Prevalence &amp; risk factors analysis of bovine mastitis in dairy herds of Rawalpindi district, Pakistan; a study to estimate severity &amp; farmers’ awareness about the disease

doi:10.21203/rs.3.rs-1753360/v1

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Prevalence & risk factors analysis of bovine mastitis in dairy herds of Rawalpindi district, Pakistan; a study to estimate severity & farmers’ awareness about the disease

https://doi.org/10.21203/rs.3.rs-1753360/v1

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Background

Bovine mastitis is inflammation of the mammary glands due to physical injury or microbial infection. The disease results in a significant reduction in milk production, hence affecting the dairy industry. The current study is designed to analyze the prevalence of mastitis, detect herd and cow-level potential risk factors, and estimate awareness level of farmers about mastitis. Total 432 cows were selected, by simple random method, from 40 herds of Rawalpindi District (Pakistan) and screened for mastitis.

Results

Based on surf field mastitis test (SFMT) result and clinical examination, 45% prevalence of mastitis was reported at herd-level. The cow-level prevalence was 18.52%, of which 1.85% and 16.66% were clinical and sub-clinical mastitis cases, respectively. The corresponding quarter-level prevalence was 7.58% and the right hind-quarter was most affected. After inoculation of 130 milk samples for bacterial isolation, S. aureus (76.9%) was the predominant etiological agent of mastitis followed by Streptococcus (61.5%). In the multivariable logistic regression model, the herd and cow-level factors significantly associated (p < 0.05) with the presence of mastitis were udder washing before milking, frequency of manure removal, floor type, lactation stage, udder condition, teat end lesion, presence of ticks, use of hormones, milking method and udder and leg hygiene. The Hosmer-Lemeshow goodness-of-fit test suggested that the model fit the data (χ2 = 4.3; p = 0.83) and the final model showed 53.75% sensitivity, 94.9% specificity, 87.27% accuracy, and 70.49% precision. The questionnaire-based survey of the farm handlers regarding the awareness of mastitis and other related factors showed some serious gaps in the perception and awareness of farmers about sub-clinical mastitis and its influence on the economy.

Conclusion

The very high prevalence of mastitis, more importantly the sub-clinical one, in the examined herds revealed the huge potential economic loss the sector suffers. Awareness and training of the farmers is essential to enhance the quantity and quality of milk. Alongside awareness, government and International agencies should also help farmers in improving their technical facilities.

Mastitis

SFMT

Udder

Sub-clinical mastitis

S. aureus

Risk factors

Awareness

Rawalpindi

Cattle are the predominant source of milk around the world. Since the last few years, the increased demand for milk has led to a 2.2% increase in milk production in Pakistan and a 3% increase around the world (1). The dairy sector also contributes a substantial part in supporting livelihood of millions especially in rural areas of the country, providing 18.5% of Pakistan’s gross domestic production (2). However, animal health issues and diseases cost almost 200 million USD to Pakistan’s economy annually, which significantly hampers the development of this sector (3). A disease called bovine mastitis is recognized as the most damaging disease of the dairy sector in Pakistan (4) and around the world (5).

Significant cost burdens linked to this disease are reduction in milk quality and production, veterinary costs, culling of chronically infected cows, and in extreme cases also the death of the animal (6). Mastitis can also be linked with serious zoonotic potential, as the shedding of bacteria and their toxins in milk (7) may find its way to affect the wellbeing of humans through the transfer of these pathogens and toxins (8, 9). Detrimental effects of mastitis in the dairy industry and human health increase pressure on dairy farmers to increase antibiotic usage which thereby, increases the pace of antibiotic resistance traits acquisition among bacteria inhabiting dairy animals (10).

The hallmark of mastitis is the inflammation of mammary glands due to microbial infections in the majority of the cases that result in an increased amount of somatic cell count (SCC), mainly due to the recruitment of neutrophils (11). The disease mainly occurs due to the interplay between various environmental factors such as milking machines, microbes, and other animal husbandry practices (12).

The bacterial infection of the teat occurs when bacteria enter the teat canal, they multiply there and release toxins, thereby causing extensive vascular damage (13). A wide range of bacterial pathogens cause bovine mastitis and epidemiologically, they have been divided into two broad classes namely: contagious and environmental pathogens (14). Udder of the infected cows serves as the main reservoir for contagious pathogens. Most common of these include Streptococcus agalactiae, Corynebacterium bovis, Staphylococcus aureus, and Mycoplasma spp. (15). Whereas, the main reservoir of the environmental pathogens is the environment in which cattle live and they cause intra-mammary infections (16). These include: Streptococcus dysgalactiae, Escherichia coli, Streptococcus uberis, and Klebsiella spp. (17, 18). The type of bacterial species infecting the animal’s udder also affects the severity of mastitis; with streptococcus and coliforms implicated in the increased disease severity (19).

The disease manifests in the form of clinical mastitis (CM) or Sub-clinical mastitis (SCM). SCM is the most pervasive form of mastitis that is 15 to 40 times more common than the CM, not visible by naked eye but there is reduced milk production and an increase in somatic cell count (20, 21, 22). The incidence of bovine mastitis can be reduced by the implementation of some preventive measures (21). In Pakistan, various farm management practices are followed, but that depends on the working attitude of farmers and these practices widely differ in rural and peri-urban parts of the country (23). The difference in attitude and behavior is the main factor that subsequently influences the occurrence of mastitis (24).

