Uberis Intramammary Infections Research Articles

Comparison of Virulence Patterns Between Streptococcus uberis Causing Transient and Persistent Intramammary Infection.

The objectives of this study were determined by two experiments including Experiment 1 (EXP1) using Streptococcus uberis obtained from a weekly longitudinal study to compare virulence patterns between transient and persistent intramammary infection (IMI), and Experiment 2 (EXP2) using a stored-known-appearance PFGE strain of a contagious S. uberis to determine a change of virulence patterns after long-term transmission. For EXP1, quarter milk samples from 31 milking cows were aseptically and longitudinally collected once a week for 10 weeks. A total of 14 S. uberis isolates from quarters with 1 and >4 weeks of duration of IMI were categorized as transient and persistent IMI, respectively. For EXP2, 11 isolates of a stored-known-appearance PFGE strain of S. uberis from our previous study (1) were randomly selected, including 5 from transient IMI (1 month) and 6 from persistent IMI (>1 month). The virulence profiles of all isolates were investigated, including sua, hasAB, hasC, gapC, pauA, and CAMP factor or cfu, using PCR. The Kaplan–Meier estimates were used to calculate the duration of IMI in EXP1. Approximately 50% of field S. uberis IMI was spontaneously cured within 1 week, while 25% was not cured within 10 weeks. From EXP1, 4 virulence patterns were found in 14 isolates. The majority of patterns for transient S. uberis did not include hasAB (63.6%), the gene relating to capsule formation. Regardless of transient or persistent IMI, a high similarity of the virulence pattern within a PFGE strain was found in EXP2. Few changes of virulence pattern within a PFGE strain were found or were related to its subsequently changing to transient IMI.

Antimicrobial Resistance Profiles and Genes in Streptococcus uberis Associated With Bovine Mastitis in Thailand.

Streptococcus uberis is recognized as an environmental mastitis pathogen in dairy cattle. The varied success rate of antibiotic treatment for S. uberis intramammary infection may be associated with the antimicrobial resistance (AMR) of these bacteria. This observational study aimed to analyze 228 S. uberis strains associated with bovine mastitis in northern Thailand from 2010 to 2017. AMR and AMR genes were determined by the minimum inhibitory concentration (MIC) using a microdilution method and polymerase chain reaction, respectively. The majority of S. uberis strains were resistant to tetracycline (187/228, 82.02%), followed by ceftiofur (44/228, 19.30%), and erythromycin (19/228, 8.33%). The MIC50 and MIC90 of ceftiofur in 2017 were 2–4-fold higher than those in 2010 (P < 0.01). Resistance to tetracycline and ceftiofur significantly increased between 2010 and 2017 (P < 0.05). The most common gene detected in S. uberis was tetM (199/228, 87.28%), followed by ermB (151/228, 66.23 %) and blaZ (15/228, 6.58 %). The association between tetracycline resistance and tetM detection was statistically significant (P < 0.01). The detection rates of tetM significantly increased, while the detection rates of tetO and ermB significantly decreased during 2010–2017. AMR monitoring for bovine mastitis pathogens, especially S. uberis, is necessary to understand the trend of AMR among mastitis pathogens, which can help create an AMR stewardship program for dairy farms in Thailand.

LuxS Gene and Biofilm Formation in Streptococcus uberis Isolated from Bovine Mastitis Cases

Background: Streptococcus uberis (S. uberis) is an environmental pathogen causing mastitis in Dairy cattle. It causes recurrent mastitis and reduction in milk production in livestock causing economic loss. The prevalence of S. uberis intramammary infections is due to ability of the organism to form biofilm in udder tissue. The present study is on in-vitro biofilm production, the correlation of luxS gene and the biofilm formation in S. uberis. Methods: A total of 91 mastitic milk samples were collected from cattle and buffaloes brought at Veterinary Hospitals and farms in Krishna, Guntur and West Godavari districts, Andhra Pradesh. The identification of the culture isolates was based on cultural and biochemical characteristics and confirmed by Polymerase Chain Reaction (PCR). The Streptococcus species cultures showing greyish, pinpointed colonies and/or aesculin hydrolysis on Edwards medium were further identified by various biochemical tests viz., catalase test, ninhydrin test, sodium hippurate hydrolysis test and type of haemolysis on 7% sheep blood agar. Confirmation of the isolates by PCR was followed by detection of biofilm formation using qualitative Congo red agar (CRA) method, quantitative microtiter plate (MTP) assay and biofilm gene (luxS) was detected using PCR.Conclusion: From this study it is suggestable that for biofilm study both phenotypic and genotypic methods should be taken together which can be influenced by various other factors also. MTP assay was a good choice for quantitative biofilm determination, which was giving a more accurate and understandable results. The results express that any of the isolates without luxS didn’t produce a strong biofilm and it is concluded that there may be other genes for regulation of biofilm production and/or luxS gene has a regulatory role for one or more genes related to biofilm formation in S. uberis.

