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Short-milking-tube infrared temperature as a subclinical mastitis detection tool in tropical dairy farms

      ABSTRACT

      Objective

      Our objective was to determine whether the infrared temperature of short-milking-tubes, as an estimator of milk temperature, differs between subclinically mastitic quarters with or without the presence of mastitis pathogens.

      Materials and Methods

      Individual mammary quarters (n = 121) with subclinical mastitis from 100 cows in 9 dairy herds in Puerto Rico were evaluated. Infrared images were taken from the short-milking-tube of each experimental quarter at 2 min after milking-unit attachment in 30-s intervals during the milking process. Relative humidity and ambient temperature were recorded at 1-min intervals using thermometer data loggers at the milking parlor during the milking process. Milk samples were collected for bacteriological analysis and SCC quantification. Molecular identification of mastitic pathogens was performed using the PathoProof Mastitis Complete-16 kit (Thermo Fisher Scientific Inc., Waltham, MA).

      Results and Discussion

      The milk temperature, measured through the short-milking-tubes with an infrared camera during the milking process, was greater in mammary gland quarters with presence of mastitic pathogens relative to quarters with no pathogens detected. Hence, the use of infrared thermography might be a useful detection tool to discriminate between subclinically mastitic quarters with or without an active infection in dairy cows.

      Implications and Applications

      With more sensitive infrared thermography cameras and sophisticated systems, the infrared temperature of all mammary gland quarters can be collected simultaneously in a single image when imaging the short-milking-tubes as opposed to imaging the skin of the mammary gland, which requires several images to capture the temperature of all 4 quarters.

      Key words

      INTRODUCTION

      Mastitis is the inflammation of the mammary gland, mainly caused by pathogenic bacteria (
      • Erskine R.J.
      • Wagner S.
      • DeGraves F.J.
      Mastitis therapy and pharmacology..
      ). If symptoms associated with mastitis are perceivable (e.g., inflammation, abnormal milk appearance, udder redness, or fever), the infection is categorized as clinical mastitis, and if otherwise, it is categorized as subclinical mastitis. However, of these 2 forms of mastitis, the subclinical form is the most economically and biologically important because it is challenging to detect (
      • Romero J.
      • Benavides E.
      • Meza C.
      Assessing financial impacts of subclinical mastitis on Colombian dairy farms..
      ). Mastitis continues to be one of the most important and common diseases of the dairy industry, negatively affecting cattle well-being (
      • Seegers H.
      • Fourichon C.
      • Beaudeau F.
      Production effects related to mastitis and mastitis economics in dairy cattle herds..
      ) and instigating economic losses associated with lower milk yields, poor milk quality, treatment costs, and culling (
      • Halasa T.
      • Nielen M.
      • Huirne R.B.
      • Hogeveen H.
      Stochastic bio-economic model of bovine intramammary infection..
      ). Yet, early mastitis detection and treatment can increase cure rates and sustain milk yields and quality (
      • Lago A.
      • Godden S.M.
      • Bey R.
      • Ruegg P.L.
      • Leslie K.
      The selective treatment of clinical mastitis based on on-farm culture results I: Effects on antibiotic use, milk withholding time and short-term clinical and bacteriological outcomes..
      ).
      Researchers have used infrared thermography (IRT) for clinical mastitis screening by imaging the skin of the mammary gland, reporting differences in temperature associated with inflammation (
      • Colak A.
      • Polat B.
      • Okumus Z.
      • Kaya M.
      • Yanmaz L.E.
      • Hayirli A.
      Short communication: Early detection of mastitis using infrared thermography in dairy cows..
      ;
      • Hovinen M.
      • Siivonen J.
      • Taponen S.
      • Hänninen L.
      • Pastell M.
      • Aisla A.-M.
      • Pyorala S.
      Detection of clinical mastitis with the help of a thermal camera..
      ;
      • Polat B.
      • Colak A.
      • Cengiz M.
      • Yanmaz L.E.
      • Oral H.
      • Bastan A.
      • Kaya S.
      • Hayirli A.
      Sensitivity and specificity of infrared thermography in detection of subclinical mastitis in dairy cows..
      ). More practical approaches for the use of IRT as a mastitis detection tool have been evaluated in tropical dairy farms.
      • Curbelo-Rodríguez J.E.
      • Almeida Montenegro A.D.
      • Ortiz-Colón G.
      • Sánchez-Rodríguez H.L.
      • Jiménez-Cabán E.
      Termografía infrarroja como herramienta para la detección de mastitis sub-clínica en ganado lechero bovino..
      collected IRT images from milk samples and milking machine components, during the milking process, to detect subclinical mastitis and predict SCC, reporting limited capabilities due to diurnal temperature variations. To date, screening tools for the detection of subclinical mastitis in tropical dairy systems remain scarce or inaccessible.
      Hence, the objectives of this study were (1) to determine whether the infrared (IR) temperature of short-milking-tubes differs between subclinically mastitic quarters with or without the presence of mastitic pathogens and (2) to evaluate whether the use of the CHROMagar Mastitis kit (CHROMagar, Paris, France) is a reliable tool for on-site farm identification of mastitic pathogens.

