Effect of bismuth subnitrate on in vitro growth of major mastitis pathogens.

The mode of action of bismuth subnitrate in teat sealant formulations as a preventative for intramammary infections during the dry period is unknown. Although previous studies proposed an action mechanism-creating a physical barrier in the teat canal to prevent bacterial invasion-it has not been proven experimentally. We hypothesized that bismuth subnitrate has an inhibitory effect on bacterial growth, in addition to its barrier effect. The objective of this study was to assess the effect of bismuth subnitrate on bacterial growth of major mastitis-causing agents. A strain of Streptococcus uberis (SR115), 2 strains of Staphylococcus aureus (SA3971/59 and SA1), and a strain of Escherichia coli (P17.14291) were tested in vitro for their ability to grow in the presence or absence of bismuth subnitrate. Disk diffusion testing, impedance measurement, and evaluation of bacterial growth in shaking conditions were the methods used to test this hypothesis. A reduction of growth in the presence of bismuth subnitrate occurred for all the strains tested. However, we observed strain and species variations in the extent of growth inhibition. These results suggest that an inhibitory effect on bacterial growth by bismuth subnitrate could partially explain the efficacy of bismuth-based formulations for preventing intramammary infections over the dry period. Further research is required to test the effect of teat sealant formulations on bacterial growth.

The ability of four strains of Streptococcus uberis to induce clinical mastitis after intramammary inoculation in lactating cows.

AIM: To compare the ability of four strains of Streptococcus uberis at two doses to induce clinical mastitis in lactating dairy cows after intramammary inoculation in order to evaluate their usefulness for future experimental infection models. MATERIALS AND METHODS: Four field strains of S. uberis (26LB, S418, and S523 and SR115) were obtained from cows with clinical mastitis in the Wairarapa and Waikato regions of New Zealand. Twenty-four crossbred lactating cows, with no history of mastitis and absence of major pathogens following culture of milk samples, were randomly allocated to four groups (one per strain) of six cows. Each cow was infused (Day 0) in one quarter with approximately 10(4) cfu and in the contralateral quarter with approximately 10(6) cfu of the same strain. The other two quarters remained unchallenged. All four quarters were then inspected for signs of clinical mastitis, by palpation and observation of the foremilk, twice daily from Days 0-9, and composite milk samples were collected from Days 0-8 for analysis of somatic cell counts (SCC). Quarters were treated with penicillin when clinical mastitis was observed. Duplicate milk samples were collected and cultured on presentation of each clinical case and on Day 4 from challenged quarters with no clinical signs. RESULTS: Clinical mastitis was diagnosed in 26/48 (54%) challenged quarters. Challenge with strain S418 resulted in more cases of mastitis (12/12 quarters) than strains SR115 (7/12), 26LB (6/12) or S523 (1/12), and the mean interval from challenge to first diagnosis of mastitis was shorter for S418 than the other strains (p<0.001). The proportion of quarters from which S. uberis could be isolated after challenge was less for strain 26LB (1/6) than SR115 (6/7) (p<0.05), and SCC following challenge was lower for strain S523 than the other strains (p<0.05). CONCLUSIONS: There were significant differences between the strains in the proportion of quarters developing clinical mastitis, the interval to mastitis onset, SCC following challenge and the proportion of clinical cases from which S. uberis could be isolated. These results illustrate the difference in the ability of S. uberis strains to cause mastitis and the severity of the infections caused. CLINICAL RELEVANCE: Experimental challenge models can be used to compare infectivity and pathogenicity of different strains of mastitis-causing bacteria, the efficacy of pharmaceutical products and host-responses in a cost-effective manner.

Susceptibility to antimicrobials of mastitis-causing Staphylococcus aureus, Streptococcus uberis and Str. dysgalactiae from New Zealand and the USA as assessed by the disk diffusion test.

