Isolation and Identification of Staphylococcus Species From Mastitic Dairy Cows and Associated Risk Factors in and Around Bahir Dar, Ethiopia

ABSTRACT

Background

Bovine mastitis remains one of the most frequent and costly diseases affecting dairy cattle, with significant impacts on health, welfare, and productivity. Staphylococcus species are among the major pathogens responsible for bovine mastitis, leading to reduced milk yield and posing a potential risk to public health. However, there has been limited research on the specific mastitis‐causing pathogens and the risk factors associated with the condition in the study area. Therefore, the objective of this study was to isolate and identify Staphylococcus species associated with bovine mastitis and to assess the risk factors linked to their occurrence.

Material and Methods

A cross‐sectional study was conducted from October 2022 to December 2023 in the Bahir Dar region to investigate the prevalence and risk factors associated with Staphylococcus species causing mastitis in dairy herds. A total of 150 lactating cows from 21 farms were included in the study, with 592 milk samples collected from the four quarters of each cow. The samples were screened via the California mastitis test and physical examination. Staphylococcus species isolates were performed on Mannitol Salt Agar, followed by biochemical testing to confirm the presence of Staphylococcus species. The STATA version 16 software was used to analyse the results of the study. An odds ratio and 95% CI were computed, and p values (p < 0.05) were considered significant. Multivariable logistic regression analysis was also used to determine the association between risk factors and cow‐level mastitis.

Result

Among the 592 milk samples collected, 119 (20.1%) samples exhibited mastitis in at least one quarter. The overall prevalence of mastitis at the farm, cow, and quarter levels was 90.5%, 48.7%, and 20.1%, respectively. Out of the total quarters examined, 33 quarters (5.5%) were positive for clinical mastitis. At the cow level, 24 cows (16%) showed clinical signs of mastitis. In addition, 86 quarters (14.52%) were positive for subclinical mastitis. At the herd level, 49 cows (32.7%) were affected by subclinical mastitis. Of the 119 mastitis‐positive samples, 80 were bacteriologically confirmed to harbour Staphylococcus species. A total of eight different Staphylococcus species were identified. Among these, Staphylococcus chromogenes was the most prevalent isolate (19%), followed by Staphylococcus aureus, Staphylococcus hyicus, and Staphylococcus epidermidis (each 15%), Staphylococcus haemolyticus (11%), Staphylococcus simulans and Staphylococcus xylosus (each 10%), and Staphylococcus intermedius (5%). The risk factors contributing to the prevalence of mastitis were identified as cows' age, lactation length, herd size, parity, and hygienic status, all of which demonstrated statistically significant associations (p < 0.05).

Conclusions

Mastitis caused by Staphylococcus is a significant issue in dairy production within the study area. Several risk factors, including age of the cows, lactation length, herd size, parity, and hygienic status, were found to be significantly associated with the prevalence of mastitis in the cows. While these factors showed a clear correlation with mastitis prevalence, breed and management type did not appear to significantly influence the occurrence of mastitis. Additionally, subclinical mastitis was more commonly observed than clinical mastitis.

Among the 592 milk samples collected, 119 (20.1%) samples exhibited mastitis in at least one quarter. Of the 119 mastitis‐positive samples, 80 were bacteriologically confirmed to harbour Staphylococcus species. A total of eight different Staphylococcus species were identified. Among these, Staphylococcus chromogenes was the most prevalent isolate (19%).

Abbreviations

AOR

Adjusted odds ratio

CI

Confidence interval

CMT

California Mastitis Test

MSA

Mannitol Salt Agar

NA

Nutrient Agar

SCM

Subclinical mastitis

VP

Voges–Proskauer

1. Introduction

In Ethiopia, dairy farming is predominantly practiced under an extensive production system, with smallholder farmers in rural areas playing a central role. However, intensive and semi‐intensive dairy production systems, particularly in areas with better market access, are becoming increasingly popular among dairy producers. Despite this growth, the dairy sector continues to face several challenges, including infectious and non‐infectious diseases, the low genetic potential of indigenous cattle breeds, inadequate feed and water resources, and the slow adoption of improved dairy production technologies (Fulasa and Deressa 2021). Among the various diseases affecting dairy cattle, mastitis is one of the most prevalent and economically important, causing substantial financial losses worldwide (Mária et al. 2026). The disease has significant adverse effects on milk production, milk quality, animal health, and welfare (Le Maréchal et al. 2011). These impacts include reduced milk yield, increased treatment costs, higher culling rates, and, in severe cases, death due to disease‐related complications (Fulasa and Deressa 2021).

