Natural variation in biomarkers indicating mastitis in healthy cows

Dairy herds are expanding and, with increasing numbers of animals in each herd, there is a need for automatic recording of indicators in milk in order to detect mastitis, inflammation of the udder. A number of biomarkers for mastitis have been suggested over the years. Mastitis usually occurs in one of the four udder quarters and since it is now possible to milk each udder quarter separately in automated milking systems, it is important to evaluate the normal variation in the biomarkers at udder quarter level. This study evaluated the normal variations between milkings for some biomarkers in clinically healthy cows, determined by repeated somatic cell count and bacteriological analysis. The biomarkers studied were serum amyloid A (SAA), haptoglobin (Hp), lactate dehydrogenase (LDH), N-acetyl-β-D-glucosaminidase (NAGase) and alkaline phosphatase (AP), parameters that have been suggested as markers for mastitis. Ten cows were monitored on 42 consecutive milking occasions through collection of udder quarter milk samples and representative cow composite milk samples, giving a total of 2100 individual milk samples. Each cow had its individual profile for the concentrations and variations in the parameters analysed. Although there was relatively large variation between cows for the biomarkers analysed, the variation between milkings in clinically healthy quarters within cows was often below 10%. The biomarker with the lowest variation in this study was LDH. The results suggest that comparing quarters within an individual cow can identify deviations from the natural variations between milkings. This could be a valuable tool instead of, or in combination with, a cut-off value for each parameter in order to detect changes in the milk indicating mastitis.     

Relationship between milk cathelicidin abundance and microbiologic culture in clinical mastitis

The availability of reliable tools to enable the sensitive and specific detection of mastitis in dairy cows can assist in developing control strategies and promote the more rational use of antibiotics. We have developed a milk cathelicidin ELISA that shows high sensitivity and specificity for dairy cow mastitis, based on latent class analysis. In this study, we investigated the effect of microbial agents on cathelicidin abundance in the milk of cows with clinical mastitis. We subjected 535 quarter milk samples (435 from quarters showing signs of clinical mastitis and 100 from healthy quarters as a control) to milk cathelicidin ELISA, somatic cell count (SCC), and microbiologic culture. Of the 435 clinical mastitis samples, 431 (99.08%) were positive for cathelicidin, 424 (97.47%) had SCC >200,000 cells/mL, and 376 (86.44%) were culture-positive. Of the 59 culture-negative samples, 58 (98.30%) were positive for cathelicidin and 55 (93.22%) had SCC >200,000 cells/mL. The abundance of cathelicidin and the extent of SCC increase depended on the causative agent: Streptococcus agalactiae and coagulase-negative staphylococci showed the highest and lowest changes, respectively. We also observed differences in behavior between the 2 markers depending on the pathogen: Streptococcus agalactiae induced the highest cathelicidin abundance, and Serratia spp. induced the highest SCC. Nevertheless, the different ability of microorganisms to induce cathelicidin release in milk did not compromise its value as a mastitis marker, given its higher sensitivity compared to SCC or microbiologic culture. All 100 negative control samples (collected from healthy quarters with SCC <100,000 cells/mL and culture-negative) were also negative for cathelicidin, corresponding to 100% specificity in the evaluated sample cohort. This study confirmed the value of the milk cathelicidin ELISA for detecting bovine mastitis, and highlighted the influence of mastitis-causing microorganisms on cathelicidin abundance. This influence did not compromise diagnostic performance; instead, it may have better reflected disease severity and evolution than SCC.     

Haptoglobin and serum amyloid A in milk and serum during acute and chronic experimentally induced Staphylococcus aureus mastitis

Local and systemic changes in the acute phase proteins, haptoglobin and serum amyloid A (SAA), were studied in six dairy cows during the acute and chronic phases of experimentally induced Staphylococcus aureus mastitis. Haptoglobin and SAA were measured in serum, and in milk from infected and healthy control udder quarters within each cow. Concentrations of haptoglobin and SAA increased rapidly in both serum and milk during the acute phase of mastitis and followed a similar pattern. Significantly raised milk concentrations of SAA were also found during chronic subclinical mastitis. Serum concentrations of SAA also tended to be higher during the chronic phase than pre-infection. Increases in milk haptoglobin and SAA were specific for the infected udder quarters. In conclusion, measurement of SAA in milk samples could be a useful tool in diagnosing mastitis.     

