2011).

The most common risk factors for mastitis in dairy herds can be divided into two groups: Animal-related risk factors and environmental and management risk factors. Animal-related risk factors include: breed, age, parity/lactation number, stage of lactation, lesions on udder/teat, swelling on udder/teat, udder shape, teat shape, udder edema, teat edema, and ease of milking. Management, environmental, and housing-related risk factors included: stimulus for milk let-down, number of animals milked by the same milker, udder washing, milking technique, dairy husbandry system, condition of floor, tick infestation, the source of drinking water, frequency of dung removal per day, farm hygiene, and husbandry practices. Other risk factors associated with mastitis included: previous exposure to mastitis, reproductive disorders, History of exposure to foot and mouth disease, and pox lesions on udder and teat (Sarker et al., 2013). Many studies report risk factors for mastitis are associated with farm management, hygiene management, the breeding environment, milking technology, feeding, calving, season, and preventive health management (Bludau et al., 2014).

In developing countries like Pakistan, owing to small herd sizes, dairy animals are mostly hand milked. Mastitis in hand-milked animals was nearly twice as frequent as in machine-milked ones (Motie et al., 1985). Infectious agents of mastitis may be transmitted from infected to healthy animals through the milker’s hand (Cuenca et al., 2024), especially because milk is often used as a lubricant for milking. Mastitis prevalence was found to be significantly influenced by stage of lactation, parity, breed, milk yield, anatomical abnormality of the udder, and some management aspects, including nutrition (Karimuribo et al., 2006; Almaw et al., 2008; Madut et al., 2009).

There are some reports on the magnitude of the disease, but information relating to its risk factors is insufficient (Kahir et al., 2008). Such information is important to envisage when designing appropriate strategies that would help to reduce its prevalence, treatment and effects (Al-Gburi and Al-Taii, 2025). However, virtually all of the published information about the risk factors for mastitis refers to dairy breeds of cattle, and little information is available for water buffaloes. Hence, the current research was done to establish the rate of sub-clinical mastitis in water buffaloes and to establish considerable animal and farm-based risk factors affecting the disease in peri-urban dairy systems in Karachi. The described aims were to measure disease burden, determine biological and management-related variables, and determine statistically significant predictors of infection. To do that, recently developed epidemiological methods such as univariate screening and multivariate logistic regression analysis were used in order to regulate confounding factors to properly estimate risk relationships, which are advised in veterinary epidemiology studies. The multivariate modelling has been extensively employed in risk factor identification in sub-clinical mastitis research and makes more valid inferences than descriptive research (Tolosa et al., 2013). The scientific importance of such approach is the production of strong epidemiological data on buffalo populations that are not overrepresented in mastitis studies. In practice, the results provide evidence-based recommendations on disease control interventions, better herd management methods, and milk quality and productivity in dairy farming systems.

Materials and Methods

Study design

A cross-sectional survey was conducted using random sampling of lactating buffaloes in the densely populated Landhi Cattle Colony, Karachi. Karachi is the largest city of Pakistan and the 12th largest in the world, with a population exceeding 20 million. Located along the Arabian Sea at the southern tip of the country. Landhi Cattle Colony, also known as Bhains Colony, is located in Bin Qasim Town and stands as Asia’s largest buffalo colony.

Milk sampling

A total of 387 animals were tested for the determination of sub-clinical mastitis. Milk samples were collected during visits to 30 different farms in the study. Before milk sampling, a thorough examination of the teats, udder, and milk was conducted. Following the examination, the udders were washed with lukewarm water, and the teats and teat orifices were disinfected using cotton wool soaked in 70% ethyl alcohol. Samples were taken in accordance with the National Mastitis Council’s suggested protocol (NMC, 2020). In a nutshell, 3 ml of milk of each quarter was cast aside and a matching amount of California Mastitis Test (CMT) reagent was added to the CMT paddle. The paddle was rotated for up to 15 seconds, and the results were classified as follows: negative (–), no gel; trace (±), slight slime; weak positive (+), soft gel; distinct positive (++), firm gel; and strong positive (+++), very thick gel.

