Subclinical Intramammary Infections Research Articles

Interleukin-6 as a Milk Marker of Clinical and Subclinical Intramammary Infections (IMI) in Cows Caused by Streptococcus spp.

The aim of the study was to evaluate the concentrations of Interleukin-6 (IL-6) in milk and serum of healthy cows (HE) and cows with mastitis caused by Streptococcus spp. The blood and milk samples were obtained from Holstein-Friesian cows (Lublin region, Poland). A total of 43 milk and serum samples from 28 cows with mastitis and 15 healthy cows were selected for study. IL-6 levels in milk from HE cows ranged from 6.09-80.24 pg/mL (median 26.6 pg/mL) and were significantly lower than in milk from both cows with clinical and subclinical mastitis (487.09 pg/mL vs. 26.6 pg/mL in CM, p < 0.001; and 165.31 pg/mL vs. 26.6 pg/mL in SCM, p < 0.001). The IL-6 concentration in the serum of HE was not significantly different from the serum IL-6 of the entire group of mastitis cows, regardless of whether the inflammation proceeded in a clinical or subclinical form (44.37 pg/mL vs. 78.09 pg/mL; 128.29 pg/mL vs. 78.09 pg/mL, respectively). The present study indicates that cows with mastitis caused by Streptococcus spp. develop a local immune response in the mammary gland in response to the pathogen. Monitoring of IL-6 levels in milk can allow early detection of mastitis, which is especially important in cases of subclinical inflammation.

Strategic administration of antioxidant multiminerals and vitamins to transitional buffaloes augments antioxidant and udder defense mechanisms in early lactation

The aim of the study was to examine the effects of repeated administrations of antioxidant multiminerals and vitamins in transition buffaloes on udder defense mechanism, antioxidant activity and occurrence of intramammary infection (IMI) in early lactation period. Forty clinically healthy pregnant buffaloes were enrolled 45 days before expected date of calving and randomly allocated into five different supplementation groups (n = 8): only basal ration (control), vitamin E and selenium (VES), multiminerals (MM), ascorbic acid (AA) and chromium (Cr) picolinate in basal diet. The udder defense mechanism was monitored by measuring phagocytic activity (PA), myeloperoxidase (MPO) and nitric oxide (NO) productions in milk leukocytes, antioxidant activity was evaluated by measuring total antioxidant capacity (TAC) in plasma and occurrence of IMI was assessed by milk cytology, bacterial count in milk and visible clinical signs of udder until day 28 post-calving. The results showed that the VES and MM supplementations exhibited significantly higher PA, MPO and NO productions of milk leukocytes till first week of lactation whereas, elevated mean TAC in plasma was maintained from day −7 to 1 of calving in MM supplementation group as compared to control group. Statistically, no significant difference in occurrences of subclinical or clinical IMI was noted across the groups until four weeks of lactation. Taken together, it is concluded that repeated administrations of VES and MM to transition buffaloes could be an effective strategy to maintain good udder health by augmenting milk leukocyte functions and antioxidant status and preventing incidence of IMI in early lactation.

Evaluation of close up antimicrobial therapies for treatment and prevention of subclinical mastitis in the herds with high prevalence of Staphylococcus aureus

This field trial was conducted to evaluate two antibiotics at a close-up period in curing the existing IMI and to prevent new clinical and subclinical Intramammary infection (IMI). Two hundred and twelve Holstein cows were assigned to one of three treatment groups: TYLO, MARB and CONT. Cows in TYLO group received 10 mg/kg Tylosin for three days at the close-up period (21 days before calving), cows in MARB group received single SC injection of 8 mg/kg SC marbofloxacin at the close-up period and cows in CONT group remained untreated. Milk samples were collected for somatic cell count, microbial culture and Total oxidant/antioxidant capacity before drying-off, and 3 and 7days post calving. Antimicrobial susceptibility test and RAPT-PCR were performed on S.aureus isolates. No significant differences were detected in total cure rate within the groups, but S. aureus cure rates in TYLO and MARB were significantly higher than in CONT (74 and 73.5 % Vs 58.1 %). No significant differences in total new IMI were observed. Furthermore, the rate of new S. aureus IMI was higher in both treatment groups than in CONT group. Clinical mastitis rate in TYLO (3.8 %) and MARB (5.8 %) was significantly lower than CONT (11.3 %). Paired S. aureus isolates from dry-off and post-calving have been clustered into 9 different RAPD types (A-I). 8 paired strains collected at dry-off were identical to those at post-calving,and 35 strains had more than 60 % dissimilarity. Administration of Tylosin or Marbofloxacin is not useful in all cases;however, they have the potential to reduce the incidence of post-calving clinical mastitis and improve S.aureus cure rate if used selectively.

