Sam Mason

Risk factors for increased rates of sole ulcers, white line disease, and digital dermatitis in dairy cattle from twenty-seven farms in England and Wales

Claw lesion treatment records were recorded by farmers on 27 dairy farms (3,074 cows, 36,432 records) in England and Wales between February 2003 and February 2004. These were combined with farm environment and management data collected using a combination of direct observations, interviews with farmers, and milk recording data. Multilevel models were constructed for the 3 most frequently reported lesions related to lameness, namely, sole ulcers, white line disease, and digital dermatitis. Risks associated with an increased incidence of sole ulcers were parity 4 or greater, the use of roads or concrete cow tracks between the parlor and grazing, the use of lime on free stalls, and housing in free stalls with sparse bedding for 4 mo or more. The risks for white line disease were increasing parity and increasing herd size, cows at pasture by day and housed at night, and solid grooved concrete floors in yards or alleys. Solid grooved flooring was also associated with an increased risk of digital dermatitis, and cows 6 or more months after calving had a decreased risk of a first case of digital dermatitis. These results improve our understanding of the specific risks for 3 important lesions associated with bovine lameness and could be used as interventions in future clinical studies targeted at the reduction of specific lesions.

Pathogen Quantitation in Complex Matrices: A Multi-Operator Comparison of DNA Extraction Methods with a Novel Assessment of PCR Inhibition

Background Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. Methodology/Principal Findings We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. Conclusions M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×105 cells g−1 using Griffiths and 4.25×106 cells g−1 using FastDNA® Spin kit.

Risk factors for herd breakdown with bovine tuberculosis in 148 cattle herds in the south west of England

Bovine tuberculosis (bTB) is caused by Mycobacterium bovis. The disease has a long latent period, heterogenous spread, can infect many species and can persist in the environment. In the UK, the rate of herd breakdowns (HBD) with bTB is increasing. A retrospective cohort study of 148 cattle herds was set up to investigate risk factors for HBD from October 2001 to November 2004. Herds were selected from farms located in the randomised badger culling trial (RBCT) and comprised holdings (24%) that were restocked with cattle after the foot and mouth disease (FMD) epidemic in 2001 and holdings (76%) that were continuously stocked throughout the FMD epidemic. Farmers were interviewed between June 2003 and February 2004. Questions on herd and farm management were asked for the period October 2001 to June 2003. Data on herd testing for bTB were sourced from the VetNet database and historic data from 1995 were used in the analysis. A discrete time survival analysis was used to examine factors associated with the risk of HBD. By the end of the study period, November 2004, 50% of study herds had experienced a HBD with bTB at least once. Farms that were restocked for less than 1 year after FMD had a reduced risk of HBD (P<0.01) compared with continuously stocked farms in the same year. This reduced risk did not persist after 1 year of restocking. Feeding vitamin and mineral lick supplements compared with not feeding these supplements also reduced the risk of HBD. Factors associated with an increased risk of HBD were storing manure and slurry indoors or in a closed container, spreading manure all year round, herds with dairy cattle compared with herds without dairy cattle, increasing herd size, purchase of cattle from markets, location of the farm in the proactive area of the RBCT compared with survey only and location of farms in Somerset and North Devon. The lower risk of HBD in the first year after restocking but not the second or third year suggests that removal of all cattle might have lowered the infectious load of M. bovis on these premises for a period of time but that this did not persist once cattle were reintroduced. Purchase of cattle from markets suggests that there was a risk of introduction or re-introduction of bTB from these cattle. Method of storage or lack of storage of slurry might aid persistence of M. bovis in the environment if M. bovis survives in slurry in some circumstances.

Seroprevalence and epidemiological characteristics of Mycobacterium avium subsp. paratuberculosis on 114 cattle farms in south west England.

Between December 2002 and April 2006, 114 cattle farms in the south west of England were visited at least once, with 100 farms visited three times. A total of 29,782 serum samples were collected from 15,736 individually identified cattle. The sera were tested for the presence of antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) using an indirect enzyme-linked immunosorbent assay (ELISA). The mean seroprevalence in herds sampled three times was 7.1%; 10.1% of cattle had at least one positive result. There were 78%, 75% and 75% dairy herds with at least one positive bovine at the first, second and third routine visits, respectively. In comparison, 44%, 42% and 46% suckler herds had at least one positive bovine for the first, second and third routine visits, respectively. In most herds (>90%), within herd seroprevalence of MAP remained stable over time. Markov chain Monte Carlo (MCMC) simulation methods were used to re-estimate the test sensitivity and specificity. The sensitivity results were 33.3% (95% CI, 28.8-37.8%), 34.5% (95% CI, 30.3-38.8%) and 34.8% (95% CI, 30.8-38.9%) for the first, second and third routine visits and the specificity results were 99.7% (95% CI, 99.3-99.9%), 99.8% (95% CI, 99.4-99.9%) and 99.7% (95% CI, 99.3-99.9%) for the first, second and third routine visits, respectively. The expected true prevalence was also estimated, 11 (21.1%) suckler herds and 1 (2.1%) dairy herd were predicted to be truly free from infection during the study period. The seroprevalence of antibodies against MAP increased with cattle age. There was a significantly higher seroprevalence of MAP in dairy breeds of cattle compared with suckler breeds of cattle. This was more pronounced in Channel Island breeds. Smaller dairy herds (<100 cattle) had a relatively lower seroprevalence of MAP than dairy herds with >/=100 cattle. In 8 (42%) of the 19 herds with > or =100 cattle born into the same herd, seropositive cattle were clustered by birth month whilst in the remaining herds clustering was not apparent. Daughters were significantly more likely to be MAP seropositive when born to a seropositive dam.