OBJECTIVE: To compare the antimicrobial susceptibility patterns of three common mastitis pathogens (Staphylococcus aureus, Streptococcus uberis and Str. dysgalactiae) isolated from milk samples from New Zealand and the USA. METHODS: A total of 182 S. aureus, 126 Str. uberis and 89 Str. dysgalactiae isolates from New Zealand (107, 106 and 41, respectively) and the USA (75, 20 and 48, respectively) were assessed using the disk diffusion test. RESULTS: Susceptibility varied among the bacterial species. All isolates were susceptible to the amoxicillin-clavulanic acid combination. Resistance to lincomycin was most frequent (susceptibility of 8.6%) across all species. Non-susceptible (i.e. resistant or intermediate) isolates of S. aureus were identified for the three non-isoxazolyl penicillins (amoxicillin, ampicillin and penicillin: 20.6% and 36.0%) and lincomycin (99.9% and 94.6%) for NZ and the USA, respectively. Resistance to erythromycin (5.3%) and tetracyclines (6.7%) was detected only in isolates from the USA. There were differences in susceptibility between Str. uberis and Str. dysgalactiae; all streptococcal isolates demonstrated resistance to aminoglycosides (neomycin 52.4% and streptomycin 27.9%) and enrofloxacin (28%). Resistance of Str. dysgalactiae to tetracycline was almost 100.0% and to oxytetracycline 89.9%. CONCLUSION AND CLINICAL RELEVANCE: Most of the isolates tested were susceptible to most of the antimicrobials commonly used for treatment of bovine mastitis, with the exception of the lincosamides. Susceptibility to a selected class-representative antimicrobial and at the genus level should be interpreted with caution. Differences between NZ and the USA confirm the value of national surveys to determine the susceptibility patterns of mastitis pathogens.

Study on the use of toltrazuril to eliminate Neospora caninum in congenitally infected lambs born from experimentally infected ewes.

To determine if toltrazuril was effective in eliminating Neospora caninum infection from congenitally infected lambs. Twenty-eight ewes were allocated to 3 groups where animals in Groups A and B were inoculated with 1 × 10(7)N. caninum tachyzoites on Day 120 of gestation and Group C was maintained as a negative control group. Lambs born from ewes in Group A were treated with toltrazuril (20mg/kg) on Days 0, 7, 14 and 21 after birth. Lambs in Groups B and C were untreated. All lambs in Groups A and B were seropositive at 12 weeks of age. At 12 weeks of age, no differences between lambs in Group A and Group B were observed in serological results (ELISA and western blot), presence of N. caninum-related brain histopathological lesions or the number of organisms detected by qPCR. Group C remained negative for serology, detection of N. caninum DNA as well as histopathology throughout the study. Results indicate that N. caninum congenitally-infected lambs had a continuing infection with N. caninum despite being treated with toltrazuril.

Vertical transmission in experimentally infected sheep despite previous inoculation with Neospora caninum NcNZ1 isolate.

Recent reports indicate N. caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the mode of transmission of neosporosis in sheep in New Zealand is limited. This study aimed to determine the rate of vertical transmission that would occur in lambs born from experimentally inoculated ewes and to determine if previous inoculation would protect the lambs from N. caninum infection. A group of 50 ewes was divided into 2 groups with one group being inoculated with 5×10(6) N. caninum tachyzoites prior to pregnancy in Year 1. In Year 2, each of these groups was subdivided into 2 groups with one from each original group being inoculated with 1×10(7) N. caninum tachyzoites on Day 120 of gestation. Inoculation of N. caninum tachyzoites into ewes prior to mating resulted in no congenital transmission in lambs born in Year 1 but without further inoculation, 7 out of 11 lambs in Year 2 were positive for N. caninum infection. Ewes that were inoculated in both years resulted in all 12 lambs born in Year 2 being positive for N. caninum infection. This indicates that previous inoculation in Year 1 did not result in any vertical transmission in that year but did not provide any protection against vertical transmission in Year 2. These results suggest that vertical transmission occurs readily once the ewe is infected.

Adaptation of a commercial ELISA to determine the IgG avidity in sheep experimentally and naturally infected with Neospora caninum.