Indirect Sensing of Subclinical Intramammary Infections in Dairy Herds with a Milking Robot.

This study determined the impact of subclinical intramammary infections (IMIs), such as the major and minor udder pathogens (MaPs and MiPs), on the somatic cell count (SCC) in cow milk and investigated the possibilities of indirect sensing of the udder pathogens using the mastitis detection index (MDi) (DeLaval, Tumba, Sweden). The MDi incorporates quarter-level milk electrical conductivity, blood in milk, and milking interval. The case group (n = 21; MDi ≥ 1.4) was compared with the control group (n = 24; MDi < 1.4) for the presence of IMIs. The microbiological investigation of udder quarter foremilk samples was performed two times with an interval of 10 to 14 days. The case and control groups differed in terms of the occurrence of MaPs and MiPs in milk. During the continuous subclinical IMI and the episodic MaP infection, a higher SCC was detected compared with the episodic MiP infection or quarters without IMI. The novel finding of this study was that by using the milk quality sensor for the sensing of subclinical IMIs, there was an indication for the successful detection of episodic MaPs. However, the sensing of the continuous subclinical IMIs was not possible in the current study and still needs to be investigated.

Serum metabolome differences associated with subclinical intramammary infection caused by Streptococcus agalactiae and Prototheca spp. in multiparous dairy cows

Mastitis is one of the most significant diseases in dairy cows and causes several economic losses. Somatic cell count (SCC) is often used as an indirect diagnostic tool for mastitis, especially for subclinical mastitis (SCM) where no symptoms or signs can be detected. Streptococcus agalactiae is one of the main causes of contagious mastitis, while Prototheca spp. is an alga inducing environmental mastitis that is not always correlated with increased milk SCC. The aim of this study was to evaluate the changes in the metabolomic profile of blood in relation to subclinical intramammary infection (sIMI) in dairy cows. In addition, differences due to the etiologic agent causing mastitis were also considered. Forty Holstein-Friesian dairy cows in mid and late lactation were enrolled in this study with a cross-sectional design. Based on the bacteriological examination of milk, the animals were divided into 3 groups: Group CTR (control group; n = 16); Group A (affected by SCM with IMI of Streptococcus agalactiae; n = 17); and Group P (affected by SCM with IMI of Prototheca spp.; n = 7). Blood samples were collected in tubes containing clot activator from jugular vein. The serum aliquot was stored until metabolomic analysis by 1H-NMR. Statistical analysis was conducted fitting a linear model with the group as fixed effect and SCC as covariate. Forty-two metabolites were identified and among them, 10 were significantly different among groups. Group A and P showed greater level of His and lactose, and lower level of acetate, Asn, and dimethylamine compared with Group CTR. Group A showed high level of Val, while the Group P showed also high level of Cit and methylguanidine, and lower level of 3-hydroxybutyrate, acetone, allantoin, carnitine, citrate, and ethanol. These metabolites were related to ruminal fermentations, energy metabolism, urea synthesis and metabolism, immune and inflammatory response, and mammary gland permeability. These results are suggestive of a systemic involvement of sIMI and that the metabolic profile of animals with SCM undergoes changes related to the etiological agent of mastitis.

Associations between subclinical intramammary infection and milk fatty acid profile at the quarter level in Holstein cattle.

Mastitis, especially the subclinical form, is the most common economic and health problem in dairy cows. Little is known about changes in milk fatty acid (FA) composition according to infection/inflammation status of the mammary gland. The aim of this study was to investigate the associations between naturally occurring subclinical intramammary infection (IMI) from different pathogens, i.e. Streptococcus agalactiae, Staphylococcus aureus, Streptococcus uberis and Prototheca spp., and the detailed milk FA profile assessed at quarter level in Holstein cows. After an initial bacteriological screening (T0) on 450 Holstein cows reared in three dairy herds, we identified 78 cows positive at the bacteriological examination. These animals were followed up at the quarter level two weeks (T1) and six weeks (T2) after T0. In total, 600 single-quarter samples were obtained at T1 and T2. Individual FAs were determined using the gas chromatography analytical method. Investigated traits were 70 individual FAs, 12 FA groups, and six desaturation indices. The associations between subclinical IMI combined with somatic cell count (SCC) and milk FA profile were investigated using a hierarchical linear mixed model (i.e., observational unit was quarter within cow) with the following fixed effects: days in milk (DIM), parity, herd, SCC, bacteriological status (BACT, positive and negative), and the SCC × BACT interaction. The random effect of individual cow nested within herd, DIM and parity was used as the error term for the latter effects. The most significant associations were detected at T2. Notably, IMI reduced the proportions of individual short-chain FA, especially 4:0 and 6:0 (-14%), but increased the proportion of the most abundant medium-chain FA (MCFA), 16:0 (+4%). A reduction in the desaturation indices was observed mostly for 14:1 index (-9%), in line with the reduction in 14:1 (-10%). Somatic cell count significantly affected 14 individual FAs. In particular, samples with high SCC (≥200 000) had significantly lower proportions of 8:0, 10:0, 11:0, 12:0, and 13:0 compared with samples with low SCC (<200 000). Increasing SCC in animals positive at the bacteriological examination were associated with a reduction in total MCFA at T2 (while in negative animals, they remained constant across SCC classes), possible evidence that elongation of the FA chain from 11 to 16 carbons is affected by a combination of infection and SCC. This study showed that subclinical IMI and SCC are mainly associated with reductions in the synthesis of FA and the desaturation process in the mammary gland.