Even though mastitis is a big problem for the dairy industry in Pakistan, knowledge and awareness about this udder disease is limited. Numerous studies (25–30) have been conducted over the years, to report the prevalence of bovine mastitis in different divisions of Pakistan. However, few of them have reported scientific evidence for the association of various farm practices that contribute to bovine mastitis. This study aimed to determine the association of different farm management conditions with disease severity and associated bacterial pathogens. For that purpose, the prevalence of mastitis was determined along with its contributing risk factors at cow level in the dairy herds located in the Rawalpindi district, and the awareness level of the farm handlers about this disease was also evaluated.

Ethical statement

This study was approved by the Board of Advanced Studies and Research (BASR) of the National University of Sciences and Technology (NUST), Islamabad. In addition, written consent was taken from the farm owners before sampling. It was made sure that milk samples for microbiological analysis were only taken from the routine milking performed at the farms, and besides milking no experimental work was conducted on the farm animals.

Study area

The study was conducted from March 2019 to March 2020 in dairy herds of the Rawalpindi district. Rawalpindi district is in the Potohar plateau in the northernmost part of the Punjab province of Pakistan. It lies between longitudes 73° 15′ 0″ east and latitude 33° 20′ 0″ north. The area of the district is around 5,286 km² (2,041 sq mi) and is situated on the southern slopes of the north-western boundaries of the Himalaya mountain series. It is rich with mountain tracts, valleys, and rivers and is known for its moderate climate due to its closeness to the foothills (31). Rawalpindi district has seven autonomous tehsils; including Rawalpindi, Taxila, Kahuta, Murree, Gujar Khan, Kallar Syedan, and Kotli Sattian. Rawalpindi city is the capital of the district and is the 4^th most populous metropolitan city of Pakistan. Punjab province is the most livestock-populated province of Pakistan (32). According to Veterinary Research Institute (VRI); the population of cows in the Rawalpindi district is around 252,298 cows, out of which 104,950 (40% of the total population) are lactating cows.

Farm type and management practices

A total of 40 farms were randomly selected from the study area. The herd size of the selected farms varied from 1 to 150 animals, of which 1 to 100 were lactating. Herd type included both single type (15%) and mixed herds (85%). For management of the farm, 34 (85%) of the herds were group barns and only 4 (10%) of the herds had bedding material. Around 52.5% of herds (21) were managed intensively and rest were managed semi-intensively. In intensive management system, animals are kept indoors all the time and given concentrated foods including crop residues and hay, etc while in semi-intensive management system, animals are free to graze in pastures and given supplementary food only in morning and evening, before milking). Moreover, all of the farms included in the study were also checked for milking method, hand and udder washing, floor type, feed sharing, post teat dipping, hygiene of the farms, use of hormones, tick control activity, manure removal, and others (Table 3) before performing mastitis detection test on cows.

Sample size determination

The sample size for the desired study was calculated according to Thursfield (33) at 5% precision, 95% confidence interval, and with 23% expected mastitis prevalence of cows (According to PRI, Rawalpindi) in the Rawalpindi district.

Based on the formula, the total number of lactating cows required for the prevalence study was 272 cows. But, for better and precise results, 432 cows and 1728 teat quarters were considered in the study.

Data collection

A questionnaire comprised of close-ended questions was used to collect the data about various animal and herd-level factors that are thought to be associated with the occurrence of mastitis in cattle. A total of 28 factors were analyzed in this study and were divided into herd-level and animal-level factors (Table 1). Leg and udder hygiene score was recorded in the form of slightly dirty, moderately dirty, and very dirty (34) (Table 2).

Table 1

Various herd-level and animal-level factors thought to be associated with the occurrence of mastitis

Sr. No

Associated with Mastitis

Description

Herd-level Factors

1. Herd location, (2) Herd Size, (3) Herd type, (4) Use of bedding material, (5) Milking practices (Machine/Manual), (6) Frequency of milking, (7) Housing, (8) Floor type, (9) Herd management system, (10) Washing practices of udder, (11) Drying of udder before milking, (12) Use of towel, (13) Use of hormones, (14) Pre/post teat dipping in sanitizing agents, (15) Standing position after milking, (16) Milking mastitic cow last, (17) Sharing of feed, (18) Manure removal routine

Animal-level Factors

2. Breed, (2) Age, (3) Lactation stage, (4) Udder position, (5) Udder condition, (6) Presence of ticks, (7) Teat end lesion, (8) History of mastitis, (9) Leg and udder hygiene score, (10) Number of people attending cow

Table 2

Criteria to assess the leg and udder hygiene score of cattle

Sr. No	Criteria	Description
1.	Slightly dirty	2-10% area of the hind legs and udder is covered in dirt
2.	Moderately dirty	10-30% area of the hind legs and udder is covered in dirt
3.	Very dirty	>30% area of the hind legs and udder is covered in dirt

Clinical examination of cow udder and milk for clinical mastitis

During farm visits, clinical examination of milk and cow udder was conducted. For milk examination; milk from each quarter was separately examined by visual inspection for the presence of any blood clots, flakes, coagulates of milk, smell, and color change. For clinical examination of the udder; the udder of each cow was first examined visually followed by palpation to detect any inflammatory swelling, atrophy of udder tissue, or fibrosis. The consistency and size of the udder were also inspected for any abnormalities such as firmness, disproportional symmetry, and blindness.

Preparation of udder and teats

The udder and the teats were thoroughly cleaned and dried before milk sample collection for the detection of mastitis. A dry towel was used to brush the surface of teats for the removal of dirt, particles of bedding material, and other filth. The teats were then swabbed with cotton and 70% ethanol (35). Teats on the far side of the udder were scrubbed first to avoid re-contamination.