Data collection and explanatory variables

Qualitative data on health-related and managemental factors were collected by means of a question guide with open-ended questions. Quantitative data were also collected on animal attendants’ socio-demographic characteristics. Dairy farms in the study area were randomly visited to collect information on mastitis. Photographs were taken for proper documentation as needed. The possible animal-based explanatory variables included in this study were parity, age, lactation stage, breed, teat length, teat diameter (apex, middle, and base), teat end to floor distance, teat and udder shape, calf suckling, milk yield, history of mastitis, body condition, milking hygiene, etc. Whereas, farm-based variables were the number of attendants at the farm, the type of housing, the management system, the bedding, the floor types, the dry cow therapy, the teat cleaning, the mastitis control program, the frequency of culling, the vaccination, the sanitation, the waste disposal, etc.

Data management and statistical analysis

Statistical analysis was conducted by the Python statistical computer software program (Python version 3.x). To assess the impact of possible risk factors, a univariate and multivariable logistic regression was used. Variables with an association with p-value < 0.001 were included in the final multivariate logistic regression model after passing the collinearity screening test. The forward stepwise multivariate logistic regression with likelihood ratio test was applied to optimize the best fitted multiple logistic regression model. The results were expressed as the odds ratio (OR) of the associated risk factor with a 95% confidence interval (CI) and a P-value. All results were considered statistically significant at a significance level of p < 0.05.

Results

Prevalence of sub-clinical mastitis (SCM)

The study evaluated the prevalence of sub-clinical mastitis in buffaloes of the Landhi cattle colony, Karachi and results were presented in Table 1. The overall prevalence of sub-clinical mastitis among the studied population (387 buffaloes) was 34.4%. The prevalence was highest (p<0.001) in animals with good body condition (48.3%), followed by those with medium (36.4%) and poor (15.3%) body condition. Buffaloes in the early stage of lactation showed a significantly (p<0.001) higher prevalence rate of 56.4%. The infection rate increased (p<0.001) with parity, reaching 69.1% in animals with more than four calvings. Animals with round-shaped udders and cylindrical teats showed higher (p<0.001) infection rates, at 54.3% and 38.4%, respectively. A prevalence of 58.9% was observed (p<0.001) in animals producing more than five litters of milk per day as compared to less than 5 litters/day (4.0%). Farms with more than two people handling the animals reported the highest prevalence (p<0.001) rate (69.9%). In relation to calf suckling, SCM was more prevalent (p<0.001, 60.6%) in farms where calves were not allowed to suckle. Higher infection rates were also observed in farms without a history of clinical mastitis (p<0.001, 47.8%) and in those without teat dipping (p>0.05, 38.6%). Sub-clinical mastitis prevalence (p<0.001) was 51.0% in animals dewormed only twice a year as compared to once (28.7%), thrice (33.2%) are not using deworming (18.7%). Farms with weekly manure removal had a prevalence (p<0.001) of 40.4%. The highest (p<0.001) infection rate (48.6%) was observed in farms that did not practice feed sharing. Surprisingly, SCM was common (54.9%) even in farms with good hygiene practices. Moreover, farms that did not use dry cow therapy reported a prevalence of 46.6%.

Discussion

The current study revealed a sub-clinical mastitis (SCM) prevalence of 34.4% among 387 examined buffaloes, indicating a substantial hidden burden of intramammary infections. Similarly, a range of 30% to 70% has been reported from South Asia (Khan et al., 2019; Sharma and Sindhu, 2007). Interestingly, SCM prevalence was highest (48.3%) among animals with good body condition, decreasing with poorer body scores. Poor body condition predisposes animals to infection by compromising immunity (Abebe et al., 2016). The higher metabolic activity and milk production in well-conditioned animals increase their susceptibility to mammary infections (Radostits et al., 2007). The infection rate was high (56.4%) in the early stage of lactation.