Molecular epidemiology of Streptococcus uberis intramammary infections: Persistent and transient patterns of infection in a dairy herd

A longitudinal observational study was carried out to explore transmission dynamics and duration of infection of Streptococcus uberis. Quarter milk samples were collected aseptically for bacterial culture from all lactating cows once a month over a 10-mo period. Molecular typing of S. uberis mastitis was performed using pulsed-field gel electrophoresis (PFGE). Molecular typing was used to determine episodes of S. uberis intramammary infection (IMI). Comparisons of spontaneous cure among PFGE types were performed using Fisher's exact chi-squared tests. Differences of duration among PFGE types and between periods of lactation were tested with Kaplan-Meier survival curves and Cox's proportional hazard model. Among a total of 851 quarter samples, 145 milk samples were detected with S. uberis presence. Based on results of PFGE, 66 episodes of S. uberis IMI were determined. From the 8 main PFGE types (A-H), PFGE type D, E, F1, F2, G, and H had only one episode indicating no evidence for transmission, subsequently defined as environmental S. uberis strains. In contrast, PFGE types A1, A2, B, C1, and C2 had at least 2 infection episodes caused by the same strain in different quarters or cows, indicating that these strains would be able to transmit to other quarters or cows. These strains were defined as contagious strains. The majority of IMI were attributable to PFGE type A1 (55%), B (17%), and A2 (11%). Spontaneous cures were observed in 35 IMI episodes. Of these 35 IMI cures, 91.4% were in IMI with duration of infection of 1 mo, n = 25, and 2 mo, n = 6. The remaining 8.6% was in IMI with duration of infection >2 mo, n = 4. Based on results from Cox's proportional hazard model, environmental S. uberis episodes were likely to have spontaneous cure with shorter duration compared with contagious S. uberis with PFGE type B (hazard ratio = 8.4). Quarters infected with S. uberis strain PFGE type A in early lactation were more likely to persist compared with those infected in late lactation (hazard ratio = 7.57). In conclusion, the majority of S. uberis IMI in this herd were transient and showed spontaneous cure. In addition to environmental S. uberis IMI, at least 3 types of contagious IMI S. uberis can be defined as (1) short duration of IMI and likely to have spontaneous cure, (2) long duration and unlikely to have spontaneous cure, and (3) wide range of duration of IMI either transient or persistent where spontaneous cure may occur depending on host defense capacity.

Comparison of the population structure of Streptococcus uberis mastitis isolates from Austrian small-scale dairy farms and a Slovakian large-scale farm

Streptococcus uberis, a major mastitis pathogen associated with intramammary infections (IMI), can be found ubiquitously in the cow's environment. Although Strep. uberis is reported to be susceptible to most antimicrobials, in practice poor responses to treatment and recurrent mastitis are observed. This can be explained by reinfection or by persistence of strains. We hypothesized that among a heterogeneous group of Strep. uberis mastitis isolates, some predominant host-adapted clones might be recurrently isolated from IMI. Therefore, the aim of this pilot study was to determine the Strep. uberis genotype variety found among small-scale dairy herds (127 Austrian dairy farms) and compare this with a large-scale herd (a Slovakian dairy farm). We determined the occurrence and strain diversity of Strep. uberis (n = 309) isolates using molecular analysis. Streptococcus uberis isolates from aseptically collected quarter milk samples were genotypically characterized using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The Strep. uberis strain set covered isolates from 4 Austrian federal areas [Lower Austria (n = 67), Upper Austria (n = 8), Salzburg (n = 51), and Styria (n = 1)] and the Bratislava Region of Slovakia (n = 1). The PFGE analysis resulted in 187 SmaI profiles with 151 unique profiles. Simpson's index of diversity was 0.988. Individual cows (n = 17) harbored up to 3 different PFGE types in the udder. Dairy cows shared distinct PFGE types within a farm. Seven PFGE types were widely distributed among Austrian dairy farms. In the Slovakian farm, 10 predominant PFGE types were recurrently isolated from the same quarters; these genotypes were assigned as persisters. We identified novel sequence types (ST) using multilocus sequence typing related to the global clonal complexes ST5 and ST143. We concluded that Strep. uberis IMI are caused by strains with a wide heterogeneity of PFGE types. This large number of unique subtypes indicates a high diversity of Strep. uberis in the environment. In the large herd, molecular epidemiological results revealed that specific strains might be involved in contagious transmission events and potentially lead to persistence.