Recent reports indicate Neospora caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the epidemiology of neosporosis in sheep is limited. This study aimed to adapt and validate a commercially available ELISA assay as an IgG avidity assay to discriminate between acute (primary and re-inoculated) and chronic N. caninum infections in sheep. In addition, it was used to compare the antibody avidity values between lambs from ewes inoculated with N. caninum either during the pregnancy or in the previous year. The avidity assay was undertaken by using 6M urea for the first wash after incubation with the primary antibody in the commercial ELISA (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia). Sequential serum samples were obtained from naïve ewes (n=16) experimentally inoculated with live N. caninum tachyzoites. All ewes were seropositive by two weeks post-inoculation and remained seropositive for 20 weeks post-inoculation. There was a linear relationship between time after inoculation and avidity values (p<0.05) over the first 24 weeks. In Week 4, all animals had avidity values <35% and by Week 8, 8/16 animals had avidity values of >35%. These results suggest that an avidity value of <35% indicates a recent primary infection while a value of >35% is indicative of a chronic infection. The assay was then validated using samples from other groups of experimentally inoculated sheep as well as samples from naturally infected ewes. When comparing sample to positive ratio (S/P) and avidity values from lambs born from recently inoculated ewes with those from ewes inoculated the previous year and re-inoculated in the current year, it was possible to differentiate the lambs at 2 weeks of age. Lambs from recently inoculated ewes had low S/P and avidity values at 2 weeks of age which increased by 12 weeks of age. In comparison, lambs from re-inoculated ewes had high S/P and avidity values at 2 weeks of age, due to maternal antibody influence but values were similar to those from lambs that were born from recently inoculated ewes at 12 weeks of age. Avidity values for four naturally infected ewes were all >60% indicating chronic infection. These results suggest that the assay is able to discriminate between recent and chronic infection in sheep as well as able to differentiate lambs with maternal immunity compared to their own de novo immunity. As such it can be utilized to understand the kinetics of N. caninum infection in sheep.

The use of liveweight change as an indicator of oestrus in a seasonally calving, pasture-fed dairy herd.

This was an observational study of 828 lactations in 542 mixed-age dairy cows that calved seasonally in a single, pasture-fed herd in New Zealand in 2008 and 2009. The study objectives were to: (i) document daily liveweight change (∆LW) before and after observed oestrus for cows subsequently diagnosed pregnant or non-pregnant and (ii) quantify the sensitivity and specificity of ∆LW as a test for oestrus. The sensitivity and specificity of ∆LW when combined with other commonly used oestrous detection methods was also evaluated. In cows that conceived as a result of service at detected oestrus, liveweight loss began 1 day before the day of detection and was greatest on the day of detection (-9.6 kg, 95% CI -11.3 kg to -7.8 kg; p < 0.01) compared with LW recorded 2 days before the day of detection. In cows that did not conceive to a service at a detected oestrus, the lowest liveweights were recorded 1 day before the day oestrus was detected (-4.3 kg, 95% CI -7.7 to -0.8 kg; p = 0.02) compared with LW recorded 4 days before the day of detection. The sensitivity and specificity of ∆LW as a means of oestrous detection were 0.42 (95% CI 0.40-0.45) and 0.96 (95% CI 0.95-0.97), respectively. When ∆LW was combined with tail paint and visual observation, the oestrous detection sensitivity and specificity were 0.86 and 0.94, respectively. Monitoring LW change holds promise to enhance the sensitivity and specificity of oestrous detection in combination with other oestrous detection methods.

The effects of liveweight loss and milk production on the risk of lameness in a seasonally calving, pasture fed dairy herd in New Zealand.

Dairy herd managers have attempted to increase and maintain profits by selectively breeding dairy cattle for high production. Selection for milk production may have resulted in a tendency for greater liveweight (LW) loss postpartum. This study aimed to: (1) determine if excessive LW loss and milk yield in the first 50 days in milk (DIM) was associated with the development of lameness after 50 DIM, and (2) estimate the incidence risk of lameness in this herd attributable to excessive liveweight loss. The dataset comprised details from 564 mixed age cows from a single, seasonally calving, pasture fed dairy herd in New Zealand. After adjusting for the confounding effects of parity, LW at calving, breed, the presence of specified disease events in the first 50 DIM and milk yield, LW loss in the first 50 DIM increased the risk of lameness after 50 DIM by a factor of 1.80 (95% CI 1.00-3.17). The risk of lameness was greatest for high yielding cows that lost excessive LW (risk ratio 4.36, 95% CI 4.21-8.19), but the effect LW loss on lameness risk at the herd level was relatively small. Based on data accumulated during the study we estimate that for this herd, there would be a 3% (95% CI 1-6%) reduction in the incidence risk of lameness if excessive LW loss was prevented. Twenty three percent of the incidence of lameness in this herd was attributable to excessive LW loss. We conclude that policies and interventions to reduce the rate and amount of LW loss in the first 50 DIM will have a non-negligible impact on the incidence risk of lameness in this herd.

Detection of Neospora caninum DNA in semen of experimental infected rams with no evidence of horizontal transmission in ewes.

