Risk factors associated with the incidence of clinical mastitis in pasture-based dairy herds in Colombia

doi:10.21203/rs.3.rs-1656864/v1

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Research Article

Risk factors associated with the incidence of clinical mastitis in pasture-based dairy herds in Colombia

https://doi.org/10.21203/rs.3.rs-1656864/v1

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Inflammation of the mammary gland is a disease of great importance in milk production that generates significant financial losses worldwide. This study pretends to determine the occurrence, predominant pathogens, and risk factors associated with clinical mastitis in the province of Ubaté. The research required the design of a twenty-four-month longitudinal epidemiological study. Data from 504 small producers, 4233 animals in production, 133 mastitis clinical cases, and 53 microbiological cultures were analyzed using binary logistic regression models to establish the association between possible risk factors and disease. Results. The disease had an incidence rate per animal and per quarter between 2.0–4.0 and 0.6-1.0 new cases per 100 animals-quarter-year at risk. The most prevalent microorganisms isolated were Coagulase-negative staphylococci and environmental streptococci. The cow and quarter level risk factors identified were dirty mammary glands, hyperkeratosis, and nipple wounds. Herd-level risk factors include workwear exclusively for milking, the prevalence of subclinical mastitis, milking method, cleanness of the workwear, the water source used during milking, use of gloves, correct use of disinfectant, pre-milking management, handwashing before milking, and foremilk disposal. Conclusions. Preventive programs should aim to control risk factors related to hygiene and milking routine since these were associated with the presentation of clinical mastitis caused by environmental microorganisms that were the most prevalent in cows affected in this study.

Incidence rate

relative risk

bovine mastitis

Risk Factors

coagulase-negative staphylococci

livestock

Dairy

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The province of Ubaté is a very important dairy region under high-altitude conditions in the northern region of Cundinamarca. Colombia. About 600,000 L of milk per day is produced in this province, representing 20% of the department's milk production and about 4% of the national production (Garzón-Nivia, 2018). Small and medium holders own about 60% of the area's cattle inventory and produce 70% of the milk (Fedegán, 2016). Inflammation of the mammary gland (MG) generates significant financial losses worldwide for the dairy industry (Andersen et al., 2010; Breen et al., 2009). Mastitis occurs subclinically and clinically, the latter being the least prevalent in dairy herds in Colombia (Ramírez et al., 2014). Clinical mastitis (CM) produces abnormalities in the milk, such as clots, lumps, changes in color and consistency, blood presence, and pus (Hammer et al., 2012). In addition, there may be signs of inflammation such as swollen and increase, in temperature, redness, pain, and eventually systemic signs of disease such as fever and dehydration, systemic illness, and endotoxemia (Hammer et al., 2012; Vliegher et al., 2018).

In some Central and South American countries, the cow and quarter prevalence of CM varies between 1.0-5.2% and 0.5–3.7%, respectively (Bonifaz et al., 2016; Gianneechini et al., 2002; Ruiz et al., 2011). In Colombia, mastitis cow prevalence varies between 1.7–3.8% and the herd prevalence between 0.3–5.7% (Calderón et al., 2008; Ramírez et al., 2011; Trujillo et al., 2009; Trujillo et al., 2011). The cumulative incidence of CM worldwide varies from 31.0 to 47.0 new cases per 100 cows per year (Bradley et al., 2007; Gao et al., 2017; Santman-Berends et al., 2015). Recently in Antioquia, Colombia, the incidence rate of CM was estimated at 13.8 cases per 100 cows-year at risk (Ramirez et al., 2018). Likewise, Uruguay and Canada reported an incidence rate of 14.4 and 13 new cases of MC per 100 cow-year at risk, respectively (Gianneechini et al., 2002; Levison et al., 2016).

A wide variety of microorganisms causes CM with different pathogenicity and frequency among herds (Gonçalves et al., 2018; Watts, 1988). Environmental microorganisms cause the highest number of clinical mastitis cases in Brazil (28% of isolations), whereas a lower number of clinical mastitis cases were associated with isolation of contagious microorganisms and Coagulase-negative staphylococci (CNS) (Gonçalves et al., 2018). In Colombia, contagious microorganisms cause 54% of CM cases, while opportunists CNS and environmental pathogens cause 25% and 16% respectively of all clinical diagnosed cases (Ramirez et al., 2018).