Mastitis in bovines remains one of the most frequent and costly diseases with significant impacts on the health, welfare, and productivity of dairy cattle. The Staphylococcus species are the primary pathogens responsible for mastitis in lactating dairy cows, leading to reduced milk yield and posing a potential risk to public health. The genus Staphylococci, particularly Staphylococcus aureus and coagulase‐negative staphylococci (CNS), are among the most common etiological agents of bovine mastitis worldwide. Staphylococcus aureus is widely regarded as a major pathogen of mastitis, typically causing clinical mastitis, whereas CNS has traditionally been considered minor pathogens, often causing subclinical mastitis (Naranjo‐Lucena and Slowey 2023). However, recent reports have highlighted increasing cases of both clinical and subclinical mastitis caused by various CNS species, establishing them as important mastitis‐causing pathogens (Aweke and Mekibib 2017; De Visscher et al. 2017; Ferreira et al. 2022; Li et al. 2015). Among the CNS species, S. chromogenes, S. epidermidis, S. haemolyticus, S. simulans, and S. xylosus have been commonly isolated from bovine milk samples (Leroy et al. 2015).

Several factors influence the occurrence of mastitis; however, relatively few studies have investigated cow‐level factors associated with its development. The risk of mastitis is higher during early lactation and increases with greater parity and higher milk production (Hisira et al. 2025). In addition, the prevalence of infected quarters increases with the age of cattle, peaking at approximately seven years of age (Radostits et al. 2007). Predisposing factors such as poor management practices, inadequate hygiene, teat injuries, and malfunctioning milking machines facilitate the entry of infectious agents and accelerate disease progression (Regecová et al. 2022). When these predisposing factors are present, pathogens can enter through the teat canal and establish infection. Major risk factors include age, stage of lactation, milk yield, hereditary influences, trauma, hygiene status, and other management‐related factors (Zigo et al. 2020).

In Ethiopia, especially in and around Bahir Dar, bovine mastitis is a significant concern for dairy farms, with a notably higher prevalence of subclinical mastitis compared to clinical mastitis. The prevalence of subclinical mastitis typically ranges from 25.4% to 73.3%, while clinical mastitis ranges from 3.2% to 26.5% (Fox et al. 1996). This high prevalence of both clinical and subclinical mastitis contributes to substantial economic losses in the dairy sector nationwide. However, there has been limited research on the specific mastitis‐causing pathogens and the risk factors associated with the condition. The objective of this study was to isolate and identify the Staphylococcus species responsible for bovine mastitis and to determine the risk factors linked to their presence. Identifying the causative agents and the associated risk factors is crucial for developing effective control strategies for mastitis, and the findings of this study are valuable for animal health professionals and policymakers.

2. Materials and Methods

2.1. Description of the Study Area

The study was conducted in and around Bahir Dar town. It is the capital city of the Amhara region and is located approximately 565 km northwest of Addis Ababa between 11°27´1.82´´ and 11°39´7.56´´ N and 37°16´36.75´´ E and 37°31´48.06´´ E (Figure 1). In the study area, there were a total of 84 dairy farms. According to the classification system proposed by Aweke and Mekibib (2017), these farms were categorised into three groups on the basis of herd size: 24 were classified as large‐scale farms, each with 21 or more lactating cows; 38 as medium‐scale farms, with herd sizes ranging from 11 to 20 lactating cows; and 22 as small‐scale farms, each with 10 or fewer lactating cows. In a study conducted by Yayeh et al. (2017), the average daily milk yield (DMY) was recorded as 7.8 L in the study area. According to Yeserah et al. (2020), there is a notable lack of standardised hygienic practices followed by producers, transporters, and processors throughout the milk production chain in the study area.

FIGURE 1.

Map of the study area (prepared by the authors using QGIS version 2.18.0).