Milk sample collection

When clinical mastitis was detected in the cow, the milk sample was collected from the infected teat by a standard milk sampling technique (36). The time chosen for milk sample collection was before milking. To prevent contamination during sampling, rear-end teats were sampled first. The first three milk streams were discarded and then approximately 10 ml milk sample was collected in a sterile falcon tube. The tube was labeled properly and kept in the icebox. Samples were transferred to the laboratory for bacterial identification. Milk samples were stored at 4ᵒ C for a maximum of 24 hours or at -20ᵒ C till further use.

Surf Field Mastitis Test (SFMT)

SFMT was used as the screening test for subclinical mastitis. It was carried out by the previously described procedure (37). A squirt of milk, about 1 ml from each quarter, was taken in each of the four shallow cups of the Mastitis detection paddle. An equal amount of the SFMT reagent (3% surf solution) was added to each cup. The mixture was gently rotated in the horizontal plan for 20-30 s. The mixture was then examined for thickening or any other visible change. The SFMT results were scored based on gel formation; 0 (negative), 1 (weakly positive), 2 (distinct positive), and 3 (strongly positive). All SFMT scores of 0 were considered as negative while SFMT scores of 1, 2, and 3 were considered as indicators of sub-clinical mastitis. Mastitis-positive cows were defined as having at least one quarter with an SFMT score of 1+ (Table: 7). The theory of gel formation (38) is summarized in Figure 1. Milk sampling was done from mastitis positive cows as described above, for bacteriological analysis. SFMT negative cows were not included in milk sampling. During sampling, milk was collected only from those quarters that were SFMT positive.

Processing of milk samples and bacteriological Assays

Bacteriological analysis was done according to National Committee for Clinical Laboratory Standards (39) and Quinn (36). Refrigerated milk samples were warmed at room temperature for 1-2 hours and vortexed thoroughly to disperse the fat and bacteria in the milk samples and left for some time to disperse the foam before inoculation. Milk sample (0.01 ml) was streaked on blood agar enriched with 5% sheep blood (HiMedia, India), plates were incubated aerobically at 37^∘ C for 24-48 h to rule out slow-growing bacteria. The plates were examined for growth, hemolytic characteristic, and morphological features including colony shape, size, opacity, and color. Suspected colonies were subcultured on nutrient agar plate (Oxoid, UK) to obtain pure cultures for further investigation. Pure colonies were subjected to primary investigation through Gram staining and cellular morphology (rods or coccus). The purified strains were further identified on genus level based on (1) their growth patterns on different selective and differential media such as; MacConkey agar (Oxoid, UK), EMB agar (Oxoid, UK), Cetrimide agar (Himedia, India), and Mannitol salt agar (Himedia, India), and (2) their biochemical tests such as catalase test, oxidase test, coagulase test, the “IMViC” tests (Indole, Methyl-Red, Vogas Proskaur, and Citrate utilization), TSI, oxidation fermentation test and CAMP test (40).

Awareness of the farm handlers regarding mastitis

Farm owners and the farmers were asked about the general concepts and information regarding mastitis, to analyze their knowledge and awareness level of this disease Figure 6. To avoid biases, the same person from the team collected the information from all farm handlers.

Preprocessing of data

All the collected data were recorded in the Microsoft Excel spreadsheet and checked for any missing value. The data was coded, and statistical analyses were performed using IBM SPSS Statistics (Version 25) software. The prevalence of clinical and subclinical mastitis was calculated as the proportion of the mastitis-positive cows against the total number of investigated cows. A dairy herd was considered as mastitis positive if at least a single animal with clinical mastitis or SFMT positive result was detected.

Bias variable identification

The association between the categorical independent variables (n=28) and dependent variable, Mastitis (0 = negative and 1 = positive) was calculated by univariable logistic regression analyses. The independent variables assessed were described in Table 1. All variables with a p-value < 0.05 in the univariable analysis were assessed for binary logistic regression model construction.

Variable selection for model construction

All variables with a p-value < 0.05 in the univariable analysis were analyzed for multicollinearity using Kruskal gamma statistics and the variables whose gamma value ranged between −0.6 and +0.6 were considered in a multivariable logistic regression analysis. The final model was built in a backward stepwise elimination procedure with reference to wald statistics. In this analysis, statistical significance was set at p < 0.05 and the variables having higher values were eliminated from the final model (Table 9).

Performance evaluation of model

The model was assessed for goodness-of-fit using the Hosmer-Lemeshow method (41). To further evaluate the performance of the binary logistic regression model; sensitivity, specificity, accuracy, and precision of the model were calculated by the given formulas:

Hygienic practices and management system of dairy farms

The data on various hygienic practices and management systems of the dairy farms were collected from 40 dairy farms selected for mastitis study in the Rawalpindi district (Table 3).