Factors associated with disease at the cow level involve the calving number, age, and days in milk (DIM) (Breen et al., 2009; Oliveira et al., 2015; Tolosa et al., 2015) as well as inadequate hygiene of the animal before milking (Breen et al., 2009), low body condition (Breen et al., 2009; Tolosa et al., 2015) and breed (Oliveira et al., 2015). Specific risk factors described at the herd level involve deficiencies in animal management within the farm. Among herd-specific risk factors that favor a high incidence of CM are inadequate milking routine (Tolosa et al., 2015), the number of milked cows (Tomazi et al., 2018), average milk production (Mungube et al., 2004), inadequate management of the dry period (Green et al., 2007). In summary, knowledge of the risk factors associated with mastitis is essential to design and formulate appropriate control and prevention strategies to mitigate the adverse effects generated by CM in the herds. Despite this, no epidemiological studies on CM have been performed recently in Colombia. This research's objectives were to estimate the incidence rate of CM and determine risk factors associated with the occurrence of this disease and estimate the prevalence of isolated microorganisms in milk samples from affected animals in smallholders’ dairy herds under high altitude conditions in the northern region of Cundinamarca

Study population

The research required the design of a twenty-four-month longitudinal epidemiological study between February 2016 and February 2018. It allowed collecting data from 504 smallholders dairy farms from twenty-three cooperatives located in the northern region of Cundinamarca in the municipalities of Carmen de Carupa, Cucunubá, Fúquene, Guachetá, Lenguazaque, Simijaca, Susa, Sutatausa, Tausa, and Ubaté. A smallholder was defined based on the number of animals in production in the herd to be less than twenty.

Clinical Definitions

A clinical case of mastitis was defined when clots, color changes (translucent, reddish), pus, fibrin, or other cellular debris appeared on the milk during milking. In addition, signs of inflammation of the mammary gland (pain, redness, heat, swollen) and the presence of systemic signs such as fever and dehydration were also considered (Hammer et al., 2012; Harmon, 2017; Klaas et al., 2017). A case of clinical mastitis was considered when an animal showed any of the alterations described above in one or more quarters. Bacterial infection occurred when one or two bacteria species were isolated from sampled milk. In addition, microbiological contamination of the sample was considered when three or more bacteria species were cultured (National Mastitis Council, 2009). Finally, nonfunctional quarters showed no milk production.

Visit protocol and sampling

An epidemiological survey was applied to each producer to establish a baseline for the study and to identify management information at three levels: the quarter, the cow, and the herd. Veterinarians diagnosed CM cases bi-monthly over 24 months; on the other hand, when producers detected CM cases on their own, an extra visit was performed for verification and sampling.

Direct observation of milk at the beginning of the milking routine allowed diagnosed clinical mastitis cases by veterinarians. Also, milk samples from affected animals during the follow-up period were collected during each visit. After teat disinfection with isopropyl alcohol (70%), five milliliters of milk from the quarters affected with MC were collected in sterile tubes, refrigerated at five degrees Celsius, and transported to the microbiology laboratory for culture, isolation, and identification of pathogenic bacteria.

Risk factor assessment

Potential risk factors and their respective levels were defined based on the literature and the authors' experience. Variables were selected and defined based on herd characteristics and management, milking routine, characteristics of the cows, hygiene, and health status of the mammary gland.

Herd level information: Variables related to herd characteristics and general management were, number of animals, average herd production, milking method, the average number of DIM, bulk tank milk somatic cell count, the prevalence of teat-papilloma (warts), teat hyperkeratosis, and teat canal– prolapse. Also, the number of milkers, use of exclusive workwear for milking, the cleanness of the milker's clothes used during the milking routine, the milking area (pasture/barn), and perceived water quality. Additionally, information about the milking routine included: handwashing before and during milking, use of ties for animal restraint, use of gloves, teat /udder washing, foremilk and checking for any symptoms of mastitis or anomalies in the milk (Yes/no), site of elimination of the squirting milk follow fore milking, pre-dip (products, contact time, method, wiping dry-off the teats) and post-dip methods and products, signs of over-milking, and information regarding dry-off (radical or progressive drying-off methods), use of antibiotic therapy or additional treatments.