Test characteristics of milk amyloid A ELISA,somatic cell count,and bacteriological culture for detection of intramammary pathogens that cause subclinical mastitis

Bovine mastitis is an important disease in the dairy industry, causing economic losses as a result of withheld milk and treatment costs. Several studies have suggested milk amyloid A (MAA) as a promising biomarker in the diagnosis of mastitis. In the absence of a gold standard for diagnosis of subclinical mastitis, we estimated the diagnostic test accuracy of a commercial MAA-ELISA, somatic cell count (SCC), and bacteriological culture using Bayesian latent class modeling. We divided intramammary infections into 2 classes: those caused by major pathogens (e.g., Escherichia coli, Staphylococcus aureus, streptococci, and lacto-/enterococci) and those caused by all pathogens (major pathogens plus Corynebacterium bovis, coagulase-negative staphylococci, Bacillus spp., Streptomyces spp.). We applied the 3 diagnostic tests to all samples. Of 433 composite milk samples included in this study, 275 (63.5%) contained at least 1 colony of any bacterial species; of those, 56 contained major pathogens and 219 contained minor pathogens. The remaining 158 samples (36.5%) were sterile. We determined 2 different thresholds for the MAA-ELISA using Bayesian latent class modeling: 3.9 µg/mL to detect mastitis caused by major pathogens and 1.6 µg/mL to detect mastitis caused by all pathogens. The optimal SCC threshold for identification of subclinical mastitis was 150,000 cells/mL; this threshold led to higher specificity (Sp) than 100,000 cells/mL. Test accuracy for major-pathogen intramammary infections was as follows: SCC, sensitivity (Se) 92.6% and Sp 72.9%; MAA-ELISA, Se 81.4% and Sp 93.4%; bacteriological culture, Se 23.8% and Sp 95.2%. Test accuracy for all-pathogen intramammary infections was as follows: SCC, sensitivity 90.3% and Sp 71.8%; MAA-ELISA, Se 88.0% and Sp 65.2%; bacteriological culture, Se 83.8% and Sp 54.8%. We suggest the use of SCC and MAA-ELISA as a combined screening procedure for situations such as a Staphylococcus aureus control program. With Bayesian latent class analysis, we were able to identify a more differentiated use of the 3 diagnostic tools. The MAA-ELISA is a valuable addition to existing tools for the diagnosis of subclinical mastitis.     

Using behavioral observations in freestalls and at milking to improve pain detection in dairy cows after lipopolysaccharide-induced clinical mastitis

This study aimed to determine the effect of lipopolysaccharide (LPS)-induced mastitis with or without nonsteroidal anti-inflammatory drug (NSAID) on dairy cows' clinical, physiological, and behavioral responses in the milking parlor and freestalls as well as the specificity (Sp) and sensitivity (Se) of behavioral responses in detecting cows with LPS-induced mastitis. Twenty-seven cows received an intramammary infusion of 25 µg of Escherichia coli LPS in 1 healthy quarter. Following LPS infusion, 14 cows received a placebo (LPS cows), and 13 cows received 3 mg/kg of body weight of ketoprofen i.m. (LPS+NSAID cows). Cow response to the challenge was monitored at regular intervals from 24 h before to 48 h postinfusion (hpi) through direct clinical observations, markers of inflammation in milk, and via point-in-time direct behavioral observations in the barn and at milking. In LPS cows, infusion induced a significant increase of plasma cortisol levels at 3 and 8 hpi, milk cortisol levels at 8 hpi, somatic cell counts from 8 to 48 hpi, IL-6 and IL-8 at 8 hpi, milk amyloid A (mAA) and haptoglobin at 8 and 24 hpi, rectal temperature at 8 hpi, and respiratory rate at 8 hpi. Their rumen motility rate decreased at 8 and 32 hpi. Compared with before the challenge, significantly more LPS cows stopped feeding/ruminating and pressed their tail between their legs at 3 and 5 hpi, increased feeding/ruminating at 24 hpi, and had the tendency to be less responsive, dropping their head, and dropping their ears at 5 hpi. At milking, compared with before challenge, significantly more LPS cows lifted their hooves at forestripping at 8 hpi. The 2 groups showed similar patterns of response for milk cortisol, somatic cell count, respiratory rate, mAA, haptoglobin, and IL-6, IL-1β, and IL-8. Compared with LPS cows, LPS+NSAID cows had significantly lower plasma cortisol levels at 3 hpi, their rectal temperature decreased at 8 hpi, their rumen motility rate increased at 8 and 32 hpi, and their heart rate increased at 32 hpi. Compared with LPS cows, a significantly larger proportion of LPS+NSAID cows were feeding/ruminating, a lower proportion had ears down at 5 hpi, and a larger proportion lied down at 24 hpi. At milking, whatever the phase of milking, for “hoof to belly,” 9 out of 14 cows did not show this behavior before infusion (Sp = 64%) and 14/14 did not kick during pre-infusion milking (Sp = 100%). Regarding sensitivity, at maximum, 5 cows out of 14 (Se = 36%) displayed “hoof to belly” after infusion. For “lifting hoof,” 14/14 did not show hoof-lifting before infusion (Sp = 100%) and 6/14 displayed it after infusion (Se = 43%) at forestripping only. In the freestall barn, 9 behaviors had a Sp >75% (at minimum, 10/14 did not show the behavior) whatever the time point but Se < 60% (at maximum, 8/14 displayed the behavior). Finally, “absence of feeding and ruminating” had Sp of 86% (12/14 ate/ruminated) and Se of 71% (10/14 did not eat/ruminate) at 5 hpi. This study shows that feeding/ruminating, tail position, and reactivity at forestripping could be used as behavioral indictors for early detection of mastitis-related pain in dairy cows.     

