Clinical Mastitis Group Research Articles

Changes in the Microbiome in Yak Mastitis: Insights Based on Full-Length 16S rRNA Sequencing.

Mastitis is an inflammation of the mammary gland that can be caused by various factors, including biological, chemical, mechanical, or physical. Microbiological culture, DNA techniques, and high-throughput next-generation sequencing have been used to identify mastitis-causing pathogens in various animal species. However, little is known about microbiota and microbiome changes linked to yak milk mastitis. This study aimed to characterize the milk microbiota of healthy and mastitis-infected yaks using full-length 16S rRNA sequencing. The results showed that the bacterial microbiota comprises 7 phyla, 9 classes, 20 orders, 39 families, 59 genera, and 72 species. Proteobacteria and Firmicutes were the predominant microbial communities, with lower abundances of Bacteroidota, Actinobacteriota, Acidobacteriota, and other minor groupings also observed. Proteobacteria dominated the clinical and subclinical mastitis groups (95.36% and 89.32%, respectively), in contrast to the healthy group (60.17%). Conversely, Firmicutes were more common in the healthy group (39.7%) than in the subclinical and clinical mastitis groups (10.49% and 2.92%, respectively). The predominant organisms found in the healthy group were Leuconostoc mesenteroides, Lactococcus piscium, Carnobacterium maltaromaticum, and Lactococcus raffinolactis. Low abundances of Staphylococcus aureus species were found in both subclinical and clinical mastitis groups, with Moraxella osloensis and Psychrobacter cibarius dominating the subclinical mastitis group and Pseudomonas fluorescens dominating the clinical mastitis group. An alpha diversity study revealed that the healthy group had a higher microbial diversity than the clinical and subclinical mastitis groups. According to beta-diversity analysis, the principal coordinate analysis identified that mastitis-infected samples significantly differed from healthy ones. The milk microbiota of healthy yaks is more varied, and specific prominent taxa within various groups can act as marker microorganisms for mastitis risk. The genera Leuconostoc and Lactococcus are promising candidates for creating probiotics.

SLC34A2 Targets in Calcium/Phosphorus Homeostasis of Mammary Gland and Involvement in Development of Clinical Mastitis in Dairy Cows.

The type II Na/Pi co-transporter (NaPi2b), encoded by the solute carrier (SLC) transporter 34A2 (SLC34A2), is responsible for calcium (Ca) and phosphorus (P) homeostasis. Unbalanced Ca/P metabolism induces mastitis in dairy cows. However, the specific role of SLC34A2 in regulating this imbalance in Holstein cows with clinical mastitis (CM) remains unclear. The aim of this study was to investigate the role of SLC34A2 and identify differentially expressed proteins (DEPs) that interact with SLC34A2 and are associated with Ca/P metabolism in dairy cows with CM. Immunohistochemical and immunofluorescence staining results showed that SLC34A2 was located primarily in the mammary epithelial cells of the mammary alveoli in both the control (healthy cows, Con/C) and CM groups. Compared to the Con/C group, the relative expression of the SLC34A2 gene and protein were significantly downregulated in the CM group. We identified 12 important DEPs included in 11 GO terms and two pathways interacting with SLC34A2 using data-independent acquisition proteomics. The PPI (protein-and-protein interaction) network results suggested that these DEPs were associated with ion metabolism and homeostasis, especially SLC34A2. These results demonstrate that SLC34A2 downregulation is negatively correlated with the occurrence and development of CM in Holstein cows, providing a basis for exploring the function and regulatory mechanism of SLC34A2 in Ca/P metabolism and homeostasis in Holstein cows with CM.