Table 3

Hygienic practices and management system of cattle farms selected for mastitis study in District Rawalpindi

Sr.No	Variables	Category	No of Herds (n=40)	Distribution (%)
1.	Locality	Rural	33	82.5
1.	Locality	Urban	07	17.5
2.	Herd size	≤ 10 cows	16	40
2.	Herd size	> 10 cows	24	60
3.	Herd type	Single type	6	15
3.	Herd type	Mixed	34	85
4.	Management system	Intensive	21	52.5
4.	Management system	Semi-intensive	19	47.5
5.	Housing	Group barn	34	85
5.	Housing	Stall barn	6	15
6.	Bedding material	Yes	4	10
6.	Bedding material	No	36	90
s7.	Floor-type	Concrete	5	12.5
		Muddy	19	47.5
		Mixed	16	40
8.	Milking method	Manual	34	85
8.	Milking method	Machine milking	6	15
9.	Feed Sharing	Yes	19	47.5
9.	Feed Sharing	No	21	52.5
10.	Hand washing before milking	With soap	23	57.5
10.	Hand washing before milking	Without soap	17	42.5
11.	Washing of udder before milking	Only teats	24	60
11.	Washing of udder before milking	Whole udder	16	40
12.	Drying of udder after washing	Yes	8	20
12.	Drying of udder after washing	No	32	80
13.	Milking mastitic cow last to avoid infection	Yes	11	27.5
13.	Milking mastitic cow last to avoid infection	No	29	72.5
14.	Pre/post teat dipping in sanitizing agents	Yes	9	22.5
14.	Pre/post teat dipping in sanitizing agents	No	31	77.5
15.	Keep the cow in a standing position for some time after milking to avoid contamination	Yes	10	25
15.		No	30	75
16.	Use of hormones to increase milk yield	Yes	26	65
16.	Use of hormones to increase milk yield	No	14	35
17.	Culling chronically infected cow	Yes	0	0
17.	Culling chronically infected cow	No	40	100
18.	Dry cow therapy	Yes	1	2.5
18.	Dry cow therapy	No	39	97.5
19.	Hygiene of the farm	Good	22	55
19.	Hygiene of the farm	Poor	18	45
20.	Tick control activity	Yes	20	50
20.	Tick control activity	No	20	50
21.	Manure removal	Daily	17	42.5
21.	Manure removal	Once a week	23	57.5

Prevalence of mastitis

The prevalence of clinical and subclinical mastitis at the herd, cow, and quarter levels is illustrated in Table 4. Based on the clinical examination of udder and milk, and SFMT results, 45% of the herds and 18.52% cows were positive for mastitis. Out of the total 18.52%, only 1.85% of cows had clinical mastitis and 16.66% were positive for subclinical mastitis. All the quarters of the cows (1728) were inspected for the presence of blindness and lesions and it was noted that 21 (1.21%) quarters were blind and thus excluded from the prevalence studies. 130 (7.61%) quarters were positive for mastitis (Figure 3) and the right hind teat (9.62%) was most infected (Table 5) (Figure 4). The prevalence of mastitis at the individual farm level is illustrated in Table 6. At the herd, cow, and quarter level, sub-clinical mastitis was the predominant one. Out of all the cows inspected for subclinical mastitis, 30.56%, 48.61%, and 20.83% were weak, distinct, and strong SFMT positive, respectively (Table 6) (Figure 5).

Table 4

Prevalence of mastitis in lactating cows at the herd, cow and quarter level

Observation	Overall mastitis in cows				Clinical mastitis		Sub-clinical mastitis
Observation	No. examined	No. of positive	Prevalence (%)	95% CI	No. observed	Prevalence (%)	No. observed	Prevalence (%)
Herd-Level	40	18	45	29.6 - 60.4	5	12.5	18	45
Cow-Level	432	80	18.52	14.84 - 22.16	8	1.85	72	16.66
Quarter-Level	1707	130	7.61	6.33 - 8.83	19	1.11	111	6.50

Table 5

Prevalence of mastitis in lactating cows at the quarter level

Quarter	Total No. of teats	Blind teats	Overall mastitis in quarter				Clinical mastitis		Sub-clinical mastitis
Quarter	Total No. of teats	Blind teats	Total teats examined	No. of positive	Prevalence (%)	95% CI	No. observed	Prevalence (%)	No. observed	Prevalence (%)
Right fore	432	5	427	29	6.79	4.4 – 9.2	3	0.70	26	6.09
Left fore	432	4	428	27	6.31	4.0 – 8.6	3	0.70	24	5.61
Left hind	432	6	426	33	7.75	5.2 – 10.2	5	1.17	28	6.57
Right hind	432	6	426	41	9.62	6.8 – 12.4	8	1.88	33	7.75

Table 6

Degree of mastitis by reaction with SFMT in dairy cows of Rawalpindi district. Cows with clinical mastitis are excluded from this study

Degree of mastitis at cow level
Sr. No	Description of reaction	Degree of mastitis	Total cows studied	No observed	Prevalence (%)	95% CI
1.	Mixture remain liquid, no gel formation	Negative (-)	424	352	83.02	79.5 – 86.5
2.	Coagulation of milk begins but no gel formation	Weak (+)	424	22	5.19	3.1 – 7.3
3.	Mixture coagulates but does not stick and distinctively forms gel)	Moderate (++)	424	35	8.25	5.6 – 10.8
4.	Mixture coagulates	Strong (+++)	424	15	3.54	1.8 – 5.2
Degree of mastitis in sub-clinical mastitis positive cows
Sr. No	Description of reaction	Degree of mastitis	Total Sub-clinical cows studied	No. observed	Prevalence (%)	95% CI
1.	Mixture remain liquid, no gel formation	Negative (-)	72	0	0	-
2.	Coagulation of milk begins but no gel formation	Weak (+)	72	22	30.56	20.0 – 47.2
3.	Mixture coagulates but does not stick and distinctively forms gel)	Moderate (++)	72	35	48.61	37.0 – 60.2
4.	Mixture coagulates	Strong (+++)	72	15	20.83	11.4 – 30.2