Evaluation of somatic cell count thresholds to detect subclinical mastitis in Gyr cows

The objectives of this study were (1) to determine the sensitivity (Se) and specificity (Sp) of somatic cell count (SCC) thresholds to identify subclinical mastitis in Gyr cows caused by major and minor pathogens; (2) to study the effects of month of sampling, rear or front mammary quarters, herd, intramammary infection (IMI), and bacterial species on SCC at quarter level; and (3) to describe the prevalence of IMI in Gyr cows in commercial dairy herds. In total, 221 lactating Gyr cows from 3 commercial dairy farms were selected. Milk samples were collected from individual quarters once a month for 1 yr from all lactating cows for SCC and bacteriological analysis. Mammary quarters were considered the experimental units and the SCC results were log₁₀-transformed. Four SCC thresholds (100, 200, 300 and 400 × 10³ cells/mL) were used to determine Se and Sp to identify infected mammary quarters. The overall prevalence of IMI in quarter milk samples of Gyr cows was 49.8%, and the prevalence of minor pathogens was higher (31.9%) than that of major pathogens (17.8%). Quarter samples with microbial isolation presented higher SCC compared with negative samples. Sensitivity and Sp of selected SCC thresholds varied according to the group of pathogen (major and minor) involved in the IMI definition. Sensitivity increased and Sp decreased when mammary quarters with only major pathogens isolation were considered positive. The use of a single SCC analysis to classify quarters as uninfected or infected in Gyr cows may not be a useful test for this breed because Se and Sp of SCC at the studied thresholds were low. The occurrence of IMI and the bacterial species are the main factors responsible for SCC variation in mammary quarters of Gyr cows. Milk samples with major pathogens isolation elicited higher SCC than those with minor pathogens.     

Milk and serum IL-4, IL-6, IL-10, and amyloid A concentrations in cows with subclinical mastitis caused by coagulase-negative staphylococci

The aim of the study was to evaluate the concentrations of cytokines IL-4, IL-6, and IL-10 and acute phase protein amyloid A in milk and in serum from cows with subclinical mastitis caused by coagulase-negative staphylococci and from healthy cows. The blood and milk samples were obtained from 35 midlactation, multiparous (between parities 2 and 4) Holstein-Friesian cows. In the milk samples from 20 cows with subclinical mastitis, the following species of Staphylococcus were detected: Staphylococcus xylosus (8 samples), Staphylococcus chromogenes (6 samples), Staphylococcus haemolyticus (2 samples), Staphylococcus simulans (2 samples), and Staphylococcus sciuri (2 samples). The results of the present study indicate that the level of IL-6 in cows suffering from subclinical mastitis tended to be high in both serum and milk (432.09 and 254.32 pg/mL) compared with the level in healthy cows (164.47 and 13.02 pg/mL, respectively). Amyloid A value also was significantly higher in milk of unhealthy cows compared with cows without subclinical mastitis (790.2 and 360.5 ng/mL). No significant differences were found in levels of amyloid A in serum of both tested groups of cows (2,680.0 and 2,720.0 ng/mL). In contrast, concentration of IL-4 was significantly lower both in serum and in milk of cows with staphylococcal mastitis (86.1 and 123.17 pg/mL) compared with control animals (413.5 and 670.2 pg/mL). The level of IL-10 also was significantly higher in milk of healthy cows than in infected cows (39.78 and 22.5 pg/mL); however, differences in serum levels of this cytokine between tested groups were significantly less important (220.6 and 175.1 pg/mL).     