      MATERIALS AND METHODS

      Dairy Herds and Environmental Parameters

      All animal procedures were conducted following the policies and procedures approved by the Institutional Animal Care and Use Committee (approval # 2012050301). Individual mammary gland quarters with subclinical mastitis (n = 121 quarters; n = 100 cows) from 9 dairy herds in Puerto Rico were selected on site using a California Mastitis Test. Participating dairy farms were chosen according to the following criteria: milking time from 0200 to 0600 h and annual average bulk tank milk SCC higher than 400,000 cells/mL. Farm municipality, number of herds per municipality, number of cows per herd, and total quarters per herd were as follows: Aguadilla (1 herd; 10 cows; 13 quarters), Hatillo (1 herd; 16 cows; 26 quarters), Hormigueros (1 herd; 7 cows; 7 quarters), Lajas (3 herds; 7, 9, and 8 cows; 7, 9, and 8 quarters, respectively), Mayagüez (1 herd; 4 cows; 6 quarters), Moca (1 herd; 19 cows; 19 quarters), and Quebradillas (1 herd; 22 cows; 26 quarters). Relative humidity (RH) and ambient temperature (AT) were recorded at 1-min intervals at the milking parlor during the milking process using 2 U23-01 HOBO Data Loggers (Onset, MA).

      Evaluation of Mammary Quarters for Subclinical Mastitis and IRT Imaging

      At the milking parlor, all teats were disinfected with commercial predip and subsequently stripped 4 to 5 times to evaluate milk appearance and to ensure that no clinical mastitic quarters were included in the study. Quarters with no perceptible clinical mastitis were subsequently tested for subclinical mastitis using a California Mastitis Test; scores ≥1 (indicating ≥400,000 SCC/mL;
      • Luedecke L.O.
      • Forster T.L.
      • Ashworth U.S.
      Relationship between California Mastitis Test reaction and leucocyte count, catalase activity, and A-esterase activity of milk from opposite quarters..
      ) were selected for milk sample collection. Duplicated milk samples (10 mL) were collected in sterile centrifuge tubes (Fisher Scientific, Pittsburgh, PA) following the Microbiological Procedures of the Diagnosis of Bovine Udder Infection and Determination of Milk Quality (

      Oliver, S. P., R. N. González, J. S. Hogan, B. M. Jayarao, and W. E. Owens. 2004. Microbiological Procedures for the Diagnosis of Bovine Udder Infection and Determination of Milk Quality. 4th ed. Natl. Mastitis Counc. Inc., Verona, WI, pp. 1–8.

      ) and kept on ice until laboratory arrival for analysis.
      Estimation of milk’s temperature was performed using a FLIR E-8 thermal imaging camera (FLIR Systems Inc., Wilsonville, OR). Images were obtained from the short-milking-tube of each experimental quarter during the milking process (Figure 1) at 30-s intervals, starting at 2 min after milking-unit attachment (to allow transference of milk’s temperature to the short-milking-tube). Thermography images were analyzed using the FLIR Tools-plus Software (FLIR Systems Inc.).
      Figure 1
      Figure 1Infrared thermography imaging of the short-milking-tubes during the milking process (A) and its corresponding regular image (B). The FLIR plus software (FLIR Systems Inc., Wilsonville, OR) was used to quantify the infrared temperature of the area of interest (B×1; experimental quarter) using quadrants of the same size in each tube and image.