2.2. Study Animals

Dairy cows (n = 150) managed with intensive (135 dairy cows) or semi‐intensive (35 dairy cows) systems in the study areas were selected for sample collection. Milk sample collection was from local and crossbred dairy cattle breeds found on 21 selected dairy farms. For convenience, the farms were grouped into three categories according to their sizes: small (farms having ≤ 10 dairy cows), medium (farms having 11 to 20), and large scale (farms having ≥ 21 lactating cows) (Aweke and Mekibib 2017). Various information regarding the potential risk factors, including management type, parity, breed, age, floor hygiene, lactation stage, and herd size of each lactating dairy cow in the selected dairy farms, was recorded before the sample was collected.

2.3. Study Design Sampling Methods

A cross‐sectional study design was employed from October 2022 to December 2023 to isolate and identify the Staphylococcus species responsible for bovine mastitis and to determine the risk factors linked to their presence. In the study area, 84 dairy farms were managed in closed houses with different floor types, such as mud, wood, and stone; with different wall types, such as earthen, wood, cement, and stone; and with different roof types, such as iron sheets and plastic sheets. Dairy cows are also managed under zero grazing. Most dairy farms clean twice a day and have ventilation. The main feeds used by dairy farms are crop residue, hay, and concentrated feed. The cows were hand‐milked twice per day. The breeding method used in dairy farms is artificial insemination. From 84 dairy farms, 21 farms (25%) were selected by using a simple random technique for sample collection. From the selected farms, all lactating cows (n = 150) were included for sampling. This included both clinically symptomatic and asymptomatic (subclinical) cows that were assessed for mastitis. A total of 592 teat quarters were examined through clinical assessment and subclinical examination. Samples for clinical mastitis were collected on the basis of the presence of observable clinical signs of mastitis, including swelling, redness, heat, and abnormal milk characteristics such as clots, discoloration, or reduced yield, whereas for subclinical mastitis, the California mastitis test (CMT) was used as a screening tool.

2.4. Sample Collection and Laboratory Analysis

2.4.1. Milk Sample Collection and California Mastitis Test

Milk from all quarters except blind teats was collected from 150 dairy cows. Before collection, quarters were soaked with 70% ethanol and dried off using a towel. Initially, one to two drops of milk were discarded, and then 15 mL of milk from each of the four quarters (left front, LF; left hind, LH; right front, RF; and right hind, RH) was collected aseptically in sterile plastic tubes (15 mL Falcon Tube). Immediately after collection, milk samples were subjected to CMT to diagnose subclinical mastitis. The California mastitis test was conducted to identify the presence of subclinical mastitis according to the standard procedure described by Mungube et al. (2005). A squirt of milk from each quarter of the udder was placed in each of the four CMT paddles, and an equal amount of the reagent, approximately 4–5 drops, was added. A gentle circular motion was applied in a horizontal plane; positive samples showed gel formation within a few seconds. The results was scored based on the gel formation and categorised as negative if there was no gel formation and recorded as 0 (zero) or positive if there was gel formation ranging from trace to +3 (Hoque et al. 2018), 1 (weak positive), 2 (distinct positive), and 3 (strong positive) based on gel formation (Kim et al. 2019). The CMT scores of 1 and 2 were considered indicators of subclinical mastitis and 3 for clinical mastitis. Positive cows had at least one quarter with a CMT score of >1. In total, 600‐quart milk samples were tested, and all CMT‐positive samples were collected aseptically into the sterile plastic tubes (15 mL/sample). Samplings were done from all quarters of CMT‐positive cows and were transported to the Bahir Dar Animal Disease Surveillance, Investigation and Diagnostic Laboratory Department of Microbiology using an icebox.

2.5. Isolation and Identification of Staphylococcus Spp

After on‐farm screening, the CMT‐positive milk samples were preserved in an icebox (Frey et al. 2013) and transported to the Department of Microbiology at the Bahir Dar Animal Disease Surveillance, Investigation, and Diagnostic Laboratory for further bacteriological examination. A total of 119 mastitis‐positive samples were analysed bacteriologically through culturing, Gram staining, and biochemical identification tests, including catalase, coagulase, and Voges–Proskauer (VP) tests, to confirm the species‐level identification of Staphylococcus. The bacteria were identified based on colony morphology, haemolytic patterns, growth on Mannitol Salt Agar and other biochemical characteristics, as described by Thorberg and Brändström (2000) (some of the techniques are shown in Annex 1 and 2). Based on these procedures, 80 out of the 119 mastitis‐positive samples were identified as Staphylococcus spp. (Table 1).

