Infrared thermography as a technique for detecting subclinical bovine mastitis

Infrared thermography is a non-invasive diagnostic tool for early detection of subclinical mastitis in pasture-raised dairy cows. The study was conducted on eight dairy farms, in Sena Madureira, Acre, in the Western Amazon, during the rainy season. One hundred and thirteen lactating crossbred cows were monitored, with measurements performed once a day before milking, from 02:00 to 06:00 in the morning. The California Mastitis Test (CMT) was subsequently applied. Data processing was performed in the R programming language and evaluated by linear models. Our results show that cow udder surface temperature by infrared thermography showed significant differences (p<0.05) between the months of the year. Cow udder temperatures of thermographic images were significantly different (p<0.05) from CMT results for animals with subclinical mastitis. CMT results showed that 45 cows (39.8%) in January, 52 cows (46%) in February and 57 cows (50.4%) in March had subclinical mastitis. Infrared thermography is a technique that can be used for the early detection of subclinical mastitis in dairy herds in a grazing system, as it detected an increase in the udder surface temperature of the same cows that were positive for CMT.


INTRODUCTION
Dairy cattle farming is one of the agricultural activities that provide food to the population, and Brazil is one of the world's largest cow milk producers, with 35.4 million liters of milk produced in 2020.The State of Acre contributed 42.6 thousand liters to that amount, with the municipality of Sena Madureira producing 2.6 thousand liters (Produção…, 2020).
Animal welfare has been increasingly recognized in importance by consumers and producers.Heat stress is easily recognized in dairy cows (Dunshea et al., 2013), as it significantly affects daily milk production (Ekine-Dzivenu et al., 2020).
Therefore, animal comfort is essential in current production systems for ethical reasons and higher productivities (Matarrese, 2013).
In this context, new precision dairy farming technologies have been adopted to improve animal welfare and zootechnical indices in different-sized farms.
However, producers should be aware of diseases that affect the mammary gland, such as bovine mastitis, causing high economic losses, as it leads to a reduction in production and changes in milk composition, in addition to promoting spending on antibiotics (Rodrigues et al., 2018).
In addition, mastitis contributes negatively to the industrial dairy process, especially at high herd infection rates, limiting milk production by up to 50% and restricting cow productivity (Martins et al., 2015).Therefore, early mastitis detection is critical to minimize losses in milk volume, ensure higher productivity, improve milk quality, and reduce economic losses, in addition to being fundamental for the effectiveness of the intramammary infection treatment (Sathiyabarathi et al., 2016).
Factors affecting comfort of dairy cattle under grazing systems and effects on animal performance, milk quality, and animal health play a key role in mastitis control and prevention.Such information allows farmers to choose the best control strategies to reduce cow udder infections and production costs, besides maintaining animal welfare.Thus, using tools that can detect it early is essential, given that mastitis is one of the main challenges for milk producers.
Thus, infrared thermography arises as an alternative to assess the impact of environmental factors on animal production, supporting decisions, promoting animal health and welfare, in addition to being able to perceive changes in the surface temperature of the mammary gland in response to different degrees of infection severity, similar to CMT (California Mastitis Test) (Leão et al., 2015).
In this sense, this study aimed to evaluate infrared thermography as a non-invasive diagnostic tool for early detection of subclinical mastitis in dairy cows raised on pasture in the Western Amazon.