Blood biochemical changes upon subclinical intramammary infection and inflammation in Holstein cattle

The aim of this study was to investigate the associations between subclinical intramammary infection (IMI) from different pathogens combined with inflammation status and a set of blood biochemical traits including energy-related metabolites, indicators of liver function or hepatic damage, oxidative stress, inflammation, innate immunity, and mineral status in 349 lactating Holstein cows. Data were analyzed with a linear model including the following fixed class effects: days in milk, parity, herd, somatic cell count (SCC), bacteriological status (positive and negative), and the SCC × bacteriological status interaction. Several metabolites had significant associations with subclinical IMI or SCC. Increased SCC was associated with a linear decrease in cholesterol concentrations which ranged from -2% for the class ≥50,000 and <200,000 cells/mL to -11% for the SCC class ≥400,000 cells/mL compared with the SCC class <50,000 cells/mL. A positive bacteriological result was associated with an increase in bilirubin (+24%), paraoxonase (+11%), the ratio paraoxonase/cholesterol (+9%), and advanced oxidation protein product concentration (+23%). Increased SCC were associated with a linear decrease in ferric reducing antioxidant power concentrations ranging from -3% for the class ≥50,000 and <200,000 cells/mL to -9% for the SCC class ≥400,000 cells/mL (respect to the SCC class <50,000 cells/mL). A positive bacteriological result was associated with an increase in haptoglobin concentrations (+19%). Increased SCC were also associated with a linear increase in haptoglobin concentrations, which ranged from +24% for the class ≥50,000 and <200,000 cells/mL (0.31 g/L) to +82% for the SCC class ≥400,000 cells/mL (0.45 g/L), with respect to the SCC class <50,000 cells/mL (0.25 g/L). Increased SCC were associated with a linear increase in ceruloplasmin concentrations (+15% for SCC ≥50,000 cells/mL). The observed changes in blood biochemical markers, mainly acute phase proteins and oxidative stress markers, suggest that cows with subclinical IMI may experience a systemic involvement.

Transcriptome-wide mapping of milk somatic cells upon subclinical mastitis infection in dairy cattle

BackgroundSubclinical intramammary infection (IMI) represents a significant problem in maintaining dairy cows’ health. Disease severity and extent depend on the interaction between the causative agent, environment, and host. To investigate the molecular mechanisms behind the host immune response, we used RNA-Seq for the milk somatic cells (SC) transcriptome profiling in healthy cows (n = 9), and cows naturally affected by subclinical IMI from Prototheca spp. (n = 11) and Streptococcus agalactiae (S. agalactiae; n = 11). Data Integration Analysis for Biomarker discovery using Latent Components (DIABLO) was used to integrate transcriptomic data and host phenotypic traits related to milk composition, SC composition, and udder health to identify hub variables for subclinical IMI detection.ResultsA total of 1,682 and 2,427 differentially expressed genes (DEGs) were identified when comparing Prototheca spp. and S. agalactiae to healthy animals, respectively. Pathogen-specific pathway analyses evidenced that Prototheca’s infection upregulated antigen processing and lymphocyte proliferation pathways while S. agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle, and carbohydrate and lipid metabolism. The integrative analysis of commonly shared DEGs between the two pathogens (n = 681) referred to the core-mastitis response genes, and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells (r2 = 0.72), followed by the udder health (r2 = 0.64) and milk quality parameters (r2 = 0.64). Variables with r ≥ 0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cytohubba plug-in. The genes in common between DIABLO and cytohubba (n = 10) were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals (sensitivity > 0.89, specificity > 0.81, accuracy > 0.87, and precision > 0.69). Among these genes, CIITA could play a key role in regulating the animals’ response to subclinical IMI.ConclusionsDespite some differences in the enriched pathways, the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response. The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.