Individual-level Information: Information gathered from each cow included: Breed, Calving Number, DIM, daily production, body condition score, hygiene of the mammary gland, history of subclinical mastitis, presence of teat-papilloma; wounds, hyperkeratosis; and teat-canal prolapse.

Statistical analysis

The databases were evaluated, updated, and analyzed after each veterinary visit controlling for outliers, missing, inaccurate or improbable values. Prevalence, cumulative incidence, and incidence rate per animal and quarter were the measures of disease occurrence used in this study (Zambrano, 2009). Excel© was the software selected for collecting data, and IBM SPSS Statistics 23 for statistical analysis (IBM SPSS® Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp).

Measures of Disease: The prevalence of clinical mastitis (%) was estimated by dividing the number of CM cases recorded during the follow-up period over the total number of animals and quarters evaluated. The prevalence of pathogens isolated from milk samples with CM resulted from dividing the number of isolates over the total samples sent to the laboratory throughout the period. Healthy milking animals at the beginning of the follow-up period were accounting for the cohort of animals at risk, which was considered to calculate the cumulative incidence. The clinical cases of mastitis in this cohort over the total animals and quarters at risk at the beginning of the follow-up allowed the calculation of the CI of clinical mastitis, expressed in new cases per 100 cows and quarters at risk per month. The Incidence Rate of clinical mastitis was calculated by dividing the number of new cases over the two months by the sum of disease-free time in months of all animals and quarters at risk during the evaluation period, these measures allowed to estimate the rate of disease spread. The results were corrected using a factor of population adjustment (10n). IR was expressed in cases for 10n cows and quarters-month at risk.

Risk factor analysis: The association between CM and the potential risk factors at the quarter, cow, and herd levels were initially determined using a univariate model, variables with a p-value < 0.2 were selected and incorporated into a binary logistic regression model. The relative risk (RR) determined the association's magnitude between disease and risk factor exposition. Any p-value <0.05 was considered statistically significant. Hosmer & Lemeshow postulates were considered to evaluate the goodness of fit for the binary logistic regression model (Iyer et al., 2013). Factors such as age and milk production were analyzed continuously and categorically to determine whether they influenced the model, causing confusion or interaction. Possible information bias obtained from the epidemiological survey was assessed through routine veterinary visits to verify the information collected.

Characteristics of the herds

During the 24-month follow-up, five hundred and four (n = 504) herds and four thousand two hundred thirty-three (n = 4233) animals were included in the dynamic cohorts, and twenty-one thousand two hundred forty-four (n = 21,244) individual clinical evaluations were part of the research. The average number of milking animals was 6.2 ± 5.4; 61% of the herds had between 1.0–5.0 milking animals, 26% between 6.0–10.0, 11% between 11.0–20.0 only 2% of the herds milked more than 21.0 cows. Rotational grazing systems with electric fences in grasslands of kikuyo grass (Pennisetum clandestinum) and inter-planting with ryegrass (Lolium sp.) was the primary form of management and maintenance of animals. Concentrate supplementation during milking, depending on the season (more likely during the summer), and availability was a common practice. Some of the producers supplemented animals with corn silage or oats silage. The observed daily average milk production was 11.8 ± 9.1 L / day (range: 2–26 L / day), all herds milked twice a day; 92% milked manually, whereas 8% used machines. Of those, 24% had a milking facility barn, and 76% used mobile milking machines.

Occurrence of clinical mastitis

Prevalence of clinical mastitis: In each two-month follow-up period, one thousand, nine hundred and twenty-nine (1929 ± 150) lactating animals were evaluated. Table 1 summarizes the cow prevalence of clinical mastitis (CPCM) and the quarter prevalence of clinical mastitis (QPCM) between 2016 and 2018. The CPCM varied between 0.5–1.1%. The prevalence was relatively low; in 2016 fluctuated between 0.7–1.1%; the following year, a slight decrease was observed (0.4–0.6%). The variation of QPCM during the follow-up showed similar variations to those observed in CPCM; these data appear in Table 1.