Milk composition and health status from mammary gland quarters adjacent to glands affected with naturally occurring clinical mastitis

Mammary gland quarters have usually been considered to be anatomically and physiologically independent, but some recent research has indicated more interdependence than previously reported. The objective of this study was to compare milk composition (fat, total protein, lactose, solids-not-fat, and chloride) and health status (somatic cell count, differential leukocyte count, and lactate dehydrogenase) of milk samples from unaffected mammary glands of an udder with a single clinically inflamed quarter to results of milk samples from healthy mammary glands of healthy cows. The study was designed as a prospective case control study with case and control cows matched by parity and days in milk. Cases were defined as cows (n = 59) experiencing clinical mastitis in a single mammary gland, and controls (n = 59) were defined as cows that had not experienced clinical mastitis during the current lactation. Quarter milk samples were collected from all mammary glands adjacent to clinically affected quarters of cases and from the same mammary glands of controls. Samples were used to assess concentration of chloride and lactate dehydrogenase, fat, total protein, solids-not-fat, somatic cell count, and differential leukocyte count. Microbiological analysis was also performed on milk samples obtained from clinically affected mammary glands (n = 59). Logistic regression models were used to assess possible associations among quarter somatic cell count (≥150,000 cells/mL) and quarter type (adjacent to case or control). Multivariate linear models were used to compare milk composition and health status between quarter types. A total of 170 quarters were enrolled per group. Milk obtained from adjacent quarters of cases contained a lesser concentration of total protein, lactose, and solids-not-fat, but had a greater concentration of fat and chloride. The somatic cell count, total leukocyte count, and absolute numbers of neutrophils, lymphocytes, and macrophages were all increased in milk obtained from adjacent quarters of case cows compared with milk obtained from quarters of control cows. The relative proportion of neutrophils was increased, whereas the proportion of macrophages was decreased in milk obtained from cases. Approximately 30% of milk samples obtained from adjacent quarters of cases had a somatic cell count ≥150,000 cells/mL compared with 12% of milk samples obtained from quarters of control cows. The position of the mammary gland was not associated with any outcomes. In conclusion, our results support previous research that indicates the immune response to intramammary infection in a single mammary gland quarter alters milk composition and health status throughout the udder.     

Determination of milk and blood concentrations of lipopolysaccharide-binding protein in cows with naturally acquired subclinical and clinical mastitis

Blood and milk concentrations of the acute phase protein lipopolysaccharide-binding protein (LBP) were evaluated in cows with naturally occurring mastitis. Blood and milk samples were collected from 101 clinically healthy dairy cows and 17 dairy cows diagnosed with clinical mastitis, and the LBP concentrations of the samples were measured by an ELISA. Concentrations of LBP were greater in the blood and milk of cows with clinical mastitis than in those with healthy quarters. Concentrations of LBP also differed between uninfected and subclinically infected quarters with low somatic cell count. Blood concentrations of LBP in cows with subclinical intramammary infections could not be differentiated from those of cows with all healthy quarters. Together, these data demonstrate that increased blood and milk concentrations of LBP can be detected in dairy cows with naturally acquired intramammary infections that cause clinical mastitis.     

Lipoprotein lipase activity of milk from cows with prolonged subclinical mastitis

The influence of prolonged subclinical mastitis on bovine milk lipoprotein lipase activity was investigated. Nine cows with at least one quarter with prolonged subclinical mastitis and at least one nonmastitic quarter were selected in various stages of lactation. Milk from subclinical quarters had a mean somatic cell count of 5.7 X 10(6) cells/ml while milk from nonmastitic quarters had an average somatic cell count of 9.4 X 10(4) cells/ml. Quarters with a subclinical infection contained the same pathogenic organisms for a minimum of 6 wk. The average milk lipoprotein lipase activity of 108.7 units/ml milk from subclinical quarters was 27.1% higher than the average enzyme activity of 79.2 units/ml milk from nonmastitic quarters. Conditions present in the mammary gland during prolonged subclinical mastitis could lead to increased milk lipoprotein lipase activity in raw milk.     