Effect of heat stress on the interaction of Streptococcus uberis with bovine mammary epithelial cells.

Heat stress (HS) negatively affects milk production and has been associated with decreased immune function, and increased rate of intramammary infections (IMI). Research has shown that HS affects gene expression, cell cycle, and cell metabolism in bovine mammary epithelial cells (BMEC). Since BMEC are an initial target of mastitis pathogens, we studied adherence to and internalisation of S. uberis into HS-BMEC, as well as the effect that this interaction has on host cells by measuring HS-BMEC viability and membrane integrity. Results reported in this Research Communication showed that HS reduced cell viability and induced membrane damage. However, these pathological changes, as well as the rate of adherence and internalisation of S. uberis into BMEC, were augmented when S. uberis was cocultured with HS-BMEC. These results may help to understand the pathogenesis of S. uberis IMI as well as the increased susceptibility of mammary glands to IMI in cows subjected to HS.

Comparison of transmission dynamics between Streptococcus uberis and Streptococcus agalactiae intramammary infections

The objectives of study were to determine the transmission parameters (β), durations of infection, and basic reproductive numbers (R0) of both Streptococcus agalactiae and Streptococcus uberis as pathogens causing mastitis outbreaks in dairy herds. A 10-mo longitudinal study was performed using 2 smallholder dairy herds with mastitis outbreaks caused by Strep. agalactiae and Strep. uberis, respectively. Both herds had poor mastitis control management and did not change their milking management during the entire study period. Quarter milk samples were collected at monthly intervals from all lactating animals in each herd for bacteriological identification. The durations of infection for Strep. uberis intramammary infection (IMI) and Strep. agalactiae IMI were examined using Kaplan-Meier survival curves, and the Kaplan-Meier survival functions for Strep. uberis IMI and Strep. agalactiae IMI were compared using log rank survival-test. The spread of Strep. uberis and Strep. agalactiae through the population was determined by transmission parameter, β, the probability per unit of time that one infectious quarter will infect another quarter, assuming that all other quarters are susceptible. For the Strep. uberis outbreak herd (31 cows), 56 new infections and 28 quarters with spontaneous cure were observed. For the Strep. agalactiae outbreak herd (19 cows), 26 new infections and 9 quarters with spontaneous cure were observed. The duration of infection for Strep. agalactiae (mean=270.84 d) was significantly longer than the duration of infection for Strep. uberis (mean=187.88 d). The transmission parameters (β) estimated (including 95% confidence interval) for Strep. uberis IMI and Strep. agalactiae IMI were 0.0155 (0.0035–0.0693) and 0.0068 (0.0008–0.0606), respectively. The R0 (including 95% confidence interval) during the study were 2.91 (0.63–13.47) and 1.86 (0.21–16.61) for Strep. uberis IMI and Strep. agalactiae IMI, respectively. In conclusion, the transmission parameter and R0 values were not different between both pathogens; however, the duration of infection for Strep. agalactiae was longer than Strep. uberis. These suggest that Strep. uberis may have a different transmission dynamic compared with Strep. agalactiae.

Protective effect of anti-SUAM antibodies on Streptococcus uberis mastitis.

In the present study, the effect of anti-recombinant Streptococcus uberis adhesion molecule (SUAM) antibodies against S. uberis intramammary infections (IMI) was evaluated using a passive protection model. Mammary quarters of healthy cows were infused with S. uberis UT888 opsonized with affinity purified anti-rSUAM antibodies or hyperimmune sera. Non-opsonized S. uberis UT888 were used as a control. Mammary quarters infused with opsonized S. uberis showed mild-to undetectable clinical symptoms of mastitis, lower milk bacterial counts, and less infected mammary quarters as compared to mammary quarters infused with non-opsonized S. uberis. These findings suggest that anti-rSUAM antibodies interfered with infection of mammary gland by S. uberis which might be through preventing adherence to and internalization into mammary gland cells, thus facilitating clearance of S. uberis, reducing colonization, and causing less IMI.

