Deeper exploration of inflammatory cell populations in milk to monitor udder health in dairy cows (original) (raw)
Related papers
2003
The presence of lymphocyte-related cutoff points that distinguish protective from non-protective anti-26 bacterial responses was investigated in bovine mastitis. Interactions among lymphocyte CD3, CD2, CD4, 27 CD8 CD 11 b and CD45r surface density, and the percent of lymphocytes expressing these markers, were 28 investigated by flow cytometry in bovine blood and milk cells before and times after experimental intra-29 mammary infusion with Staphylococcus aureus. The somatic cell count (SCC) and bacterial counts in milk 30 cultures were also recorded. The surface density of these markers per cell (measured as median fluorescent 31 intensity or MFI) provided confidence intervals that differentiated non-mastitic from mastitic animals and, 32 among inflammed cows, identified very early (1 day post-infusion) from later cases (1-2 week[s] post-33 infusion). Critical values for immune markers were observed ("immune thresholds"). Above them, no 34 bacterial counts and SCC <500,000 cells/ml were found. Regardless of time from infection onset, protective 35 thresholds were also observed when milk CD2+ and CD45r+ lymphocytes exceeded 73% and 21%, 36 respectively. All 6 markers showed identical MFI thresholds for both outcome indicators (SCC and CFU). When individual time points and multi-factor interactions were considered, the value of the immune threshold increased over time for the percentage of milk CD3+ cells, and decreased for the percents of CD2+, CD 11 b+ and CD45r+ cells, and for the marker surface density per cell of all markers. Findings indicated that determination of time of infection (early versus late inflammation) is necessary to make inferences on the effect of the immune response, information provided by immune data. Because measurement of immune thresholds also facilitates predictions on outcomes to future bacterial invasions, it may be applicable for animal selection against bovine mastitis. There are, conceptually, at least three levels of scale for the measurement of anti-bacterial immune responses. For bovine mastitis, they are: i) the somatic cell count (SCC) or mixed leukocyte level, in which Lymphocyte immune thresholds in bovine mastitis 3 47 no individual leukocyte is accounted for; ii) the leukocyte differential1evel, in which lymphocytes, 48 monocytes/macrophages and polymorphonuclear cells (PMN) are individually evaluated; and iii) the sub-49 cellular level in which cell functions are considered, which can be characterized by, at least, two dimensions. These dimensions are the percentage of cells of a given type showing specific surface molecules relevant in immune responses (antigen differentiation markers), and (for cells expressing a given immune marker), the marker surface density per cell. While research on bovine mastitis has emphasized the first level, relatively few studies have considered the remaining levels. A major objective of immunological research is to describe the events that determine the outcome of host-pathogen interactions (i.e., whether a bacterial invasion will lead to bacterial multiplication and, eventually, infection, or, alternatively, to a response that will prevent bacteria from colonizing the host's tissues). The central event of the immune response is the process by which the T cell receptor (TCR) is stimulated in numbers and speed such that a cascade of responses will be triggered promptly and efficaciously. In humans, approximately 8000 TCR molecules per lymphocyte must be activated before T cells reach certain critical activation status such that the immune response begins (Viola & Lanzavecchia, 1996). However, the threshold for TCR triggering also appears to be influenced by non-CD3 molecules 62 (Grossman et al., 2001; Tanchot et al., 2001). While previous studies have assessed immune thresholds, 63 (i.e., variables representing early phenomena, like cell proliferation and cytokine synthesis), only a few 64 studies have included outcome indicators, such as infections .(Hesse et al., 2001). Neither immune thresholds 65 nor the effect of non-CD3 lymphocyte molecules on CD3 triggering have been determined in dairy cattle. 66 While multiple immune markers have been measured simultaneously in other species (Falcioni et al., 67 1996), interactions among multiple immune markers have not been conducted in the bovine species. 68 Although individual lymphocyte phenotype percentages have been determined in relation to bovine mastitis 69 (Taylor et al. 1994; Rivas et al. [2000, 2001a,b, 2002]), previous studies did not assess relationships Lymphocyte immune thresholds in bovine mastitis 4 70 between multiple immune markers (predictors) and the outcome to bacterial invasion (infection vs. 71 protection). Measurement of cell percentage showing specific immune markers and/or marker density per 72 cell may identify healthy from non-healthy individuals (Bikoue et al., 1996; Resino et al., 2000). 