      Analysis of Milk Samples

      Somatic cell counts of milk samples were immediately quantified upon laboratory arrival (within 6 h from collection time) using a DeLaval cell counter (Tumba, Sweden), and those with concentrations ≥200,000 SCC/mL were used for bacteriological analysis. Milk samples were then cultured using the CHROMagar Mastitis kit (CHROM), and a duplicate sample was stored at −80°C until delivery to the Dairy Herd Improvement Association laboratory (DHIA, Manheim, PA) for molecular identification of mastitic pathogens using the PathoProof Mastitis Complete-16 kit (PtoPrf-16; Thermo Fisher Scientific Inc., Waltham, MA). The output of cultures from CHROM and PtoPrf-16 were evaluated and compared. To accomplish this, gram-positive and gram-negative CHROM plates were prepared following the instructions from the manufacturer (

      CHROMagar. 2017. CHROMagar™ ECC: Instructions for Use. NT-EXT-016. 33. CHROMagar, Paris, France.

      ). The inoculation process was performed following the recommendations of

      Oliver, S. P., R. N. González, J. S. Hogan, B. M. Jayarao, and W. E. Owens. 2004. Microbiological Procedures for the Diagnosis of Bovine Udder Infection and Determination of Milk Quality. 4th ed. Natl. Mastitis Counc. Inc., Verona, WI, pp. 1–8.

      . Colony growth in CHROM plates was evaluated after 48 h of incubation, allowing gram family categorization. If both gram families were present in a milk sample, the predominant bacterium was reported.

      Experimental Design and Statistical Analysis

      The study was conducted as a complete randomized design with repeated measures taken from the same cows. The PtoPrf-16 bacteriological results were categorized as detection [gram positive (n = 80) and gram negative (n = 5)] or no detection [no pathogen detection (n = 31)]. Associations between the IR temperature of the milk through the short-milking-tubes and the results of PtoPrf-16 were analyzed using the PROC GLIMMIX in SAS (SAS Institute Inc., Cary, NC). The final statistical model included quarter and PtoPrf-16 bacteriological result as fixed effects and cow nested within herd as random variable.
      The SCC was converted to somatic cell score (SCS) for statistical analysis using the following formula: log2(SCC/100,000) + 3 (
      • Ali A.K.A.
      • Shook G.E.
      An optimum transformation for somatic cell concentration in milk..
      ). Means among IR temperature of the short-milking-tubes, PtoPrf-16 results, and SCS were compared using LSMEANS with a Tukey adjustment. Correlations between SCC, IR temperature of the short-milking-tubes, AT, and RH were analyzed using PROC CORR in SAS. Sensitivity and specificity of CHROM was evaluated using PtoPrf-16 as comparative test in contingency tables with PROC FREQ. Sensitivity of gram-positive or gram-negative detection by CHROM was defined as the number of cases in which both CHROM and PtoPrf-16 detected the same gram family. Moreover, specificity of CHROM was defined as the number of cases in which no bacterial colonies were observed in CHROM plates and PtoPrf-16 did not detect any of the targeted mastitic pathogens.

      RESULTS AND DISCUSSION

      IR Temperature of the Short-Milking-Tubes, Milk SCC, and Environmental Parameters