Mastitis risk factors

Several herd and cow level factors described in Table 1 were considered to analyze their association with mastitis by the univariable logistic regression analysis. Among these factors herd type, breed, lactation stage, management system, bedding material, floor type, milking method, milking routine, washing of udder before milking, drying of udder after washing, udder position, udder condition, presence of ticks, teat end lesions, pre/post teat dipping in sanitizing agents, keep the cow in standing position for some time after milking to avoid contamination, milking mastitic cow last to avoid infection, use of hormonal boost to increase milk yield, use of towel, history of mastitis, udder and leg hygiene score, feed Sharing, manure removal and no of people attending cows were found to be significantly associated (p < 0.05) with the presence of mastitis. While the location of the farm, herd size, age and housing did not show any significant effect (p > 0.05) on the occurrence of mastitis (Table 7). Among the factors in the initial univariable analysis, breed of the cows (herd type gamma = 0.95, lactation gamma = 0.98, bedding material gamma = 0.99, etc.), management system, bedding material and milking routine was dropped from further analysis due to multi-collinearity with other independent variables. Only those gamma values were considered that were below 0.6 and those variables were removed that have multi-collinearity with at least two other independent variables. Lactation stage and udder and leg hygiene score were showed multi-collinearity but still considered for final model based on biological plausibility and thus retained. The final model was built in backward stepwise elimination procedures about wald statistics and the statistical significance was set at p < 0.05. Thus, the variables subjected to multivariable logistic regression analysis were lactation stage, floor type, milking method, washing of udder, udder condition, presence of ticks, teat end lesion, use of hormones to increase milk yield, manure removal, and udder and leg hygiene score (Table 8). All the variables entered in the final model remained significant predictors of mastitis in cows (p < 0.05).

Table 7

Univariable logistic regression analysis of the association of mastitis at cow level, with different risk factor

Sr. No	Variables	Category	Cows examined (N = 432)	No. of positive for mastitis (Frequency)	Prevalence (%)	x²	p-value
1.	Udder condition	Normal	363	38	10.47	97.903	˂0.001
		Swelling	53	33	62.26
		Atrophy	16	9	56.25
2.	Teat end lesion	Yes	62	35	56.45	69.031	˂0.001
		No	370	45	12.16
3.	Use of hormones to increase milk yield	Yes	241	73	30.29	50.061	˂0.001
		No	191	07	3.66
4.	Washing of udder before milking	Only teats	242	72	29.75	46.017	˂0.001
		Whole udder	190	08	4.21
5.	Keep the cow in standing position for some time after milking to avoid contamination	Yes	190	08	4.21	46.017	˂0.001
		No	242	72	29.75
6.	Udder and leg hygiene score	Slightly dirty	148	09	6.08	44.052	˂0.001
		Moderately dirty	178	30	16.85
		Very dirty	106	41	38.68
7.	Milking mastitic cow last to avoid infection	Yes	170	07	4.12	38.525	˂0.001
		No	262	73	27.86
8.	Pre/post teat dipping in sanitizing agents	Yes	170	07	4.12	38.525	˂0.001
		No	262	73	27.86
9.	Bedding material	Yes	134	5	3.73	28.150	˂0.001
		No	298	75	25.17
10.	Herd type	Single type	137	6	4.38	26.580	˂0.001
		Mixed	295	74	25.08
11.	Presence of ticks	Yes	45	21	46.67	26.377	˂0.001
		No	387	59	15.24
12.	Udder position	Normal	356	52	14.61	20.521	˂0.001
		Pendulous	76	28	36.84
13.	Use of towel	Yes	153	13	8.50	15.769	˂0.001
		No	279	67	24.01
14.	Manure removal	Daily	183	20	10.93	12.120	˂0.001
		Once a week	249	60	24.10
15.	Floor type	Concrete	164	17	10.37	14.040	0.001
		Muddy	103	29	28.15
		Mixed	165	34	20.61
16.	Drying of udder after washing	Yes	150	15	10.0	11.051	0.001
		No	282	65	23.05
17.	History of mastitis	Yes	120	34	28.33	10.607	0.001
		No	312	46	14.74
18.	Milking method	Manual	323	49	15.17	9.511	0.002
		Machine milking	109	31	28.44
19.	Lactation stage	Early	144	38	26.39	11.945	0.003
		Mid	165	18	10.91
		Late	123	24	19.51
20.	Feed sharing	Yes	303	66	21.78	7.163	0.007
		No	129	14	48.27
21.	No of people attending cows	Only 1	135	15	11.11	7.140	0.008
		≥ 2 people	297	65	21.89
22.	Milking routine	2 times a day	202	28	13.86	5.454	0.020
		3 times a day	230	52	22.61
23.	Management system	Intensive	312	66	21.15	5.170	0.023
		Semi-intensive	120	14	11.67
24.	Breed	Local	187	26	13.90	4.654	0.031
		Cross	245	54	22.04
25.	Housing	Group barn	382	75	19.63	2.179	0.099
		Stall barn	50	5	10.0
26.	Herd size	≤ 10 cows	73	09	12.33	2.230	0.135
		> 10 cows	359	71	19.78
27.	Age	≤ 5 years	210	35	16.67	0.929	0.335
		> 5 years	222	45	20.27
28.	Location of Farm	Rural	375	69	18.40	0.026	0.871
		Urban	57	11	19.30

Table 8

Multivariable logistic regression analysis of the association of mastitis at cow level, with different risk factors