Recent reports from New Zealand indicate Neospora caninum has a possible role in causing abortions in sheep. Transmission of N. caninum via semen has been documented in cattle. This study aimed to investigate if horizontal transmission through semen was also possible in sheep. Initially, 6-month old crossbred ram lambs (n=32), seronegative to N. caninum, were divided into 4 equal groups. Group 1 remained uninoculated whilst the remainder were inoculated with N. caninum tachyzoites intravenously as follows: Group 2 - 50 tachyzoites; Group 3 - 10(3) tachyzoites; Group 4 - 10(7) tachyzoites. Semen samples were collected weekly for 8 weeks for the detection of N. caninum DNA and quantified using quantitative PCR (qPCR). Plasma collected 1 month post-inoculation was subjected to ELISA (IDEXX Chekit) and Western blot. At 2 weeks post-infection, three rams from Group 1 (uninoculated) and three rams from Group 4 (10(7)tachyzoites/ml) were mated with two groups of 16 ewes over two oestrus cycles. Ewe sera collected 1 and 2 months post-mating were tested for seroconversion by ELISA and Western blot. All experimentally infected rams seroconverted by 1 month with ELISA S/P% values ranging from 11% to 36.5% in Group 2, 12-39.5% in Group 3 and 40-81% in Group 4. However, none of the ewes mated with the experimentally infected rams seroconverted. For the Western blot, responses towards immunodominant antigens (IDAs) were observed in ram sera directed against proteins at 10, 17, 21, 25-29, 30, 31, 33 and 37 kDa. Rams in Group 2, 3 and 4 were noted to have at least 3 IDAs present. None of the ewes showed any of the 8 prominent IDAs except for the one at 21 kDa which was seen in 30 out of 32 ewes in both groups. N. caninum DNA was detected intermittently in the ram's semen up to 5 weeks post-inoculation with the concentrations ranging from that equivalent to 1-889 tachyzoites per ml of semen. Low concentrations of N. caninum DNA were also detected in the brain tissue of two rams (Groups 1 and 4). These results suggest that although N. caninum DNA can be found in the semen of experimentally infected rams, the transmission of N. caninum via natural mating is an unlikely event.

The effect of clinical lameness on liveweight in a seasonally calving, pasture-fed dairy herd.

This study investigated the effect of lameness on liveweight (LW) in pasture-fed dairy cattle. The data comprised 222,446 averaged daily LW measurements from 828 lactations of 542 mixed-age cows in a seasonally calving, pasture-fed New Zealand dairy herd. The LW measurements for individual cows were aggregated into weekly averages and analyses conducted to evaluate the effect of a diagnosis of lameness on LW change after controlling for the effect of week in milk, parity, LW at calving, breed, calendar month, and season. In lame cows, LW decreased for up to 3 wk before lameness was diagnosed and for up to 4 wk after treatment. Total LW loss arising from a single lameness episode was, on average, 61 kg (95% confidence interval: 47 to 74 kg). The results from this study demonstrate how LW records for individual animals can be used to enhance a herd manager's ability to detect lame cows and present them for treatment. The methods presented here show how daily LW monitoring might be used as a tool for early detection of lameness in dairy cattle.

Efficacy of a Neospora caninum killed tachyzoite vaccine in preventing abortion and vertical transmission in dairy cattle.

A clinical trial was undertaken to assess the efficacy of Bovilis(®) Neoguard, a killed Neospora caninum tachyzoite vaccine on 5 commercial dairy farms in New Zealand with a history of Neospora-associated abortion. Cattle were enrolled in the trial at 30-60 days of gestation and randomly allocated to treatment or control groups. Treatment consisted of 5 mL doses of Bovilis Neoguard administered subcutaneously at enrolment then 4 weeks later. Isotonic saline was administered to the control group. Of 2246 cattle enrolled in the trial, 10.7% of cows and 12.6% of heifers were seropositive to N. caninum. Sampling of a randomly selected proportion of enrolled animals 6 weeks after the second treatment showed that 188/232 (81.0%) vaccinated with Bovilis(®) Neoguard had seroconverted, while 11/130 (8.5%) cows and 10/36 (27.8%) heifers in the control group had seroconverted. Forty-eight vaccinated and 63 control animals aborted. On one farm 12.5% of control animals and 6.1% of vaccinated animals aborted (vaccine efficacy 0.61; p=0.03). On another farm with a high level of abortion 8.4% of control animals and 8.7% of vaccinates aborted. On the remaining 3 farms fewer abortions occurred than expected. A modified Poisson regression approach was used to calculate relative risks for abortion and vertical transmission. Overall vaccine efficacy was 0.25 (p=0.12). Heifer replacement calves from the animals enrolled in the trial were sampled for antibodies to N. caninum at 6-9 months of age. Fourteen of 17 calves from vaccinated, seropositive cows were seropositive as were 13/23 calves from seropositive cows in the control group. The interaction between dam serostatus and treatment group was significant (p=0.05) with vaccination increasing the risk of vertical transmission. It was concluded that vaccination after conception prevented 61% abortions in one of five herds and that vaccination may have increased the risk of early embryonic death.