Bhutto et al. (2012) reported elevated susceptibility to SCM in early lactation due to hormonal and metabolic stress. Additionally, SCM prevalence rose with parity, peaking at 69.1% in animals with more than four calvings. This trend reflects cumulative exposure to pathogens over successive lactations and potential anatomical changes in the teat canal that compromise its barrier function (Kavitha et al., 2009). Udder and teat morphology also influenced SCM risk. Buffaloes with round-shaped udders and cylindrical teats had higher infection rates (54.3% and 38.4%), respectively, supporting the hypothesis that such shapes may favour milk accumulation and pathogen colonization (Saravanan et al., 2021). A higher prevalence (58.9%) was noted in animals producing more than five litters of milk per day. High-producing animals are often more prone to SCM due to increased stress on the mammary gland (Sahoo et al., 2014). Moreover, farms with more than two handlers showed a significantly higher SCM prevalence (69.9%), highlighting the role of human-mediated transmission and the importance of personnel hygiene and milking practices (Dego and Tareke, 2003). Buffaloes not allowed to suckle their calves showed a higher SCM rate (60.6%).

Table 1: Prevalence of Sub-clinical mastitis in Buffaloes of cattle colony Landhi, Karachi (n=387).

Variable	Description	No. of animals examined	No. of CMT positive animals	Prevalence (%)	p-value
Body Condition	Poor	124	19	15.3	0.001
	Medium	110	40	36.4	0.001
	Good	153	74	48.3
Stage of Lactation	Early	138	78	56.4	0.001
	Mid	118	12	10.2
	Late	131	43	32.9
Parity	1	90	2	2.2	0.001
	2	93	6	6.5
	3	85	50	58.6
	4	75	45	59.6
	>4	43	30	69.1
Udder shape	Non- Pendulous	110	14	12.7	0.001
	Pendulous	97	35	36.1
	Cylindrical	112	47	42.1
	Round	68	37	54.3
Teat shape	Round Cylindrical Bowl	135 151 101	46 58 29	34.1 38.4 28.7	0.534
Milk yield per day	>5	214	126	58.9	0.001
	<5	173	7	4.0
No. of people attending animals	1 2 >2	116 167 104	0 60 73	0.0 36.0 69.9	0.001
Use of calf suckling	Yes No	179 208	7 126	3.9 60.6	0.001
History of Mastitis	Yes No	132 255	11 122	8.3 47.8	0.001
Dipping status	Yes No	198 189	60 73	30.3 38.6	0.227
Deworming program	No De-worming Once a year Twice a year Thrice a year	75 94 110 108	14 27 56 36	18.7 28.7 51.0 33.2	0.011
Manure Removal	Daily Once a week	234 153	71 62	30.4 40.4	0.001
Feed Sharing	Yes No	179 208	32 101	17.9 48.6	0.001
Farm Hygiene	Poor Good Excellent	131 171 85	31 94 8	23.8 54.9 9.4	0.001
Dry cow therapy	Yes No	132 255	14 119	10.6 46.6	0.001
	TOTAL	387	133	34.36

Note: Variables having p-value < 0.001 were included in the final multivariate logistic regression model.

Natural suckling may assist in teat canal cleaning and improve milk ejection, thereby reducing the risk of infection (Sharma et al., 2011). The study also reported higher SCM prevalence in farms with no history of clinical mastitis (47.8%). Farms not practicing teat dipping showed a 38.6% prevalence, reinforcing the importance of post-milking teat disinfection as a preventive measure (NMC, 2020). Animals dewormed only twice a year had a prevalence of 51.0%, suggesting that inadequate parasite control may compromise immunity and increase susceptibility to SCM. Similarly, a 40.4% prevalence in farms where manure was removed weekly reflects the impact of environmental hygiene on udder health. Unexpectedly, even farms with good hygiene practices reported a 54.9% prevalence, indicating that hygiene alone, without proper udder health management (e.g., dry cow therapy), may not be suitable to control SCM (Zadoks et al., 2001). The study observed a 46.6% prevalence in farms that did not use dry cow therapy, underscoring the value of this approach in eliminating existing infections and preventing new ones during the dry period (Bradley and Green, 2000). Notably, non-practice of feed sharing was also associated with a higher prevalence (48.6%), possibly due to stress or resource competition among animals within individual feeding systems. Sub-clinical mastitis (SCM) poses a significant threat to dairy production, particularly in buffaloes, due to its asymptomatic nature and adverse impact on milk quality and yield. The present study assessed a range of animal- and management-related risk factors for SCM among buffaloes in the Landhi Cattle Colony, Karachi.