Comparative efficacy of local and systemic antibiotic treatment in lactating cows with clinical mastitis

The intramuscular administration of penethamate hydriodide over 3 consecutive days and the intramammary administration of an ampicillin/cloxacillin combination were compared in lactating cows suffering from infectious clinical mastitis in one quarter, through an open, randomized, controlled multicenter field trial. Clinical examinations were carried out on d 1 (immediately before treatment), 3, 8, 17, and 22. Milk samples were taken from affected quarters for bacteriological analysis on d 1, 17, and 22, and from all quarters for somatic cell count (SCC) determination on d 1, 8, 17, and 22. There was no significant difference in bacteriological and clinical cure rates between the 2 treatment groups. The systemic treatment with penethamate resulted more frequently in a reduction of the milk SCC below the threshold of 250,000 cells/mL. This also occurred in the adjacent quarters not affected by clinical mastitis but with an SCC above 250,000 cells/mL before treatment. These findings suggest that the parenteral treatment with penethamate provides collateral cure on the quarters of the cows affected by subclinical mastitis. The number of quarters per cow affected by clinical or subclinical mastitis should be considered when selecting an antibiotic treatment by the local or systemic route.     

Somatic cell count status across the dry period as a risk factor for the development of clinical mastitis in the subsequent lactation

The objective of this study was to determine the risk of clinical mastitis in the first 120 d in lactation based on previous somatic cell count (SCC) history in a herd with a very low prevalence of contagious pathogens. A total of 218 cows from a university herd were enrolled at dry-off. Duplicate quarter milk samples were collected from all quarters at dry-off, postcalving (2 to 9 d in milk), and before treatment of all first cases of clinical mastitis that occurred during the first 120 d of the subsequent lactation. Quarter SCC statuses across the dry period were defined based on comparison of quarter SCC between the date of dry-off and the postcalving sampling periods. The relationship between the probability of developing clinical mastitis in the first 120 d of lactation and SCC status across the dry period and other explanatory variables was assessed using logistic regression. In the first 120 d postcalving, 68 first cases of clinical mastitis occurred in 47 cows. Of quarters that experienced a microbiologically positive clinical case, the same microorganism was never isolated from milk samples obtained at dry-off or consistently isolated from milk samples collected at all sampling periods. Coagulase negative staphylococci were the most prevalent pathogens isolated from subclinical intramammary infection, whereas gram-negative pathogens were the most common pathogen associated with clinical cases. Quarters that had at least 1 case of mastitis during the previous lactation were 4.2 times more likely to have a first case of clinical mastitis in the current lactation than quarters that did not have clinical mastitis in the previous lactation [odds ratio (OR) = 4.2 (1.8, 10.0)]. Quarters of cows of greater than fourth parity were 4.2 times more likely to have a first case of clinical mastitis than quarters of cows of second parity [OR = 4.2 (1.4, 10.0)]. Quarters with SCC ≥200,000 cells/mL at dry-off and postcalving were 2.7 times more likely to experience a first case of mastitis than quarters with SCC <200,000 cells/mL at both periods [OR = 2.7 (0.97, 7.67)].     

Effect of clinical and subclinical mastitis on lipid composition of teat canal keratin.

Two experiments were conducted to investigate lipid composition of teat canal keratin when different conditions of bacterial colonization and quarter inflammation were present. In Experiment 1, 11 multiparous cows with subclinical mastitis (bacteria present but no visible inflammation) in at least one quarter were selected for study. Quarters that were sampled and found negative for bacterial growth were classified as control. In Experiment 2, 10 multiparous cows with clinical mastitis in one or more quarters were selected. Milk samples from inflamed quarters were cultured to identify mastitis pathogens; these quarters were classified as clinical; all other quarters were classified as control. Teat canal keratin was collected from all quarters just before an a.m. milking, samples were weighed, and lipid determinations were conducted by TLC. Keratin from subclinical quarters compared with keratin from control quarters did not differ in either neutral lipid or fatty acid composition. Total lipid was significantly higher in keratin from teats of clinical quarters than in keratin from control quarters (27.8 vs. 21.5 microgram/mg). Neutral lipid composition of keratin was similar between teats from clinical quarters and teats from control quarters. In Experiment 2, quarter foremilk samples were also obtained to determine lipid composition. The FFA in milk from clinical quarters contained fewer short-chain fatty acids, whereas polyunsaturated fatty acids were significantly higher in milk from clinical quarters.     