Table 1

Prevalence of Clinical Mastitis per animal and per quarter in the Province of Ubaté, Cundinamarca, 2016–2018.
	I				II						III
	Jun	Ag	Oct	Dec	Feb	Apr	Jun	Ag	Oct	Dec	Feb
Cow cases	21	14	13	12	10	7	9	13	14	10	10
Quarters cases	27	23	24	21	14	8	10	22	16	12	11
Nonfunctional Quarter	34	31	43	36	42	39	35	36	38	30	26
CPCM (%)	1.1	0.8	0.8	0.7	0.5	0.4	0.5	0.6	0.6	0.5	0.5
QPCM (%)	0.4	0.3	0.4	0.3	0.2	0.1	0.1	0.3	0.2	0.2	0.1
(n) Cows	1865	1810	1643	1796	1920	1883	2000	2071	2157	2004	2075
(n) Quarters	7460	7240	6572	7184	7680	7532	8000	8284	8628	8016	8300
CPCM: Prevalence of Clinical Mastitis per cow; QPCM: Prevalence of Clinical Mastitis per quarter

Cumulative incidence of clinical mastitis: CI per animal per year range 2.0–8.0 new cases per 100 animals at risk. The cumulative incidence of MC per quarter ranges 1.0–3.0 new infected quarters per 100 quarters at risk per year.

Incidence rate of clinical mastitis

On average, forty new cases of CM occurred in the cohort in one year. The IRCM varied between 0.11–0.36 new cases per 100 animals-month at risk and 0.02–0.17 new cases per 100 quarter-month at risk. Figure 1 presented the observed changes in the incidence rate in the population evaluated.

To extend the discussion of the outcome of this measure, the annual IRCM was estimated between 2.0–4.0 new cases per 100 animals-year at risk and between 0.6–1.1 new quarters with CM for 100 quarter-year at risk.

Etiological Agents of Clinical Mastitis

A total of n = 53 cases of CM occurred between July 2016 and June 2017. The most frequent bacteria isolated were coagulase-negative staph (CNS) (43%), environmental streptococci (32%), contagious microorganisms (17%), and coliform bacteria (2%). In 6% of the cases, there was no bacterial isolation. Table 2 summarizes the microbiological cultures carried out in milk samples from animals with CM in the province of Ubaté.

Table 2

Prevalence of isolated microorganisms from the milk samples of quarters with Clinical Mastitis in the Province of Ubaté, July 2016 – June 2018
Type of Isolated Microorganism	Total (n)	Prevalence (%)
Coagulase-negative staphylococci	23	43.0
Environmental Staphylococci	17	32.0
Contagious	9	17.0
Environmental Coliform	1	2.0
No isolation	3	6.0
TOTAL	53	100.0

Factors associated with the occurrence of clinical mastitis

Table 3 summarizes the quarter and cow level risk factors associated with the incidence of CM in dairy herds in the province of Ubaté. This disease's risk is greater in those quarters that had wounds or alterations in the nipples. Additionally, inadequate hygiene of the mammary gland during the milking routine increased the risk of the occurrence of CM. In addition, the milking method was considered a factor that increased risk of CM. Table 4 summarizes the risk factors that affect the incidence of CM at the herd level, including management factors and milking routine.

Table 3

Individual-level Risk factors -associated with the incidence of Clinical Mastitis in the Province of Ubaté, Cundinamarca, 2016–2018
Variable	Category	N	RR	95% CI.		p
Variable	Category	N	RR	Inferior	Superior	p
Milking Method	Manual	1455
Milking Method	Mechanical	411	5.7	2.4	12.9	0.000*
MG Hygiene	Clean	1696
MG Hygiene	Dirty	170	4.1	1.6	10.2	0.004*
Hyperkeratosis Teat Tip	Absent	1787
Hyperkeratosis Teat Tip	Present	24	14	3.3	44.1	0.001*
Teat-wounds	Absent	2239
Teat-wounds	Present	11	19.9	2.2	73.5	0.009*
RR (Relative Risk)
95% IC (Interval of confidence at 95%)
p < 0.05 is statistically significant
No association was observed with the interaction terms included in the model.