Multivariate analysis showed that medium or good body condition, mid and late stages of lactation, parity ≥3, pendulous, cylindrical, or round udder shapes, milk yield <5 L/day, more than one person attending animals, use of calf suckling, a history of mastitis, deworming twice a year, feed sharing, good or excellent farm hygiene, and use of dry cow therapy were significant independent risk factors associated with sub-clinical mastitis in buffaloes (P < 0.05, Table 2).

Using univariate and multivariate logistic regression analyses. A total of 13 risk factors were initially identified as significantly associated with SCM in the univariate analysis, and variables with p < 0.001 were further examined in a multivariate model after controlling for multicollinearity. The final logistic regression model revealed several notable risk factors. Among them, calf suckling was found to significantly increase the risk of SCM (OR=15.5; p < 0.001). Calf suckling can facilitate the transmission of pathogens from the calf’s oral cavity to the udder, potentially introducing or spreading intramammary infections (Khan et al., 2015). Moreover, suckling may reduce the opportunity for proper post-milking teat disinfection, thereby enhancing the risk of infection (Radostits et al., 2007). Low milk yield (<5 litters/day) was another strong predictor of SCM (OR=14.56; p < 0.001). Low-yielding animals may receive less attention from farm workers, leading to suboptimal hygiene and udder care, thus increasing susceptibility to infections (Bhutto et al., 2012). Furthermore, reduced milk yield may also indicate underlying or chronic udder infections not yet clinically evident. The mid-lactation stage was also significantly associated with SCM (OR=5.56; p < 0.001). This finding aligns with earlier reports suggesting that animals in mid-lactation are more prone to mastitis due to physiological stress and peak milk production that may compromise immune defences (Ali et al., 2011). The changing udder environment during this stage can also facilitate pathogen colonization. Previous history of mastitis was another strong predictor (OR=5.74; p < 0.001), which supports existing literature. Animals with a history of mastitis are more likely to suffer recurrent infections due to persistent pathogens, damaged udder tissue, or weakened local immunity (Abrahamsen et al., 2014). This emphasizes the need for proper record-keeping and culling decisions in mastitis control programs. Additionally, sharing feed among animals emerged as a significant management-related risk factor (OR=2.716; p < 0.001). Shared feeding practices may enhance the risk of cross-contamination and increase contact among animals, thereby facilitating the transmission of contagious mastitis pathogens such as Staphylococcus aureus (Tolosa et al., 2013).

The major limitation of the current study is that most existing research focuses on risk factors in dairy cattle, making it difficult to directly compare or correlate these findings with the results observed in buffaloes. Moreover, our study was conducted using animals from an urbanized cattle colony, so the identified risk factors may not be directly comparable to those present in rural farming systems.

CONCLUSION

The incidence of sub-clinical mastitis is extremely high in Landhi Cattle Colony, Karachi, which contributes to a serious threat to the quality and quantity of milk. Animal and management related factors have a strong correlation with the disease especially calf suckling, low milk yield, mid lactation stage, past history of mastitis, and share of the feed. Infection risk was also augmented by poor hygiene practices, absence of dry cow therapy and poor farm management. SCM can be effectively controlled through the enhancement of milking hygiene, regular screening, implementation of dry cow therapy and creation of awareness programs to the farmers that can lead to the reduction of economic losses and the enhancement of the health of the udders.

Table 2: Multiple logistic regression model for risk factors associated with the prevalence of sub-clinical mastitis in buffaloes of cattle colony Landhi, Karachi