73 The objectives of this study were: 1) to evaluate whether assessment of surface density in 74 conjunction with percentage oflymphocytes expressing immune markers could identify cutoff points that 75 differentiated mastitic from non-mastitic animals and, if so, to determine whether immune markers could 76 identify (early vs. late) inflammatory phases; 2) to identify cutoff points that characterize the outcome of 77 the host-pathogen interaction (infection vs. protection), 3) to explore whether cutoff points vary as a 78 function of immune marker interactions and/or time and, 4) to assess whether immune markers may predict 79 the outcome of future bacterial invasions. 80 81 MATERIALS and METHODS 82 Animals 83 Five first-lactation, non-periparturient Holstein heifers were investigated. They had no history of mastitis. At least three consecutive microbiological tests yielded no bacterial growth of specific pathogens from milk 85 samples, and no individual mammary gland quarter showed somatic cell counts (SCC) > 200,000 cells/mi. Intra-mammary infusions Staphylococcus aureus ribotype 116-232-S3 (Rivas et al., 1997), a strain isolated from a New York commercial herd, was cultured in sterile Todd-Hewitt broth at 37 C until the exponential growth phase was reached. The number of colony-forming units (CFU) was determined, and cultures were diluted to 200 91 CFU/ml in sterile Todd-Hewitt broth and kept at 4 C until infused. After the morning milking on day 0, 1 ml of inoculum (200 CFU in Todd-Hewitt broth) was infused into each ofthe right front and left hind
Translational Animal Science
The use of milk leukocyte differential (MLD) test has been proposed as a complement to somatic cell count (SCC) to assess the presence and the severity of intramammary infection. However, detailed information regarding the behavior of MLD under different physiological or pathological stages of the cow is nonexistent. The objective was to analyze the association between milk leukocyte proportions provided by a commercial automated MLD test and multiple cow and quarter-level variables. The study population consisted of 104 Holstein cows (32 primiparous and 72 multiparous) in one farm. Cows were categorized by days in milk as early (<50 DIM; n=29), middle (50–250 DIM; n=25), and late lactation (>250 DIM; n = 50). Milk from 416 quarters was collected and analyzed for lymphocytes (LYM), neutrophils (NEU), and macrophages (MAC) counts using an automated milk fluorescence microscopy system. Concurrently, a sterile composite milk sample was collected from each cow for pathogen identif...
Frontiers in Veterinary Science, 2021
Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription-polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 10 3 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.
Preventive Veterinary Medicine
The objective of this study was to investigate the new differential somatic cell count (DSCC) as a supplementary indicator to SCC for the identification of intramammary infection (IMI) in dairy cows at the end of the lactation period. Different approaches for identification of cows with IMI (i.e. often based on SCC) and targeted antimicrobial treatment of those rather than of all cows have been developed (i.e. selective dry cow treatment). Recently, DSCC representing the proportion of polymorphonuclear neutrophils and lymphocytes, has been introduced as an additional indicator for the presence of IMI. We used the last dairy herd improvement (DHI) samples taken within 42 d prior to dry-off as well as hand-stripped samples collected within 5 days prior to dryoff to measure DSCC and SCC. The bacteriological status was determined using quarter foremilk samples collected close to drying off. In total, 582 cows were dried off during our study but not all of them could be included in the data analysis for different reasons (e.g. incomplete data, samples too old for reliable determination of SCC and DSCC, contamination). Eventually, the final data set comprised of 310 cows of which 64 and 149 were infected with major and minor pathogens, respectively, and 97 were uninfected. The area under receiver-operating characteristics curves (AUC) were calculated to compare the diagnostic abilities of the different parameters. The AUC for identification of IMI by major pathogens when using the combination of DSCC and SCC was 0.64 compared to 0.62 for SCC alone and 0.62 for DSCC alone. The different parameters were further compared based on test characteristics and predictive values. For example, classifying cows as infected based on a cutoff of 200,000 cells/ml for SCC alone and in terms of using DSCC combined with SCC based on either > 60% and/ or > 200,000 cells/ml, the sensitivity changed from 47 to 66% and the specificity from 74 to 54%. At the same time, the negative predictive value changed from 84 to 86% and the positive predictive value from 32 to 27%. Test characteristics and predictive values of the parameters DSCC and SCC were similar using DHI and handstripped samples. In conclusion, our study provides first indications on test characteristics and predictive values for the combination of DSCC and SCC. However, more work on this subject and the actual practical application is needed.