Effect of cooling during the dry period on immune response after Streptococcus uberis intramammary infection challenge of dairy cows

Heat stress in the dry period affects the immune status of dairy cows in the subsequent lactation. We hypothesized that cooling during the dry period improves immune response to postpartum intramammary infection (IMI) by environmental pathogens such as Streptococcus uberis. Cows were dried off 46 d before expected calving and assigned to cooling (CL, n=15) or heat stress (HT, n=15). Cooled cows were housed with sprinklers, fans, and shade, whereas the HT group had only shade. All cows were cooled postpartum. Rectal temperature (RT) and respiration rate (RR) were recorded thrice weekly during the dry period. From −46 to 42 d relative to calving, dry matter intake was recorded daily, and both body weight (BW) and body condition score (BCS) weekly. Milk yield and composition were recorded daily after calving. Streptococcus uberis IMI was induced at 5 d postpartum in a subset of cows (CL, n=5; HT, n=5). Blood was collected at 0, 12, 18, 24, and 36h after IMI. Hematological analysis was performed, and neutrophils isolated for RNA extraction. Immune response genes (TLR2, IL1-β, IL6, IL8, IL10, and TNFα) were assessed by real-time, reverse transcription-PCR. Relative to HT cows, CL cows had lower RT and RR during the dry period. The CL cows also consumed more feed prepartum but not postpartum, gained more BW prepartum but lost more BW in lactation, and had higher BCS score prepartum and a lower BCS postpartum. During 40wk of lactation, CL cows produced more milk (33.8 vs. 30.0kg/d) than HT cows but milk composition was not affected. Cows in the CL group had greater white blood cell counts and more neutrophils than HT cows during IMI. From 0 to 36h post-IMI, TNFα mRNA expression decreased, whereas that of IL6 and IL8 increased in both treatments. Additionally, CL cows had lower IL10 mRNA expression at 18h post-IMI. Expression of TLR2 mRNA decreased over time in both treatments. However, CL cows had greater overall TLR2 mRNA expression than HT. No differences were detected for mRNA expression of IL1-β, IL6, IL8, or TNFα. Cooling cows during the dry period alters immune function and neutrophil response to IMI in early lactation.

Quarter, cow, and farm risk factors for intramammary infections with major pathogens relative to minor pathogens in Thai dairy cows

A cross-sectional study was carried out from May to September 2011 on 35 smallholder dairy farms in Chiang Mai, Thailand, to identify the quarter, cow, and farm factors that relate to intramammary infections (IMI) from major specified pathogens, compared to infections from minor pathogens. Data on general farm management, milking management, and dry cow management were recorded for each herd. Quarter milk samples were collected from either clinical or subclinical mastitis quarters. Dependent variables were binary data defining the specified major pathogens, including Streptococcus agalactiae (7.1%), Streptococcus uberis (9.4%), Streptococcus dysgalactiae (4.0%), and other streptococci (16.7%), as a case, and all minor pathogens as a control, in each dependent variable. The occurrence of S. agalactiae IMI was lower in first-parity cows and cows with short milking time. Cows with body condition score (BCS) <2.5 had higher occurrence of S. agalactiae IMI. The occurrence of S. uberis IMI was higher in quarters with California mastitis test (CMT) score 2, score 3, and having clinical mastitis and in farms with increasing age of vacuum system. Quarters with CMT score 3, having clinical mastitis, cow with manual milking after detaching milking cluster, and farms with high bulk milk somatic cell counts (BMSCC >500,000 cells/ml) had higher occurrence of S. dysgalactiae IMI. For other streptococci, quarters having clinical mastitis, BCS <2.5, and pulling down of milking cluster while milking increased occurrence of other streptococci IMI relative to minor pathogen IMI. These results highlight the importance of individual cow factors, milking characteristics, and BMSCC in determining the risk of IMI from major pathogens.