At the individual level, high prevalence of teat-prolapse and teat-wounds showed a higher risk incidence of CM (RR = 27.1, 95% CI (4.9–72.9), p = 0.000) and (RR = 17.1, 95% CI (2.4–63.4), p = 0.006) respectively. Similarly, an association was found between the high prevalence of the subclinical form of the disease and the incidence of CM (RR = 7.6, 95% CI (1.5–30.7), p = 0.014).

At the herd level, herds with mechanical milking were almost seven times more likely to have a high incidence of disease than those where cows were milked manually (RR = 6.8, 95% CI (2.0–20.6), p = 0.002). Also, herds with more than one milker were almost two times more likely to have a high incidence of CM than herds, where only one person was responsible for milking (RR = 5.7, 95% CI (1.2–23.4), p = 0.029). Similarly, those herds in which milkers' workwear was dirty showed a higher occurrence of new cases of CM (RR = 2.7, 95% CI (1.5–3.8), p = 0.003).

Table 4

Herd-level risk factors associated with the incidence of CM in herds of the Province of Ubaté, Cundinamarca, 2016–2018.
Variable		Category	n	RR	95% CI.		p
Variable		Category	n	RR	Inferior	Superior
Characteristics and General Management of the Herd	Prevalence of Teat-prolapse	‹5%	358
	Prevalence of Teat-prolapse	≥ 5%	13	27.1	4.9	72.9	0.000*
	Prevalence of teat Wounds	≥ 5%	361
	Prevalence of teat Wounds	› 5%	14	17.1	2.4	63.4	0.006*
	Exclusively workwear For milking	Yes	33
	Exclusively workwear For milking	No	270	7.6	1.6	29.7	0.012*
	Prevalence of Subclinical mastitis	› 20%	285
	Prevalence of Subclinical mastitis	≥ 20%	78	7.6	1.5	30.7	0.014*
	Milking method	Manual	308
	Milking method	Mechanical	33	6.8	2.0	20.6	0.002*
	Cleanness of the workwear	Clean	224
	Cleanness of the workwear	Dirty	57	6.5	1.3	26.4	0.020*
	Source of water used during milking	Aqueduct	243
	Source of water used during milking	River, pool, fenced, other	81	5.7	1.5	19.3	0.011*
	Number of Milkers	1	202
	Number of Milkers	›1	173	5.7	1.2	23.4	0.029*
	Prevalence of teat-Papilloma	› 5%	270
	Prevalence of teat-Papilloma	≥ 5%	51	4.3	1.4	12.4	0.011*
	The average number of Milking cows	≤ 5	190
	The average number of Milking cows	≤ 5	122	4.0	1.3	12.0	0.018*
Milking Routine	Use of gloves	Yes	252
	Use of gloves	No	51	16.1	1.6	69.6	0.020*
	Pre-milking management	Yes	67
	Pre-milking management	No	214	15.7	3.1	51.8	0.001*
	Handwashing Before milking	Yes	222
	Handwashing Before milking	No	153	11.7	2.0	46.8	0.008*
	Foremilk disposal	Dark bottom, other	212
	Foremilk disposal	Floor	40	8.7	1.4	38.3	0.019*
	Milking dry teats	Yes	277
	Milking dry teats	No	64	5.2	1.6	15.6	0.006*
	Foremilk	Yes	238
	Foremilk	No	133	5.2	1.3	18.5	0.021*
RR (Relative Risk)
95% IC (Confidence Interval of 95%)
*p < 0.05 is statistically significant
No association was found with the interaction terms included in the model

