Identifying gram-negative and gram-positive clinical mastitis using daily milk component and behavioral sensor data.

Opportunities exist for automated animal health monitoring and early detection of diseases such as mastitis with greater on-farm adoption of precision technologies. Our objective was to evaluate time series changes in individual milk component or behavioral variables for all clinical mastitis (CM) cases (ACM), for CM caused by gram-negative (GN) or gram-positive (GP) pathogens, or CM cases in which no pathogen was isolated (NPI). We developed algorithms using a combination of milk and activity parameters for predicting each of these infection types. Milk and activity data were collated for the 14 d preceding a CM event (n = 170) and for controls (n = 166) matched for breed, parity, and days in milk. Explanatory variables in the univariate and multiple regression models were the slope change in milk (milk yield, conductivity, somatic cell count, lactose percentage, protein percentage, and fat percentage) and activity parameters (steps, lying time, lying bout duration, and number of lying bouts) over 7 d. Slopes were estimated using linear regression between d -7 and -5, d -7 and -4, d -7 and -3, d -7 and -2, and d -7 and -1 relative to CM detection for all parameters. Univariate analyses determined significant slope ranges for explanatory variables against the 4 responses: ACM, GN, GP, and NPI. Next, all slope ranges were offered into the multivariate models for the same 4 responses using 3 baselines: d -10, -7, and -3 relative to CM detection. In the univariate analysis, no explanatory variables were significant indicators of ACM, whereas at least 1 parameter was significant for each of GN, GP, and NPI models. Superior sensitivity (Se) and specificity (Sp) estimates were observed for the best GP (Se = 82%, Sp = 87%) and NPI (Se = 80%, Sp = 94%) multiple regression models compared with the best ACM (Se = 73%, Sp = 75%) and GN (Se = 71%, Sp = 74%) models. Sensitivity for the GN model was greater at the baseline closest to the day of CM detection (d -3), whereas the opposite was observed for the GP and NPI model as Se was maximized at the d -10 baseline. Based on this screening of relationships, milk and activity sensor data could be used in CM detection systems.

The effect of J5 bacterins on clinical, behavioral, and antibody response following an Escherichia coli intramammary challenge in dairy cows at peak lactation.

Vaccination against coliform mastitis has become part of mastitis control programs in the past 3 decades, as a means of reducing the severity of clinical mastitis. Our study objective was to evaluate the effect of 2 commercially available vaccines on clinical, behavioral, and antibody response following Escherichia coli intramammary challenge in cows near peak lactation. Cows (n = 12 per group) were vaccinated with vaccine 1 (V1) or vaccine 2 (V2) at dry-off, 21 d pre-calving, and 14 d post-calving. Twelve cows served as unvaccinated controls (CTL). Cows were challenged with E. coli in a rear quarter at approximately 100 d in milk. Milk samples were collected pre- and post-challenge to enumerate E. coli and determine somatic cell count. Serum was collected before each vaccination and at d 0, 1, 2, 3, 6, 30, and 60 relative to challenge, to study antibody response. Milk IgA and tumor necrosis factor-α concentrations were determined in whey. Vaginal temperature, cow activity, and milk yield and components were monitored post-challenge. Bacterial count, somatic cell score, milk yield and component decline, vaginal temperature, activity measures, and antibody and cytokine response were analyzed for treatment differences. The effects of parity, breed, and a repeated measure of time were also tested. Seven cows had to be removed from the study post-challenge for antibiotic treatment (CTL and V1, n = 3 each; V2, n = 1), 2 of which were euthanized (both CTL). Vaccinated cows exhibited fever (vaginal temperature ≥39.4°C) 3 h earlier than CTL cows, but we found no differences between treatments for bacterial count, somatic cell score, or milk yield reduction. Vaccinated cows spent more time lying per rest bout 2 d post-challenge, but total daily lying time was not different from CTL cows during the 7 d post-challenge. The vaccines differed in antibody response: V1 cows had greater serum IgG1 and IgG2 post-challenge. A parity effect was also evident: primiparous cows had lower bacterial counts, somatic cell score and a smaller milk yield decline than multiparous cows, but also had lower antibody production. Immunization with either J5 bacterin did not reduce clinical signs of mastitis in cows challenged at 100 d in milk, demonstrating that the effects of J5 vaccination had diminished at peak lactation.

Lack of efficacy of homeopathic therapy against post-calving clinical mastitis in dairy herds in the Waikato region of New Zealand.