Relationship between previous history ofStreptococcus uberisinfection and response to a challenge model

Streptococcus uberis is the most common cause of clinical mastitis at calving in pasture-based dairy cows. Results of experimental inoculations were compared with cows' previous history of infection to help define a model for susceptibility to Str. uberis mastitis. Cows used had either no apparent history of intramammary infection (IMI) by Str. uberis or other major mastitis pathogens throughout their productive lifetime ('apparently uninfected'; AUI), or had a confirmed history of Str. uberis IMI ('historically infected'; HI). Cows were exposed to Str. uberis in sequential steps: dipping of the teat end (DIP; n=53 cows); a teat canal inoculation (TCI; n=33 cows); and, finally, intramammary inoculation challenge (IC; n=7 cows). Only cows that remained free of infection at each step progressed to the next phase. Infection rates were similar between AUI or HI cows following the DIP (9 and 17% respectively), or the TCI (75 and 68% respectively). Physical and biochemical traits of cows were examined. Analysis of traits prior to inoculations implied that HI cows produced more milk fat, while AUI cows tended to have longer teat canals. Analysis of traits for cows that became infected following DIP, implied that there was a positive association with milk fat production and negative association with somatic cell count (SCC), while there was a positive association with the duration of p.m. milking, and negative association with SCC in those cows that became infected following TCI. Only AUI cows became infected following the IC inoculation. Similarity in response to experimental inoculation between the two groups suggests that the current dip or teat canal inoculation (using a 3-mm depth of inoculation) models are not good predictors of natural resistance to Str. uberis. However, a population of cows was identified that remained uninfected after DIP, TCI and IC, and may comprise a resistant phenotype.

Peripartum infection with Streptococcus uberis but not coagulase-negative staphylococci reduced milk production in primiparous cows

The effect of an intramammary infection (IMI) at calving on the milk yield of heifers during their first 200 d in milk (DIM) was estimated by comparing monozygotic twins, where one member had a naturally occurring IMI detected at the first milking after calving and the other twin did not. Data collected weekly over a full lactation for 29 twin pairs were used to estimate the effects of a peri-calving Streptococcus uberis IMI on milk yield and composition. Data for 19 twin pairs were used to estimate the effects of pericalving coagulase-negative staphylococci (CNS) IMI. A heifer with a Strep. uberis IMI produced 200kg (7%) less milk during the first 200 d of lactation compared with her uninfected twin, with significant differences evident throughout the 200-d period. Similar milk losses were recorded for heifers that developed CM or remained subclinical. An elevated milk SCC for infected heifers was only apparent for the first month (d 2–30), although SCC tended to remain high during the second (d 31–60) and third (d 61–90) months. Milk protein concentrations were greater in the Strep. uberis-infected twin over the 200-d period, whereas fat and lactose concentrations showed little change. An IMI caused by Strep. uberis was associated with a lower milk yield, whereas an IMI by CNS was not, despite CNS-infected twins having a higher SCC than their uninfected twin for the first 30 d of lactation.

Technical note: Development of a challenge model for Streptococcus uberis mastitis in dairy heifers

A challenge model for experimentally inducing Streptococcus uberis mastitis in bred dairy heifers was developed. Qualifying heifers (n=7) exhibited antibody titers of <1:10,000 against Strep. uberis antigens and were free of intramammary infections (IMI). Two contralateral quarters of each heifer were assigned to receive an infusion of Strep. uberis (1,000 to 2,000cfu); remaining quarters served as unchallenged controls. For a successful challenge and infection, 3 of 4 consecutive mammary secretion samples had to culture positive for Strep. uberis. Six of the 7 heifers were challenged successfully in both infused quarters with a mean dose of 1,080cfu; once confirmed, infections were treated with a one-time infusion of nonlactating cow therapy. Before challenge, mammary secretion leukocyte counts averaged 8.4×106/mL in all quarters. At 24h after challenge, leukocyte count increased to 18.4×106/mL in challenged quarters, peaking on d 5 at 24.3×106/mL; unchallenged quarters remained at ≤10.4×106/mL, but increased to 15.2×106/mL on d 7 and then decreased. Before challenge, macrophages predominated (81%) in mammary secretions followed by lymphocytes (15.3%) and neutrophils (3.7%). By 24h after challenge, neutrophils increased in challenged quarters and predominated for the duration of the trial (65.3 to 70%), whereas macrophages predominated in unchallenged control quarters (65.2 to 75.2%). The challenge model was successful in establishing Strep. uberis IMI in 85.7% of animals, and IMI were controlled (100% cure) by administering nonlactating cow therapy. All heifers calved free of IMI and antimicrobial residues, with milk production similar to that of herd mates and with somatic cell counts (SCC) <200,000 cells/mL.