Causes of abortion on New Zealand dairy farms with a history of abortion associated with Neospora caninum.

AIM: To investigate the cause of abortion in cows from farms with a history of abortion associated with Neospora caninum. METHODS: Cows and primiparous heifers (n = 2,246) that were enrolled in a clinical trial of a vaccine for N. caninum, and 403 cows and heifers that conceived >6 weeks after the planned start of mating (PSM), were monitored for abortion on five dairy farms with a history of abortion associated with N. caninum. When abortion was detected the fetus and/or placenta were submitted for histopathological examination and maternal paired sera were collected for the detection of antibodies to N. caninum using the indirect fluorescent antibody test (IFAT), bovine viral diarrhoea virus (BVDV) using ELISA, and Leptospira spp. using the microscopic agglutination test (MAT). RESULTS: At the start of the clinical trial, 189/1,760 (10.7%) of cows and 61/486 (12.6%) of heifers had IFAT titres ≥1:200 for N. caninum. Abortions were detected in 111 (4.9%) enrolled and 37 (9.2%) non-enrolled cows and heifers. The relative risk of abortion was 4.21 (95% CI 2.92-6.08) times greater in enrolled animals that were seropositive to N. caninum than seronegative animals (p < 0.01). Among all animals, histopathological lesions suggestive of protozoal abortion were seen in nine, and of bacterial infection in 15, of the 40 cases where the fetus and/or placenta were recovered. IFAT titres for N. caninum ≥1:1,000 were detected in 66 cows at abortion, including 7/9 cases where there were fetal lesions indicative of N. caninum infection. In nine cows that aborted, there was evidence of seroconversion to BVDV and eight cows had MAT titres for Leptospira spp. ≥1:200. Histopathology revealed dual infectious aetiologies in two cases and, in another 17 cases, there was serological evidence of recent exposure to a second infectious agent capable of causing abortion in conjunction with N. caninum lesions in the fetus or fetal bacteraemia. CONCLUSIONS: Multiple infectious aetiologies occur at herd-level and in individual cows on farms with a history of abortion associated with N. caninum. A diagnosis of abortion due to N. caninum infection was made in 9/34 (26.5%) cases where fetal histopathology was undertaken. CLINICAL RELEVANCE: Thorough and ongoing diagnostic investigations should be undertaken in herds with higher than expected abortion losses to identify all causative agents as control measures could be implemented that may also reduce the risk of abortion in cows infected with N. caninum.

Culture results from milk samples submitted to veterinary diagnostic laboratories from August 2003 to December 2006 in New Zealand.

AIMS: To determine the pattern of isolation of major mastitis-causing organisms isolated from milk samples submitted to five veterinary diagnostic laboratories in New Zealand. METHODS: The culture results of 25,288 milk samples that were collected from dairy cows throughout New Zealand from August 2003 to December 2006 and submitted to a group of veterinary diagnostic laboratories were assembled, reviewed and summarised. Logistic regression was used to analyse the effect of year, region (i.e. North vs South Island), and season on the probability of isolating the two most common organisms. RESULTS: The most commonly isolated mastitis causing organisms from all samples were: Streptococcus uberis (23.6%), Staphylococcus aureus (23.5%), coagulase-negative staphylococci (CNS; 7.2%), Strep. dysgalactiae (6.2%), Bacillus spp. (4.0%), and coliforms (3.7%). The percentage of samples with isolates of Strep. uberis or Staph. aureus was affected by island, year and season (p<0.001). For most of the year, except in late winter and early spring when Strep. uberis was much more common, the percentage of isolates of Strep. uberis and Staph. aureus were not apparently different despite the former being an environmental pathogen and the other a contagious one. CONCLUSION: The pattern of isolation of major mastitis-causing organisms, as determined from culture of milk samples submitted to diagnostic laboratories in New Zealand, has changed significantly over the last 40 years, with a substantial increase in the percentage of isolates that are Strep. uberis and a decrease in isolates of Strep. agalactiae. There is a clear seasonal pattern to the isolation of both Strep. uberis and Staph. aureus, particularly the former. CLINICAL RELEVANCE: Knowledge of the aetiological agents causing bovine mastitis on a farm is of value in determining the choice of treatment. This dataset shows that, although there is seasonal pattern to the isolation of mastitis-causing organisms in New Zealand, both Strep. uberis and Staph. aureus are isolated throughout the year, so bacteriology is of value in determining aetiology even in late winter/early spring.