A four year longitudinal sero-epidemiological study of bovine herpesvirus type-1 (BHV-1) in adult cattle in 107 unvaccinated herds in south west England

BackgroundBovine herpesvirus type-1 (BHV-1) is an important pathogen of cattle that presents with a variety of clinical signs, including the upper respiratory tract infection infectious bovine rhinotracheitis (IBR). A seroepidemiological study of BHV-1 antibodies was conducted in England from 2002 – 2004: 29,782 blood samples were taken from 15,736 cattle from 114 herds which were visited on up to three occasions. Antibody concentration was measured using a commercial ELISA. Farm management information was collected using an interview questionnaire, and herd size and cattle movements were obtained from the cattle tuberculosis testing database and the British Cattle Movement Service. Hierarchical statistical models were used to investigate associations between cattle and herd variables and the continuous outcome percentage positive (PP) values from the ELISA test in unvaccinated herds.ResultsThere were 7 vaccinated herds, all with at least one seropositive bovine. In unvaccinated herds 83.2% had at least one BHV-1 seropositive bovine, and the mean cattle and herd BHV-1 seroprevalence were 42.5% and 43.1% respectively. There were positive associations between PP value, age, herd size, presence of dairy cattle. Adult cattle in herds with grower cattle had lower PP values than those in herds without grower cattle. Purchased cattle had significantly lower PP values than homebred cattle, whereas cattle in herds that were totally restocked after the foot-and-mouth epidemic in 2001 had significantly higher PP values than those in continuously stocked herds. Samples taken in spring and summer had significantly lower PP values than those taken in winter, whereas those taken in autumn had significantly higher PP values than those taken in winter. The risks estimated from a logistic regression model with a binary outcome (seropositive yes/no) were similar.ConclusionThe prevalence of BHV-1 seropositivity in cattle and herds has increased since the 1970s. Although the study population prevalence of BHV-1 was temporally stable during study period, the associations between serological status and cattle age, herd size, herd type, presence of young stock and restocked versus continuously stocked herds indicate that there is heterogeneity between herds and so potential for further spread of BHV-1 within and between herds.

Herd and individual animal risks associated with bovine tuberculosis skin test positivity in cattle in herds in south west England.

The aim of this study was to investigate the cattle-exposure factors associated with the risk of a bovine animal reacting to a bovine tuberculosis (bTB) skin test at a whole herd test. There were 148 study farms enrolled. These were located in six counties of the south west of England in an area considered endemic for bovine tuberculosis (bTB): 24% were restocked after foot and mouth disease (FMD) in 2001; all farms were located within the Randomised Badger Culling Trial (RBCT) area. Data on cattle on these farms were sourced from the bTB Vetnet database from 1996 to 2004 and from the British Cattle Movement Scheme database. Individual animal records were created that included data on whether or not an animal became a reactor at a full-herd bTB test between 1 June 2001 and 19 August 2004, their prior exposure to cattle with bTB (defined by presence at a bTB test where at least one reactor was detected), whether the animal was homebred, the farm history of bTB and the farm restocking status. Data from 144 farms were used, 4 farms had no data. Cattle were more likely to react to the bTB skin test when they had been present at a previous bTB herd test (or tests) where other cattle had reacted to the skin test. This positively correlated with age and the number of bTB tests an animal had had. Cattle on restocked farms were less likely to react to the skin test compared with cattle on continuously stocked farms. These results highlight the likely importance of exposure to infected cattle at a previous test as a source of infection to cattle that subsequently became reactors and suggest that there was a lower risk of exposure to bTB to cattle in newly formed herds.

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis.

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 105 cells g−1 and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.

Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity

Background Environmental mycobacteria (EM) include species commonly found in various terrestrial and aquatic environments, encompassing animal and human pathogens in addition to saprophytes. Approximately 150 EM species can be separated into fast and slow growers based on sequence and copy number differences of their 16S rRNA genes. Cultivation methods are not appropriate for diversity studies; few studies have investigated EM diversity in soil despite their importance as potential reservoirs of pathogens and their hypothesized role in masking or blocking M. bovis BCG vaccine. Methods We report here the development, optimization and validation of molecular assays targeting the 16S rRNA gene to assess diversity and prevalence of fast and slow growing EM in representative soils from semi tropical and temperate areas. New primer sets were designed also to target uniquely slow growing mycobacteria and used with PCR-DGGE, tag-encoded Titanium amplicon pyrosequencing and quantitative PCR. Results PCR-DGGE and pyrosequencing provided a consensus of EM diversity; for example, a high abundance of pyrosequencing reads and DGGE bands corresponded to M. moriokaense, M. colombiense and M. riyadhense. As expected pyrosequencing provided more comprehensive information; additional prevalent species included M. chlorophenolicum, M. neglectum, M. gordonae, M. aemonae. Prevalence of the total Mycobacterium genus in the soil samples ranged from 2.3×107 to 2.7×108 gene targets g−1; slow growers prevalence from 2.9×105 to 1.2×107 cells g−1. Conclusions This combined molecular approach enabled an unprecedented qualitative and quantitative assessment of EM across soil samples. Good concordance was found between methods and the bioinformatics analysis was validated by random resampling. Sequences from most pathogenic groups associated with slow growth were identified in extenso in all soils tested with a specific assay, allowing to unmask them from the Mycobacterium whole genus, in which, as minority members, they would have remained undetected.

A four year longitudinal sero-epidemiology study of Neospora caninum in adult cattle from 114 cattle herds in south west England: Associations with age, herd and dam-offspring pairs

BackgroundNeosporosis caused by the protozoan parasite Neospora caninum, is an economically important cause of abortion, stillbirth, low milk yield, reduced weight gain and premature culling in cattle. Consequently, a seroepidemiological study of N. caninum antibodies was conducted in England with 29,782 samples of blood taken from 15,736 cattle from 114 herds visited on three occasions at yearly intervals. Herds were categorised into lower (< 10%) and higher (≥ 10%) median herd seroprevalence. Hierarchical models were run to investigate associations between the sample to positive (S/P) ratio and herd and cattle factors.ResultsNinety-four percent of herds had at least one seropositive cow; 12.9% of adult cattle had at least one seropositive test. Approximately 90% of herds were seropositive at all visits; 9 herds (8%) changed serological status between visits. The median N. caninum seroprevalence in positive herds was 10% (range 0.4% to 58.8%). There was a positive association between the serostatus of offspring and dams that were ever seropositive. In the hierarchical model of low seroprevalence herds there was no significant association between S/P ratio and cattle age. There was a significantly lower S/P ratio in cattle in herds that were totally restocked after the foot-and-mouth epidemic of 2001 compared with those from continuously stocked herds and cattle purchased into these herds had a higher S/P ratio than homebred cattle. In the model of high seroprevalence herds the S/P ratio increased with cattle age, but was not associated with restocking or cattle origin.ConclusionThere were no strong temporal changes in herd seroprevalence of N. caninum but 90% of herds had some seropositive cattle over this time period. Vertical transmission from seropositive dams appeared to occur in all herds. In herds with a high seroprevalence the increasing S/P ratio in 2–4 year old cattle is suggestive of exposure to N. caninum: horizontal transmission between adult cattle, infection from a local source or recrudescence and abortions. Between-herd movements of infected cattle enhance the spread of N. caninum, particularly into low seroprevalence herds. Some restocked herds had little exposure to N. caninum, while in others infection had spread in the time since restocking.

Evaluating observer agreement of scoring systems for foot integrity and footrot lesions in sheep

BackgroundA scoring scale with five ordinal categories is used for visual diagnosis of footrot in sheep and to study its epidemiology and control. More recently a 4 point ordinal scale has been used by researchers to score foot integrity (wall and sole horn damage) in sheep. There is no information on observer agreement using either of these scales. Observer agreement for ordinal scores is usually estimated by single measure values such as weighted kappa or Kendall’s coefficient of concordance which provide no information where the disagreement lies. Modeling techniques such as latent class models provide information on both observer bias and whether observers have different thresholds at which they change the score given. In this paper we use weighted kappa and located latent class modeling to explore observer agreement when scoring footrot lesions (using photographs and videos) and foot integrity (using post mortem specimens) in sheep. We used 3 observers and 80 photographs and videos and 80 feet respectively.ResultsBoth footrot and foot integrity scoring scales were more consistent within observers than between. The weighted kappa values between observers for both footrot and integrity scoring scales ranged from moderate to substantial. There was disagreement between observers with both observer bias and different thresholds between score values. The between observer thresholds were different for scores 1 and 2 for footrot (using photographs and videos) and for all scores for integrity (both walls and soles). The within observer agreement was higher with weighted kappa values ranging from substantial to almost perfect. Within observer thresholds were also more consistent than between observer thresholds. Scoring using photographs was less variable than scoring using video clips or feet.ConclusionsLatent class modeling is a useful method for exploring components of disagreement within and between observers and this information could be used when developing a scoring system to improve reliability.