Evaluation of test-day milk somatic cell count to predict intramammary infection in late lactation grazing dairy cows

Use of selective dry cow antimicrobial therapy requires to precisely differentiate cows with an intramammary infection (IMI) from uninfected cows close to drying-off to enable treatment allocation. Milk somatic cell count (SCC) is an indicator of an inflammatory response in the mammary gland and is usually associated with IMI. However, SCC can also be influenced by cow-level variables such as milk yield, lactation number and stage of lactation. In recent years, predictive algorithms have been developed to differentiate cows with IMI from cows without IMI based on SCC data. The objective of this observational study was to explore the association between SCC and subclinical IMI, taking cognizance of cow-level predictors on Irish seasonal spring calving, pasture-based systems. Additionally, the optimal test-day SCC cut-point (maximized sensitivity and specificity) for IMI diagnosis was determined. A total of 2,074 cows, across 21 spring calving dairy herds with an average monthly milk weighted bulk tank SCC of ≤200,000 cells/mL were enrolled in the study. Quarter-level milk sampling was carried out on all cows in late lactation (interquartile range = 240-261 d in milk) for bacteriological culturing. Bacteriological results were used to define cows with IMI, when ≥1 quarter sample resulted in bacterial growth. Cow-level test-day SCC records were provided by the herd owners. The ability of the average, maximum and last test-day SCC to predict infection were compared using receiver operator curves. Predictive logistic regression models tested included parity (primiparous or multiparous), yield at last test-day and a standardized count of high SCC test-days. In total, 18.7% of cows were classified as having an IMI, with first parity cows having a higher proportion of IMI (29.3%) compared with multiparous cows (16.1%). Staphylococcus aureus accounted for the majority of these infections. The last test-day SCC was the best predictor of infection with the highest area under the curve. The inclusions of parity, yield at last test-day, and a standardized count of high SCC test-days as predictors did not significantly improve the ability of last test-day SCC to predict IMI. The cut-point for last test-day SCC which maximized sensitivity and specificity was 64,975 cells/mL. This study indicates that in Irish seasonal pasture-based dairy herds,with low bulk tank SCC control programs, the last test-day SCC (interquartile range days in milk = 221-240) is the best predictor of IMI in late lactation.

High genetic diversity and zoonotic potential of Staphylococcus aureus strains recovered from bovine intramammary infections in Colombians dairy herds

Genotyping of Staphylococcus aureus isolated from mastitis has become a fundamental tool to understand its complex epidemiology and to evaluate spillover events. The aim of this study was to describe the frequency of genotypes of the S. aureus strains isolated from intramammary infections by spa typing technique, and to evaluate the association between genotypes and the ability to form biofilm under in vitro conditions. Sixty-six strains of S. aureus recovered from bovines intramammary infections on 56 dairy herds located in 14 municipalities of the department of Antioquia were characterized. The majority of strains (65/66) were isolated from milk samples collected from dairy cows with subclinical intramammary infections. Nineteen different spa types were found in this study, t521 (19.70%), t267 (15.15%), and t605 (12.12%) being the most frequent. The strains from the t605 spa type showed the highest biofilm production. The high frequency of spa types with zoonotic potential found in this study, identified cattle as an important reservoir of theses clones for people in close proximity, such as milkers and consumers of unpasteurized dairy products.

Detection of Methicillin-Resistant Staphylococcus aureus in Clinical and Subclinical Mastitis in Ruminants and Studying the Effect of Novel Green Synthetized Nanoparticles as One of the Alternative Treatments.