Investigation of milk microbiota of healthy and mastitic Sahiwal cattle

BackgroundSahiwal cattle is an indigenous cattle breed of Pakistan and mastitis is one of the major problems faced by Sahiwal cattle which hinders its production potential. The study was designed to investigate the milk microbiota of healthy and mastitic Sahiwal cattle as part of a multistep project to develop probiotics for the mitigation and control of mastitis. Milk samples of Sahiwal cattle (healthy clinical mastitis and subclinical mastitis) reared under similar husbandry and management practices were processed for 16S rRNA gene base metagenomics analysis.ResultsResults revealed that Proteobacteria were dominant in the healthy group and subclinical mastitis group (56.48% and 48.77%, respectively) as compared to the clinical mastitis group (2.68%). In contrast, Firmicutes were abundant in the clinical mastitis group (64%) as compared to the healthy and subclinical mastitis groups (15.87% and 38.98%, respectively). Dominant species assigned in the healthy group were Ignavibacterium album, Novosphingobium capsulatum, Akkermansia muciniphila and Lactobacillus fermentum.The clinical mastitis group was dominated by Streptococcus dysgalactiae and Corynebacterium bovis, while subclinical mastitis group included Lactobacillus fermentum and uncultured acidobacteriales and Akkermansia muciniphila as dominant species. Alpha diversity indices showed higher microbial diversity in the healthy group compared to the clinical and sub-clinical mastitis groups.ConclusionIt is concluded that the milk microbiota of healthy sahiwal cattle has higher diversity and dominant taxa in the different groups may be used as signature microbes for mastitis susceptibility. Akkermansia muciniphila is one of candidate specie that was identified and may be used for development of probiotics.

Exploring the Milk Microbiota of Healthy and Mastitic Nili Ravi Buffalo Using 16S rRNA Gene Base Metagenomic Analysis.

The Nili Ravi, a buffalo breed from Pakistan, significantly contributes to the dairy industry. Mastitis is one of the major challenges affecting milk production in this breed. The objective of the current study was to identify the bacterial communities and diversity in healthy and mastitic milk of this breed. Milk samples (n = 14) were collected from Nili Ravi buffaloes with different udder health statuses, i.e., healthy (5), subclinical mastitis (4), and clinical mastitis (5). The DNAs were extracted, subjected to partial amplification of 16S rDNA (V3 and V4 regions), and sequenced using the Illumina platform. The results revealed variations in the bacterial communities in the milk of animals with different udder health statuses. Proteobacteria was the predominant phylum in the healthy group, while clinical and subclinical mastitis milk had a higher abundance of Firmicutes. Dominant bacterial genera in the healthy group were Streptococcus (11.60%), Herbaspirillum (7.65%), and Staphylococcus (4.70%), whereas the clinical mastitis group was dominated by Streptococcus (33.96%), Staphylococcus (7.87%), and Corynebacterium (2.68%), and the subclinical mastitis group was dominated by Bacillus (15.70%), Corynebacterium (6.70%), and Staphylococcus (6.58%). Assignment of operational taxonomic units at the species level resulted in most species being assigned to uncultured or unknown bacteria or remaining unassigned. Alpha diversity indices indicated lower microbial diversity in the clinical mastitis group, while beta diversity indices showed a scattered pattern of sample clustering in PCA plots among different groups. It is concluded that bacterial diversity in the milk of Nili Ravi buffaloes suffering from clinical mastitis is lower compared to healthy and subclinical mastitis cases. It is concluded that the variations in the microbiota of healthy and mastitic milk may be further investigated and exploited as signature microbes associated with the udder health status of Nili Ravi buffalo.

Comparative Proteomic Analysis of Milk-Derived Extracellular Vesicles from Dairy Cows with Clinical and Subclinical Mastitis.

Extracellular vesicles (EVs) are membranous vesicles found in biological fluids with essential functions. However, milk-derived EV proteins from clinical mastitis (CM) and subclinical mastitis (SM) cows have yet to be studied in detail. In this study, milk-derived EVs of CM, SM, and Healthy cows were extracted using a combination of acetic acid/ultracentrifugation and density gradient ultracentrifugation and analyzed using a shotgun proteomic by data-independent acquisition mode. A total of 1253 milk exosome proteins were identified and quantified. Differently enriched (DE) proteins were identified as given a Benjamini−Hochberg adjusted p < 0.05 and a fold change of at least 2. There were 53 and 1 DE proteins in milk-derived EVs from CM and SM cows compared with healthy cows. Protein S100-A9, Protein S100-A8, Chitinase-3-like protein 1, Haptoglobin, Integrin beta-2, and Chloride intracellular channel protein 1 were more abundant in the CM group (adjusted p < 0.05). Still, their enrichment in the SM group was not significant as in the Healthy group. The enrichment of DE proteins between CM and Healthy group was consistent with elevated GO (Gene Ontology) processes—defense response, defense response to Gram-positive bacterium, granulocyte chemotaxis also contributed to Reactome pathways—neutrophil degranulation, innate immune system, and antimicrobial peptides in the CM group. These results provide essential information on mastitis-associated proteins in milk-derived EVs and indicate the biological functions of milk-derived EVs proteins require further elucidation.

