Identifying the seasonal origins of human campylobacteriosis.

Human campylobacteriosis exhibits a distinctive seasonality in temperate regions. This paper aims to identify the origins of this seasonality. Clinical isolates [typed by multi-locus sequence typing (MLST)] and epidemiological data were collected from Scotland. Young rural children were found to have an increased burden of disease in the late spring due to strains of non-chicken origin (e.g. ruminant and wild bird strains from environmental sources). In contrast the adult population had an extended summer peak associated with chicken strains. Travel abroad and UK mainland travel were associated with up to 17% and 18% of cases, respectively. International strains were associated with chicken, had a higher diversity than indigenous strains and a different spectrum of MLST types representative of these countries. Integrating empirical epidemiology and molecular subtyping can successfully elucidate the seasonal components of human campylobacteriosis. The findings will enable public health officials to focus strategies to reduce the disease burden.

Putative household outbreaks of campylobacteriosis typically comprise single MLST genotypes.

During a 15-month period in Scotland a small but important number of human Campylobacter cases (3·2%) arose from 91 putative household outbreaks. Of the 26 outbreaks with known strain composition, 89% were composed of the same MLST which supports the potential use of MLST in public health epidemiology. The number of cases associated with household outbreaks is much larger than general outbreaks and there is some evidence to indicate that there may be secondary transmission, although this is relatively rare.

Stability of common biochemistry analytes in equine blood stored at room temperature.

REASONS FOR PERFORMING STUDY: Time delays between collection of blood samples and biochemical analysis of equine blood are unavoidably common in equine practice. The effect that delays may have on the accuracy of results of blood biochemical analyses is not well established. HYPOTHESIS: Delays in processing of blood of up to 72 h results in alterations in measured levels of common biochemical analytes that are of potential clinical relevance. Separation of serum prior to storage is protective against the effects of time delays. METHODS: Samples of clotted blood, separated serum and oxalate fluoride plasma from 20 horses were stored and analysed at 0, 24, 48 and 72 h. Graphical exploration of each analyte was undertaken. General linear models with fixed effects were fitted for the whole blood data. The mean bias and 95% limits of agreement were calculated, using bootstrapped data, to assess agreement between pairs of samples analysed at 0 h and other time points. Bland-Altman plots were used to explore general trends in the data. Paired t tests were used to compare the results from whole blood and separated serum. RESULTS: Delays in processing equine blood resulted in significant increases in measured concentrations of aspartate aminotransferase, creatine kinase, lactate dehydrogenase, total bile acids and magnesium. A significant decrease in concentration was identified for glucose (serum and oxalate fluoride preserved plasma). Separation of serum immediately following clot formation resulted in nonsignificant increases in accuracy for some analytes. CONCLUSIONS AND PRACTICAL SIGNIFICANCE: Delays in processing of blood samples may result in biochemical changes of clinical relevance in individual cases; however, in the majority of cases, where delays are only a few days and a number of analytes are assessed concurrently, delays are unlikely to have an effect on the interpretation of results. Separation of serum following clot formation is of limited benefit. Clinical samples in which a delay in processing has occurred may be interpreted with reference to the data presented.

Exploring the longitudinal dynamics of herd BVD antibody test results using model-based clustering.

Determining the Bovine Viral Diarrhoea (BVD) infection status of cattle herds is a challenge for control and eradication schemes. Given the changing dynamics of BVD  virus (BVDV) antibody responses in cattle, classifying herds based on longitudinal changes in the results of BVDV antibody tests could offer a novel, complementary approach to categorising herds that is less likely than the present system to result in a herd's status changing from year to year, as it is more likely to capture the true exposure dynamics of the farms. This paper describes the dynamics of BVDV antibody test values (measured as percentage positivity (PP)) obtained from 15,500 bovines between 2007 and 2010 from thirty nine cattle herds located in Scotland and Northern England. It explores approaches of classifying herds based on trend, magnitude and shape of their antibody PP trajectories and investigates the epidemiological similarities between farms within the same cluster. Gaussian mixture models were used for the magnitude and shape clustering. Epidemiologically meaningful clusters were obtained. Farm cluster membership depends on clustering approach used. Moderate concordance was found between the shape and magnitude clusters. These methods hold potential for application to enhance control efforts for BVD and other infectious livestock diseases.