Mastitis is an important disease in dairy animals worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of clinical and subclinical intramammary infections. In the current study, we isolated bacteria from 150 mastitic milk samples. Multiplex PCR was used to detect the methicillin resistance genes in S. aureus to detect the occurrence of MRSA isolates. Green synthesized titanium dioxide nanoparticles (TiO2 NPs) using aqueous leaf extracts of Artemisia herb Alba (A. herb Alba TiO2 NPs). The antibacterial efficacy of these nanoparticles was evaluated (in vitro and in vivo) against collected MRSA isolates using the disc diffusion method and SPF rats. Out of 150 mastitic milk samples, the frequency of S. aureus was 38 (25.3%), that of E. coli was 45 (30%), that of Klebsiella spp. Was 7 (4.7%), and that of Streptococcus spp. Was 11 (7.3%). Among 38 positive isolates of S. aureus, MRSA was 16 (42.1%) by antimicrobial sensitivity testing (AST) and 14 (38.8%) by multiplex PCR. The MRSA isolates were shown to have 100% resistance to penicillin and methicillin, 87.5% resistance to gentamicin, 50% resistance to cefoxitin and amoxicillin, and 75% resistance to ampicillin and ampicillin/sublactam with low resistance against erythromycin, ciprofloxacin, tetracycline, and levofloxacin by AST, respectively. A. herb Alba TiO2 NP formation was observed by changing the colour from white to dark green. The UV spectrum revealed absorbance peaks at 240-250 nm, and their sizes ranged from 42-66 nm and 11 to 45 nm by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). ‏A. herb Alba TiO2 NP suspensions were evaluated against MRSA, with the highest zone of inhibition (43 ± 0.45 mm) at a concentration of 40 μg/ml. Hematological parameters and histological examination after oral administration of 20 mg/kg of A. herb Alba TiO2 NPs indicated that A. herb Alba TiO2 NPs can be used as a new antimicrobial against resistant bacteria (MRSA) with consideration of the dose and methods of synthesis of plant-based compounds.

Streptococcus agalactiae and Prototheca spp. induce different mammary gland leukocyte responses in Holstein cows

In this study, we investigated the association between natural subclinical intramammary infection (IMI) caused by Streptococcus agalactiae and Prototheca spp. and milk differential cell counts assessed by cytofluorimetric analysis in Holstein cows. After an initial bacteriological screening on 188 animals and a second assessment carried out 2 wk later aimed at confirming the bacteriological status, we collected milk samples from 47 animals and performed (1) milk composition analyses; (2) somatic cell counts and differential somatic cell counts (DSCC); and (3) cytofluorimetric analyses. Before statistical analyses, animals with co-infections were filtered out. Bacteriological status (negative, positive for Strep. agalactiae, or positive for Prototheca spp.) significantly affected the investigated traits. Compared with culture-negative samples, those that were positive for Strep. agalactiae and Prototheca spp. had higher SCS (+61% and +49%, respectively), DSCC (+4% and +19%, respectively), log polymorphonuclear neutrophil (PMN)-lymphocyte (LYM) counts (+59% and +71%, respectively), and log macrophage (MAC) counts (+63% and +72%, respectively). The individual leukocyte populations determined by cytofluorimetric analysis confirmed that mastitis infection increased the proportion of PMN in the milk samples compared with culture-negative samples, particularly when caused by Strep. agalactiae (+51%). In the case of MAC, the 2 pathogens behaved in opposite ways: Strep. agalactiae increased MAC by 41%, whereas Prototheca decreased MAC by 25%. Prototheca infection strongly increased the proportion of total T lymphocytes (TL; +87%) and T-helper lymphocytes (+83%). Accordingly, the (PMN+MAC):TL ratio increased with Strep. agalactiae infection (+95%) and decreased with Prototheca infection (−43%) compared with culture-negative samples. These results suggest the prevalence of an adaptive immune response and chronic inflammation in Prototheca infection, and an innate immune response to Strep. agalactiae. This knowledge might provide an important contribution to the development of novel and effective diagnostics and therapeutics.

Quarter-level analyses of the associations among subclinical intramammary infection and milk quality, udder health, and cheesemaking traits in Holstein cows

In this study, we investigated associations among subclinical intra-mammary infection (IMI) and quarter-level milk composition, udder health indicators, and cheesemaking traits. The dataset included records from 450 Holstein cows belonging to three dairy herds. After an initial screening (T0) to identify animals infected by Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, and Prototheca spp., 613 quarter milk samples for 2 different sampling times (T1 and T2, 1 mo after T1) were used for analysis. Milk traits were analyzed using a hierarchical linear mixed model including the effects of days in milk, parity and herd, and bacteriological and inflammatory category [culture negative with somatic cell count (SCC) <200,000 cells/mL; culture negative with SCC ≥200,000 cells/mL; or culture positive]. All udder health indicators were associated with increased SCC and IMI at both sampling times. The largest effects were detected at T2 for milk lactose (-7% and -5%) and milk conductivity (+9% and +8%). In contrast, the increase in differential SCC (DSCC) in samples with elevated SCC was larger at T1 (+17%). Culture-negative samples with SCC ≥200,000 cells/mL had the highest SCC and greatest numbers of polymorphonuclear-neutrophils-lymphocytes and macrophages at both T1 and T2. Regarding milk cheesemaking ability, samples with elevated SCC showed the worst pattern of curd firmness at T1 and T2. At T2, increased SCC and IMI induced large decreases in recoveries of nutrients into the curd, in particular recovered protein (-14% and -16%) and recovered fat (-12% and -14%). Different behaviors were observed between Strep. agalactiae and Prototheca spp., especially at T2. In particular, samples that were positive for Strep. agalactiae had higher proportions of DSCC (+19%) compared with negative samples with low SCC, whereas samples that were positive for Prototheca spp. had lower DSCC (-11%). Intramammary infection with Prototheca spp. increased milk pH compared with culture-negative samples (+3%) and negative samples that had increased SCC (+2%). The greatest impairment in curd firmness at 30 min from rennet addition was observed for samples that were positive for Prototheca spp. (-99% compared with negative samples, and -98% compared with negative samples with high SCC). These results suggest that IMI caused by Prototheca spp. have detrimental effects on milk technological traits that deserve further investigation of the mechanisms underlying animals' responses to infection.