AIM: To compare clinical and bacteriological cure rates of clinical mastitis following treatment with either antimicrobials or homeopathic preparations. METHODS: Seven spring-calving herds from the Waikato region of New Zealand were used to source cases of clinical mastitis (n = 263 glands) during the first 90 days following calving. Duplicate milk samples were collected for bacteriology from each clinically infected gland at diagnosis and 25 (SD 5.3) days after initial treatment. Affected glands were treated with either an antimicrobial formulation or a homeopathic remedy. Generalised linear models with binomial error distribution and logit link were used to analyse the proportion of cows that were clinical treatment cures and the proportion of glands that were classified as bacteriological cures, based on initial and post-treatment milk samples. RESULTS: Mean cumulative incidence of clinical mastitis was 7% (range 2-13% across herds) of cows. Streptococcus uberis was the most common pathogen isolated from culture-positive samples from affected glands (140/209; 67%). The clinical cure rate was higher for cows treated with antimicrobials (107/113; 95%) than for cows treated with homeopathic remedies (72/114; 63%) (p < 0.001) based on the observance of clinical signs following initial treatment. Across all pathogen types bacteriological cure rate at gland level was higher for those cows treated with antimicrobials (75/102; 74%) than for those treated with a homeopathic preparation (39/107; 36%) (p < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: Using herds located in the Waikato region of New Zealand, homeopathic remedies had significantly lower clinical and bacteriological cure rates compared with antimicrobials when used to treat post-calving clinical mastitis where S. uberis was the most common pathogen. The proportion of cows that needed retreatment was significantly higher for the homeopathic treated cows. This, combined with lower bacteriological cure rates, has implications for duration of infection, individual cow somatic cell count, costs associated with treatment and animal welfare.

Effect of disinfecting teats post-milking or pre- and post-milking on intramammary infection and somatic cell count.

AIMS: To determine the effects of (a) post-milking teat disinfection compared with no disinfection and (b) pre- and post-milking teat disinfection compared with post-milking disinfection alone, on the incidence of new intramammary infection (IMI), somatic cell count (SCC) and teat skin abnormalities in dairy cows. METHODS: In Experiment 1, dairy cows in five dairy herds were randomly allocated to a post-milking teat disinfection group (n=230), that was sprayed with an iodine-based disinfectant (TeatguardPlus) for a complete lactation, or to a non-disinfected group (n=239). In Experiment 2, cows were randomly allocated to post-milking teat disinfection (n=239) or both pre- and post-milking teat disinfection (n=235), using a chloramine-T-based disinfectant (Teatsweet) for both treatments, from calving to 118-127 days in milk. The incidence of new IMI was determined by aseptic sampling of all quarters at calving, during lactation, and at trial end or at drying-off, with clinical mastitis cases sampled on detection. SCC and teat skin abnormalities were measured at 2-monthly intervals during lactation. In both experiments, disinfectant was applied by spray application. RESULTS: Cows that received post-milking teat disinfection had a lower incidence of new IMI caused by Staphylococcus aureus, Streptococcus uberis, Corynebacterium spp and coagulase negative staphylococci, had lower bulk milk SCC during lactation, and had fewer teat skin abnormalities compared with the non-disinfected cows (p < 0.05). Pre-milking teat disinfection, in addition to post-milking teat disinfection, did not reduce the incidence of new IMI for any pathogens and did not reduce SCC (p> 0.05). CONCLUSIONS: Post-milking teat disinfection applied as a spray is a key component in mastitis control in New Zealand. There was no benefit from the addition of pre-milking disinfection. CLINICAL RELEVANCE: This study confirms previous findings of the effectiveness of post-milking teat disinfection in reducing the incidence of IMI caused by the common mastitis-causing pathogens in New Zealand, and presents the first results of a controlled study examining pre-milking teat spraying undertaken in New Zealand commercial dairy herds.

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 200 kg (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.

Experimentally induced intramammary infection with multiple strains of Streptococcus uberis.

The effect of infusing a mixture of 5 Streptococcus uberis strains into mammary quarters of 10 lactating cows was investigated. All 5 strains, which included 2 originally isolated from the dairy environment and 3 from clinical cases of mastitis, were capable of establishing an intramammary infection when infused individually. However, when the 5 strains were infused together, a single strain predominated in 7 out of 10 quarters. One strain in particular prevailed in 4 mammary quarters and was also found to inhibit the growth of the other 4 strains with deferred antagonism on esculin blood agar. The genes required for the production of bacteriocins nisin U and uberolysin were identified in this strain, whereas the other 4 strains contained only uberolysin genes. Direct competition may have occurred between strains within the mammary gland but competition was not apparent when cultured together in UHT milk, where no strain predominated. Although the mechanism is unknown, these results imply that a selection process can occur within the mammary gland, leading to a single strain that is detected upon diagnosis of mastitis.