The distribution of the pathogenic nematode Nematodirus battus in lambs is zero-inflated.

Understanding the frequency distribution of parasites and parasite stages among hosts is essential for efficient experimental design and statistical analysis, and is also required for the development of sustainable methods of controlling infection. Nematodirus battus is one of the most important organisms that infect sheep but the distribution of parasites among hosts is unknown. An initial analysis indicated a high frequency of animals without N. battus and with zero egg counts, suggesting the possibility of a zero-inflated distribution. We developed a Bayesian analysis using Markov chain Monte Carlo methods to estimate the parameters of the zero-inflated negative binomial distribution. The analysis of 3000 simulated data sets indicated that this method out-performed the maximum likelihood procedure. Application of this technique to faecal egg counts from lambs in a commercial upland flock indicated that N. battus counts were indeed zero-inflated. Estimating the extent of zero-inflation is important for effective statistical analysis and for the accurate identification of genetically resistant animals.

Equine parascarosis under the tropical weather conditions of Ethiopia: a coprological and postmortem study.

A cross-sectional coprological survey in the regions of Ada, Akaki, Bereh and Boset, and a retrospective postmortem investigation were conducted to study the epidemiology of Parascaris equorum in donkeys and horses in Ethiopia. Faecal samples from 803 working donkeys and 402 horses were collected, and the numbers of worms recovered from 112 donkeys examined postmortem between 1995 and 2004 were analysed. There was a high prevalence of infection and faecal egg output of P equorum in both donkeys and horses, and the severity of the infection in donkeys was increased irrespective of their age. The prevalence of the infection in the donkeys was 51.1 per cent and in the horses 16.2 per cent, and the prevalence in the donkeys examined postmortem was 55 per cent. There was no significant difference between different age groups of donkeys in either the prevalence or the intensity of the infection. The prevalence of the infection was significantly higher in the Ada and Akaki regions than in the Bereh and Boset regions.

An empirical comparison of isolate-based and sample-based definitions of antimicrobial resistance and their effect on estimates of prevalence.

Antimicrobial resistance is primarily a problem in human medicine but there are unquantified links of transmission in both directions between animal and human populations. Quantitative assessment of the costs and benefits of reduced antimicrobial usage in livestock requires robust quantification of transmission of resistance between animals, the environment and the human population. This in turn requires appropriate measurement of resistance. To tackle this we selected two different methods for determining whether a sample is resistant - one based on screening a sample, the other on testing individual isolates. Our overall objective was to explore the differences arising from choice of measurement. A literature search demonstrated the widespread use of testing of individual isolates. The first aim of this study was to compare, quantitatively, sample level and isolate level screening. Cattle or sheep faecal samples (n=41) submitted for routine parasitology were tested for antimicrobial resistance in two ways: (1) "streak" direct culture onto plates containing the antimicrobial of interest; (2) determination of minimum inhibitory concentration (MIC) of 8-10 isolates per sample compared to published MIC thresholds. Two antibiotics (ampicillin and nalidixic acid) were tested. With ampicillin, direct culture resulted in more than double the number of resistant samples than the MIC method based on eight individual isolates. The second aim of this study was to demonstrate the utility of the observed relationship between these two measures of antimicrobial resistance to re-estimate the prevalence of antimicrobial resistance from a previous study, in which we had used "streak" cultures. Boot-strap methods were used to estimate the proportion of samples that would have tested resistant in the historic study, had we used the isolate-based MIC method instead. Our boot-strap results indicate that our estimates of prevalence of antimicrobial resistance would have been considerably lower in the historic study had the MIC method been used. Finally we conclude that there is no single way of defining a sample as resistant to an antimicrobial agent. The method used greatly affects the estimated prevalence of antimicrobial resistance in a sampled population of animals, thus potentially resulting in misleading results. Comparing methods on the same samples allows us to re-estimate the prevalence from other studies, had other methods for determining resistance been used. The results of this study highlight the importance of establishing what the most appropriate measure of antimicrobial resistance is, for the proposed purpose of the results.