Sulfur Amino Acid Metabolism and the Role of Endogenous Cystathionine-γ-lyase/H2S in Holstein Cows with Clinical Mastitis.

Simple SummaryEndogenous hydrogen sulfide (H2S) has been implicated in many physiological and pathological processes, particularly in inflammatory responses and adaptive immunity. In this study, we identified the candidate differentially expressed proteins (DEPs) associated with H2S metabolism in Holstein cows with clinical mastitis (CM). The results revealed 17 DEPs included in 44 Gene Ontology (GO) terms and five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to sulfide metabolism and indicated the important role of cystathionine-γ-lyase (CTH)/H2S in CM. Our findings can support research into the function and regulatory mechanism of CTH/H2S in Holstein cows and provide a basis for the prevention and treatment of CM.H2S plays an important role in various inflammatory diseases. However, the role of H2S and synthetic enzymes in Holstein cows with CM is unknown. The aim of this study was to identify DEPs associated with sulfide metabolism and further investigate their roles in dairy cows with CM. From 3739 DEPs generated by data-independent acquisition proteomics, we identified a total of 17 DEPs included in 44 GO terms and five KEGG pathways related to sulfide metabolism, including CTH and cystathionine-β-synthase (CBS). Immunohistochemical and immunofluorescence staining results showed that CTH and CBS proteins were present mainly in the cytoplasm of mammary epithelial cells. Endogenous H2S production in the serum of the CM group was significantly lower than that of the healthy Holstein cows. CTH and CBS mRNA and protein levels in the mammary glands of the CM group were significantly downregulated compared to those of the healthy group. These results indicate that CTH and H2S were correlated with the occurrence and development of CM in Holstein cows, which provides important insights into the function and regulatory mechanism of CTH/H2S in Holstein cows.

Effect of retained placenta and clinical mastitis on reproduction parameters, immune response, and steroidogenic receptors gene expression in postpartum crossbred dairy cows.

The purpose of this study was to evaluate the effect of retained placenta (RP) and clinical mastitis (CM) on the reproductive efficiency of crossbred dairy cows during the postpartum period and the effect in some innate immune system indicators. For this, two experiments were carried out. In the first, a total of 232 cows were evaluated and divided as: healthy control (n = 184), RP (n = 22), and CM (n = 26) groups. The RP and CM was evaluated until 30 days postpartum (DPP) and reproductive rates were measured. In experiment 2, cows were divided in control (n = 10), RP (n = 10), and CM (n = 30) groups. Between 40 and 50 DPP, clinical, gynecological examination and endometrial cytobrush were performed to evaluate subclinical endometritis (SE) and gene expression of interleukins 1β (IL-1β) and 6 (IL-6), chemokine ligand 5 (CCL5), estrogen α (ESR1), and progesterone (PGR) receptors by qRT-PCR analysis. In experiment 1, the conception rate at 1st artificial insemination (AI) was lower in RP and CM groups and pregnancy rate at 150 days decreased in CM group. Calving-to-1st AI interval and days open were shorter in healthy cows. In experiment 2, the occurrence of SE was 26.7% and higher in RP and CM groups. The expression of IL-1β increased in RP and CM groups, while IL-6 was less expressed in RP group. The CCL5, ESR1, and PGR were similar between groups. In conclusion, cows with RP and CM had their reproductive efficiency negatively affected and had they initial pro-inflammatory response improved by the increase of IL-β.