Vaccines against bovine mastitis in the New Zealand context: what is the best way forward?

Mastitis is an important animal health disease which constitutes a serious problem for the dairy industry in New Zealand. Mastitis reduces milk yield and quality, necessitates the use of antibiotic therapy, with associated risks of contaminating the raw milk supply, and imposes a serious economic burden, currently estimated at NZ$300 million per year. Mastitis is caused by a variety of infectious agents. In the New Zealand context, with cattle grazing on pasture, Streptococcus uberis is a major bacterial pathogen, responsible for a significant proportion of clinical cases, especially during early lactation and the dry period. Other pathogens of significance include Staphylococcus aureus, Streptococcus dysgalactiae and Escherichia coli, as well as so-called 'minor pathogens', namely coagulase-negative staphylococci(CNS). Current strategies aimed at reducing cases of mastitis include improved hygiene in the farm environment, particularly with regards to the health and cleanliness of teats. Once mastitis occurs, antibiotic therapy is a favoured option, and as a prophylactic tool, in the form of dry-cow therapy, has also shown value. Prevention of mastitis using immunological tools such as vaccines lags behind the major vaccine breakthroughs that have been achieved in preventing and/or reducing the severity of numerous infectious diseases in animals. In this review, the current state of research in the area of development of vaccines against mastitis is summarised, with particular emphasis on bacteria important to the dairy farming industry in New Zealand. Few, if any, effective vaccines have been designed to prevent or mitigate intramammary infections. It is argued that novel approaches must be considered to search for vaccine candidates, and vaccines need to be designed and constructed within the special framework of their uses, in the mammary gland which offers a unique immunological environment. In addition, effective vaccines against mastitis due to Strep. uberis may be more likely to emerge from strategies that target the cell-mediated arm of the immune response rather than strategies that target specific antibody responses.

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. 16 kg of dry matter (DM)/d] and reduced milking frequency (once, 1x vs. twice, 2x/d), on the behavior and udder characteristics of dairy cattle were assessed in late lactation and the early dry period. Milking cows 1x instead of 2x in the week before dry off reduced milk yield (7.0 vs. 8.9 +/- 0.95 kg/d for 1x and 2x, 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 16 kg of DM/d, respectively), udder firmness after dry off (7.3 vs. 8.0 +/- 0.24 g force for 8 and 16 kg of DM/d, respectively), milk leakage (2 d after dry off, 14% of cows offered 8 kg of DM/d were leaking milk compared with 42% cows offered 16 kg 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 16 kg of DM/d, respectively). Despite these benefits, cows offered only 8 kg of DM/d spent less time eating (7.3 vs. 8.3 +/- 0.28 h/d for 8 and 16 kg DM/d, respectively), more time lying (8.8 vs. 7.3 +/- 0.24 h/d), and vocalized more before dry off than cows offered 16 kg of DM/d (0.8 vs. 0.2 +/- 0.15 calls/min for 8 and 16 kg 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.

Field observations on the variation of Streptococcus uberis populations in a pasture-based dairy farm.

Microbiological and molecular tools were used to monitor Streptococcus uberis populations on farm tracks and paddocks on a dairy farm during different seasons of a year to identify and profile potential environmental niches of Strep. uberis in a pasture-based dairying system. Farm tracks of high or low cow traffic were sampled every 2 wk for an entire year and Strep. uberis numbers were enumerated from a selective medium. During each season of the year, paddocks were sampled for the presence of Strep. uberis before and after grazing by dairy cows. Farm tracks of high cow traffic generally had greater concentrations of Strep. uberis isolated compared with tracks with less cow traffic, but there was also significant variation in the concentrations of Strep. uberis contamination among seasons, being highest in winter and lowest in summer. The bacterium was detected in paddocks only after cow grazing had occurred, but the bacteria could still be detected in soil for up to 2 wk following grazing in winter. Multilocus sequence typing showed great heterogeneity, with some commonality between farm track and milk isolates, which may help explain cow-to-environment or environment-to-cow transmission of the bacterium in the dairy setting.