Lymphocyte Non-Protective and Protective Responses in Infectious Bovine Mastitis
2003
The presence of lymphocyte-related cutoff points that distinguish protective from non-protective anti-26 bacterial responses was investigated in bovine mastitis. Interactions among lymphocyte CD3, CD2, CD4, 27 CD8 CD 11 b and CD45r surface density, and the percent of lymphocytes expressing these markers, were 28 investigated by flow cytometry in bovine blood and milk cells before and times after experimental intra-29 mammary infusion with Staphylococcus aureus. The somatic cell count (SCC) and bacterial counts in milk 30 cultures were also recorded. The surface density of these markers per cell (measured as median fluorescent 31 intensity or MFI) provided confidence intervals that differentiated non-mastitic from mastitic animals and, 32 among inflammed cows, identified very early (1 day post-infusion) from later cases (1-2 week[s] post-33 infusion). Critical values for immune markers were observed ("immune thresholds"). Above them, no 34 bacterial counts and SCC <500,000 cells/ml were found. Regardless of time from infection onset, protective 35 thresholds were also observed when milk CD2+ and CD45r+ lymphocytes exceeded 73% and 21%, 36 respectively. All 6 markers showed identical MFI thresholds for both outcome indicators (SCC and CFU). When individual time points and multi-factor interactions were considered, the value of the immune threshold increased over time for the percentage of milk CD3+ cells, and decreased for the percents of CD2+, CD 11 b+ and CD45r+ cells, and for the marker surface density per cell of all markers. Findings indicated that determination of time of infection (early versus late inflammation) is necessary to make inferences on the effect of the immune response, information provided by immune data. Because measurement of immune thresholds also facilitates predictions on outcomes to future bacterial invasions, it may be applicable for animal selection against bovine mastitis. There are, conceptually, at least three levels of scale for the measurement of anti-bacterial immune responses. For bovine mastitis, they are: i) the somatic cell count (SCC) or mixed leukocyte level, in which Lymphocyte immune thresholds in bovine mastitis 3 47 no individual leukocyte is accounted for; ii) the leukocyte differential1evel, in which lymphocytes, 48 monocytes/macrophages and polymorphonuclear cells (PMN) are individually evaluated; and iii) the sub-49 cellular level in which cell functions are considered, which can be characterized by, at least, two dimensions. These dimensions are the percentage of cells of a given type showing specific surface molecules relevant in immune responses (antigen differentiation markers), and (for cells expressing a given immune marker), the marker surface density per cell. While research on bovine mastitis has emphasized the first level, relatively few studies have considered the remaining levels. A major objective of immunological research is to describe the events that determine the outcome of host-pathogen interactions (i.e., whether a bacterial invasion will lead to bacterial multiplication and, eventually, infection, or, alternatively, to a response that will prevent bacteria from colonizing the host's tissues). The central event of the immune response is the process by which the T cell receptor (TCR) is stimulated in numbers and speed such that a cascade of responses will be triggered promptly and efficaciously. In humans, approximately 8000 TCR molecules per lymphocyte must be activated before T cells reach certain critical activation status such that the immune response begins (Viola & Lanzavecchia, 1996). However, the threshold for TCR triggering also appears to be influenced by non-CD3 molecules 62 (Grossman et al., 2001; Tanchot et al., 2001). While previous studies have assessed immune thresholds, 63 (i.e., variables representing early phenomena, like cell proliferation and cytokine synthesis), only a few 64 studies have included outcome indicators, such as infections .(Hesse et al., 2001). Neither immune thresholds 65 nor the effect of non-CD3 lymphocyte molecules on CD3 triggering have been determined in dairy cattle. 66 While multiple immune markers have been measured simultaneously in other species (Falcioni et al., 67 1996), interactions among multiple immune markers have not been conducted in the bovine species. 