A stochastic modelling approach to determine the effect of diverse Staphylococcus aureus strains on the economic and epidemiological outcomes of mastitis intervention strategies in dairy cattle

Staphylococcus aureus (S. aureus) strains with considerable genetic and phenotypic differences have previously been identified. The economic and epidemiologic impact of S. aureus mastitis has been investigated, but none of these studies took differences between strains into account. Here we aimed to investigate how differences between S. aureus strains affect the economic and epidemiologic outcome of various intervention strategies against clinical and subclinical intramammary infections. Five S. aureus strains were modelled using a stochastic bio-economic model simulating a dairy herd of 200 cows using single-day time steps. The strain characteristics of the five simulated S. aureus strains (general, contagious, spill-over, clinical and persistent) were based on divergent phenotypes as described in literature. Outcomes of the model included incidence (both clinical and subclinical), number of antibiotic treatment days, number of culled cows, and net income. Intervention strategies against clinical and subclinical intramammary infections were based on (variations of) intramammary antibiotic treatment, testing, and culling. Both single and multiple pathogen (intramammary infection caused by S. aureus, Escherichia coli, and non-aureus staphylococci) scenarios were simulated to determine the effect of the five S. aureus strains on the impact of 19 different intervention strategies. The results showed that the incidence (both clinical and subclinical), number of treatment days, number of culled cows, and net income varied considerably for the different S. aureus strains. Comparison of the model outcomes within and between strains showed that for most intervention strategies the relative impact differed per strain. However, the intervention strategy with the best outcome for most variables and strains was the culling of cows with a recovery probability lower than 50%. This shows that the relative economic and epidemiologic impact of most of the modelled intervention strategies were strain-dependent, while some intervention strategies were not strain-dependent. From this, we conclude that, depending on the intervention strategy applied on a farm, it could be advantageous to type S. aureus to determine whether it would be economically and epidemiologically beneficial for the existing intervention strategy to be changed.

The untargeted lipidomic profile of quarter milk from dairy cows with subclinical intramammary infection by non-aureus staphylococci

This observational study determined the lipidome of cow milk during subclinical intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted approach. Among the pathogens causing bovine IMI, NAS have become the most frequently isolated bacteria from milk samples. Although the application of system biology approaches to mastitis has provided pivotal information by investigating the transcriptome, proteome, peptidome, and metabolome, the milk lipidome during mammary gland inflammation remains undisclosed. To cover this gap, we determined the milk lipidome of 17 dairy cows with IMI caused by NAS (NAS-IMI), and we compared the results with those of healthy quarter milk from 11 cows. The lipidome was determined following a liquid chromatography-quadrupole time-of-flight mass spectrometry approach. Sixteen subclasses of lipids were identified in both groups of animals. From 2,556 measured lipids, the abundance of 597 changed more than 10-fold in quarter milk with NAS-IMI compared with healthy quarters. The results demonstrate the influence of NAS-IMI on the milk lipidome, implying significant changes in lipid species belonging to the family of triacylglycerols and sphingomyelins, and contribute to the understanding of inflammatory processes in the bovine udder, highlighting potential novel biomarkers for improving mastitis diagnostics.