The study findings allowed us to elucidate the determining factors in the occurrence of clinical mastitis at the quarter, cow, and herd-level in smallholders in the province of Ubaté in Colombia. The identified risk factors are essential to establish specific control and prevention measures. The prevalence of CM varied between 0.5–1.1%, while the prevalence per quarter showed variations between 0.1–0.4%, which was lower when compared with studies conducted in Antioquia and the Cundinamarca-Boyacá Highlands, where the prevalence was reported per individual to be between 2.2–4.7% and per quarter between 0.6–1.3% (Calderón et al., 2008; Ramírez et al., 2011; C. M. Trujillo et al., 2011). In this study, the population of interest belonged to small producers with 6.2 ± 5.4 milking animals, with a predominance of manual milking at 92%, and with average milk production of 11 L / day. It is essential to point out that adequate management during the milking routine was marginal at the beginning and improve during the execution of the study; additionally, producers showed little knowledge of control and preventative CM measurements. In contrast, studies in other regions of the Colombia estimated a higher prevalence of CM in medium-sized herds (minimum 29 animals), with a higher proportion of mechanical milking (> 60%), with technical assistance, and some knowledge of bovine mastitis (Calderón et al., 2008; Martínez, 2006; Ramírez et al., 2011). The differences in the results could be consistent with the methodology and epidemiological design implemented in these studies, and the differences in herd management.

There are currently no known studies that report the cumulative incidence of CM in Colombia. International studies have reported a high cumulative incidence of CM, 30–47 new cases per 100 animals at risk per year (Gao et al., 2017; Santman-Berends et al., 2015), attributed to factors such as rainfall, high level of production, and housing (Gianneechini et al., 2002). In intensive systems, the animals are under confined conditions with a higher risk of infection attributable to inadequate management of bedding, the increased animal density, and the lack of hygiene of premises (Gianneechini et al., 2002; Klaas et al., 2017). In Ubaté, intensive-housed management does not exist, and the cows grazed throughout the year. This could explain the low cumulative incidence observed (2–8 new cases / 100 animals at risk per year) in this research; therefore, it suggests a lower risk of CM of cows kept on pasture compared to intensive production systems.

The IRCM observed (2–4 new cases per 100 animals/year at risk) contrasts with a recent study in northern Antioquia, which estimated 13.8 new cases of CM / 100 animals-year at risk (Ramirez et al., 2018). Different studies carried out under confined conditions reported an IRCM range of 23–52 cases 100 cows-year at risk (Hammer et al., 2012; Karimuribo et al., 2006; Kivaria et al., 2007). Interestingly, pasture-based systems represent a low risk for CM compared to free or tie stalls (Morton et al., 2014). In housing management, the hygienic conditions of stalls, bedding, and hallways are challenging, and therefore favor the proliferation of bacteria and pathogenic environmental streptococci associated with clinical mastitis (Klaas et al., 2017).

At the individual level, some CM risk factors could suggest an increased susceptibility among animals. Hyperkeratosis at the tip of the teat caused a higher risk of CM (OR = 2.19, 95% CI: 1.26–3.83 p < 0.05) because it alters the physical barrier of the teal canal protection (Breen et al., 2009). Another study also demonstrated a higher bacterial load on the skin of the teat in the presence of hyperkeratosis (Paduch et al., 2012). Furthermore, the incidence of other lesions such as teat-prolapse and papillomas determines a greater susceptibility of ascendant bacterial infections into the mammary gland (Kivaria et al., 2007; Tolosa et al., 2015). Together, our findings confirm that the disruption of the sphincter anatomy coupled with the disruption of the teat skin may alter the innate defense mechanisms of the mammary gland leading to a high rate of bacterial colonization and infection (Meglia et al., 2001). Moreover, teat skin lesions are associated with the proliferation of coagulase-negative staphylococci, the most prevalent pathogen cultured in the present study (43.0%) (De Visscher et al., 2014). The high bacterial load resulting from skin disruption combined with the lack of a proper routine of disinfection of the teat may favor CM's incidence in this study. The udder hygiene score allows assessing the influence of organic matter, mud, or dirt as a risk factor for clinical mastitis (Kivaria et al., 2007). Animals with more than 50% of the MG covered with dirt had a higher risk of having CM (RR = 4.1, 95% CI (1.6-10-2), p = 0.004). This observation is consistent with the results of Breen et al., 2009, and highlights the importance of controlling situations that promote environmental microorganisms' proliferation (Klaas et al., 2017; Ruegg, 2012).