Levels of milk glycosaminoglycans in Holstein cows with clinical and subclinical mastitis

Mastitis is an inflammation of the mammary gland whose diagnosis is based on clinical criteria and the determination of inflammatory markers. The disease is mainly caused by bacteria causing intra-mammary infections. The etiological agent/s are detected by milk culture or by molecular biology approaches. However, these methodologies are unpractical for on-farm diagnosis, where rapid decisions must be made when infected animals are detected. For this reason, the levels of glycosaminoglycans (GAGs) in milk were evaluated as a tool for the diagnosis of mastitis. GAGs levels were determined by the dimethylmethylene blue assay (DMMB) method in milk from healthy animals and in milk from animals with mastitis caused by different etiological agents. Low levels of GAGs (1.93 ± 0.40 mg) were found in milk samples obtained from healthy animals (H), while intermediate (2.4 ± 0.61 mg) and high levels (2.87 ± 0.7 mg) were found in milk obtained from cows with subclinical (SM) and clinical mastitis (CM), respectively. Statistically significant differences were found between the SM and H groups (p < 0.001) and between CM and the other groups (p < 0.0001). The highest levels of GAGs were detected in the CM group, in which coagulase negative Staphylococcus (CNS) and Streptococcus spp. were isolated. Animals infected by S. aureus had the highest levels of GAGs in the SM group. The sensitivity (Se) and specificity (Sp) for the assessment of GAGs levels for CM diagnosis were 89.7 % and 81.6 %, respectively. For the diagnosis of SM, Se and Sp were 73.2 % and 95 %, respectively. The area under the curve for the diagnosis of CM was 0.902 (p < 0.0001), while that for SM was 0.833 (p < 0.0001). Results suggest that the measurement of GAGs levels in milk can be used as an indicator of udder health in dairy cattle, since increased levels of GAGs were associated with mastitis.

INFRARED THERMOGRAPHY TO MAP THE UDDER HEALTH STATUS OF ZEBUINE DAIRY COWS

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; Infrared thermography is a diagnostic imaging technique that detects the emission of heat by infrared radiation, indicating the temperature of a particular area. &lt;strong&gt;Objective&lt;/strong&gt;: The objective of this study was to measure the magnitude of the udder temperature variation by infrared thermography for mastitis diagnosis of zebuine dairy cows in a tropical pasture-based system. &lt;strong&gt;Methodology:&lt;/strong&gt; The udder temperature of Holstein-Gyr crossbred cows was measured with a FLIR&lt;sup&gt;®&lt;/sup&gt; camera. Milk samples were collected for CMT and somatic cell count determination. The surface temperature of the mammary gland and rectal temperature were grouped according to udder health status of animals and were submitted to analysis of variance and correlation analysis. &lt;strong&gt;Results:&lt;/strong&gt; The udder temperatures were higher for the clinical mastitis group (37.2±0.7 °C) than the subclinical mastitis (36.6±0.9 °C) and healthy udder (36.3 ±1.0 °C) groups. A weak positive correlation (r=0.18) was observed between somatic cell count (SCC) and udder temperature. A strong positive correlation (r=0.68) was observed between rectal temperature and udder temperature. &lt;strong&gt;Implications:&lt;/strong&gt; The results of this work can be applied in the future in precision animal husbandry, generating more efficient strategies and practices for the diagnosis and control of mastitis, since it is a promising tool for easy and quick diagnosis of mastitis. &lt;strong&gt;Conclusions:&lt;/strong&gt; Infrared thermography can be an effective method for diagnosing mastitis in cows. However, its use should be made with the complementary California mastitis test.&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;

A novel subtraction diversity array distinguishes between clinical and non-clinical Streptococcus uberis and identifies potential virulence determinants