TABLE 1.

Biochemical characteristics used for identification of Staphylococcus species.

No.	Staphylococcus species	Growth on MSA	Colony characteristics on NA	Haemolysis	Coagulase	Mannitol fermentation	VP test	References
1	S. aureus	+	Yellow colonies	β‐haemolysis	+	+	+	Thorberg and Brändström (2000)
2	S. intermedius	+	White colonies	α‐haemolysis	+	Delayed +	−	Thorberg and Brändström (2000)
3	S. chromogenes	+	White colonies	−	−	−	−	Thorberg and Brändström (2000)
4	S. epidermidis	+	White colonies	−	−	−	+	Thorberg and Brändström (2000)
5	S. hyicus	+	White colonies	α‐haemolysis	+	−	−	Thorberg and Brändström (2000)
6	S. simulans	+	White colonies	α‐haemolysis	−	+	−	Thorberg and Brändström (2000)
7	S. haemolyticus	+	White colonies	β‐haemolysis	−	+	+	Thorberg and Brändström (2000)
8	S. xylosus	+	White colonies	−	−	+	−	Thorberg and Brändström (2000)

MSA, Mannitol Salt Agar; NA, Nutrient Agar; VP, Voges–Proskauer; +, positive reaction; −, negative reaction; β‐haemolysis, complete haemolysis; α‐haemolysis, partial haemolysis.

Note: ‘Delayed +’ indicates a delayed positive reaction.

2.6. Data Analysis

After coding, the data was entered into an MS Excel spreadsheet and checked for accuracy. The data was properly recorded in a Microsoft Excel spreadsheet at each step of the laboratory work, from the isolation and identification process of Staphylococcus spp. The STATA version 16 software was used to analyse the results of the study. Descriptive statistics were used to calculate the proportions and frequencies of all explanatory variables. An odds ratio and 95% CI were computed, and p values (p < 0.05) were considered significant. Multivariate logistic regression analysis was also used to determine the association between risk factors and cow‐level mastitis. Univariable logistic regression analysis was performed before fitting variables into the multivariable logistic regression model to assess the association between each predictor and the outcome and to screen candidate variables for the multivariable analysis. Confounding and interaction effects were evaluated in the final model. A backward stepwise elimination procedure was used to build the final model while assessing potential confounding. Confounding was considered present if the coefficients of the remaining variables changed by at least 25% after the removal of a nonsignificant variable (p > 0.05) from the model.

3. Results

3.1. Mastitis and Associated Risk Factors

A total of 150 lactating cows, excluding 8 blind teats, comprising 592 functional quarters, were subjected to examination to assess the prevalence of mastitis. A total of 592 quarters were evaluated through clinical examination, with 33 quarters (5.5%) testing positive for clinical mastitis. In terms of cows, 24 cows (16%) exhibited clinical signs of mastitis, corresponding to 5.5% of the individual quarters. This indicates that a minority of cows in the herd were showing overt clinical symptoms of the disease. CMT, a commonly used method to detect subclinical mastitis, revealed that 86 quarters (14.52%) tested positive for elevated somatic cell counts, indicative of subclinical mastitis. At the cow level, 49 cows (32.7%) were CMT positive, suggesting a substantial burden of subclinical mastitis within the herd.

The study identified several potential risk factors associated with the occurrence of Staphylococcus spp. mastitis. Among these, age, lactation stage, herd size, and parity showed positive associations with mastitis (AOR > 1), indicating increased odds of disease. In contrast, breed and management type were not significantly associated with mastitis and showed AOR values < 1, suggesting no increased risk in the studied population. Additionally, the AOR value for hygienic status (< 1) indicates that good hygiene has a protective effect against mastitis, whereas poor hygiene increases the risk of disease occurrence. The current study indicated that cows managed in large herd sizes had approximately 1.878 times higher odds of developing mastitis compared to cows in small herd sizes. A statistically significant association was observed between herd size and the occurrence of mastitis (AOR = 1.878, p = 0.023, and 95% CI = 1.902–3.231). The odds ratio for floor hygiene (AOR = 0.164, p = 0.001, and 95% CI = 0.067–0.4038) is less than 1, indicating a protective effect of the reference category (likely good hygiene). This suggests that good floor hygiene is associated with a significantly reduced risk of mastitis, whereas poor floor hygiene increases the likelihood of mastitis occurrence. Regarding parity, the odds ratio (AOR = 1.983, p = 0.020, and 95% CI = 1.113–3.531) indicates that cows with high parity (>4 calves) had approximately 1.983 times higher odds of developing mastitis compared to cows with lower parity (≤2 calves). This association was statistically significant. Cows in late lactation stages (≥7 months) had approximately 2.18 times higher odds of developing mastitis compared to cows in early lactation stages (≤2 months). This association was statistically significant (AOR = 2.18, p = 0.003, 95% CI = 1.193–3.983). Similarly, the odds ratio for age (AOR = 2.567, p = 0.011) indicates that cows older than 5 years had approximately 2.6 times higher odds of developing mastitis compared to cows aged less than 5 years, and this association was statistically significant (Table 2).