Evaluation of fully oxidised β-carotene as a feed ingredient to reduce bacterial infection and somatic cell counts in pasture-fed cows with subclinical mastitis

ABSTRACT Aims To assess the effect of oral supplementation with fully oxidised β-carotene (OxBC) on bacteriological cure, the incidence of clinical mastitis, and somatic cell counts (SCC) in pasture-fed cows with subclinical intramammary infection. Methods Cows from four dairy herds were enrolled in early lactation if they had quarter-level SCC >200,000 cells/mL and they had a recognised bacterial intramammary pathogen in one or more quarters. They were randomly assigned to be individually fed from Day 0, for a mean of 40 days, with 0.5 kg of a cereal-based supplementary feed that either contained 300 mg of OxBC (treatment; n = 129 quarters) or did not (control; n = 135 quarters). Quarter-milk samples were collected on Days 21 and 42 for microbiology and SCC assessment. Bacteriological cure was defined as having occurred when the bacteria present on Day 0 were not isolated from samples collected on Days 21 or 42. A new intramammary infection was defined as a bacterial species isolated either on Day 21 or 42 differing from that isolated on Day 0. Clinical mastitis was diagnosed and recorded by herdowners up to Day 42. Results The bacteriological cure rate was greater for quarters from cows in the treatment group (13.9 (95% CI = 4.1–23.7)%) than for cows in the control group (6.9 (95% CI = 4.8–9.1)%; p = 0.02). The percentage of quarters that developed a new intramammary infection at Day 21 or 42 was greater for cows in the treatment group (17.9 (95% CI = 6.7–29.1)%) than for cows in the control group (13.0 (95% CI = 4.3–21.8)%; p < 0.01). The prevalence of quarters that were infected on Day 42 was less in cows in the treatment group (79.9 (95% CI = 62.3–97.6)%) than the control group (88.2 (95% CI = 78.4–97.9)%; p = 0.009). The incidence of quarters diagnosed with clinical mastitis by Day 42 was lower in cows from the treatment group (1/129 (0.78 (95% CI = 0.02–4.24)%)) than in cows from the control group (6/135 (4.44 (95% CI = 1.65-9.42)%; p = 0.03)). Mean quarter-level SCC was not statistically different between treatment groups (p = 0.34). Conclusions and clinical relevance Feeding 300 mg/cow/day of OxBC resulted in a higher bacteriological cure rate, a lower prevalence of intramammary infection, and a lower incidence of clinical mastitis compared to untreated controls. However new intramammary infections increased in treated cows, and the magnitude of the increased bacteriological cure was low, resulting in 80% of cows remaining infected at Day 42. Therefore treatment with OxBC should be considered as an adjunct to other mastitis control measures.

Pathogen-specific changes in composition and quality traits of milk from goats affected by subclinical intramammary infections.

We investigated the effects of pathogens associated with subclinical intramammary infections on yield, composition and quality indicators of goat milk. By means of a longitudinal study, individual half udder milk samples (n = 132) were collected at different lactation periods and assessed for milk yield and physicochemical composition, somatic cell count (SCC), total bacteria count (TBC) and microbiological culture. Staphylococci species accounted for the great majority of the isolates (96.1%). Intramammary infections significantly reduced fat and total solids in goat milk and increased both SCC and TBC. However, these indicators were significantly higher in udder halves affected by S. aureus compared with other staphylococci species.

Longitudinal study on the effects of intramammary infection with non-aureus staphylococci on udder health and milk production in dairy heifers

We conducted a longitudinal study to evaluate the effect of non-aureus staphylococci (NAS) causing subclinical intramammary infections (IMI) on quarter milk somatic cell count (qSCC) and quarter milk yield (qMY). In total, 324 quarters of 82 Holstein Friesian heifers were followed from calving to 130 d in milk (DIM) and were sampled 10 times each at 14-d intervals. The IMI status of each quarter was determined based on bacterial culture results at the current and previous or next sampling day, or both. The qSCC was determined on each sampling day and the average qMY on sampling day was available through stored daily milk weight data in the management program of the automatic milking system. A transient IMI (tIMI) was defined as a case where a specific pathogen was isolated from a quarter on only one sampling day and not on the previous or next sampling day. When the same bacterial strain, as defined by random amplification of polymorphic DNA-PCR, was isolated from the same quarter on multiple sampling days, it was defined as a persistent IMI (pIMI) status on those sampling days; a pIMI episode was defined as the combination of multiple consecutive pIMI statuses with the same bacterial strain on different sampling days. During this study, 142 subclinical IMI with NAS occurred in 116 different quarters from 64 animals, yielding in total 304 NAS isolates belonging to 17 different species. The prevalence of NAS was highest in the first 4 DIM. Overall, the predominant species was Staphylococcus chromogenes (52% of the isolates), followed by S. epidermidis (9.2%), S. xylosus (8.2%), and S. equorum (5.9%). Staphylococcus chromogenes was the only species for which an effect on qSCC and qMY could be analyzed separately; the other NAS species were considered as a group because of their low prevalence. Eighteen out of 40 IMI (45%) caused by S. chromogenes persisted over at least 2 sampling days, whereas only 10 of 102 (9.8%) IMI caused by other NAS species persisted for at least 2 sampling days. The average duration of pIMI episodes was 110.4 d for S. chromogenes and 70 d for the other NAS species. Remarkably, 17 of the 18 pIMI episodes with S. chromogenes started within the first 18 DIM. The qSCC was highest in quarters having a pIMI with a major pathogen, followed by quarters having a pIMI with S. chromogenes, and a pIMI with other NAS. Transient IMI with other NAS or with a major pathogen caused a small but significantly higher qSCC, whereas the qSCC in quarters having a tIMI with S. chromogenes was not statistically different compared with noninfected quarters. No significant differences in qMY were observed between quarters having a pIMI or tIMI with S. chromogenes or with the other NAS species compared with noninfected quarters, despite the higher qSCC. Quarters having a pIMI with major pathogens showed significantly lower daily milk production. Surprisingly, quarters that cured from an IMI with S. chromogenes had a significantly lower qMY than noninfected quarters.