Application of udder surface temperature by infrared thermography for diagnosis of subclinical mastitis in Holstein cows located in tropical highlands

Several reports have indicated that udder surface temperature (UST) can be a useful indicator of subclinical mastitis (SCM). The objective was to evaluate UST by infrared thermography (IRT) as a diagnostic tool for SCM and intramammary infection (IMI), and to assess the influence of environmental conditions in the potential diagnosis of this disease in dairy cows located at high-altitude tropical regions. A total of 105 cows (397 quarters) from 3 dairy farms with mechanical and manual milking methods were enrolled in the study. Subclinical mastitis was diagnosed when quarter samples had a somatic cell count (SCC) ≥200 × 10³ cells/mL, microbial growth (MG) was defined when a major pathogen (≥1 cfu/plate) or Corynebacterium spp. (≥10 cfu/plate) was isolated, and IMI was defined as the presence of MG and SCC ≥100 × 10³ cells/mL. Infrared images were taken with a thermal camera placed 1 m away from the udder, and shots of the rear and left and right lateral view were made during the morning milking, before any manipulation of the udder and employing dark cardboard on the contralateral side to avoid artifacts in the background. A multilevel mixed effects linear regression model clustered within cows and herd was performed to evaluate the associations with UST. Clinical performance was evaluated using the Youden index to establish the optimum UST thresholds, which were set at 32.6°C for any case definition when milking was by hand, at 33.7°C for MG, and at 34°C for SCM and IMI in machine-milked quarters. Sensitivity (Se), specificity (Sp), area under curve (AUC), and positive likelihood ratio (+LR) were also assessed. Test agreement was assessed by kappa coefficient (κ). The UST of healthy quarters ranged between (95% CI) 32.4 and 32.6°C, lower than SCM quarters (n = 88) at 32.9°C (95% CI: 32.7–33.1 °C), MG quarters (n = 56) at 33.5°C (95% CI: 33.3–33.7°C), and IMI quarters (n = 50) at 33.5°C (95% CI: 33.2–33.7 °C). The UST was also related to the milking method: higher temperatures were observed for hand milking (n = 90) compared with machine milking (n = 185). No relation between environmental conditions such as wind speed, atmospheric temperature, relative humidity, and temperature-humidity index and UST were observed during this study. For hand milking, the optimal UST threshold was 32.6°C; for SCM, Se = 0.53, Sp = 0.89, AUC = 0.71, κ = 0.4; for MG, Se = 0.83, Sp = 0.93, AUC = 0.88, κ = 0.77; and for IMI, Se = 0.82, Sp = 0.92, AUC = 0.87, κ = 0.74. The machine milking threshold for SCM resulted in Se = 0.42, Sp = 0.97, AUC = 0.70, κ = 0.47; for MG, Se = 0.82, Sp = 0.89, AUC = 0.85, κ = 0.60; and for IMI, Se = 0.82, Sp = 0.98, AUC = 0.90, κ = 0.79. These findings suggest that UST determined by IRT is higher in machine-milked cows and in quarters with MG and IMI than in healthy quarters; therefore, UST by IRT is a reliable, clinically useful method for MG and IMI diagnosis.     

A method for assessing loss in milk yield due to subclinical mastitis

A method for assessing the loss in quarter milk yield due to subclinical mastitis is proposed. It is based upon two indices calculated from quarter milk yields and results from the assessment of mastitis obtained by measurement of the electrical conductivity of milk. The results were obtained from the cows through repeated quarter-milkings during several months of lactation. The grade of abnormality of a quarter with subclinical mastitis was expressed by the first index, the mean quarter difference of electrical conductivity of milk produced by the quarter (mQdEC, 25 degrees C). The reduction in milk yield of a quarter presumed to be due to subclinical mastitis was expressed by another index, the quarter yield ratio, R, which is that percentage of normal milk yield produced by a quarter with mastitis. Examination of the relationship between these two indices showed that subclinical mastitis caused a reduction in milk yield in the affected quarters. From the linear regression, it was found that approximately 3% reduction in quarter milk yield occurred for every 1 X 10(-4)S/cm increase in the mQdEC of the quarter with presumed subclinical mastitis.     