68 Although individual lymphocyte phenotype percentages have been determined in relation to bovine mastitis 69 (Taylor et al. 1994; Rivas et al. [2000, 2001a,b, 2002]), previous studies did not assess relationships Lymphocyte immune thresholds in bovine mastitis 4 70 between multiple immune markers (predictors) and the outcome to bacterial invasion (infection vs. 71 protection). Measurement of cell percentage showing specific immune markers and/or marker density per 72 cell may identify healthy from non-healthy individuals (Bikoue et al., 1996; Resino et al., 2000). 73 The objectives of this study were: 1) to evaluate whether assessment of surface density in 74 conjunction with percentage oflymphocytes expressing immune markers could identify cutoff points that 75 differentiated mastitic from non-mastitic animals and, if so, to determine whether immune markers could 76 identify (early vs. late) inflammatory phases; 2) to identify cutoff points that characterize the outcome of 77 the host-pathogen interaction (infection vs. protection), 3) to explore whether cutoff points vary as a 78 function of immune marker interactions and/or time and, 4) to assess whether immune markers may predict 79 the outcome of future bacterial invasions. 80 81 MATERIALS and METHODS 82 Animals 83 Five first-lactation, non-periparturient Holstein heifers were investigated. They had no history of mastitis. At least three consecutive microbiological tests yielded no bacterial growth of specific pathogens from milk 85 samples, and no individual mammary gland quarter showed somatic cell counts (SCC) > 200,000 cells/mi. Intra-mammary infusions Staphylococcus aureus ribotype 116-232-S3 (Rivas et al., 1997), a strain isolated from a New York commercial herd, was cultured in sterile Todd-Hewitt broth at 37 C until the exponential growth phase was reached. The number of colony-forming units (CFU) was determined, and cultures were diluted to 200 91 CFU/ml in sterile Todd-Hewitt broth and kept at 4 C until infused. After the morning milking on day 0, 1 ml of inoculum (200 CFU in Todd-Hewitt broth) was infused into each ofthe right front and left hind
Veterinary Immunology and Immunopathology, 2015
The transition period is known to be the most critical phase in the life of high yielding dairy cow. Changes in the immune functions have been observed during the transition period which may account for the onset of clinical and subclinical (e.g. inflammatory response) problems at calving or at the beginning of lactation however this relationship has not yet been adequately investigated. Thus, to establish the potential of the periparturient dairy cow's immune system to respond to stimuli, two challenges [an ex vivo whole blood stimulation assay (WBA) with lipopolysaccharides and a carrageenan skin test (CST)] were performed in addition to characterizing the metabolic and inflammatory profile. The WBA was performed using 0, 0.01 and 5 g LPS/mL on whole blood and CST was administered by subcutaneous injection of 0.7 mL solution containing 4.2 mg of carrageenan to the shoulder region of the cows. These tests were performed on 10 Holstein-Friesian cows at −45 ± 2, −20 ± 2, −3, 3, 7, 28 ± 2 days from parturition (DFP). Cows were also monitored for health status, body condition score, milk yield. The results demonstrate a higher production of IL-1 and IL-6 from leukocytes after LPS stimulation around calving (from −3 to 3 DFP) compared to −45 DFP (P < 0.05). Moreover, IL-6 (but not IL-1) was able to reach close to the maximum response at the lower stimulus intensity (0.01 g LPS/mL), maintaining a higher response over a longer time in early lactation. The release of higher levels of IL-6 in the transition period, with low LPS dose, suggests its crucial role in the regulation of inflammatory response around calving. The response of cows to CST decreased a few days before calving (−3 DFP) compared with response at −45 and 28 DFP (P < 0.05), and remained low in the first week of lactation. This result suggests the reduction of the functionality of some vascular factors, which decreases diapedesis. Overall, the WBA and CST tests confirm changes in immunocompetence around calving. These tests are able to better describe the changes of the innate immune response at a local and systemic level, mainly when combined with conventional metabolic and inflammatory indices.