Interestingly, the isolation frequency of coliforms in this study was low (2.0%) and did not seem to represent a problem compared to infections caused for environmental streptococci (32.0%). In the grazing systems developed in the Province of Ubaté, the milking area is the same where animals stay all day long. So, it is common to deal with unfavorable conditions such as waterlogging and accumulation of mud and manure in places where animals grouped, such as the milking site. For this reason, it is possible to explain the high prevalence of environmental streptococci find in these herds (Gianneechini et al., 2002). It is important to note that the presence of organic matter covering either the udder or the teats may impair the pre-and post-dip disinfectant effect and therefore increase the risk of bacterial survival and infection (Munoz et al., 2008).

Other observed factors that may contribute to the risk of clinical infections commonly associated with poor milking hygiene were the lack of hygiene of the milkers' hands, the paucity of fully dry-off the teats before milking, and the use of dirty workwear or clothes not suitable for milking. Amusingly, factors such as exclusively workwear for milking, cleanness of the workwear, and water source used during milking were associated with the incidence of clinical mastitis in this research. There is no other reported association between clinical mastitis and these factors in the literature, however, they should be considered additional environmental streptococci sources.

Herds using milking machines were at a higher risk of developing CM (RR = 6.8, 95% CI (2.0-20.6), p = 0.002). Contagious microorganisms infect healthy animals through the teat cups or the operators' hands during milking (Klaas et al., 2017; Ruegg, 2012). Likewise, the transmission of infection may increase due to the inappropriate use of milking gloves and disinfectants and milking equipment with little maintenance and poorly calibrated (Keefe, 2012; Ruegg, 2012). The high prevalence of contagious microorganisms (17.0%) observed in conjunction with inadequate handwashing, the non-use of milking gloves, and the use of milking machines with low maintenance validate the importance of these factors in the transmission of the disease. A proper milking routine reduces the transmission of this type of microorganism, preventing the appearance of new disease cases. During this study, it was possible to assess that the average interval of maintenance and calibration of the milking machine was two years, which without a doubt, may increase opportunities and sources of bacterial exposure. On the other hand, an unappropriated use of detergents for washing the milking equipment may facilitate de growth of contagious microorganisms. An observed common practice in the present study was washing the milking system with alkaline and acid detergents and hot water just once a week. The lack of hygiene (De Visscher et al., 2014), coupled with the execution of inadequate practices of maintenance, washing, and disinfection of the equipment (National Mastitis Council, 2015; Ruegg, 2012), have been determining factors in the clinical presentation of mastitis by these pathogens.

This study's estimated CM incidence was relatively low (range 2.0–4.0 new cases per 100 animals-year at risk). Nevertheless, clinical infections were caused primarily by coagulase-negative staphylococci (43%) and environmental streptococci (32%). These findings highlight the importance of establishing prevention and control programs oriented to target identified risk factors such as exclusive workwear for milking, the prevalence of subclinical mastitis, milking method, cleanness of the workwear, source of water used during milking, use of gloves, correct use of disinfectant, pre-milking management, handwashing before milking and foremilk disposal.

Acknowledgements

The authors are thankful for funding from el Sistema General de Regalias and La Gobernación de Cundinamarca, Colombia. They are also thankful to Smallholders in La Provincia de Ubaté who spent their invaluable time to provide the data for the current study

Author contributions

Conceptualization: D.J.G.A., J.Z.V, and C.J.E. Methodology: D.J.G.A and J.Z.V. Data analysis: D.J.G.A. and J.Z.V. Investigation: D.J.G.A and M.V.G. Resources: D.J.G.A and M.V.G. Data curation: D.J.G.A. and J.Z.V. Writing—original draft preparation: D.J.G.A. Writing—review and editing: J.Z.V. and C.J.E. Visualization: D.J.G.A., J.Z.V., and C.J.E. All authors have revised and agreed to the submitted version of the manuscript.

Funding

This research was funded by el Sistema General de Regalias and La Gobernación de Cundinamarca, Colombia.

Availability of data and material

The data presented in the current study are available on request from the corresponding author.

Ethical consideration

The bioethical committee of Facultad de Medicina Veterinaria y de Zootecnia de la Universidad Nacional de Colombia approved this study for animal research (CB-043-2015).

Conflict of interest

The authors declare they have no conflicts of interest regarding the work presented in this study.

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Editorial decision: Major revision with re-assessment
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Risk factors associated with the incidence of clinical mastitis in pasture-based dairy herds in Colombia

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