Streptococcus uberis is an important bovine mastitis pathogen, but not all isolates have equal capacity to cause disease. The aims of this study were to identify possible virulence-associated genes that could be used to identify isolates with enhanced virulence. DNA from a pool of putative commensals was subtracted from a clinical pool resulting in a set of DNA sequences (probes) that were enriched in the clinical mastitis group. The probes were hybridised with DNA from a collection 29 isolates from cases of clinical mastitis and isolates not associated with disease. Hybridization revealed five major clusters. The first cluster (7 isolates) consisted almost entirely of commensals, while the second (7 isolates) was mixed. The remaining three clusters contained 15 S. uberis isolates from cows with clinical mastitis. Twenty-six probes were selected for sequencing based on principal component analysis (PCA) or their presence mainly in clinical isolates. PCA identified five probes with clear differences in intensity between signals from clinical isolates and commensals; these probes could represent novel virulence determinants. Manual inspection of arrays identified genes prominent among clinical isolates that specify carbohydrate and lipid metabolism (possible role in the growth or survival of S. uberis in milk) and genes specifying hypothetical proteins, possibly novel virulence factors. The common occurrence, among clinical isolates, of probes having homology with transposases and insertion sequences suggests recent acquisition of factors that could be associated with virulence. These results suggest the existence of a subset of S. uberis with enhanced virulence, due possession of virulence-associated gene sequences.

Paraoxonase-1 activity and lipid profile in dairy cows with subclinical and clinical mastitis

ABSTRACT The aim of this study was to investigate serum paraoxonase-1 (PON1) activity and lipid status in cows with subclinical and clinical mastitis in order to evaluate systemic inflammatory and oxidative stress responses. The study was conducted on a total of 90 Holstein–Friesian dairy cows kept in farms in eastern Croatia. Cows were assigned into three groups: the cows suffering from clinical mastitis (CLM), the cows with subclinical mastitis (SCM) and control (CTL) group. In collected sera, PON1, lipid status and calcium concentration were measured. Total cholesterol (CHOL), high-density lipoprotein cholesterol (HDL-C) and calcium concentrations were significantly lower in the CLM group of cows compared to the SCM and CTL groups (P < 0.05). There were no significant differences in the lipid status and calcium level between the CTL and SCM groups. PON1 activity was significantly lower in both the SCM and CLM groups compared with CTL indicating that PON1 could be considered as a potential biomarker for the diagnosis of subclinical form of the disease.

Surface expression of CD11b, CD62L, CD44 receptors on blood and milk neutrophils during subclinical and clinical mastitis in Sahiwal cows

Surface expression of cell adhering molecules and CD44 was studied in Sahiwal (SW) cows suffering naturally from subclinical (SCM) and clinical mastitis (CM). Cows were divided into groups, viz. healthy (12), SCM (12) and CM (12) groups on the basis of CMT scoring, bacteriological culture, gross morphological changes in the milk and by counting milk SCC after screening of 136 SW cows. Bacteriological culture indicated infection of Staphylococcus aureus as the causative agent of SCM and CM. Healthy cows showed significantly higher CD62L expression as compared to the SCM and CM group of animals in both blood and milk neutrophils but no significant difference was found between blood and milk neutrophils. The results revealed a significant upregulation of CD11b positive cells in the CM group of cows. Significantly higher expression of CD44 was found in the neutrophils of both blood and milk of SCM group as compared to CM group. From the study, it was evident that neutrophils exhibit dynamic interplay between the integrins (CD11b) and selectins (CD62L) along with CD44 during SCM and CM Staphylococcal mastitis to offer host protection against the foreign invaders.

Neutrophil dynamics in the blood and milk of crossbred cows naturally infected with Staphylococcus aureus.

Aim:The present study was designed to evaluate the neutrophil dynamics in terms of the functional competence during subclinical mastitis (SCM) and clinical mastitis (CM).Materials and Methods:A total of 146 Karan fries cows were screened and were divided into three groups as control (n=12), SCM, n=12 and CM, n=12 groups on the basis of California mastitis test scoring, bacteriological evaluation, gross and morphological changes in milk and by counting milk somatic cell count (SCC). Both blood and milk polymorphonuclear neutrophils (PMNs) were isolated in the study. Phagocytic activity (PA) was studied by spectrophotometrically; neutrophil extracelluar traps (NETs) were studied by scanning electron microscopy (SEM); CD44 was quantified by flow cytometry and apoptosis was studied by fluorescent microscopy.Results:Significantly (p<0.05) higher SCC, PA was found in milk of CM cows as compared to SCM and control cows. Significantly lower (p<0.05) apoptosis was observed in PMNs isolated from both blood and milk of CM group of cows when compared to control and SCM group. The milk neutrophils of CM group of cows formed NETs as evidenced from the SEM images. Surface expression of CD44 revealed a significantly (p<0.05) lower expression in milk neutrophils of CM group of cows when compared to SCM and control group of cows.Conclusion:The study indicated a positive correlation between delayed neutrophil apoptosis, persistent staying of neutrophils at the site of infection along with formation of NETs as the strategies to fight against the pathogens in the udder during Staphylococcal mastitis. The study forms a strong base for future molecular research in terms of neutrophil recruitment and neutrophil removal from the site of infection.