      The fixed effects of RH, AT, quarter, herd, or their interactions did not affect the IR temperature of the short-milking-tubes (P > 0.05). This could mean that the collection of IRT images during milking times from 0200 to 0600 h was effective in reducing the effects of diurnal temperature variations on IR temperature of the short-milking-tubes. However, the variability of cows within herds was the most important variance component in the model (Z-value = 4.79). Previous studies have reported differences in milk temperature attributed to milk yield, protein content, milk flow, and fluctuations, which could account for this variability (
      • King J.O.L.
      Milk temperatures of dairy cows during milking..
      ;
      • Gil Z.
      Milk temperature fluctuations during milking in cows with subclinical mastitis..
      ). Factors such as walking distance from pens to the milking parlor (i.e., cow activity) have also been reported to affect cow temperature and, therefore, milk temperature (
      • Colak A.
      • Polat B.
      • Okumus Z.
      • Kaya M.
      • Yanmaz L.E.
      • Hayirli A.
      Short communication: Early detection of mastitis using infrared thermography in dairy cows..
      ;
      • Curbelo-Rodríguez J.E.
      • Almeida Montenegro A.D.
      • Ortiz-Colón G.
      • Sánchez-Rodríguez H.L.
      • Jiménez-Cabán E.
      Termografía infrarroja como herramienta para la detección de mastitis sub-clínica en ganado lechero bovino..
      ). For instance, 2 h of physical activity increases cow udder skin temperature by approximately 1.0°C (
      • Berry R.J.
      • Kennedy A.D.
      • Scott S.L.
      • Kyle B.L.
      • Schaefer A.L.
      Daily variation in the udder surface temperature of dairy cows measured by infrared thermography: Potential for mastitis detection..
      The IR temperature of the short-milking-tubes from quarters with bacterial detection and no detection were significantly different: 33.61 ± 0.16 and 33.02 ± 0.25°C (Delta T = 0.59°C), respectively (P = 0.0386; Figure 2. In general, immune responses associated with pathogen invasion trigger immunological events associated with a temperature increase at the mammary gland level (
      • Strandberg Y.
      • Gray C.
      • Vuocolo T.
      • Donaldson L.
      • Broadway M.
      • Tellam R.
      Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells..
      ;
      • Wellnitz O.
      • Arnold E.T.
      • Bruckmaier R.M.
      Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland..
      ;
      • Wellnitz O.
      • Bruckmaier R.M.
      The innate immune response of the bovine mammary gland to bacterial infection..
      ). Moreover, immunological responses associated with an increment in temperature could be related to the amount of SCC in milk (
      • Wellnitz O.
      • Arnold E.T.
      • Bruckmaier R.M.
      Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland..
      ). However, in our study, milk SCS did not differ between bacteriological groups (P = 0.27; ​Table 1
      Figure 2
      Figure 2Infrared temperature (°C, mean ± SEM) of the short-milking-tubes of mammary gland quarters with subclinical mastitis grouped by bacteriological results according to the PathoProof Mastitis Complete-16 kit (Thermo Fisher Scientific Inc., Waltham, MA). The infrared temperatures of the short-milking-tubes from mastitic bovine mammary quarters with mastitic pathogens present were higher (P = 0.0386) relative to those of the short-milking-tubes from quarters where no pathogens were detected.
      Table 1Mean somatic cell score (SCS) and SCC ± SEM between PathoProof Mastitis Complete-16 assay bacteriological results
      PtoPrf-16
      Results of PathoProof Mastitis Complete-16 kit (Thermo Fisher Scientific Inc., Waltham, MA).
      nSCSSCC/mL
      No detection317.22 ± 0.162,158,516 ± 185,100
      Detection856.59 ± 0.151,719,858 ± 119,642
      1 Results of PathoProof Mastitis Complete-16 kit (Thermo Fisher Scientific Inc., Waltham, MA).
      This could be explained by the fact that only quarters with subclinical mastitis were included in this study. Activation of inflammatory responses by pathogens not recognized by PtoPrf-16, such as Pseudomonas, Streptococcus parauberis,Streptococcus salivarius, Enterococcus saccharolyticus, among others, could also be attributed to the SCC similarities (

      Carrillo-Casas, E. M., and R. E. Miranda-Morales. 2012. Bovine mastitis pathogens: Prevalence and effects on somatic cell count. Pages 360–374 in Milk Production—An Up-to-Date Overview of Animal Nutrition, Management and Health. Narongsak Chaiyabutr, IntechOpen, London, UK. 10.5772/51032.

      ). In fact, 26.7% of the milk samples evaluated had no bacterial presence (subclinical mastitic milk; data not shown). This could be related to remnant immune cells from a successfully resolved infection, injury, or chemical irritation in mammary quarters with no-detection results (
      • Stevenson W.G.
      Injury as a cause of mastitis..
      ).
      The average IR temperature of the most predominantly detected bacteria are presented in Figure 3. From the resulting 85 milk samples with PtoPrf-16 results, 39.2, 18.6, and 13.1% corresponded to Staphylococcus aureus, coagulase-negativeStaphylococcus, and Corynebacterium bovis, respectively. No differences in IR temperatures (P = 0.57) or SCS (P = 0.70) were detected among these pathogens. No correlations between the IR temperature of the short-milking-tubes and somatic cells (SCC and SCS) or environmental parameters such as RH and AT were observed (P > 0.05). The lack of association between the IR temperature and environmental parameters could be explained by the selected milking times to collect IRT images of the short-milking-tubes, as mentioned previously.
      Figure 3
      Figure 3Infrared temperature (IRT; °C, mean ± SEM) of the short-milking-tubes and corresponding somatic cell scores (SCS; mean ± SEM) of mammary gland quarters with subclinical mastitis grouped by the more predominant pathogens identified. PathoProof = PathoProof Mastitis Complete-16 kit (Thermo Fisher Scientific Inc., Waltham, MA); CNS = coagulase-negativeStaphylococcus; C. bovis = Corynebacterium bovis; S. aureus = Staphylococcus aureus.