Milk L-lactate concentration is increased during mastitis

A study was undertaken in cattle to evaluate changes in milk L-lactate in relation to mastitis. A healthy, rear quarter of the udder of each of ten cows in mid-lactation was infused with 1000 colony-forming units (cfu) of Streptococcus uberis following an afternoon milking. Foremilk samples were taken at each milking from control and treated quarters and antibiotic treatment was applied following the onset of clinical mastitis or after 72 h. One cow did not become infected. Six quarters showed clinical symptoms of mastitis within 24-40 h and this was associated with a more than 30-fold increase in milk L-lactate (to 3.3 mM) and an increase in somatic cell count (SCC) from 4.5 x 10(3) to 1 x 10(7) cells/ml. Three cows were subclinical, with cell counts ranging from 1.5 x 10(6) to 1 x 10(7) cells/ml. In these animals, milk lactate ranged from 0.7 to 1.5 mM in the infected quarters up to 40 h post-infection, compared with less than 0.1 mM in control quarters. Milk was examined from 137 cows in mid-lactation which were known to have mastitis. Foremilk samples were taken aseptically from control and infected quarters of cows on commercial farms. Mean milk L-lactate concentrations and SCC were 0.14 +/- 0.02 mM and 1.85 +/- 0.3 x 10(5) cells/ml, respectively, in control (bacteriologically negative) samples. However, L-lactate concentrations exceeded 2.5 mM in the presence of some types of infection, the level of the lactate response being closely related to the impact of the infection on SCC. L-Lactate concentrations were relatively elevated in milk samples taken post partum, declining from 0.8 to 0.14 mM oyer the first few days of lactation. In conclusion, milk L-lactate has potential as an indicator of clinical and subclinical mastitis in dairy cows.     

Haptoglobin and serum amyloid A in relation to the somatic cell count in quarter, cow composite and bulk tank milk samples

Milk somatic cell count (SCC) is the gold standard in diagnosis of subclinical mastitis, and is also an important parameter in quality programmes of dairy cooperatives. As routine SCC analysis is usually restricted to central laboratories, much effort has been invested in the search for alternative biomarkers of mastitis and milk quality, including the presence in the milk of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid A (SAA). The aim of this study was to investigate relationships between Hp, SAA and SCC in quarter, cow composite, and bulk tank milk samples. Cows (n=165), without any clinical signs of disease or abnormalities in the milk or udder, from three different dairy farms, were used. Cow composite milk samples from all cows delivering milk at the sampling occasion were taken once in each herd. In one of the farms, representative quarter milk samples (n=103) from 26 cows were also collected. In addition, bulk tank milk samples from 96 dairy farms were included in the study. Samples were analysed for Hp, SAA and SCC, and relationships between the parameters were evaluated at quarter, cow and tank milk levels using Chi-square analysis. Milk samples were categorized according to their SCC, and the presence, or no presence, of SAA and Hp, based on the detection limits of the screening methods (0.3 mg/l and 1.0 mg/l for SAA and Hp, respectively). Hp and SAA were found in milk at quarter, cow composite and bulk tank levels. A large proportion (53%) of the animals had detectable milk concentrations of APP, and SAA was detected more frequently, and at higher concentrations than Hp, regardless of sample type. SAA was detected in as many as 82% of the bulk tank milk samples. Significant relationships were found between Hp, SAA and SCC at quarter and cow composite milk levels, but only between SAA and SCC at bulk tank milk level. Detectable levels of APP were more common at high SCC.     

Systemic treatment of subclinical mastitis in lactating cows with penethamate hydriodide

A randomized controlled field trial was performed to evaluate the efficacy of a 3-d treatment regimen with i.m. penethamate hydriodide compared with no treatment in lactating cows with subclinical mastitis. To be included, a cow had to have 2 somatic cell counts (SCC) 300,000 cells/mL at the last 3 monthly controls, 1 or more quarters with SCC >250,000 cells/mL, and the same bacterial species isolated in 2 consecutive samples 2 to 4 d apart. A total of 151 quarters from 92 cows were monitored for 2 mo following treatment. Quarter milk samples were examined for bacteriological cure (BC) and SCC at 14, 28, and 60 d after treatment. Bacteriological cure was defined as not having the same bacterial species isolated from the quarter milk samples taken at 14 and 28 d posttreatment as in the samples taken before treatment. Systemic treatment with penethamate resulted in BC in 59.5% of quarters and 52.2% of cows, compared with 16.7 and 10.9% in the untreated cows. Somatic cell count decreased significantly in the penethamate-treated cows, steadily in the case of BC and transiently when the infections persisted. This study confirms that systemic treatment of subclinical mastitis with penethamate is effective and that BC of infected quarters has a sustained positive effect on milk SCC during the 2 mo following treatment.     