Analysis of genetic polymorphism of IL8R receptor gene a marker associated with bovine mastitis among crossbred cattle

A total of 60 crossbred cattle were screened for subclinical and clinical mastitis in and around Jabalpur using California mastitis test (CMT) followed by somatic cell count (SCC). The DNA was isolated and 311 bp fragments of IL8R gene was amplified using PCR. PCR-SSCP (Single Strand conformational polymorphism) method was used to identify genetic polymorphism of 311 bp amplicon of IL8R gene. PCR-SSCP revealed five SSCP patterns, SSCP pattern 1 (P1) was present in highest percentage 63.6% in healthy group, SSCP pattern 3 (P3) was present in highest percentage 64.2% in subclinical mastitis group while SSCP patterns (P2 and P5) were present in highest frequency 100% and 100% respectively in clinical mastitis group. Cattles exhibiting pattern P2, P5 and P3 were significantly (p<0.05) associated with susceptibility to clinical and subclinical mastitis in crossbred cattle while those exhibiting pattern P1 were significantly (p<0.05) associated with mastitis resistance. The animals exhibiting SSCP pattern P1 may have IL-8R haplotypes that are good genetic markers for increased mastitis resistance while animals exhibiting pattern P2, P5 and P3 may have IL-8R haplotypes as good genetic markers for clinical and subclinical mastitis susceptibility. Thus, animals exhibiting these patterns can be selected or culled at an early age for future breeding.

Formation of NET, phagocytic activity, surface architecture, apoptosis and expression of toll like receptors 2 and 4 (TLR2 and TLR4) in neutrophils of mastitic cows.

Neutrophils employ both oxidative and non oxidative mechanisms to destroy pathogens. Function of neutrophils coming in milk during mammary invasion is not clearly understood in dairy animals. Therefore, the present study was designed in 36 Sahiwal cows to see the changes occurring in the neutrophil activity of cows suffering from subclinical (SCM) and clinical mastitis (CM). Cows were divided into three groups as healthy (n = 12), SCM (n = 12) and CM (n = 12) groups on the basis of CMT scoring, gross morphological changes in milk, bacteriological examination of milk and by counting milk SCC. Significantly (P < 0.05) higher milk SCC, neutrophil percent and significantly (P < 0.05) lower viability of both blood and milk neutrophils were observed in CM group of cows as compared to SCM and control group of cows. Phagocytic activity (PA) was significantly (P < 0.05) higher in milk neutrophils of SCM and CM cows as compared to control cows. Toll like receptors 2 and 4 in blood and milk neutrophils were found to be significantly (P < 0.05) higher, whereas, apoptosis in terms of altered mitochondrial transmembrane potential, Caspase 3 and 7 activities were found to be significantly (P < 0.05) lower in CM cows. Alterations in surface architecture of neutrophils in terms of formation of pseudopods was observed by scanning electron microscope (SEM) and found to be higher in CM cows. Blood neutrophils were found to be spherical as compared to milk neutrophils. Formation of neutrophil extracellular trap (NETs) were found milk neutrophils of CM cows, whereas, SCM and healthy cows did not exhibit NET formation. The study indicated a positive correlation between lower neutrophil apoptosis and higher expression of TLR2 and TLR4 with the formation of NETs and change in surface architecture. Formation of NET like structures seemed to be an effective mode of defense employed by neutrophils of cows suffering from clinical mastitis.