Molecular typing of Streptococcus uberis strains isolated from cases of bovine mastitis.

The discriminatory power of two polymerase chain reaction-based DNA fingerprinting methods, random amplified polymorphic DNA and repetitive extragenic palindrome were compared by subtyping 128 isolates of Streptococcus uberis cultured from cows in six different dairy herds in New Zealand. The typing results demonstrated that the majority of isolates possessed unique fingerprint profiles except on occasions where multiple isolates were obtained from individual cows. On these occasions, individual quarters of the mammary gland were generally, but not exclusively, infected by the same strain of bacteria. Both random amplified polymorphic DNA and repetitive extragenic palindromic typing assays were simple to perform, relatively inexpensive ($11.00 per reaction), and provided reliable and reproducible results. Furthermore, when these assays were used in conjunction with each other, they provided a means of confirmation of the specific DNA fingerprint patterns obtained.

Effect of localised antibiotic infusions applied to the teat-canal and teat sinus at drying-off on mastitis in the dry-period and at calving.

An experiment using three New Zealand herds and a total of 632 cows, examined the effect of localised prophylactic treatments with antibiotic at drying-off on the incidence of new intramammary infection during the dry period and at calving. Antibiotic was infused either into the teat canal (0.22 g of dry-cow formulation) or the teat sinus (3.1 g of lactating-cow formulation) of uninfected quarters to eliminate any bacteria present in these locations at the last milking of lactation. These treatments were compared with a negative control (nil treatment) and a positive antibiotic control (infusion of 3.6 g of dry-cow formulation). All antibiotic formulations used the same active ingredient, sodium cloxacillin. No significant reduction in new dry period clinical mastitis was observed for the two localised treatments whereas the positive control treatment achieved 100% reduction in new clinical mastitis compared with untreated control quarters. A 41% reduction (P < 0.05) in new Streptococcus uberis infections at calving was associated with the teat canal antibiotic treatment, compared with an 82% reduction (P < 0.001) for the positive antibiotic control. Both localised treatments showed a reduced incidence of new intramammary infection (P < 0.001) when pooled across periods and pathogens. Teats receiving either the teat canal antibiotic treatment or a full infusion of long acting dry-cow antibiotic had a lower incidence of open teat canals (P < 0.05) at 3 weeks after drying-off.

Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils.

The physiological effects of intramammary infusions of recombinant bovine cytokines in six lactating dairy cows on the quality and yield of milk and the bactericidal activity of milk neutrophils were investigated. Recombinant bovine interleukin-2 (rboIL-2) and interferon-gamma (rboIFN-gamma) were produced in the yeast Pichia pastoris. Two animals were given rboIL-2 (2 x 10(5) units) in two quarters, two animals were given rboIFN-gamma (6.5 x 10(5) units) in two quarters, and the other two cows received a dose of rboIL-2 in one quarter and rboIFN-gamma in a second quarter. In addition, each animal was given phosphate-buffered saline (PBS) in the other two quarters as a control. Somatic cell counts and conductivity of the fore milk were monitored before and after infusion. Neutrophils were isolated from quarter milk samples 36 h after infusion of cytokine or PBS and their bactericidal activities against Staphylococcus aureus were measured in vitro with a colorimetric assay. Quarters infused with rboIL-2 or rboIFN-gamma showed significant but transitory increases in both milk somatic cell counts and conductivity when compared with preinfusion values and with control quarters. There were minimal effects on daily milk yield. Neutrophils isolated from milk from quarters infused with rboIL-2 showed enhanced bactericidal activity against Staph. aureus. The bacterial killing from rboIL-2 treated quarters was significantly greater, with a mean of 63.5% compared with a mean of 5.4% for neutrophils taken from uninfected quarters to which PBS had been administered. The bactericidal activities for quarters treated with rboIFN-gamma and infected quarters treated with PBS were 15.0 and 30.0% respectively. The results indicate that intramammary infusions of rboIL-2 and rboIFN-gamma to lactating cows are well tolerated, and that rboIL-2 can activate milk neutrophils and augment their bactericidal activity.

Effect of milking frequency and pasture intake on milk yield and composition of late lactation cows.