Factors	Description	Coefficient β	Standard Error	Odds ratio	95% Cl	p-value
Body Condition	Poor (Reference)	.....	.....	.....	.....	..... 0.005*
	Medium	0.957	0.308	0.421	0.23 - 0.77	..... 0.005*
	Good		0.284	0.317	0.18 - 0.55	0.000**
Stage of Lactation	Early (Reference)	.....	.....	.....	.....	.....
	Mid	3.190	0.334	5.558	2.89 - 10.69	0.000**
	Late		0.226	1.722	1.11 - 2.68	0.016*
Parity	1 (Reference)	.....	.....	.....	.....	.....
	2	-0.373	0.830	0.344	0.068 - 1.75	0.200 NS
	3		0.737	0.038	0.009 - 0.16	0.000**
	4		0.739	0.037	0.009 - 0.16	0.000**
	>4		0.753	0.032	0.007 - 0.14	0.000**
Udder shape	Non- Pendulous (Reference)	.....	.....	.....	.....	.....
	Pendulous	-0.124	0.345	0.353	0.18 - 0.70	0.002*
	Cylindrical		0.333	0.303	0.16 - 0.58	0.000**
	Round		0.349	0.234	0.12 - 0.46	0.000**
Milk yield per day	>5 (Reference)	.....	.....	.....	.....	.....
	<5	2.900	0.401	14.56	6.61 - 32.04	0.000**
No. of people attending animals	1 (Reference) 2 >2	..... 0.203	.....	..... 0.011 0.006	..... 0.07 - 0.19 0.03 - 0.09	..... 0.001* 0.000**
Use of calf suckling	No (Reference) Yes	..... 4.100	..... 0.401	..... 15.50	..... 7.06 - 34.01	..... 0.000**
History of Mastitis	No (Reference) Yes	..... 0.903	..... 0.333	..... 5.74	..... 2.98 - 11.01	..... 0.000**
Deworming program	No De-worming (Reference) Once a year Twice a year Thrice a year	..... -0.23	..... 0.364 0.334 0.349	..... 0.650 0.367 0.560	..... 0.32 - 1.33 0.19 - 0.71 0.28 - 1.11	..... 0.236 NS 0.002* 0.096 NS
Manure Removal	Once a week (Reference) Daily	..... 0.289	..... 0.203	..... 1.335	..... 0.89 - 1.98	..... 0.153 NS
Feed Sharing	No (Reference) Yes	..... 1.00	..... 0.227	..... 2.716	..... 1.74 - 4.24	..... 0.000**
Farm Hygiene	Poor (Reference) Good Excellent	..... 0.99	..... 0.237 0.420	..... 0.430 2.514	..... 0.27 - 0.69 1.10 - 5.73	..... 0.000** 0.028*
Dry cow therapy	No (Reference) Yes	..... 0.854	..... 0.302	..... 4.40	..... 2.43 - 7.96	..... 0.000**

NS = Non significant difference. * = The result is significant at p < 0.05. ** = The result is highly significant at p < 0.001

Acknowledge ment

The authors are thankful to the dairy farmers and farm attendants of Landhi Cattle Colony, Karachi, for their cooperation during sample collection and data recording. The authors also acknowledge the Department of Veterinary Medicine, Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University Tandojam, Pakistan, for providing technical guidance and research facilities to conduct this study.

Novelty Statement

This study provides updated epidemiological information on the prevalence of sub-clinical mastitis and its associated animal- and farm-level risk factors in dairy buffaloes of Landhi Cattle Colony, Karachi. The findings highlight key management practices influencing disease occurrence and offer evidence-based recommendations for improving udder health, milk quality, and dairy productivity in peri-urban buffalo production systems of Pakistan.

Author’s Contribution

Muhammad Ajmal: Conceived and designed the study, collected field data and milk samples, performed statistical analysis, interpreted results, and prepared the manuscript.

Amjad Hussain Mirani: Supervised the research work, contributed to study design, data interpretation, and manuscript revision.

Jam Kashif Zaman: Assisted in sample collection, laboratory work, and data compilation.

Zubair Ahmed Leghari : Assisted in data analysis, interpretation of findings, and manuscript editing.

Khalique ur Rehman Bhutto: Provided technical guidance, critically reviewed the manuscript, and approved the final version for publication.