PGh9:ISS1 transpositional mutations in Streptococcus uberis UT888 causes reduced bacterial adherence to and internalization into bovine mammary epithelial cells

Streptococcus uberis is an important mastitis pathogen that affects dairy cows worldwide. In spite of the economic impact caused by the high prevalence of S. uberis intramammary infections (IMI) in many well-managed dairy herds, pathogenic strategies and associated virulence factors of S. uberis are not well understood. It has been shown that S. uberis attaches to and internalizes into mammary epithelial cells and can survive inside cells for extended periods of time. We hypothesize that early attachment to and internalization into mammary epithelial cells is a critical step for the establishment of intramammary infection. The aim of this study is to identify and characterize chromosomally encoded virulence factors of S. uberis that allow early bacterial attachment to and internalization into mammary epithelial cells. A common approach used to identify virulence factors is by generating random insertion mutants that are defective in adherence to and internalization into mammary epithelial cells using pGh9:ISS1 mutagenesis system. A random insertion mutant library of S. uberis strain UT888 was created using a thermo-sensitive plasmid pGh9:ISS1 carrying ISS1 insertion sequence. Integration of the insertion sequence into the chromosome of these mutant clones was confirmed by PCR and Southern blot. Southern blot analysis of mutant clones also showed that insertional integration was random. Of 1000 random chromosomal insertion mutants of S. uberis strain UT888 screened, 32 had significantly reduced ability to adhere to and internalize into mammary epithelial cells. Chromosomal mapping of insertion sequence integration sites in some of these defective mutants showed integration into penicillin binding protein 2A (pbp2A), sensor histidine kinase, tetR family regulatory protein, phosphoribosylaminoimidazole carboxylase catalytic subunit (purE), lactose phosphotransferase, phosphoribosylamine glycine ligase (purD), and other genes involved in metabolic activities. These proteins may have a significant role in early bacterial colonization of the mammary gland during infection.

Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell proliferation and inhibition of PPARgamma signaling as potential mechanism for the negative relationships between immune response and lipid metabolism.

BackgroundInformation generated via microarrays might uncover interactions between the mammary gland and Streptococcus uberis (S. uberis) that could help identify control measures for the prevention and spread of S. uberis mastitis, as well as improve overall animal health and welfare, and decrease economic losses to dairy farmers. The main objective of this study was to determine the most affected gene networks and pathways in mammary tissue in response to an intramammary infection (IMI) with S. uberis and relate these with other physiological measurements associated with immune and/or metabolic responses to mastitis challenge with S. uberis O140J.ResultsStreptococcus uberis IMI resulted in 2,102 (1,939 annotated) differentially expressed genes (DEG). Within this set of DEG, we uncovered 20 significantly enriched canonical pathways (with 20 to 61 genes each), the majority of which were signaling pathways. Among the most inhibited were LXR/RXR Signaling and PPARα/RXRα Signaling. Pathways activated by IMI were IL-10 Signaling and IL-6 Signaling which likely reflected counter mechanisms of mammary tissue to respond to infection. Of the 2,102 DEG, 1,082 were up-regulated during IMI and were primarily involved with the immune response, e.g., IL6, TNF, IL8, IL10, SELL, LYZ, and SAA3. Genes down-regulated (1,020) included those associated with milk fat synthesis, e.g., LPIN1, LPL, CD36, and BTN1A1. Network analysis of DEG indicated that TNF had positive relationships with genes involved with immune system function (e.g., CD14, IL8, IL1B, and TLR2) and negative relationships with genes involved with lipid metabolism (e.g., GPAM, SCD, FABP4, CD36, and LPL) and antioxidant activity (SOD1).ConclusionResults provided novel information into the early signaling and metabolic pathways in mammary tissue that are associated with the innate immune response to S. uberis infection. Our study indicated that IMI challenge with S. uberis (strain O140J) elicited a strong transcriptomic response, leading to potent activation of pro-inflammatory pathways that were associated with a marked inhibition of lipid synthesis, stress-activated kinase signaling cascades, and PPAR signaling (most likely PPARγ). This latter effect may provide a mechanistic explanation for the inverse relationship between immune response and milk fat synthesis.