TABLE 2.

Multivariable logistic regression analysis of risk factors associated with cow‐level mastitis.

Independent variable	Category	No. examined	No. positive	Positive (%)	AOR	p‐value	95% CI
Breed	Local (Ref.)	10	1	10.0	1.00	−	−
	Crossbred	140	60	42.8	0.97	0.171	0.034–2.734
Age (years)	≤5 (Ref.)	98	29	29.6	1.00	−	−
	>5	52	32	61.5	2.567	0.011	1.005–6.555
Lactation length (months)	≤2 (Ref.)	36	10	27.7	1.00	−	−
	3–6	68	24	35.3	1.798	0.042	1.128–3.213
	≥7	46	27	58.7	2.180	0.003	1.193–3.983
Herd size (cows)	≤10 (Ref.)	46	12	26.1	1.00	−	−
	11–20	55	22	40.0	1.356	0.035	1.023–2.674
	>20	49	27	55.1	1.878	0.023	1.502–3.231
Management type	Semi‐intensive (Ref.)	15	4	26.7	1.00	−	−
	Intensive	135	57	42.2	0.658	0.693	0.082–5.258
Parity (calves)	≤2 (Ref.)	60	14	23.3	1.00	−	−
	3–4	55	26	47.3	1.327	0.040	1.103–2.671
	>4	35	21	60.0	1.983	0.020	1.113–3.531
Hygienic status	Poor (Ref.)	85	47	55.3	1.00	−	−
	Good	65	14	21.5	0.164	<0.001	0.067–0.404

AOR, adjusted odds ratio; CI, confidence interval; Ref., reference category.

Note: Percentages are calculated as the number of mastitis‐positive cows divided by the total number of cows in each category. AORs, p‐values and 95% CIs are from the multivariable logistic regression model. Statistically significant associations are shown in bold in the manuscript.

3.2. Isolation Rate Mastitis Pathogen

Based on the findings of the CMT and clinical examination, an overall 90.5% clinical positivity for bovine mastitis was present in the study areas at the farm level. However, overall cow‐level and quarter‐level mastitis were 48.7% and 20.1%, respectively, based on CMT and physical scoring, of which 16% and 5.57% were clinical mastitis and 32.7% and 14.52% were subclinical mastitis, respectively (Table 3).

TABLE 3.

Occurrence of mastitis at farm, cow and quarter levels.

Observation level	No. examined	No. positive	Overall mastitis, n (%)	Clinical mastitis, n (%)	Subclinical mastitis, n (%)
Farm level	21	19	90.5	−	−
Cow level	150	73	48.7	24 (16.0)	49 (32.7)
Quarter level	592	119	20.1	33 (5.6)	86 (14.5)

Note: Prevalence was calculated as the number of positive farms, cows or quarters divided by the corresponding number examined. At the cow level, clinical and subclinical mastitis categories are presented as proportions of all cows examined. At the quarter level, clinical and subclinical mastitis are presented as proportions of all quarters examined. The number of examined quarters was 592 because eight quarters were not available for examination.

Bacteriology examination indicates, an overall 80.95% (17/21) isolation rate of farm‐level mastitis was caused by Staphylococcus species in the study areas, of which clinical and subclinical mastitis was 11.33% and 29.37% at cow level and 3.7% and 10%, quarter level respectively (Table 4).

TABLE 4.

Isolation of Staphylococcus species from mastitis cases at farm, cow and quarter levels.