MATERIAL AND METHODS
All animal management practices were approved by the Ethics Committee on the Use of Animals/UFAC (CEUA) (Process No. 23107.016410/2019-06).The study was carried out during the rainy Amazon winter period, from January to March 2020, in Sena Madureira, State of Acre, in the Western Amazon.Eight farms with an average area of 39 ha for the dairy production system were used to collect the data.Sena Madureira is located at latitude 09°03′57″ S and longitude 68°39′25″ W and 150 m above sea level.The climate is tropical, according to the Köppen classification.Throughout the year, temperatures range from 20 to 33 °C, rarely below 13 °C or above 37 °C (Condições…, 2021).
One hundred and thirteen crossbred lactating cows with the genetic profile Holstein x Girolando, Holstein x Nellore, and Holstein x Zebu and an average daily production of 4.8 L/day were evaluated on the eight farms.Data were collected three times (January, February, and March) 30 days apart.Milking is carried out manually on seven farms (87.5%) and mechanically on one (12.5%).Milking management had no modifications for this research to avoid stressful conditions.The cows were raised on pasture and brought to the pen only at milking time.Three thermogram evaluations of the udder of 113 cows (Bos taurus taurus x Bos taurus indicus) were performed in the pen in the morning before milking (between 2:00 am and 6:00 am according to the farm routine).On each farm, the data were collected once a day (one day in January, another in February, and the last day in March 2020).Images were obtained using a FLIR E63900 T198547 infrared camera, with an emissivity of 0.95, thermal resolution of 160 x 120, temperature range from 20 to 250 °C, and accuracy of ±2% or 2 °C.Captures were made at one meter from the right lateral view of the entire body, forehead, eye, and hind leg and 0.6 m from the udder, both from the right and left antimere.

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The images were captured before starting the milking process to avoid a possible change in the surface temperature of the animal's body after milking.The camera operator acquired at least ten images, thus ensuring a clear image, without any movement of the animal that could partially hide the udder.The images were analyzed by the FLIR Tools ® software, which allowed importing, editing, and analyzing the images using polygons to assess the temperatures of desired areas.
A total of 452 mammary quarters of crossbred lactating cows (n=113) were performed using the California Mastitis Test (CMT) for the diagnosis of subclinical mastitis, following the methodology recommended by Simões et al. (2016).An appropriate tray was used, 2 mL of milk was milked from each mammary quarter, and about 2 mL of reagent was immediately added, mixing with circular movements, and visualizing the result by gelatinization.
Scores were used in the quantitative analysis to characterize the inflammatory reaction and in the qualitative analysis from the crosses according to the intensity to measure the degree of mastitis severity in the mammary quarters through CMT: score 0, no precipitation was formed (healthy); one teat = 0.25; two teats = 0.50; three teats = 0.75; four teats = 1.0.To evaluate the degree of gel formation was performed through the quantification of crosses.The numerical classification was performed aiming at the statistical analysis.
All processing was performed in the R programming language (R Core Team, 2019), in which the parameters were evaluated by generalized linear models (GLM) with a Gaussian distribution.Month-and farminfluenced parameters were evaluated by the Tukey's test at 5% significance level.Monthinfluenced parameters were individually evaluated on each farm.