Effect of milking frequency and feeding level before and after dry off on dairy cattle behavior and udder characteristics

The effects of 2 common dry-off management procedures, feed restriction [8 vs. 16kg of dry matter (DM)/d] and reduced milking frequency (once, 1× vs. twice, 2×/d), on the behavior and udder characteristics of dairy cattle were assessed in late lactation and the early dry period. Milking cows 1× instead of 2× in the week before dry off reduced milk yield (7.0 vs. 8.9 ± 0.95 kg/d for 1× and 2×, respectively), but had little effect on behavior before or after cessation of milking. In comparison, feed restriction reduced milk yield (6.9 vs. 9.1 ± 0.95 kg/d for 8 and 16kg of DM/d, respectively), udder firmness after dry off (7.3 vs. 8.0 ± 0.24 g force for 8 and 16kg of DM/d, respectively), milk leakage (2 d after dry off, 14% of cows offered 8kg of DM/d were leaking milk compared with 42% cows offered 16kg of DM/d), and the likelihood of Streptococcus uberis intramammary infection (nonclinical mastitis; 12.5 vs. 62.5% of groups with at least 1 cow with a new intramammary infection for 8 and 16kg of DM/d, respectively). Despite these benefits, cows offered only 8kg of DM/d spent less time eating (7.3 vs. 8.3 ± 0.28 h/d for 8 and 16kg DM/d, respectively), more time lying (8.8 vs. 7.3 ± 0.24 h/d), and vocalized more before dry off than cows offered 16kg of DM/d (0.8 vs. 0.2 ± 0.15 calls/min for 8 and 16kg of DM/d, respectively). These behavioral changes indicate that this level of feed restriction may cause hunger. Information is needed about alternative dry-off procedures that maintain the health benefits and comfort associated with lower milk yield before dry off but prevent hunger, such as feeding low quality diets ad libitum.

Heifer teats sprayed in the dry period with an iodine teat sanitizer have reduced Streptococcus uberis teat-end contamination and less Streptococcus uberis intra-mammary infections at calving

Heifers managed under pastoral conditions are at risk from Streptococcus uberis mastitis infections at calving. A total of 397 heifers from six farms around New Zealand were enrolled in a study to identify and enumerate S. uberis on teat-ends of heifers in the peri-partum period, and to understand the effect of teat-spraying in the pre-calving period on the prevalence and incidence of S. uberis mastitis post-calving. Heifers were randomly assigned to Control or Sprayed groups. Sprayed heifers were teat-sprayed once, three times a week (Monday, Wednesday and Friday) with a commercial iodine-based teat sanitizer, starting at 3 weeks prior to calving and ending at day of calving. Across three farms, all glands of cows in both groups were sampled at calving to determine S. uberis intra-mammary infection (IMI) prevalence. For all farms, clinical mastitis (CM) cases detected during the week after calving were sampled and submitted for bacteriological analysis. Swabbing of teat-ends of 54 heifers from one farm showed that heifers had a pre-existing S. uberis contamination averaging 610 colony-forming units per swab (cfu/swab), at 3 weeks prior to calving. At calving, teat-end contamination was 560 cfu/swab for Sprayed heifers and 1775 cfu/swab for Control heifers. Two weeks after calving, teat-end contamination was similar between both groups, at 30 cfu/swab. The prevalence of S. uberis IMI was significantly lower in the Sprayed (3.5% glands) vs. the Control (7.4%) heifers in the first week after calving. There was a trend for Sprayed heifers (3.6% heifers) to have a lower incidence of S. uberis CM compared with Control heifers (7.4% heifers). It is concluded that teat-spraying in the dry period is a management option that could contribute to controlling heifer S. uberis mastitis in the transition period.

Relationship Between Milk Lactoferrin and Etiological Agent in the Mastitic Bovine Mammary Gland