Acute-phase inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) levels in serum and milk of cows with subclinical mastitis caused by Streptococcus species and coagulase-negative Staphylococcus species

The aim of the study was to investigate the concentrations of acute-phase inter-α-trypsin inhibitor heavy chain 4 (ITIH4) in serum and milk of cows with subclinical mastitis caused by Streptococcus spp. (STR) and coagulase-negative Staphylococcus spp. (CNS) and healthy cows. The blood and milk samples were obtained from 60 mid-lactation, multiparous Holstein-Friesian cows from 7 herds in the Lublin region of Poland. In the milk samples from 40 cows with subclinical mastitis, Streptococcus spp. and CNS were isolated. The ITIH4 was significantly higher in serum of cows with subclinical mastitis caused both by STR and CNS compared with healthy cows. One hundred percent of animals infected with Streptococcus spp. and 89% of animals infected with Staphylococcus spp. showed ITIH4 concentration in sera higher than 0.5 mg/mL. The concentration of ITIH4 in milk also was significantly higher in cows with subclinical mastitis caused by Streptococcus spp. and Staphylococcus spp. compared with the control group. Seventy percent of cows infected by STR and CNS showed ITIH4 concentration in milk higher than 2.5 μg/mL. Milk ITIH4 concentration higher than 5 µg/mL was found in 55% of animals infected with Streptococcus spp. and in 40% of animals infected with Staphylococcus spp. No statistically significant differences were observed in ITIH4 concentrations both in serum and in milk between the studied unhealthy animal groups. These results suggest that ITIH4 may be used in the future as a novel diagnostic marker in serum and in milk of subclinical mastitis in cows.     

Randomized controlled field trial comparing quarter and cow level selective dry cow treatment using the California Mastitis Test

Selective use of antibiotic dry cow treatment can be implemented at the cow or quarter level, with the latter having the potential to further reduce antibiotic use. Our objective was to compare these 2 approaches in 6 herds in the United Kingdom in which environmental mastitis predominated. Eight hundred seven cows were enrolled and categorized as having a high cell count (n = 401) or low cell count (n = 406) in the last 3 mo of lactation and clinical mastitis history. All quarters of all enrolled cows received an internal teat sealant. Within each category, cows were randomly allocated to 1 of 3 groups; in one group antibiotic treatment was allocated at cow level (i.e., all 4 quarters received antibiotic), whereas in the 2 remaining groups antibiotic treatment was allocated at quarter level, based on California Mastitis Test (CMT) findings. Two different thresholds, score 1 and 2, were used to determine likely infection status. Quarter milk samples were collected at dry off and postcalving for bacteriological culture and somatic cell count (SCC). Cows were monitored for clinical mastitis from dry off until 100 d in milk. Cow level SCC and milk yield data were collated from farm records. Within each category, the 2 quarter level treatment groups were compared with cow level treatment at dry off. Leaving quarters untreated with intramammary antibiotic in cows in the high cell count group, with a CMT <2 or <1, reduced antibiotic use by 55% and 31%, respectively, and resulted in no difference in the odds of being infected with any pathogen postcalving, but was associated with a higher SCC at the first test day. Intramammary antibiotic treatment of quarters with a CMT ≥1 in cows in the low cell count category at dry off was not associated with any reduction in the odds of being infected with a major pathogen postcalving but was associated with a decrease in the odds of being infected with a minor mastitis pathogen postcalving. The use of antibiotics in quarters of cows categorized as low cell count at dry off, increased the proportion of quarters treated with antibiotic from 0% at cow level to 31% (CMT ≥ 1) and 12% (CMT ≥ 2) at quarter level, only resulting in a reduction in SCC of around 20,000 cells/mL at the first test day, if all quarters with CMT score ≥1 were treated with antibiotic. No differences in clinical mastitis incidence and milk yield in the first 100 d in milk were detected between any of the treatment groups. These study findings support selective quarter level dry off treatment only in cows with cow level SCC >200,000 cells/mL at dry off.