Automatic recording of daily walkover liveweight of dairy cattle at pasture in the first 100 days in milk.

Daily walkover liveweight (WoLW) records (n=79,697) from 463 pasture-fed dairy cows from a single dairy herd in the lower North Island of New Zealand were recorded over the first 100 d of lactation. The aims of this study were to (1) describe LW records retrieved by a standalone automatic Wo daily weighing system; (2) describe the frequency and nature of outlier LW records measured by the system and develop an approach for excluding identified outlier LW records; (3) quantify the agreement between cow LW measured using the Wo system and those measured statically; and (4) describe the autocorrelation between daily LW measurements to provide an indication of how frequently management decisions need to be reviewed to effectively monitor cow LW change in the early-lactation period. The standard deviation of daily LW measurements across parities was 17 kg, on average. A near perfect association between LW measured statically and WoLW (concordance correlation coefficient 0.99, 95% CI 0.99-1.0) was observed. After controlling for the effect of LW at calving and long-term LW change using a mixed-effects linear regression model, the autocorrelation between WoLW recorded on successive days was 0.21, decaying to zero by 8 d. This study shows that by using a standalone automatic Wo weighing system positioned in the exit race of a rotary milking parlor, it was possible to record LW of individual cows on a daily basis and, with controlled cow flow over the weighing platform (allowing for sufficient succession distance to prevent congestion), results were similar to those recorded using conventional, static weighing techniques using the same scales. Based on the autocorrelation analyses, we recommend that LW are recorded on a daily basis to allow changes in physiological status such as the onset of acute illness or estrus to be detected. For managerial purposes, such as using LW change as a guide for adjusting the herd feeding program, we recommend a 7-d decision interval to effectively monitor significant changes in cows' recorded daily LW measurements.

Efficacy of a novel internal dry period teat sealant containing 0.5% chlorhexidine against experimental challenge with Streptococcus uberis in dairy cattle.

The incidence of clinical mastitis and infection status at calving was assessed in quarters treated with 1 of 2 internal teat sealants at the time of dry off. Two contralateral quarters per cow (n=63 cows) were treated with a sealant that contained 0.5% chlorhexidine; the other quarters were treated with a commercial teat sealant. Ten cows were untreated (controls). On d 2, 4, and 16 after dry off, cows were challenged with Streptococcus uberis S210 strain. Cows were examined daily for 34 d after drying off and cases of clinical mastitis were recorded. Milk samples were collected for culture from any quarters that developed clinical mastitis during the first 34 d after drying-off and from all quarters on d -5 and 0 relative to treatment and at the first and twentieth milking after calving. The incidence of clinical mastitis during the examination period was lower in treated quarters (n=7/252; 1.5%; lower incidence for those treated with chlorhexidine-containing teat sealant n=3/126; 1.2%) than in untreated quarters (n=13/40; 26.8%). The protection against intramammary infection after calving, adjusted for the effect of cow, was higher in quarters treated with the novel teat sealant (89/105; 15.2%; 95% CI=9.6-23.4) than in those treated with the commercial teat sealant (71/104; 31.7%; 95% CI=23.5-41.3) and untreated controls (6/28; 78.6%; 95% CI=59.8-90.0), respectively. Quarters treated with teat sealants were less likely to have an intramammary infection after calving and had a lower incidence of clinical mastitis during the early dry period than did untreated controls in this challenge study.

Dose-titration challenge of young pregnant sheep with Neospora caninum tachyzoites.