Mastitis detection in sheep by infrared thermography

This study aims to evaluate the use of an infrared thermograph for mastitis diagnosis in sheep. Thirty-seven Santa Inês ewes were evaluated weekly through infrared images obtained with thermograph FLIR System Series-i®. Milk was collected for somatic cell count and milk compound level determination. The clinical mastitis group had the highest fat and protein level, as well as the lowest lactose level. The udder temperatures were higher for subclinical mastitis group. The udder temperature data was able to correctly classify the animals into the mastitis groups and the canonical analysis showed that these temperatures clearly differentiated the subclinical mastitis groups from the others. Therefore, this study showed that udder infrared temperatures can be used as diagnostic method to mastitis in sheep.

Naturally occurring mastitis effects on timing of ovulation, steroid and gonadotrophic hormone concentrations, and follicular and luteal growth in cows

The effects of naturally occurring subclinical chronic or clinical short-term mastitis on time of ovulation, plasma steroid and gonadotropin concentrations, and follicular and luteal dynamics were examined in 73 lactating Holstein cows. Cows were sorted by milk somatic cell count and bacteriological examination into an uninfected group (n=22), a clinical mastitis group (n=9; events occurring 20±7 d before the study), and a subclinical chronic mastitis group (n=42). In addition, uninfected and mastitic cows were further sorted by their estrus to ovulation (E-O) interval. About 30% of mastitic cows (mainly subclinical) manifested an extended E-O interval of 56±9.2h compared with 28±0.8h in uninfected cows and 29±0.5h in the other 70% of mastitic cows. In mastitic cows with extended E-O interval, the concentration of plasma estradiol at onset of estrus was lower than that of uninfected cows or mastitic cows that exhibited normal E-O intervals (3.1±0.4, 5.8±0.5, and 5.5±0.5 pg/mL, respectively). The disruptive effect of mastitis on follicular estradiol probably does not involve alterations in gonadotropin secretion because any depressive effects of mastitis on pulsatile LH concentrations were not detected. Cortisol concentrations did not differ among groups. The preovulatory LH surge in mastitic cows with delayed ovulation varied among individuals, being lower, delayed, or with no surge noted compared with the normal LH surge exhibited by uninfected cows or mastitic cows with normal E-O interval (6.8±0.7 ng/mL). The diameter of the second-wave dominant follicle was larger and the number of medium follicles was smaller in uninfected and subclinical cows with normal intervals compared with subclinical cows with extended intervals (13.4±0.5 vs. 10.9±0.9mm, and 3.8±0.2 vs. 6.7±0.14 follicles, respectively). Mid-luteal progesterone concentrations were similar in uninfected and mastitic cows. These results indicate for the first time that around 30% of cows with subclinical chronic mastitis exhibit delayed ovulation that is associated with low plasma concentrations of estradiol and a low or delayed preovulatory LH surge.

Enhanced Erythrocytic Lipid Peroxides and Reduced Plasma Ascorbic Acid, and Alteration in Blood Trace Elements Level in Dairy Cows with Mastitis

Oxidative stress has been associated in several inflammatory conditions and incriminated in the pathogenesis of many diseases. However, little information is available on the status of plasma antioxidant levels, essential components of important antioxidant enzymes such as copper, zinc and selenium in blood, and the end product of oxidative damage to the erythrocytic polyunsaturated fatty acids in inflammatory udder conditions. Blood samples were collected from three groups of dairy cows, with 21 in each group: animals with healthy udder, clinical mastitis, and subclinical mastitis. These animals were randomly selected from a herd on the basis of the California mastitis test, somatic cell count and total bacterial count. The mean plasma ascorbic acid concentration was significantly lower in cows with subclinical (p = 0.004) and clinical mastitis (p = 0.000) and the erythrocytic lipid peroxide levels were significantly (p = 0.000) higher in clinical mastitis as compared to controls. There was a significant decrease in mean blood zinc concentration in subclinical (p = 0.005) and clinical mastitis (p = 0.000), but an increase in mean blood copper level in the clinical mastitis group. It was concluded that the blood antioxidant status declines in inflammatory udder conditions, suggesting that incorporation of antioxidants may help in better management of mastitis in dairy cows.