Klasterisasi Staphylococcus aureus Resisten Neutrofil Berdasar Assesory Gene Regulator

Staphylococcus aureus is recognized worldwide as a major pathogen causing subclinical intramammary infections in dairy cows and food poisoning due to its ability to produce enterotoxin. The study aimed to identify enterotoxins of S. aureus and clustering the enterotoxins based on assessory gene regulator (agr). Virulence of S. aureus to the host was characterized based on the response of polymorphonuclear cells to the infection. Twelve S. aureus could be isolated from milk cows in central of dairy farming in Sumedang West Java. The identification of S. aureus was based on cultural and biochemical tests and an amplification of a specific section of the 23S rRNA gene. The sensitivity test against antibiotics revealed that some isolates of S. aureus were resistant to penicillin and methycillin. By PCR amplification one or more staphylococcal enterotoxin genes could be observed five genes in combinations of sea (216 bp), seb (478 bp), seh (375 bp), sei (576 bp), and sej (142 bp). Clustering of S. aureus based on the assesory gene regulator could be grouped into 4 clusters for agr1 (1 isolat), agr2 (2 isolates), in combination for agr1 and agr2 (1 isolate), and for non agr (2 isolates). Based on the response of polymorphonuclear cell in vitro and in vivo assays, revealed that S. aureus strain I-2 (agr1 cluster) and P1 (agr1+agr2 cluster) were more resistant to polymorphonuclear cells and could survive intracellularly, indicated that these strains could be used as proper candidates to develop dignostic tool based on agr against staphylococcal mastitis.

A simulation study to investigate the added value in using differential somatic cell count as an additional indicator for udder health management in dairy herds

Mastitis is one of the most costly diseases in dairy herds worldwide. Somatic cell count (SCC) is widely used as an indicator for subclinical intramammary infections (IMI) that may eventually cause mastitis in dairy herds. Differential somatic cell count (DSCC) has recently been introduced as an additional indicator for IMI. The objective of this study was to investigate the value of using DSCC as an additional indicator to select cows for testing and subsequent intervention for subclinical mastitis during the lactation. We parameterized an existing bio-economic simulation model for dairy herds to include DSCC. Then, we simulated three Danish dairy cattle herd situations with different pathogen distributions where the main pathogens were 1) Staphylococcus aureus, 2) Streptococcus agalactiae, and 3) Streptococcus uberis. In these herds, we simulated two different selection strategies for testing (bacterial culture) for subclinical IMI and various intervention strategies for test positive cases. The first selection strategy considered only SCC; cows were selected for testing if they had a low SCC measurement followed by two high SCC measurements. In the second selection strategy, cows additionally had to have a high DSCC measurement. Results showed that both selection strategies led to a similar net income and to a similar number of clinical and subclinical cases for all investigated intervention strategies. However, when using DSCC in the selection of animals, the number of treatment days and the number of cows culled in relation to IMI was reduced: The median annual number of treatment days was reduced by 25–38 days in herd 1, by 25–42 days in herd 2, and by 30–48 days in herd 3, depending on the intervention strategy. The median annual number of cows culled in relation to IMI was reduced by up to 8 cows (10 cows in herd 3) for one of the intervention strategies. Subject to limitations associated with model assumptions, these results suggest that considering DSCC when selecting cows for testing can reduce IMI related culling and the use of antibiotics without changing in-herd prevalence nor resulting in economic loss.