      Specificity and Sensitivity of CHROMagar Plates Compared with PtoPrf-16

      The PtoPrf-16 results were used to determine the specificity and sensitivity of the CHROMagar through contingency tables (Table 2). Colonies with colors differing from the CHROM’s colony identification legend were not further identified with differential tests. This discrepancy could have been caused by the growth of pathogens not included in CHROM targets. The CHROM had a specificity (gram-negative and gram-positive plates) of 52.17% and a sensitivity of 73.0 and 20% for gram-positive and gram-negative plates, respectively, when using PtoPrf-16 as a comparative test. Differences in bacteriological results among these tests could be attributed to several factors. As mentioned above, 26.7% of mastitic quarters had no bacterial detection when using PtoPrf-16. Hence, the use of additional tests to identify specific mastitic pathogens should be considered for future validation studies.
      Table 2Contingency table of CHROMagar versus PathoProof Mastitis Complete-16 kit results
      The PathoProof Mastitis Complete-16 kit (PtoPrf-16; Thermo Fisher Scientific Inc., Waltham, MA) results were used to determine the specificity and sensitivity of the CHROMagar (CHROMagar, Paris, France) through contingency tables.
      PtoPrf-16CHROMagarTotal
      Gram positiveGram negativeNo detection
      Gram positive4611663
      (73.0%)
      Gram negative2125
      (20.0%)
      No detection1011223
      (52.2%)
      Total58132091
      1 The PathoProof Mastitis Complete-16 kit (PtoPrf-16; Thermo Fisher Scientific Inc., Waltham, MA) results were used to determine the specificity and sensitivity of the CHROMagar (CHROMagar, Paris, France) through contingency tables.

      APPLICATIONS

      Milk’s temperature, measured through the short-milking-tubes, using an infrared camera, was greater in bovine mammary gland quarters with mastitic pathogens present relative to those with no pathogens detected. Therefore, the use of infrared thermography, as a noninvasive and practical approach for detection of subclinical mastitis, could be a valuable tool to discriminate mastitic quarters among healthy ones. With more sensitive infrared thermography cameras and sophisticated infrared thermography systems, the infrared temperature of each quarter in a single image can be collected when imaging the short-milking-tubes as opposed to imaging the skin of the mammary gland, which requires 3 images to capture the temperature of all quarters (i.e., front quarters separately and the rear quarters in a single image). It has to be noted, however, that this approach may not be extrapolated to other milking times, climates, or management systems. Due to large variation of infrared temperature of the short-milking-tubes, a large number of observations is needed to use this approach as a discriminatory mastitis tool (e.g., automated image collection and analysis system).
      To our knowledge, the specificity and sensitivity of CHROMagar plates have not been published previously. The main purpose of evaluating the capacity of detection of mastitic pathogens using CHROM, according to molecular testing (PathoProof Mastitis-16), was to provide suggestions to dairy farmers from areas with poor accessibility to milk quality laboratories about practical mastitis diagnostic strategies to maximize precision. One of the advantages of using CHROM plates, as an on-farm mastitis detection tool, is its discriminatory capacity between gram families (i.e., sensitivity of 73.0% for gram positives). This tool could be an asset to farmers who tend to treat mastitis cases indiscriminately. However, farmers have to be trained in strategies for aseptic milk sample collection, management, and culturing techniques to reduce false-positive results.

      ACKNOWLEDGMENTS

      These results are based on work funded by the National Institute of Food and Agriculture, USDA, under grant number 2011-36100-0609. We acknowledge the help of the participating dairy farmers and the collaborative efforts of the Puerto Rico Extension Service Dairy Group for visit coordination with the dairy farmers.

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