Detection of genes with moderate effects on disease resistance using ovine mhc and resistance to nematodes as an example.

Detecting some of the genes that influence disease resistance would improve our understanding of the processes that cause disease and also simplify disease control. Genes within the major histocompatibility complex (mhc) are strong candidates for disease resistance and they have been intensely studied for the last 30 years. Recently, several groups working independently have reported the existence of alleles within the mhc that are associated with enhanced resistance to nematode infection. This article uses hindsight to describe some of the potential pitfalls that hinder the search for valid disease resistance genes. The search requires a good understanding of disease biology, molecular genetics, statistical genetics and especially, the design and analysis of experiments. The power to detect mhc effects is quite low and is quite sensitive to the frequency of the putative resistance alleles.

Risk factors for hide contamination of Scottish cattle at slaughter with Escherichia coli O157.

In the slaughter processing of cattle, contaminated hides have been identified as one of the major sources of Escherichia coli O157 carcase contamination. Logistic regression analysis was applied to data collected in a large scale study in Scotland involving 222 cattle forming 34 groups sent for slaughter from 30 farms to 10 slaughterhouses. Aspects of individual animal characteristics, farm management practices and slaughterhouse features were examined to identify potential risk factors for hide contamination at harvest. Two models were developed, the first in which slaughterhouse was modelled as a fixed effect, and a second model where slaughterhouse and farm groups were modelled as random effects. In the first model, there was a significantly increased risk of a carcase testing positive for E. coli O157 on the hide if either the hide of the carcase immediately before or after it on the line was contaminated (OR 3.6; 95% CI: 1.4-9.9). If both adjacent carcases had contaminated hides, the odds ratio for the study carcase having a contaminated hide rose to 11.5 (95% CI: 4.4-32.5). If animals were held in lairage, receiving hay as feed appeared to have a protective effect on hide contamination. Transportation to the slaughterhouse by haulier, as opposed to transport by the farmer, was associated with a 5.4 increase in the odds of E. coli O157 contamination. The use of a crush in the lairage, often employed when reading ear tags, was also found to significantly increase the odds of hide contamination with E. coli O157. In the second model, the inclusion of slaughterhouse and farm group as random effects resulted in two of the previously identified factors being associated with hide contamination. If at least one of the adjacent carcases on the line had a contaminated hide, the associated odds ratio was 6.6 (95% CI: 2.8-15.9), which rose to 22.7 (95% CI: 9.3-55.5) if both adjacent hides were contaminated. Receiving hay in lairage was found to be important to the model, although not significant in itself (OR 0.005; 95% CI: 1.2e(-6)-20.7). These results suggest that modifiable risk factors for hide contamination exist. However, in order best to reduce the prevalence of hide contamination at slaughter, individual slaughterhouse risk assessment and intervention strategies are appropriate.

Sucrose concentration in blood: a new method for assessment of gastric permeability in horses with gastric ulceration.