Bovine mastitis is one of the most deleterious diseases for dairy herds and is mainly caused by contagious and environmental bacterial pathogens. Among contagious bacteria, Staphylococcus aureus is the most prevalent, whereas the main environmental mastitis pathogens are Streptococcus uberis and Escherichia coli. Bovine lactoferrin (bLF) is an approximately 80-kDa glycoprotein present in milk that participates in the innate response of the mammary gland against bacterial infection. The objectives of the current study were to analyze potential changes in bLF milk concentration, which would constitute a response of the mammary gland toward mastitis induced by different etiologic agents, and to evaluate a possible relation between this response and pathogen susceptibility to bLF. Microbiology analysis and bLF quantification in milk from different bovine mammary gland quarters were performed. Infected quarters presented greater concentrations of bLF compared with those from microbiologically negative quarters. Analysis of individual pathogen contributions showed that most of this increase was attributable to Strep. uberis intra-mammary infection. The ability of mammary gland cells to synthesize bLF in response to Strep. uberis challenge was demonstrated by immunodetection of the protein in in vitro infection experiments. Susceptibility of Strep. uberis, E. coli, and Staph. aureus to the antimicrobial activity of bLF was determined by growth inhibition assays conducted with 4 different isolates of each species. Whereas Staph. aureus and E. coli were shown to be susceptible to this protein, Strep. uberis appeared to be resistant to the antimicrobial activity of bLF. Molecular typing of the 4 Strep. uberis isolates used throughout this study showed that this result was representative of the species and not exclusive of a particular strain. Results presented herein suggest that different bacteria species may elicit different mammary gland responses mediated by bLF secretion and that Strep. uberis has probably adapted to this immune reaction by developing resistance to bLF inhibitory action.

Interleukin-1β infusion in bovine mammary glands prior to challenge with Streptococcus uberis reduces bacterial growth but causes sterile mastitis

Dairy cows are especially vulnerable to intramammary infection by the bacterial pathogen Streptococcus uberis in the dry period. Use of immunotherapeutic agents at drying off could increase cellular defences in the gland and prevent establishment of new S. uberis infections. This study investigated the potential of infusing recombinant bovine interleukin-1 beta (rbIL-1beta) in the mammary glands as a prophylactic agent against subsequent intramammary challenge with S. uberis in the early dry period. Immediately after the last milking at commencement of the dry period, one cow from each of 10 monozygous twinsets was infused with 10 microg of rbIL-1beta in two quarters and the other twin was infused with the carrier agent, sterile phosphate buffered saline. Twenty-four hours later, the quarters were infused with 10(3) colony-forming units (CFU) of S. uberis. Bacteriology, somatic cell count (SCC), concentrations of specific cytokines and antibody responses were monitored in mammary gland secretions and sera for the next 21 days. Infusion of rbIL-1beta into mammary glands at commencement of the dry period was associated with less new S. uberis intramammary infections, as determined by the number of quarters with bacterial growth. However, high SCC in quarters following infusion of rbIL-1beta masked the full beneficial effect of this procedure.

Extended Ceftiofur Therapy for Treatment of Experimentally-Induced Streptococcus uberisMastitis in Lactating Dairy Cattle

Streptococcus uberisis an important cause of mastitis in dairy cows throughout the world, particularly during the dry period, the period around calving, and during early lactation. Strategies for controlling Strep. uberismastitis are poorly defined and are currently inadequate. Objectives of the present study were to evaluate efficacy of ceftiofur, a new broad-spectrum cephalosporin antibiotic, for treatment of experimentally induced Strep. uberisintramammary infections (IMI) in lactating dairy cows during early lactation and to determine whether extended therapy regimens enhanced efficacy of ceftiofur. Efficacy of extended ceftiofur intramammary therapy regimens was investigated in 37 mammary quarters of 23 dairy cows that developed clinical mastitis following experimental infection with Strep. uberisduring early lactation. Cows that developed clinical mastitis during the challenge period were allocated randomly to 3 groups representing 3 different ceftiofur treatment regimens: 2-d (n=7 mammary quarters), 5-d (n=16 mammary quarters), and 8-d (n=14 mammary quarters) treatment regimens. For all groups, 125mg of ceftiofur hydrochloride was administered via intramammary infusion. A bacteriological cure was defined as an experimentally infected quarter that was treated and was bacteriologically negative for the presence of Strep. uberisat 7, 14, 21, and 28 d posttreatment. Percentage of Strep. uberisIMI eliminated was 43, 88, and 100% for the 2-, 5-, and 8-d ceftiofur treatment regimens, respectively. Both the 5- and 8-d ceftiofur extended therapy treatment regimens had significantly higher bacterial cure rates than the standard 2-d ceftiofur treatment regimen. The bacterial cure rate of the 8-d ceftiofur extended therapy group was marginally better (P=0.052) than the 5-d ceftiofur extended therapy group. Results of this study indicate that ceftiofur therapy was effective for eliminating Strep. uberisexperimental IMI, and 5- and 8-d extended ceftiofur therapy regimens were more effective than the standard 2-d treatment.