Generative AI and AI assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Abebe R, Hatiya H, Abera M, Megersa B, Asmare K (2016). Bovine mastitis: prevalence, risk factors and isolation of Staphylococcus aureus in dairy herds at Hawassa milk shed, South Ethiopia. BMC Vet. Res., 12: 270. https://doi.org/10.1186/s12917-016-0905-3

Abrahamsen M, Persson Y, Kanyima BM, Båge R (2014). Prevalence of subclinical mastitis in dairy farms in urban and peri-urban areas of Kampala, Uganda. Trop. Anim. Health Prod., 46: 99–105. https://doi.org/10.1007/s11250-013-0455-7

Al-Gburi NM, Al-Taii NA (2025). Virulence and antimicrobial resistance profiles of Enterococcus spp. isolated from clinically mastitic versus apparently healthy bovine milk. J. Anim. Health Prod., 13: 803–811. https://doi.org/10.17582/journal.jahp/2025/13.3.803.811

Ali HR, Ali SF, Abd-Algawad RH, Sdeek FA, Arafa M, Kamel E, Shahein MA (2022). Impact of udder infections on biochemical composition of milk in context of pesticides exposure. Vet. World, 15: 797–808. https://doi.org/10.14202/vetworld.2022.797-808

Ali L, Khan A, Naz S, Zaman MA (2011). Prevalence of subclinical mastitis in dairy buffaloes of Punjab, Pakistan. J. Anim. Plant Sci., 21: 477–480.

Almaw G, Zerihun A, Asfaw Y (2008). Bovine mastitis and its association with selected risk factors in smallholder dairy farms in and around Bahir Dar, Ethiopia. Trop. Anim. Health Prod., 40: 427–432. https://doi.org/10.1007/s11250-007-9115-0

Bhutto AL, Murray RD, Woldehiwet Z (2012). Udder shape and teat-end hyperkeratosis and their relationship with subclinical mastitis in dairy cows. Vet. Rec., 170: 261.

Bilal MQ, Iqbal MU, Mohammad G, Avais M, Sajid MS (2004). Factors affecting the prevalence of clinical mastitis in buffaloes around Faisalabad district (Pakistan). Int. J. Agric. Biol., 6: 185–187.

Bludau MJ, Maeschli A, Leiber F, Steiner A, Klocke P (2014). Mastitis in dairy heifers: prevalence and risk factors. Vet. J., 202: 566–572. https://doi.org/10.1016/j.tvjl.2014.09.021

Bradley AJ, Green MJ (2000). A study of the incidence and significance of intramammary enterobacterial infections acquired during the dry period. J. Dairy Sci., 83: 1957–1965. https://doi.org/10.3168/jds.S0022-0302(00)75072-7

Cuenca M, Reinoso L, González J, García D (2024). Etiology of subclinical bovine mastitis in Biblian-Ecuador. J. Anim. Health Prod., 12: 100–107. https://doi.org/10.17582/journal.jahp/2024/12.1.100.107

Dego OK, Tareke F (2003). Bovine mastitis in selected areas of southern Ethiopia. Trop. Anim. Health Prod., 35: 197–205. https://doi.org/10.1023/A:1023352811751

FAO (2001). The State of Food and Agriculture 2001. Food and Agriculture Organization, Rome.

Farooq AA, Inayat S, Akhtar MS, Mushtaq M (2008). Prevalence of mastitis and antibiotic sensitivity of bacterial isolates recovered from Nili-Ravi buffaloes. J. Anim. Plant Sci., 18: 76–77.

Hossain MM, Lee SI, Kim IH (2014). Effect of dietary Korean aged garlic extract by Leukonostoc citreum SK2556 on production, hematological status, meat quality, relative organ weight, targeted Escherichia coli colony and excreta gas emission in broilers. Anim. Feed Sci. Technol., 198: 333–340. https://doi.org/10.1016/j.anifeedsci.2014.09.015

Hussain R, Javed MT, Khan A, Muhammad G (2013). Risk factors associated with subclinical mastitis in water buffaloes in Pakistan. Trop. Anim. Health Prod., 45: 1723–1729. https://doi.org/10.1007/s11250-013-0421-4

Kahir MA, Islam MM, Rahman AKM, Nahar A, Rahman MS, Son HJ (2008). Prevalence and risk factors of subclinical bovine mastitis in some dairy farms of Sylhet district. Korean J. Vet. Serv., 31: 497–504.

Karimuribo ED, Fitzpatrick JL, Bell CE, Swai ES, Kambarage DM, Ogden NH, Bryant M, French NP (2006). Clinical and subclinical mastitis in smallholder dairy farms in Tanzania. Prev. Vet. Med., 74: 84–98. https://doi.org/10.1016/j.prevetmed.2006.01.009

Kavitha KL, Rajesh K, Suresh K, Sreedevi B, Satyanarayana ML (2009). Epidemiological studies of subclinical mastitis in dairy cows. Indian Vet. J., 86: 858–860.