Observation level	No. examined	No. positive for Staphylococcus spp.	Overall isolation, n (%)	Clinical mastitis, n (%)	Subclinical mastitis, n (%)
Farm level	21	17	81.0	−	−
Cow level	150	61	40.7	17 (11.3)	44 (29.3)
Quarter level	592	80	13.5	22 (3.7)	58 (9.8)

Note: Percentages represent the proportion of farms, cows or quarters positive for Staphylococcus spp. among those examined at the respective level. At the cow level, 17 (11.3%) cows had clinical mastitis and 44 (29.3%) had subclinical mastitis caused by Staphylococcus spp. At the quarter level, 22 (3.7%) quarters had clinical mastitis and 58 (9.8%) had subclinical mastitis associated with Staphylococcus spp.

The overall occurrence of cow‐ and quarter‐level Staphylococcus species in mastitis cases was 40.67 and 13.5%, respectively. In both cases, the pathogen was predominantly associated with subclinical mastitis rather than clinical mastitis. From 592 samples, 80 Staphylococcus species isolates were detected, of which 58 (9.8%) isolates were from SCM and 22 (3.7%) from CM samples. The number of isolates also varied according to udder quarter location and remained highest in left rear (LH) quarters (24 (16.33%)), followed by right front (23 (15.54%)), right rear (18 (12.16%)), and left front (15 (10.06%)) quarters, respectively (Table 5). Generally, hindquarters were more affected than front quarters, with the prevalence of 42 (14.23%) and 38 (12.8%), respectively (Table 6).

TABLE 5.

Quarter‐level prevalence of overall mastitis and isolation of Staphylococcus spp.

Quarter	No. examined	Overall mastitis, n (%)	Staphylococcus spp.‐positive, n (%)
Right hind (RH)	148	30 (20.3)	18 (12.2)
Right front (RF)	148	30 (20.3)	23 (15.5)
Left front (LF)	149	25 (16.8)	15 (10.1)
Left hind (LH)	147	34 (23.1)	24 (16.3)
Total	592	119 (20.1)	80 (13.5)

RH, right hind; RF, right front; LF, left front; LH, left hind.

Note: Percentages were calculated using the number of quarters examined in each anatomical position as the denominator.

TABLE 6.

Comparison of mastitis prevalence and Staphylococcus spp. isolation between front and hind quarters.

Quarter position	No. examined	Overall mastitis, n (%)	Staphylococcus spp.‐positive, n (%)
Front quarters	297	55 (18.5)	38 (12.8)
Hind quarters	295	64 (21.7)	42 (14.2)
Total	592	119 (20.1)	80 (13.5)

Note: Percentages were calculated using the number of quarters examined in each position as the denominator.

Based on the bacteriological methods, 80 (13.5%) presumptive Staphylococcus species isolates were identified. The frequencies of the different Staphylococcus species are presented in Figure 2. Overall, S. chromogenes was the predominant species, accounting for 19% of the total isolates, followed by S. aureus, S. hyicus, and S. epidermidis (each 15%), S. haemolyticus (11%), S. simulans and S. xylosus (each 10%), and S. intermedius (5%).

FIGURE 2.

The frequency of Staphylococcus spp. in milk samples.

4. Discussion

The study was conducted in and around Bahir Dar town and aimed at isolation and identification and their risk factors associated with Staphylococcus species isolated from bovine mastitis. Cattle mastitis remains a serious and common disease in animals with significant economic losses in the dairy industry worldwide (Atasever 2012; Momtaz et al. 2010); therefore, knowing that mastitis‐causing bacteria and their risk factors are determinants is crucial to controlling intramammary infection (El‐Behiry et al. 2015). The present study is one of the few bovine mastitis studies conducted in and around Bahir Dar, in which the overall prevalence of mastitis at both cow and quarter levels, as well as the associated risk factors, were assessed.

The present study revealed that 80.95% of the dairy farms had at least one cow affected by Staphylococcus‐associated mastitis. However, direct comparison of the farm‐level prevalence with previous studies conducted in and around Bahir Dar was not possible because similar farm‐level investigations have not been reported.