RESULTS AND DISCUSSION
The udder surface temperature of the cows (Bos taurus taurus x Bos taurus indicus) evaluated with the infrared thermography technique had significant differences (p<0.05) between months of the year (January, February, and March 2020).The cows (Bos taurus taurus x Bos taurus indicus) that had an increase in udder temperature over the months were individually evaluated on each farm.
Increases in cow udder surface temperature may be correlated with the possibility of mastitis.According to Sathiyabarathi et al. (2016), the inflammatory response in mastitis is initially associated with an increase in udder temperature due to inflammatory processes in the mammary gland, characterized by pathological changes in the glandular tissue (Peres Neto and Zappa, 2011).
Similarly, Sathiyabarathi et al. ( 2016) carried out a study on a farm under subtropical climate conditions where the temperature rises to 36°C in the summer and reaches 15°C during the winter and found that the average udder surface temperatures of the affected quarters were 37.9 and 38.2°C, respectively.In the current research, the udder temperature obtained through thermographic imaging for cows (Bos taurus taurus x Bos taurus indicus) affected with subclinical mastitis ranged from 38 to 40°C (Fig. 1).The change in udder temperature of cows (Bos taurus taurus x Bos taurus indicus) captured through thermographic images was significant (p<0.05) by the California Mastitis Test (CMT) for cows that had subclinical mastitis.It evidences that the analysis of thermographic images can show changes in the udder surface temperature of dairy cows from extensive grazing systems diagnosed with subclinical mastitis through CMT (Table 1).Therefore, thermography can be applied to detect subclinical mastitis.The CMT results showed that 45 cows (39.8%) in January, 52 cows (46%) in February and 57 cows (50.4%) in March had subclinical mastitis.Farm H presented 29 cows with subclinical mastitis (25.7%) (Table 2).The low technological level of the studied farms and the excess of mud in the environment may be responsible for the higher prevalence of mastitis in lactating cows (Bos taurus taurus x Bos taurus indicus).
The high rate of mastitis found in this study corroborates that in Linhares et al. (2021).These authors found high incidence of mastitis of a clinical nature in eight farms (44%) when carrying out a study to analyze the applicability of some management tools, such as SWOT matrix, GUT matrix, brainstorming, PDCA, Ishikawa diagram, and 5W2H, in improving milk quality in 18 family-run farms in the city of Senador Guiomard in the State of Acre.The high rate of mastitis (25.7%) on farm H may be due to its ineffective management, especially in the drainage system in the milking parlor.
Water accumulation may generate mud with the trampling of the cows.Besides, this farm had a mechanical milking machine in its production system.Its malfunction and improper use can increase the risk of new mastitis cases by increasing bacterial transfers between cows while milking, increasing lesions on the skin and tip of the teats, and facilitating the introduction of bacteria through the teat canal (Gentilini and Santos, 2019).
Environmental hygiene may be one of the main factors contributing to the prevalence of mastitis in lactating cows on this property.According to Mariano et al. (2015) the microorganisms involved in this disease remain in the environment and infections usually occur between milking practices.According to Simões et al. (2016), the proliferation of bacteria in the udder by pathogens leads to an immediate inflammatory response and the cells migrate, causing an increase in somatic cells in the milk, negatively affecting milk quality.
In general, good milking practices for all farms (100%) are still a great challenge to become a reality in the region.In this region, culturally, the calf is released first, and only then does the cow have its legs tied when performing manual milking.In addition, producers claim that the process of carrying out prophylactic measures and mastitis detection takes time.

CONCLUSION
During the experiment, the highest number of cows (Bos taurus x Bos taurus indicus) affected by subclinical mastitis was 57 and was observed in March.Among the farms evaluated, farm H had the highest number of affected cows.The animals were diagnosed using the California Mastitis Test -CMT.
The udder temperature of cows affected by mastitis captured through thermographic images ranged from 38 to 40 ºC.Infrared thermography is a technique that can be used for early detection of subclinical mastitis in dairy herds in a grazing system, as IRT detected an increase in udder surface temperature in the same cows that CMT was positive for subclinical mastitis.
Knowledge about the factors that affect dairy cattle raised in grazing systems and the effects that can cause physiological changes plays an essential role to prevent and control mastitis.Aware producers will be able to adopt management and prophylactic measures for decision-making.

Table 1 .
Udder surface temperature obtained through thermographic images of 113 lactating crossbred cows (Bos taurus taurus x Bos taurus indicus) on eight farms from January to March in the municipality of Sena Madureira, State of Acre, Brazil different lowercase letters in the column and uppercase letters in the row differ statistically by the Tukey test at a 5% probability (P<0.05).
Polat et al. (2010)compared infrared thermography and somatic cell count with CMT to identify subclinical mastitis.The authors reported that infrared thermography can be used to detect subclinical mastitis by measuring the udder surface temperature with a diagnosis similar to that of CMT because the CMT scores increased as the udder skin surface temperature (USST) increased linearly (USST, °C = 33.45+1.08× CMT score; R 2 =0.75 P<0.0001), as shown by infrared thermography.