Sr. No	Variables	Category	OR	SE	z	p	95% CI for OR
1.	Washing of udder	Whole udder	Ref	-	-	-	-
1.	Washing of udder	Only teats	10.696	0.754	9.886	0.002	2.442 – 46.681
2.	Manure removal	Daily	Ref	-	-	-	-
2.	Manure removal	Once a week	4.234	0.592	5.952	0.015	1.328 – 13.500
3.	Lactation stage	Early	Ref	-	12.327	0.002	-
		Mid	4.557	0.434	12.226	˂0.001	1.947 – 10.662
		Late	2.168	0.449	2.973	0.085	0.900 – 5.222
4.	Floor-type	Muddy	Ref	-	11.444	0.003	-
		Concrete	0.158	0.618	8.897	0.003	0.047 – 0.532
		Mixed	1.022	0.426	0.003	0.958	0.444 – 2.355
5.	Milking method	Manual	Ref	-	-	-	-
5.	Milking method	Machine milking	2.790	0.444	5.345	0.021	1.169 – 6.657
6.	Udder condition	Normal	Ref	-	19.653	˂0.001	-
		Swelling	2.155	0.761	1.019	0.313	0.485 – 9.577
		Atrophy	0.216	0.510	9.061	0.003	0.079 – 0.586
7.	Presence of ticks	No	Ref	-	-	-	-
7.	Presence of ticks	Yes	0.306	0.486	5.951	0.015	0.118 – 0.729
8.	Teat end lesion	No	Ref	-	-	-	-
8.	Teat end lesion	Yes	0.250	0.494	7.866	0.005	0.095 – 0.659
9.	Use of hormones to increase milk yield	No	Ref	-	-	-	-
9.	Use of hormones to increase milk yield	Yes	0.292	0.560	4.819	0.028	0.097 – 0.877
10.	Udder and leg hygiene score	Slightly dirty	Ref	-	18.623	˂0.001	-
		Moderately dirty	0.246	0.388	13.045	˂0.001	0.115 – 0.526
		Very dirty	0.153	0.522	12.912	˂0.001	0.055 – 0.426

Performance evaluation of model

The Hosmer-Lemeshow goodness-of-fit test suggested that the model fit the data (χ2 = 4.3; p = 0.83) (Table 8). To evaluate the performance of the binary logistic regression model; the sensitivity, specificity, accuracy, and precision of the model were calculated from a 2*2 classification table (Table 9). According to this table, the final model showed 53.75% sensitivity, 94.9% specificity, 87.27% accuracy and 70.49% precision.

Table 9

A 2*2 classification table obtained from binary logistic regression model for its performance evaluation

		Predicted
		Mastitis		Percentage correct
Observed		0	1	Percentage correct
Mastitis	0	334 (TN)	18 (FP)	94.9
Mastitis	1	37 (FN)	43 (TP)	53.8
Overall Percentage				87.3

Where;

TP: True Positive – Diseased cases that are correctly identified as diseased

FP: False Positive – Normal cases that are incorrectly identified as diseased

TN: True Negative – Normal cases that are correctly identified as normal

FN: False Negative – Diseased cases that are incorrectly identified as normal

Microbiological Cultures

The bacterial isolation rate and their prevalence are shown in Table 10. The predominant isolated bacteria were S. aureus species with an isolation rate of 76.9% followed by Streptococcus with an isolation rate of 61.54%, E. coli with an isolation rate of 11.54%, Klebsiella with an isolation rate of 10.77%, E. aerogenes with an isolation rate of 10% and P. aeruginosa with an isolation rate of 7.69%. M. luteus was the least isolated which accounts for only 6.1%.

Table 10

Table representing the percentage prevalence of bacterial species isolated from mastitis positive cows

Sr. No	Isolated Bacterial strains	No of isolated Bacterial strains	Percentage of isolated Bacterial strains (%)
1.	S. aureus	100	76.92
2.	Streptococcus sp.	80	61.54
3.	E. coli	15	11.54
4.	Klebsiella sp.	14	10.77
5.	E. aerogenes	13	10
6.	P. aeruginosa	10	7.69
7.	M. luteus	08	6.1
8.	S. epidermidis	08	6.1
9.	Coagulase Negative Staphylococci	07	5.38

Awareness of the farm handlers regarding bovine mastitis

The farm handlers’ awareness about mastitis was accredited as all of them were aware of this disease and has encountered this udder disease of cows in their farms before. But, only 50% of the interviewees know sub-clinical mastitis and 55% have the awareness that common clinical symptoms such as swelling, fever and lesions be involved in the onset of mastitis. A total of 35% of respondents doesn’t know that milking the diseased cow last can somehow prevent the infection. Most of the farm handlers (95%) don’t know that culling the chronically infected cow can prevent the spread of the mastitis in healthy animals and the same is the case with dry cow therapy, where only 10% of respondents know the benefits of this therapy. A total of 70% of farm handlers know that mastitis affects milk quality and yield. To analyze their knowledge regarding preventive measures; questions were asked about udder washing and hand washing before milking, and all of them know that udder washing is important and 90% of them have the knowledge that handwashing before milking can prevent disease spread. One alarming situation reported was that most of the owners don’t know about the side effects of using hormones before milking as only 35% of the respondents have the awareness of the harmful effects of hormones usage (Figure 6).