A urine sucrose test has recently been reported to be a reliable method of detecting gastric ulcers in horses; however, technical difficulties associated with urine collection have limited the practical value of the test. The objective of this pilot study was to determine whether gastric sucrose permeability, as evaluated by serum sucrose concentration, could be used to detect gastric mucosal injury in horses. Twelve adult horses with naturally acquired gastric ulceration were studied. After a 20-hour nonfeeding period, each horse was dosed with 250 g of sucrose via nasogastric intubation. Blood samples were collected at 0, 15, 30, 45, 60, and 90 minutes, and horses underwent gastroscopy 4 hours later. The severity of gastric ulceration in each horse was defined by means of a 4-point ulcer-scoring system, and the relationship with serum sucrose concentration was analyzed by means of a linear mixed-effects model. Serum sucrose concentration was measured by liquid chromatography operating in tandem with electrospray mass spectrometry. After nasogastric administration of table sugar, horses with moderate to severe gastric ulceration had significant increase in serum sucrose concentration at 30, 45, 60, and 90 minutes, relative to earlier times (P < .05). Peak sucrose concentration was observed at 45 minutes, and was correlated with ulcer severity (Spearman's rank correlation coefficient = 0.898, P < .05). These data indicate that determination of sucrose concentration in equine serum may be a useful test for identifying horses with endoscopically visible gastric ulceration and has potential use as a noninvasive method for screening and monitoring horses engaged in racing training and other performance-related disciplines.

The evolution and maintenance of polymorphism in the major histocompatibility complex.

Lambs with the G2 allele at the ovine major histocompatibility complex (mhc) class II locus DRB1 has previously been shown to have lower faecal nematode egg counts than lambs with the I allele at this locus. This association has been confirmed in separate cohorts from the same farm. Other alleles within the mhc have also shown associations with nematode resistance in other breeds of sheep. Therefore, variation in the mhc is responsible for part of the observed genetic variation in resistance to nematode infection. In addition to the specific effect of particular alleles, heterozygotes are also more resistant than homozygotes. This heterozygote advantage is capable of maintaining the high levels of polymorphism observed within the mhc.

Treatment of persistent intramammary infections with Streptococcus uberis in dairy cows.

A survey was conducted of the prevalence of environmental pathogens, especially Streptococcus uberis, as causes of clinical mastitis in dairy cows. The response of intramammary infections with S uberis to conventional treatment was monitored by taking milk samples for bacteriology and somatic cell counting seven, 14 and 21 days after the treatment. The results showed that 51 per cent of the infections failed to respond, and the odds of cases failing to respond was significantly increased when the individual quarter somatic cell count seven days after the treatment was greater than 201,000 cells/ml. Ninety-six per cent of the suspected S uberis isolates identified by culture were confirmed as S uberis by using the api 20 Strep system. Restriction endonuclease fingerprinting was used to type the strains of S uberis isolated from 75 milk samples from 32 cows. Analysis showed that 96 per cent of the cases of S uberis that failed to respond to conventional treatment were persistent infections with one strain rather than reinfections with different strains. The persistent cases of S uberis were treated further with an extended course of intramammary preparations containing either procaine penicillin with dihydrostreptomycin or cefquinome. There was no significant difference between the cure rates achieved by the two preparations, and 55 per cent of the cases that had failed to respond to conventional treatment responded to the additional treatment.

Frozen storage of Escherichia coli O157 in buffered peptone water and its detection on bovine carcasses.

The adaptation of a standard Escherichia coli O157 isolation method involving immunomagnetic separation and a period of frozen storage was investigated. A series of experiments was designed to test the recovery of a bovine strain of E. coli O157 from buffered peptone water after a period of frozen storage at -80 degrees C. The effects of the addition of glycerol at 5 and 10%, freezing time, the number of freeze-thaw cycles, the method of freezing and the method of thawing, the inclusion of a resuscitation-and-incubation step, and the sensitivity of the isolation method were investigated. The most effective method of storing frozen samples for 6 months and recovering strains of E. coli O157 after storage was found to involve 6 h of incubation of sample material in buffered peptone water at 37 degrees C before frozen storage at -80 degrees C with 10% glycerol, a rapid thaw after frozen storage, and resuscitation at 27 degrees C for 1 h and incubation at 37 degrees C for 1 h to allow freeze-injured and stressed bacteria to recover with a period of growth prior to immunomagnetic separation isolation. There was no significant decrease in log counts of a bovine strain E. coli O157 over 6 months of frozen storage in buffered peptone water with 10% glycerol. With this method, it was possible to isolate E. coli O157 from naturally infected bovine carcasses after a period of frozen storage.