Reduced Levels of Total Leukocytes and Neutrophils in Norwegian Cattle Selected for Decreased Mastitis Incidence

Two groups of Norwegian Cattle showing significant genetic differences in clinical mastitis susceptibility were examined for hematological changes at three stages of lactation. Blood samples were taken from 91 healthy Norwegian Cattle cows and heifers belonging either to a high protein yield group or to a low clinical mastitis group and analyzed for hematological properties and serum cortisol levels at three stages of lactation. All animals were free of intramammary infections at the time of sampling.All cows had increased total white blood cells, neutrophils, and neutrophil/lymphocyte ratio as parturition approached, with peak levels at parturition. Cows selected for low clinical mastitis had lower levels of total white blood cells and neutrophils compared with cows selected for high protein yield throughout all three periods. The difference was significant prepartum. Cows from the low clinical mastitis group also had lower neutrophil/lymphocyte ratios and lower serum cortisol levels than those in the high protein yield group. A significant positive correlation was found between cortisol and total white blood cells and neutrophils, respectively.We conclude that selection for lower mastitis incidence in the low clinical mastitis group is associated with a significant decrease in total white blood cells and neutrophils in blood prepartum. Results from the present study, and the genetic difference in mastitis incidence observed in the groups, indicates that increased leukocyte mobilization at certain stages of lactation may be associated with increased susceptibility to mastitis.

Norfloxacin pharmacokinetics in lactating cows with sub-clinical and clinical mastitis.

Single-dose pharmacokinetics of norfloxacin after intravenous administration of norfloxacin nicotinate at 10 mg norfloxacin/kg body weight was investigated in cows with healthy udders and in cows with chronic subclinical and postacute clinical mastitis. An HPLC method was used to determine the norfloxacin concentrations in serum and milk. Significant differences were observed in norfloxacin pharmacokinetics when administered to cows with infected udder quarters. The clearance (Cl) values were 10.4+/-2.5, 13.2+/-1.9 and 14.2+/-2.1 mL/min/kg (mean +/-SD) in the control (healthy udder) cows and in cows with subclinical and postacute clinical mastitis, respectively. There appeared to be a trend of increasing clearance according to severity of the disease. The volume of distribution at steady state (Vss) in the respective groups was 3.1+/-0.7, 2.2+/-0.6 and 1.3+/-0.2 L/kg. The volume of distribution was significantly decreased in the cows with postacute clinical mastitis. The half-lives (t1/2) and mean residence times (MRT) of norfloxacin were 353, 206 and 115 min (harmonic means) and 306+/-76, 168+/-39 and 95+/-9 min in control cows or in cows with subclinical and postacute clinical mastitis, respectively. The half-lives in the clinical mastitis group were significantly shorter than in the control group and the mean residence times were significantly shorter in the two mastitis groups when compared to the control group. Norfloxacin concentrations in milk were extremely high when compared to the respective serum concentrations. The area under the concentration vs. time curve (AUC) of norfloxacin in milk was 23899+/-6206 mg/L x min in the control cow group. The AUC in milk was significantly lower in the infected udder quarters of the mastitis groups (5075+/-1887 mg/L x min and 7484+/-4645 mg/L x min in the subclinical and the clinical group). The AUC values were significantly lower in milk from the infected udder quarters of the cows with chronic subclinical and postacute clinical mastitis when compared to the values in milk from the healthy quarters of the same udder. Norfloxacin was marginally bound to serum protein. The binding was concentration dependent and was 19, 13 and 6% at 0.2, 1.0 and 8.4 mg/L, respectively. Binding to milk protein was 46-51% and concentration independent. An in vitro dialysis model was used to simulate drug transport between serum and milk as a function of protein binding. The results showed that the rate of norfloxacin disposition from milk to serum was slower than from serum to milk, which was in agreement with the findings obtained in the pharmacokinetic study. Norfloxacin was poorly soluble in organic solvents and our results suggest that changes in the degree of ionization of the drug in different body fluids considerably affect its disposition.