The current study aimed to find out the prevalence of clinical and subclinical mastitis in the Rawalpindi district of Pakistan and to identify the risk factors responsible for the transmission and spread of mastitis. Another focus of this study was to evaluate the knowledge of the farm handlers’ regarding mastitis. This is one of the few bovine mastitis studies in Pakistan which involved a large number of lactating cows (n=432) selected from 40 dairy herds. The study revealed that 45% of the herds had at least a single cow suffering from mastitis. Due to the lack of similarly designed studies within Pakistan, it was difficult to compare the herd-level mastitis results. But similar studies were done abroad and one study in Ethiopia showed a very high prevalence of herd-level mastitis (74.7%) (34). Few other studies reported 21.7% in Tanzania (42), 29% in South Wales (43), and 49.3% mastitis in Zimbabwe (44).

The overall cow-level prevalence was 18.52% and it was largely accounted from sub-clinical mastitis (16.66%) and a smaller part (1.85%) accounted from clinical mastitis. This finding is in contrast with the previous research conducted in Pakistan that showed a much higher rate of mastitis including 47.58% in district Gojra (25), 51.88% in Tehsil Burewala (26), 24.71% in district Lahore (27), 53%ss in Dera Ismail Khan (28), 21.96% sub-clinical mastitis in the Cholistan (29), and 67.3% sub-clinical mastitis in Pothohar region (30). This finding is also in contrast with the previous research conducted in some other countries whose dairy management is somehow similar to ours including 51.6% in Tanzania (42), 42.9% in Egypt (45), 21.1% in Zimbabwe (44), 51.8% in Rewanda (46), 38.89% in Sudan (47), 85.3% in Nigeria (48), and 87.9% in Uganda (49). The reason for less prevalence of mastitis in the studied area might be that this region is right next to the Capital territory and most of the studied farms were commercial and well managed. The dominance of sub-clinical mastitis is alarming as it is 15-40 times more prevalent than clinical mastitis and its duration is long (6,45). The possible reason could be the little attention paid by the handlers as the infected animals show no obvious sign of infection and milk yield is also normal.

The observation of a higher rate of prevalence in the hind-quarters of the cows is similar to several other studies (34, 50-53). In contrast to this finding, a study in Nigeria reported a higher prevalence in fore-quarters (48). The justification of this observation might be the lower position of the hind-quarters than the fore ones thus making them prone to contamination and injuries that can lead to mastitis. According to some studies, the hind-quarters produce more milk than the fore one and that can also be a possible reason for the higher prevalence of mastitis in the hind-quarters (54).

The present study showed that the likelihood of mastitis was 10.69 times higher in cows that were milked after only teats’ washing than the cows with whole udder washing. Udder washing helps in the stimulation of udder muscles to get better milk flow and reduction in machine-on time. Pre-milking sanitation is usually considered an important factor in the prevention of mastitis and a study suggests that washing and drying of udder dramatically reduce the development of mastitis especially by environmental pathogens and improves udder health (55).

The current study showed that the odds of finding a cow with mastitis was 4.2 times higher in herds where manure removal is not done daily. The frequency of manure removal is a potential threat to the spread and transmission of SCM as manure is a rich source for the incubation of the environmental pathogens involved in SCM occurrence. This finding is in line with the previous studies (26,56,57).

Like this study, several other studies also showed the trend of prevalence increment in the early lactation stage (34,45,46,52,58). In early lactating cows, the prevalence of mastitis can be linked to, 1. diapedesis of neutrophils into the mammary gland take longer time than adult cows (15), 2. absence of dry cow therapy in the region, 3. reduced antioxidant defense mechanisms with increased oxidative stress in early lactation (59), 4. due to sensitiveness of teat muscles to any external pressure when hand milking is adopted. Contrary to this study, scientists in Kenya reported a significantly higher prevalence rate at the mid-lactation stage (60) while few scientists found a higher prevalence rate at the late lactation stage (61). There is still ambiguity that what is the actual source of the variation in the lactation stages, but it could be related to the disparities in parity, breed, age, and management practices of the sampled cows.

Floor cleanliness is directly proportional to the enhanced mastitis occurrence, respiratory problems, lameness, and hygiene of cow (62,63). Animals that were kept in muddy floor type showed a higher prevalence of mastitis occurrence, as these floors are difficult to manage and clean than concrete ones, and it is also shown in the previous study (64).

According to our study, the odds of finding a positive CMT result was 2.79 times higher in cows that were milked by machines. Unwashed or soiled milking clusters of machines and milking cups’ attachment to insufficiently wiped udder can result in cross-contamination of bacteria among cows and hence a major source of mastitis spread among herd (65). Moreover, if not properly used, milking machines can cause teat lesions hence increased susceptibility to mastitis (66).

The cows with swelled udder are prone to mastitis due to the immune system impairment in udder tissue along with the defect in the blood circulation. The inflammation disrupts the blood circulation in udder tissue that leads to the edema and leakage of milk from teats thus making the cow prone to mastitis (67). The cows with swelled udder are also prone to the open teat canal antepartum and that can lead to the increased risk of mastitis (68,69). Several studies have also shown the association between udder condition and mastitis (67,70,71).

According to the current study, the likelihood of cows with tick infestation to develop mastitis was 0.31 times higher. Ticks generally infest the udder leads to the lesions of skin and teat that help in bacterial entry to teat canals. Several other studies also showed a correlation between tick infestation and mastitis occurrence (72-75). It is strongly suggested that the ticks should be removed from the body of the cows by gentle rotation and firm move without damaging the skin and tissue of the udder (76).

The teat end lesion is an important risk factor as it can be recurrently colonized by various bacterial species (77). The odds of finding a positive SFMT result was 0.25 times higher in cows with teat end lesions. Several studies are in line with this finding (78,79).