A descriptive study of 'kangaroo gait' in sheep in northern Britain.

Two questionnaires were undertaken to obtain improved knowledge of the signalment, incidence and risk factors of the condition termed 'kangaroo gait' (KG; a forelimb locomotor disorder of adult female sheep). The first survey was mailed to 221 veterinary surgeons in practices in northern Britain which provided veterinary care for sheep, and the second was mailed to 438 sheep-flock owners and managers located in the Scottish Borders, who were recruited via their veterinary surgeons. Responses of 84.7 and 30.7% were achieved, respectively. In total, 29.7% of veterinary practices and 24.1% of flock owners and managers had identified one or more cases of KG in their practice area or flocks since the first case was reported in 1976. Almost all respondents indicated that adult females were affected-usually during lactation (and less commonly during late gestation). KG was more common among commercial crossbreeds and less common among sheep grazing hill pasture (than sheep grazing upland or lowland pasture). Cases usually occurred between March and June while sheep were at grass, although it was unclear whether the temporal pattern was associated with grazing or lambing patterns. Stocking densities were significantly higher among affected flocks than among non-affected flocks. The annual number of individuals and flocks identified as being affected had increased since the condition first was identified. Most cases of locomotor disorders resembling KG were not reported to veterinary surgeons.

Morphometry of bovine dilated cardiomyopathy.

Bovine dilated cardiomyopathy (BDCM) is a primary disease of the myocardium that has been described in Holstein-Friesian cattle worldwide in the last 20 years. The principal morphological changes in the myocardium are interstitial fibrosis and increased variability in cardiomyocyte size. Sections of heart muscle from nine cases of BDCM and nine unaffected controls matched for age, sex and breed were studied by means of a computer-assisted image analyser to measure the degree of fibrosis, and the cardiomyocyte cellular and nuclear cross-sectional area and length. The amount of connective tissue in the hearts of BDCM cases was increased by 6.7 times, the nuclear transverse cross-sectional area by 1.9 times, and the cardiomyocyte length and cross-sectional area by 1.7 and 1.6 times, respectively. This resulted in an estimated 2.5-fold increase in mean cardiomyocyte volume. Animals with clinical signs of BDCM showed a mean loss of 51% of the total number of cardiomyocytes as compared with controls. Of the five parameters studied, the percentage of fibrosis was found to be the most consistent discriminator for BDCM. It is possible that the degree of fibrosis could be used to distinguish BDCM from other cardiac diseases of cattle.

Use of clinical information to predict the characteristics of bacteria isolated from clinical cases of bovine mastitis.

Farmers recorded the clinical signs of cows with clinical mastitis and submitted milk samples for bacteriological examination, so that the clinical signs could be correlated with the bacteriological findings. Odds ratios for the demeanour of the cow, the appearance of the milk, milk yield, udder texture, and the administration of parenteral antibiotics were calculated for mastitis cases classified in terms of their microbiology as either enterobacteriaceae, major Gram-positive pathogens, minor pathogens, 'no growths' or 'all other pathogens'. Animals infected with enterobacteriaceae had the highest odds of being reported as having a reduced milk yield, swollen or hard udders, watery milk and/or being systemically sick. A logistic regression model was used to predict the Gram-staining characteristics of the bacteria causing clinical mastitis. The clinical findings found to be significant predictors in the model were the demeanour of the cow and its milk yield. The regression model was used as a basis for a predictive test. Using a test data set, the sensitivity of the test was 28 per cent, its specificity was 96 per cent, the positive predictive value was 74 per cent and the negative predictive value was 80 per cent. The overall accuracy of these predictions was 79 per cent.

Quantifying the sources of variability in equine faecal egg counts: implications for improving the utility of the method.