Khan MZ, Khan A, Avais M (2019). Prevalence of subclinical mastitis in buffaloes and associated risk factors in Pakistan. Pak. J. Zool., 51: 1125–1131.

Khan MZ, Khan A, Avais M, Khan JA (2015). Identification of risk factors for subclinical mastitis in buffaloes in Southern Punjab, Pakistan. Pak. J. Agric. Sci., 52: 1101–1105.

Madut NA, Gadir AEA, El-Jalii IM (2009). Host determinants of bovine mastitis in semi-intensive production systems. J. Cell Anim. Biol., 3: 71–77.

Motie A, Ramudit S, Mohabir R (1985). Subclinical mastitis in dairy cattle in Guyana. Trop. Anim. Health Prod., 17: 245–246. https://doi.org/10.1007/BF02356987

National Mastitis Council (2020). Procedures for evaluating teat disinfectants. NMC, USA.

Neelam J, Singh VK, Joshi VG, Chhabra R, Singh (2022). Virulence and antimicrobial resistance gene profiles of Staphylococcus aureus associated with clinical mastitis in cattle. PLoS One, 17: e0264762. https://doi.org/10.1371/journal.pone.0264762

Persson Y, Nyman AKJ, Grönlund-Andersson U (2011). Etiology and antimicrobial susceptibility of udder pathogens from cases of subclinical mastitis in dairy cows in Sweden. Acta Vet. Scand., 53: 36. https://doi.org/10.1186/1751-0147-53-36

Radostits OM, Gay CC, Hinchcliff KW, Constable PD (2007). Veterinary Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and goats. Saunders Elsevier.

Rehman A, Jingdong L, Chandio AA, Hussain I (2017). Livestock production and population census in Pakistan. Inform. Process Agric., 4: 168–177. https://doi.org/10.1016/j.inpa.2017.03.002

Rola JG, Larska M, Grzeszuk M, Rola J (2015). Association between antibody status to bovine herpesvirus 1 and quality of milk in dairy herds in Poland. J. Dairy Sci., 98: 781–789. https://doi.org/10.3168/jds.2014-8781

Sahoo NR, Singh RK, Kumar A (2014). Subclinical mastitis in dairy animals: incidence, economics, and predisposing factors. Int. J. Livest. Res., 4: 71–83. https://doi.org/10.1155/2014/523984

Saravanan R, Kumar PS, Manoharan G (2021). Relationship of udder and teat morphology with intramammary infection in dairy cattle. Indian J. Anim. Sci., 91: 123–127.

Sarker SC, Parvin MS, Rahman AA, Islam MT (2013). Prevalence and risk factors of subclinical mastitis in lactating dairy cows. Trop. Anim. Health Prod., 45: 1171–1176. https://doi.org/10.1007/s11250-012-0342-7

Schukken YH, González RN, Tikofsky LL, Schulte, H.F., Santisteban, C.G., Welcome, F.L., Bennett, G.J., Zurakowski, M.J., Zadoks, R.N., and other co-authors (2009). CNS mastitis: Nothing to worry about? Vet. Microbiol., 134: 9–14. https://doi.org/10.1016/j.vetmic.2008.09.014

Sharma N, Maiti SK, Sharma KK (2011). Subclinical mastitis in buffaloes in India. Int. J. Dairy Sci., 6: 163–167.

Sharma N, Sindhu N (2007). Occurrence of clinical and subclinical mastitis in buffaloes in Haryana. Indian J. Anim. Sci., 77: 471–473.

Tolosa T, Verbeke J, Piepers S, Supré K, De Vliegher S (2013). Risk factors associated with subclinical mastitis. Prev. Vet. Med., 112: 68–75. https://doi.org/10.1016/j.prevetmed.2013.06.009

Zadoks RN, Allore HG, Hagenaars TJ, Barkema HW, Schukken YH (2001). Mathematical model of Staphylococcus aureus control in dairy herds. Epidemiol. Infect., 127: 397–416. https://doi.org/10.1017/S0950268802007483