Pregnant ewe lambs (7-8 months old at breeding) were inoculated intravenously at 90 days after joining with the ram with 50, 5 x 10(3), 10(6), or 10(8)Neospora caninum tachyzoites and outcomes were compared to a control group. Seroconversion was measured by indirect fluorescent antibody test (IFAT) at fortnightly intervals and by enzyme-linked immunosorbent assay (ELISA) at 28 days post-challenge. Seroconversion (by IFAT) occurred in all animals except for 2 sheep in the lowest dose group, including the 9 sheep in the control group. IFAT antibody titres rose and fell rapidly, peaking at 1:800 and in 8 animals was negative within 7 days of abortion. ELISA results more closely reflected infection dose and outcome of pregnancy than IFAT serology did. All 10 ewes in each of the 2 highest dose groups aborted, 5 of 10 ewes in the mid-dose group aborted and no ewes in the lowest dose or control groups aborted. Histological lesions consistent with N. caninum infection were seen in the brains of all 25 aborted lambs, as well as in 2 live-born premature lambs from the group receiving 5 x 10(3) tachyzoites and 2 clinically normal lambs (one from the control group). Polymerase chain reaction (PCR) detected N. caninum DNA in a variety of tissues from lambs, aborted foetuses and dams including 3 of 9 ewe-lamb pairs from the control group. The results from this study showed a strong relationship between challenge dose of N. caninum tachyzoites, the sample to positive (S/P) percentage in an ELISA test 28 days after challenge and pregnancy outcome. IFAT results did not correlate well with the level of challenge or the outcome of pregnancy and their relevance in studies of this kind should be questioned.

The incidence and aetiology of clinical bovine mastitis on 14 farms in Northland, New Zealand.

AIM: To estimate the incidence of clinical mastitis and the frequency of isolation of mastitis-causing organisms from clinical cases in one lactation season (July 2005 to May 2006) on 14 dairy farms from the Northland region of New Zealand. METHODS: Cases of clinical mastitis were determined by trained farm personnel who recorded the identity of affected cows. Pooled milk samples from affected quarter(s) were aseptically collected by the farm personnel, for microbiology. Mean numbers of affected cows and quarters were compared at the population and farm level per 305 cow-days-at-risk (DAR). RESULTS: One or more cases of clinical mastitis occurred in 559/3,765 (14.8%) lactating cows. The average incidence of clinical mastitis was 0.19 cases per 305 DAR. The incidence in rear quarters (56.2%) was 1.3 times (p=0.027) that of front quarters (43.8%). The incidence of clinical mastitis and numbers of affected quarters were significantly influenced by the stage of lactation (higher in early lactation), age (higher in older cows) and farm. At the cow level, the most common isolates were Staphylococcus aureus (23.7%) and Streptococcus uberis (23.3%). No causative organisms were identified in 19.9% of the samples. Each cow had an average of 1.8 quarters affected during a case of clinical mastitis. CONCLUSIONS: This study demonstrated a higher incidence of staphylococcal clinical mastitis on dairy farms from Northland than has been reported in other regions of New Zealand.

Comparison of a camera - software system and typical farm management for detecting oestrus in dairy cattle at pasture.

AIM: To compare the sensitivity, specificity, predictive values and accuracy of detection of oestrus using a novel oestrus detection strip (ODS) and a camera-software device (CSD) with typical farm management practices of visual observation and use of tail paint in dairy cattle at pasture. METHODS: Dairy cows (n = 480) in a seasonal-calving herd managed at pasture under typical commercial conditions in New Zealand were stratified by age, body condition score and days in milk, then randomly allocated to one of two groups prior to the planned start of mating (PSM). Tail paint was applied to all cows and oestrus detected by visual observation of oestrous behaviour and removal of paint, by farm staff. One group (n = 240) was fitted with ODS and also monitored for signs of oestrus using a CSD, while the Control group (n = 240) was monitored using tail paint and visual observations only. Cows detected in oestrus were artificially inseminated (AI), and pregnancy status determined using rectal palpation and ultrasonography, 51-52 days after the end of a 55-day A period. Results of pregnancy diagnosis were used to confirm the occurrence of oestrus, and the sensitivity, specificity, predictive value and accuracy of detection of oestrus compared between oestrus detection methods. RESULTS: The sensitivity and accuracy of oestrus detection in the Control group, using visual observation and tail paint, were low. Compared with the Control group, detection of oestrus using the ODS and CSD resulted in greater sensitivity (85% vs 78%; p = 0.006), specificity (99.6% vs 98.0%; p < 0.001), positive predictive value (PPV; 88% vs 51%; p < 0.001) and overall accuracy (99.0% vs 98.0%; p < 0.001). Negative predictive value (NPV) did not differ significantly between groups (99.4% vs 99.3%; p = 0.28). Pregnancy rate to first service was higher in the CSD group than in the Control group (72% vs 39%; p < 0.05). Use of the CSD significantly increased the cumulative proportion of cows pregnant to AI over the breeding period (p = 0.044). CONCLUSION AND CLINICAL RELEVANCE: The ODS and CSD was satisfactory for detection of oestrus in seasonal calving dairy herds grazing on pasture and could improve the sensitivity and accuracy of detection of oestrus in herds where these are low.