The faecal egg count (FEC) is the most widely used means of quantifying the nematode burden of horses, and is frequently used in clinical practice to inform treatment and prevention. The statistical process underlying the FEC is complex, comprising a Poisson counting error process for each sample, compounded with an underlying continuous distribution of means between samples. Being able to quantify the sources of variability contributing to this distribution of means is a necessary step towards providing estimates of statistical power for future FEC and FECRT studies, and may help to improve the usefulness of the FEC technique by identifying and minimising unwanted sources of variability. Obtaining such estimates require a hierarchical statistical model coupled with repeated FEC observations from a single animal over a short period of time. Here, we use this approach to provide the first comparative estimate of multiple sources of within-horse FEC variability. The results demonstrate that a substantial proportion of the observed variation in FEC between horses occurs as a result of variation in FEC within an animal, with the major sources being aggregation of eggs within faeces and variation in egg concentration between faecal piles. The McMaster procedure itself is associated with a comparatively small coefficient of variation, and is therefore highly repeatable when a sufficiently large number of eggs are observed to reduce the error associated with the counting process. We conclude that the variation between samples taken from the same animal is substantial, but can be reduced through the use of larger homogenised faecal samples. Estimates are provided for the coefficient of variation (cv) associated with each within animal source of variability in observed FEC, allowing the usefulness of individual FEC to be quantified, and providing a basis for future FEC and FECRT studies.

Assessing the probability of acquisition of meticillin-resistant Staphylococcus aureus (MRSA) in a dog using a nested stochastic simulation model and logistic regression sensitivity analysis.

Meticillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial and community-acquired pathogen with zoonotic potential. The relationship between MRSA in humans and companion animals is poorly understood. This study presents a quantitative exposure assessment, based on expert opinion and published data, in the form of a second order stochastic simulation model with accompanying logistic regression sensitivity analysis that aims to define the most important factors for MRSA acquisition in dogs. The simulation model was parameterised using expert opinion estimates, along with published and unpublished data. The outcome of the model was biologically plausible and found to be dominated by uncertainty over variability. The sensitivity analysis, in the form of four separate logistic regression models, found that both veterinary and non-veterinary routes of acquisition of MRSA are likely to be relevant for dogs. The effects of exposure to, and probability of, transmission of MRSA from the home environment were ranked as the most influential predictors in all sensitivity analyses, although it is unlikely that this environmental source of MRSA is independent of alternative sources of MRSA (human and/or animal). Exposure to and transmission from MRSA positive family members were also found to be influential for acquisition of MRSA in pet dogs, along with veterinary clinic attendance and, while exposure to and transmission from the veterinary clinic environment was also found to be influential, it was difficult to differentiate between the importance of independent sources of MRSA within the veterinary clinic. The implementation of logistic regression analyses directly to the input/output relationship within the simulation model presented in this paper represents the application of a variance based sensitivity analysis technique in the area of veterinary medicine and is a useful means of ranking the relative importance of input variables.

Epidemiological features of fasciolosis in working donkeys in Ethiopia.

A cross-sectional coprological survey in the tropical regions of Ada, Akaki, Bereh and Boset, and a retrospective post-mortem investigation were conducted to study the epidemiology of fasciolosis in working donkeys in Ethiopia. Faecal samples from 803 donkeys were collected, and the number of liver flukes recovered from 112 donkeys at post-mortem between 1995 and 2004 were analysed. There was a high prevalence of fasciolosis irrespective of the age of the donkeys. The overall prevalence of the infection was 44.4% in coprologically examined donkeys, and the prevalence in the donkeys examined post-mortem was 41.9%. The infection prevalence was significantly higher in Bereh and Ada regions than in Akaki and Boset regions. Bereh with 72.6% and Boset with 21.5% showed a significantly higher and lower infection prevalence, respectively, than the rest of the regions (P<0.001). There was no significant difference between different age groups of donkeys in the infection prevalence (P>0.05) but infection intensity was significantly higher in donkeys 8 years old and above (P<0.0001). Both Fasciola hepatica and Fasciola gigantica were identified.