The overall cow‐level isolation rate of mastitis due to Staphylococcus species was 40.7%, of which the majority of the cows, 29.37%, were suffering from subclinical mastitis (SCM), and the rest, 11.33%, of cows were affected with clinical mastitis. This finding is comparable with the report of (Mellenberger 2001). But it is higher than the report of Molalegne Bitew et al. (2010), who reported 28.2% prevalence in Bahir Dar town from Zebu cows, and the report of (Lo and Wang 2011), 34.9% in southern Ethiopia. The prevalence of quarter‐level mastitis in this study was 13.5% (75.0% SCM and 25% CM). This quarter‐level mastitis prevalence was also higher than (Hailemelekot et al. 2021), who reported 10.43% prevalence in Bahir Dar town from Zebu cows, but similar to (Molalegne Bitew et al. 2010), who reported 13.6% in small‐scale dairy farms of Adama town. However, it was higher than the result of (Lo and Wang 2011), who reported 2.4% in small‐scale dairy farms of Selalle, and lower than the report of (Getahun et al. 2008), who reported 29.0% in Adama dairy farms. Differences in husbandry practices like good hygienic practices and other interventions between different areas might be the reason for the difference in prevalence reported by various researchers.

The association between mastitis and age of cows was significant. Old cows in this study were more susceptible to mastitis infection than young and adult cows. The increasing occurrence of mastitis with increasing age was in agreement with the findings by Kerro Dego and Tareke (2003), who found that the risk of clinical and subclinical mastitis increases significantly with the advancing age of the cow. This might be due to the increased opportunity of infection with time and the prolonged duration of infection, especially in a herd without a mastitis control program (Ayano et al. 2013).

The isolation rate of mastitis was higher in poor hygienic status than in good ones. This finding is in agreement with reports made by different authors in different parts of the country (Almaw et al. 2008; Mekonnen and Tesfaye 2010) who found hygienic status as one of the most associated risk factors for mastitis. This is due to associations with poor sanitation and cows, which were maintained in dirty and muddy common barns with bedding materials that favour the proliferation and transmission of mastitis pathogens.

Lactation length was found to be a risk factor for mastitis, and the prevalence was higher in the late stage than in mid‐ and early lactation. The result of the current study is in line with the finding of Getahun et al. (2008) and Almaw et al. (2008) that the prevalence of mastitis was higher in the late stage of lactation. But it disagrees with El‐Behiry et al. (2015) and Mellenberger (2001), who reported mastitis prevalence was higher in early lactation. The variation could be attributed to different management practices (floor hygiene, dry cow therapy practices) in different study areas. The highest prevalence during late lactation is an indication of infection, probably due to increased exposure frequency of teats to the causative agents.

As the cows increased in their parity, it showed that they were more highly affected by mastitis than cows with low parity. This is in agreement with Almaw et al. (2008), Molalegne et al. (2010), and Abebe et al. (2016), which identified parity as a risk factor for mastitis in the study conducted at different parts of Ethiopia. The association might be due to the increased opportunity of infection with time and the prolonged duration of infection, especially in a herd without a mastitis control program, and also as animals produce more and more calves, there is risk of contracting bacteria responsible for mastitis, and as age goes on, the level of immunity reduces.

In the current study, the isolation rate of subclinical mastitis was higher than clinical mastitis at both the cow level and quarter level in all mastitis cases and mastitis due to Staphylococcus species. In most reports, including the present study, clinical mastitis is far lower than subclinical mastitis. This could be attributed to little attention given to subclinical mastitis, as the infected animal shows no obvious clinical symptoms and secretes normal milk. Therefore, farmers are not well informed about invisible loss from subclinical mastitis. In Ethiopia, the subclinical forms of mastitis received little attention, and efforts have been concentrated on the treatment of clinical cases (Abebe et al. 2016; Ismael 2018).

In this study, the breed was not significantly influenced by the occurrence of mastitis. In contrast to this study, the breed has a significant influence on the occurrence of mastitis (Almaw et al. 2009) in Gondar town and its surroundings (Sori et al. 2005) in Gondar town and its surroundings, in and around Sebeta. Mastitis occurrence among breeds might reflect the differences in management (floor hygiene, dry‐cow therapy, and culling of older cows) rather than a true genetic difference (Radostits et al. 2007).