The current study revealed an association between the use of hormones and mastitis occurrence. There is not much data available on this effect and it's still ambiguous but there is a study that is in line with this finding that hormones do affect the mastitis occurrence (80). In past, most of the researchers and dairy industries believed that some hormones especially oxytocin were helpful in milk production enhancement during lactation with no health issues but further experimentation suggested that continuous use of the hormones lead to reproductive disarray, reduced lactation period, poor milk quality, increased abortion rate, delayed puberty, and low conception rate. Moreover, the animal becomes habitual of the drug, and let down of milk will be difficult without its administration (81-84).

The present study showed an association between cows’ legs and udder hygiene score and enhanced occurrence of mastitis. Based on the score used, all the examined cows belong to slightly to very dirty legs and udders and there was a significant increase in the occurrence of mastitis as the dirtiness of cows increase. The uncleanliness of cows was the result of poor management and hygienic conditions (accumulation of urine and manure, presence of flies and ticks, and lack of wastage drainage) of the farm. Several other studies also agree with this fact (34,46,49,60).

According to the microbiological finding of this study, S. aureus was isolated from 76.9% mastitic milk samples followed by Streptococcus (61.5%). This finding is in line with other studies where S. aureus also showed as the predominant pathogen of mastitis (34,48,85-88). This can be linked to the absence of pre/post milking dipping agents, absence of dry cow therapy, invariable hand milking practices, and lack of culling of chronically infected cows.

In Pakistan, this study was the first of its kind as no previous data was available regarding the farmers’ awareness of mastitis that is crucial in the control and prevention of this disease (78,79). This study showed that there were huge gaps in farmers’ awareness and perception about the disease severity, preventive measures, economic effects, and other related issues. Among the 40 surveyed farms, all the farmers were aware of mastitis and all of them had encountered mastitis in their farm that emphasizes the prevalence of the disease while only 50% of respondents were aware of subclinical mastitis. This contrasts with the other studies, one such study reported 97% of clinical mastitis cases in the farm (89), and another study suggested only 62.1% awareness regarding clinical mastitis in the Southern Highlands of Tanzania (90). Almost 35% of respondents knew that milking mastitic cow last can prevent the disease. This is in line with the finding of the study done in Hawassa where almost 32% of farmers were aware of this (91).

Economics of mastitis decision-making was carried out by the costs of clinical and subclinical mastitis cases concerning the cost of the management system. This study along with previous studies suggested that not only in developing countries but also developed countries like Netherland where good dairy habits are followed, farmers generally underestimated the economic losses due to mastitis (34). The focus of most of the farmers was the quantity of the milk rather than quality that is the basic soundness criteria of milk for human consumption.

Culling is one of the best options to get rid of the mastitic cows and is defined as the departure of the animal from the herd because of slaughter, salvage, sale, or death (92). Unfortunately, only 5% of respondents recognized the importance of culling. Dry cow therapy is an important antibiotic therapy that is used to treat existing intramammary infections and to prevent new infections to occur in the dry period (93). About 10% of farmers were aware of dry cow therapy.

Almost 70% of farmers believed that multiparous cows are at more risk of developing mastitis than primiparous cows. This may be due to the fact the primiparous cows have a more defined immune system than multiparous cows. This study agreed with the previous study that also stated greater parity number had significantly higher chances of mastitis (94).

Mastitis is one of the most economically significant diseases of the dairy industry worldwide. The present study has shown that sub-clinical mastitis is a widely prevalent disease of the dairy herds at both cow and herd-level. The study suggested that there are several risk factors associated with the occurrence of mastitis in cows including early stage of lactation, udder and leg hygiene scores, injured teat ends, and swollen udder. Besides, the use of hormones, frequency of manure removal, udder washing, floor type, and presence of ticks also affect the occurrence of mastitis. The study also reveals that S. aureus is the predominant etiological agent of mastitis in the Rawalpindi district followed by Streptococcus. Farm owners'/attendants responses showed that they lack sufficient awareness and perception about bovine mastitis including subclinical form, prevention methods of diseases, the importance of culling of chronically infected cows, effect on the quality of milk, dry cow therapy, and side effects on the use of hormones. Therefore, a wide variety of awareness programs should be arranged throughout the country with the help of government and international agencies to teach the farmers about the prevention and management of mastitis at a broad level. Awareness and training of the farmers is essential to enhance the quantity and quality of milk. Alongside awareness, government and International agencies should also help farmers in improving their technical facilities.

SCC: Somatic cell count

CM: Clinical mastitis

SCM: Sub-clinical mastitis

SFMT: Surf field mastitis test

Ethics approval and consent to participate

Consent for publication

Not applicable.

Availability of data and materials

All data generated and analyzed during this study are included in this published article [and its Additional files].

Competing interests

The authors declare that they have no competing interests.

Funding

This study was funded by Higher Education Committee (HEC), Pakistan under the NRPU (National research program for universities) program.

Authors’ contributions

AJ and FA designed the study. AJ and AZA collected all the required data. AJ, AZA, and QA performed the experimental work. AJ and ZH analyzed and interpreted the data via statistical tools. MTU helped during sampling. AJ drafted the manuscript. FA critically and substantially revised the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors express special gratitude towards farm owners and managers included in the study for their collaboration and support during the study period. Members of the veterinary department in the surveyed district are also appreciated for their cooperation in providing information. Authors are also thankful to the editor and anonymous reviewers for their constructive comments that helped in improving the quality of this paper.

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Prevalence & risk factors analysis of bovine mastitis in dairy herds of Rawalpindi district, Pakistan; a study to estimate severity & farmers’ awareness about the disease

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