Neospora caninum: quantification of DNA in the blood of naturally infected aborted and pregnant cows using real-time PCR.

This study quantified Neospora caninum DNA in the blood and brain of pregnant and aborted heifers by monitoring PCR product formation in real-time using SYBR Green I, a double-stranded DNA-binding dye. Primers were designed to amplify a 188 bp product specific to N. caninum from the Nc-5 gene fragment of N. caninum. Similarly, a 71 bp product was amplified from the 28S rRNA gene of bovine genomic DNA that served as a control. Agarose gel electrophoresis and analysis of the melting curve for PCR products showed that both primer pairs were specific to their targets. Standard curves were generated for both bovine and N. caninum genomic DNA, and were used to compute the relative concentration of parasite to bovine DNA in the test samples. The concentration of N. caninum DNA in 1 ng of bovine genomic DNA obtained from blood ranged between 0.097 ng at the 1st week of the observation and 0 ng at the 15th week in aborted cows. In pregnant cows, the values ranged between 0.080 ng at the 1st week and 0.155 ng at the 15th week of observation. There was a sustained decrease of DNA concentration in the aborted group after abortion and an increase in DNA concentration in the pregnant group. Comparison of parasite DNA in blood and brain of infected heifers showed a higher DNA concentration in brain than in blood. This study shows that N. caninum DNA can be quantified to obtain the relative concentration of parasite DNA to host genomic DNA in blood. This technique allows testing of a large number of samples at one time and can be done without the need for slaughter of tested animals.

Associations between pregnancy outcome and serological response to Neospora caninum among a group of dairy heifers.

AIM: To monitor pregnancy in a group of rising 2-year-old dairy heifers on a farm on which abortion due to Neospora caninum was known to occur in previous years. METHODS: A prospective cohort study group of 164 rising 2-year-old heifers was pregnancy-tested and blood-sampled at 4-5-week intervals throughout gestation. Sera were tested for antibodies to N. caninum at 3-4-month intervals, using an enzyme-linked immunosorbent assay (ELISA). When loss of pregnancy was detected, an N. caninum indirect fluorescent antibody test (IFAT) was conducted retrospectively on stored sera collected the month before abortion, the month abortion was detected, and for the following 2 months, from heifers that aborted. All fetal and placental material detected following abortion was subjected to gross post-mortem and histopathological examination. RESULTS: Eleven of 18 (61%) heifers that were seropositive and 4/146 (3%) heifers that were seronegative to N. caninum by ELISA, aborted. The relative risk for abortion among ELISA-positive heifers was 23.6. Abortion occurred predominantly between Days 120 and 152 gestation among the ELISA-positive heifers and throughout gestation among the ELISA-negative heifers. IFAT titres rose around the time of abortion in most of the heifers that were previously seropositive by ELISA, but dropped rapidly again in post-abortion samples. IFAT titres among 4/6 ELISA-positive heifers that did not abort increased, but later in gestation than the time other heifers aborted. IFAT titres remained negative in heifers that aborted that were ELISA negative. CONCLUSIONS: Heifers that were seropositive to N. caninum by ELISA had a much greater risk of abortion than seronegative heifers. Most seropositive heifers showed evidence of a reactivation of infection during pregnancy. High (> or =1:2,000) N. caninum IFAT titres also occurred in non-aborting heifers. CLINICAL RELEVANCE: Culling of replacement heifers seropositive to N. caninum may be a cost-effective strategy for minimising risk of abortion. Pregnancy testing heifers before 5 months gestation may overestimate the number that calve in N. caninum-infected herds, but would assist in documenting the occurrence of abortion. Reliance on a high (>1:2,000) IFAT titre to rule-in N. caninum as a cause of abortion is likely to produce false-positive results.