Out of 592 screened teats, 80 (13.5%) teat quarters were infected with mastitis due to Staphylococcus species. The left hind (16.33%) and right front (15.54%) were the most infected teat quarters during the study. The prevalence of mastitis at the quarter level was higher in the left hindquarters than in the front quarter, especially since the left hindquarters were more affected. This is because the hindquarters are highly predisposed to contamination with dirt. In addition to this, a large amount of milk is produced from hindquarters, and as a result, the pressure on the teat canal forces the canals to be opened widely, which allows the entrance of microbes. The observation of blind quarters in this study might be an indication of a serious mastitis problem on the farms and of the absence of culling that should have served to remove a source of mammary pathogens for the cows. This finding agrees with the report from Abebe et al. (2016), but contrary to the study of Nigeria (Shittu et al. 2012), it reported more bovine mastitis in the left front.

The most commonly isolated CNS species in the study herds, regardless of inflammatory reaction in the udder quarter, were S. chromogenes, S. epidermidis, S. haemolyticus, S. simulans, and S. xylosus. The findings are in line with a Danish study of early lactation heifers in multiple farms (Aarestrup and Jensen 1997). But in a Canadian study in herds with a high prevalence of CNS, S. hominis, S. sciuri and S. xylosus were the most prevalent species, followed by S. epidermidis and S. warneri (Davidson et al. 1992). From which, S. chromogenes (19%), S. epidermidis (15%) and S. haemolyticus (11%) were the most prevalent CNS species isolated from mastitis‐affected cows. The isolation rate of these CNS species was agreed upon in the previous study of the United States, Canada, and Argentina, which was reported by De Visscher et al. (2017), Hasan et al. (2022), Fazal et al. (2023), and Raspanti et al. (2016), regardless of their percentage. Generally, the present study indicates that the age of cows, hygienic status, parity, breed, and herd size should be considered important factors in dairy farming systems, as they have a significant effect on the occurrence of mastitis in dairy cows. Therefore, dairy farm owners should implement appropriate biosecurity and biosafety measures to reduce the risk of mastitis. In addition, animal health professionals should regularly educate and advise dairy farmers on mastitis prevention and conduct periodic farm supervision to improve herd health and reduce the occurrence of mastitis.

5. Conclusion and Recommendations

Mastitis caused by Staphylococcus spp. represents a significant challenge to dairy production in the study area. The present study identified several key risk factors strongly associated with the prevalence of mastitis in cows, including age, lactation stage, and herd size, parity, and hygienic status of the farm. In contrast, breed and management type were not found to have a statistically significant association with mastitis prevalence. Notably, subclinical mastitis was more prevalent than clinical mastitis. In addition, the hindquarters were more frequently affected than the front quarters. From a bacteriological perspective, eight different Staphylococcus species were identified as presumptive causative agents in the affected cows. To reduce the prevalence of Staphylococcus‐associated mastitis and improve milk production in the study area, appropriate control strategies such as improving hygienic status, implementing dry cow therapy, and culling older cows should be adopted.

6. Limitation of the Study

In this study, the identification of Staphylococcus species was performed using conventional bacteriological methods because species‐specific primers required for molecular identification were not available at the time of the study. However, this manuscript represents the second part of the research. The first part, which included molecular detection of virulence genes and antimicrobial susceptibility testing, has already been published in a BMC journal. Therefore, these analyses are not included in the present manuscript. Nevertheless, the absence of molecular confirmation of Staphylococcus species identification and antimicrobial susceptibility testing in this manuscript should be considered a limitation of the study. In addition, due to time and budget constraints, the study included a relatively small number of dairy farms and lactating cows, which may limit the generalisability of the findings.

Author Contributions

DD conceptualised the study, designed the methodology, conducted the tests, and wrote the original paper. LT and MA participated while doing my laboratory work. HT, BT and TS contributed to manuscript editing and final preparation. All the authors contributed to the article and approved the submitted version.

Funding

The authors have nothing to report.

Ethics Statement

Bahir Dar University's ethical review committee evaluated ethical issues related to research work and approved its ethical soundness and acceptability. Thus, Bahir Dar University sent a letter of cooperation to the study area. The purpose and procedures of the study were properly explained to the farm owner and study participants, and milk samples were collected from dairy farms whose owners were willing to participate. All methods were performed according to the relevant guidelines and regulations.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: vms371282‐sup‐0001‐SuppMat.docx

Data Availability Statement

The raw data collected during this study have been included as a supporting file alongside this manuscript. Additional information regarding the data can be obtained by contacting the corresponding author.