Mastitis is one of the most important bovine diseases causing economic losses to dairy producers and it is a consequence of the activity of various internal factors that cooperate to destroy the invading microorganisms. The local immune response during the bacterial infection is fundamental in effectively designing therapies and control measures to help eradicate bovine mastitis (Oviedo-Boyso et all., 2007). Many studies have examined the intramammary neutrophil, lymphocyte, and monocyte function by in vitro "testing", but a gap still persists in relating the local, innate and adaptive immune function to the systemic process of immune defense of the mastitic animals. The study aimed to establish a connection between non-specific (phagocytosis, total Ig, circulating immune complexes) and specific (lymphocytes) effectors involved in the overall immune protection of the intensively farmed animals, diagnosed with various degrees of clinical mastitis, and the species of bacteri...
Journal of King Saud University - Science, 2020
Mastitis an inflammation of mammary gland in dairy herds is a key concern of economic losses worldwide caused by bacteria and its toxins. In this study, we investigated pro-inflammatory and anti-inflammatory cytokines (IL-2, IL-1b, IL-6, TGF-b, IL-10, TNF-a), lactoferrin and albumin, and milk composition in normal dairy cows and lactating cows with symptoms of sub-clinical and clinical mastitis. Lactating cows with clinical mastitis showed marked increase in IL-2, IL-6, IL-1b, TNF-a and a decrease in anti-inflammatory levels of TGF-b, IL-10, milk parameters fat, protein, SNF, lactose and increase in pH compared to normal lactating cows. Subclinical lactating cows showed significant alteration in TNF-a, IL-1b when compared to normal lactating cows. There was no significant difference between IL and 2 and IL-6 in normal and subclinical lactating cows. The subclinical cows also did not exhibit significant difference in TGF-b, albumin, milk fat, protein and pH when compared with normal lactating cows. Findings in the present study indicate that cytokines together with proteins lactoferrin and albumin can be considered as prospective markers in early detection of subclinical and clinical mastitis.
Canadian journal of veterinary research = Revue canadienne de recherche veterinaire, 2002
The number and function of bovine mammary-gland phagocytes were assessed in 8 lactating cows, each tested at least twice within an 8-mo period (total number of observations, 20). Macrophages and polymorphonuclear (PMN) cells were evaluated by conventional cytology, flow cytometry, fluorescent microscopy, and somatic-cell count (SCC). Phagocytosis was evaluated from the uptake of fluorescent beads and expressed as median fluorescence intensity (MFI). Two major subpopulations of phagocytes, of low or high MFI (LFI or HFI), were observed, and there were up to 4 sub-subpopulations within the HFI subpopulation of both macrophages and PMN cells. Fluorescent microscopy identified phagocytes containing up to 4 beads per cell. Cows showing < or = 72.3% phagocytes by cytology were regarded as non-mastitic (11 observations), and those showing > or = 80.7% phagocytes were considered to be mastitic (8 observations). Phagocyte MFI was negatively associated with mastitis; that is, the higher...
The expression profiles of inflammatory cytokines viz. interleukins (IL)-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor, interferon-γ and tumor necrosis factor-α in response to subclinical mastitis in indigenous cattle breed Kankrej (n = 6), Gir (Bos indicus) (n = 12) and crossbred (Bos taurus × Bos indicus) (n = 7) were investigated using quantitative real time PCR. Significant correlation (p < 0.05) was observed between total bacterial load and somatic cell count (SCC) in all three breeds of cattle. All the cytokines were observed to be up-regulated compared to cows with healthy quarters, however, level of their expression varied among three breeds of cattle. In Kankrej most cytokines were found to be transcribed to higher levels than in other two breeds; the milk had higher load of bacteria but not so high SCC, implying that Kankrej has a higher inherent resistance against mastitis. The results of present study indicated that mammary glands of crossbred cattl...