Twenty-four monozygous twinsets in late lactation (> 210 d in milk) were used to examine the effects of feed restriction and milking frequency prior to drying off on milk yield and composition in a pastoral dairying system. Cows were assigned to one of four treatment groups for 26 d and were milked either twice or once daily and given either unrestricted or restricted access to feed. Dry matter intakes averaged 16 or 8 kg per cow per day, and diets comprised ryegrass and white clover pasture supplemented with 15% pasture silage. Feed restriction and once daily milking reduced milk yield and increased concentrations of milk fat and protein. Somatic cell count was increased by feed restriction only. Production losses caused by feed restriction were nearly threefold higher than were those for once daily milking. Yields of components that were mammary synthesized and serum derived were reduced by feed restriction, in accordance with milk volume reduction. Plasma lactose concentration increased with once daily milking only and indicated enhanced permeability of mammary tight junctions. Both feed restriction and once daily milking compromised milk quality, but increased leakage of serum components into milk via mammary tight junctions was deemed to occur only for once daily milking.

Influence of pulsationless milking on teat canal keratin growth and turnover.

In two separate experiments, the effects of pulsationless milking and milking vacuum on the rate of keratin removal from the teat canal were determined. Sixteen cows were milked with or without pulsation for either a single milking or for eight milkings. Milking without pulsation removed 10% of keratin present before milking, significantly less than milking with pulsation, which removed 32%. After eight milkings (4 d) without pulsation, up to 20% more keratin had accumulated within the teat canal but the rate of keratin regeneration reduced significantly upon return to pulsation milking. In a second experiment, ten cows were milked at 45 or 55 kPa and without pulsation. Only the absence of pulsation significantly reduced keratin loss during milking. Keratin loss during milking appears to be controlled by liner compression rather than by the rate of milk flowing through the teat canal. Pulsationless milking may increase penetrability of the teat canal to bacteria by reducing the natural rate of keratin removal during milking and reducing the rate of keratin regeneration.

Effect of milking frequency on milk somatic cell count characteristics and mammary secretory cell damage in cows.

OBJECTIVES: To examine whether once daily milking (ODM) increases somatic cell count (SCC) and causes changes in the proportion of macrophages, lymphocytes, or neutrophils in milk, and to examine whether ODM causes mammary secretory cell damage. DESIGN: During 3 successive periods, cows were milked twice daily (days 1 through 4), once daily (days 5 through 10), and twice daily (days 11 through 14); thus, each cow served as its own control. Milk samples were collected during each milking for SCC, bovine serum albumin (BSA; except days 1 and 14), and N-acetyl-beta-D-glucosaminidase (NAGase) activity analyses. Differential cell counts were determined on days 2 (period 1), 5 and 8 (period 2), and 11 (period 3) in morning milk samples. ANIMALS: 8 multiparous Jersey cows. PROCEDURES: Cell counter (SCC), staining and counting (differential SCC), ELISA (BSA), and enzyme activity (NAGase). RESULTS: Once daily milking increased SCC (total and concentration), percentage of neutrophils, and BSA (content and total mass); during subsequent twice daily milking, SCC decreased, but BSA content and percentage of neutrophils remained increased. Activity of NAGase was not affected by ODM. CONCLUSIONS: ODM increases SCC, but this is not associated with damage to mammary secretory cells. The ODM-related sustained increase in BSA content coincided with an increase in neutrophil numbers, suggesting a causal relation.

Physical characteristics of the bovine teat canal and their influence on susceptibility to streptococcal infection.

Physical characteristics of the bovine teat canal were examined for their influence on susceptibility to intramammary infection. All quarters of 18 cows were inoculated with 2 x 10(5) cfu Streptococcus agalactiae (Trial 1) and 20 cows with 10(5) cfu Str. uberis (Trial 2) 3-4 mm into the teat canal every 3 d for 12 d. Incidence of quarter infection was similar for both pathogens, 30/72 (42%) in Trial 1 and 32/80 (40%) in Trial 2. Logistic regression analysis showed that probability of infection by Str. agalactiae increased significantly with an increase in quarter peak flow rate (P < 0.05) whereas probability of infection increased for Str. uberis with a decrease in teat canal length (P < 0.05). A significantly higher (P < 0.001) incidence of infection by Str. uberis was observed in quarters that contained a low wet weight (< 1.8 mg) of removable keratin compared with those that contained > 1.8 mg keratin, but there was no correlation between weight of keratin and length of the teat canal. Infections by Str. uberis took significantly less (P < 0.05) time to show a rise in somatic cell count above 7.5 x 10(5) cells/ml than Str. agalactiae. The results provide evidence that these pathogens use different mechanisms to pass through the teat canal.