Clinical and laboratory evaluation of a real-time PCR for Clostridium difficile toxin A and B genes.

Many laboratories use enzyme immunoassays (EIAs) for the diagnosis of Clostridium difficile infection (CDI). More recently, polymerase chain reaction (PCR)-based diagnosis has been described as a sensitive test. Real-time PCR for the detection of C. difficile toxin A and B genes was evaluated. A prospective evaluation was performed on stool samples from 150 hospitalized adult patients and 141 healthy volunteers. PCR was compared to toxigenic culture (TC), direct cytotoxicity test (CTT), ImmunoCard® Toxin A and B (Meridian Bioscience), and enzyme-linked immunosorbent assay (ELISA) (Vidas). The results were correlated with clinical data using a standardized questionnaire. The diagnostic yield of the PCR was further evaluated after implementation. Using toxigenic culture as the gold standard, the sensitivity and specificity of PCR were 100 and 99.2%, respectively. Patients were categorized as follows: TC/PCR-positive (n = 17) and negative TC (n = 133). The differences in these groups were more frequent use of antibiotics and leukocytosis (p < 0.05). The diagnostic yield of PCR was evaluated during a period of 6 months and showed an increase of positive patients by 50%. PCR for the detection of toxigenic C. difficile has a high sensitivity and can rule out CDI, but cannot differentiate CDI from asymptomatic carriage. Clinicians should be aware of this in order to prevent inappropriate treatment and delay of other diagnostics.

Performance of two tube coagulase methods for rapid identification of Staphylococcus aureus from blood cultures and their impact on antimicrobial management.

Test parameters and clinical impact of the direct tube coagulase test (DTCT) for rapid identification of Staphylococcus aureus from blood culture were investigated. The sensitivity of the DTCT at 4 h using saline dilution was 96%, compared with 93% using serum separator tubes; specificity was 100% for both methods. Among 32 patients with S. aureus bacteraemia, treatment modifications were based on microbiology results from the primary source of infection in 12 patients, on a Gram's stain from blood culture in seven patients, and on the DTCT in nine patients. The DTCT is a valuable adjunct in the routine microbiology laboratory because of its good performance, technical simplicity and low cost.

Natural postnatal Neospora caninum infection in cattle can persist and lead to endogenous transplacental infection.

A serological follow-up study of 3.5 years duration was done of a dairy herd that had experienced a mass seroconversion to Neospora caninum following a point source exposure shortly before the 17th of January 2000. A total of 913 blood samples of 244 animals at seven sampling dates were used to investigate the seroprevalence dynamics in the herd. Most postnatally infected cattle remained seropositive during the period of investigation but 11 animals became seronegative after 6-27 months indicating transient infection. Six animals seroconverted later than the main group of 45 animals and 5 animals became seronegative after at least two seropositive records possibly due to a low infection dose or difference in the haplotypes of the infected animals. In total 58% (14/24) of the offspring of postnatally infected dams was seropositive. Nine of 16 (56%) daughters originating from inseminations after the postnatal infection of their dams were seropositive indicating endogenous transplacental infection.

Herd and cow-level prevalence of sole ulcers in The Netherlands and associated-risk factors.

A cross-sectional study was performed, to (re)evaluate the prevalence of sole ulcers (SUs) and the risk factors involved in dairy herds that perform claw trimming on a routine base. Twenty hoof trimmers collected data on the presence or absence of claw disorders on the hind claws of dairy cows during whole herd trimming in 383 herds in The Netherlands. A questionnaire was used to acquire information on management and housing. Additional data on parity, breed and stage of lactation of the cows were provided by the Dutch Herd Book Organization. Sole ulcer was present in 85% of the herds and in 5.6% (S.E. 0.2) of the study population (n=22,454 cows). The within-herd prevalence varied from 0% (14.6% of herds) to 26% (0.3% of herds) and in herds with SU was most frequently diagnosed 1-5% of the trimmed cows (45% of all herds). The risk for SU increased significantly with increasing parity. Cows that were at or after peak of lactation (>60 days in milk, DIM) had higher odds on SU in comparison to cows that were between 0 and 60 DIM (OR=0.6). The presence of other non-infectious claw disorders appeared to predispose to SU. Based on an estimation of the population attributable fraction, it was concluded that if sole hemorrhages, chronic laminitis (CL), white line disease and interdigital hyperplasia were the cause of SU and if those diseases could have been prevented in the study population, 35.4, 5.8, 11.6 and 4.6% of the SU cases respectively could have been prevented. Because of this relation, an effective intervention strategy of SU should focus on an integral approach of reducing the first stages of the bovine laminitis complex, whereas SU and CL are considered to be a later stage. Cows trimmed during late summer were at a higher risk for SU than cows trimmed in other periods of the year. Finally, cows in herds with purchased heifers were at a higher risk (OR=1.4) for SU than cows in herds that did not purchase heifers. A comparable positive effect was visible in herds with mattress bedding.

Supranational comparison of Neospora caninum seroprevalences in cattle in Germany, The Netherlands, Spain and Sweden.

Herd, within-herd and animal prevalences for Neospora caninum in beef and dairy cattle were compared between four countries. In randomly selected herds from regions of Germany, The Netherlands, Spain and Sweden that were representative for the cattle production of these countries, all animals > or = 2 years were examined serologically by enzyme-linked immunosorbent assays (ELISAs) with high test specificity (> 98.0%). In a previous study, the ELISAs had been validated against each other. Single reacting animals within a herd were confirmed by immunobloting. At the time of sampling, animal (age, breed, herdtype, sex, lactation stage) and herd data (region) were collected. Considerable differences in N. caninum herd, within-herd, and overall animal prevalence estimations were observed between countries, regions, herdtype, age categories and breeds. Herd prevalences, based on confirmation of single reactors, for dairy herds were estimated to be 16% (95%CI: 10-24%) in Sweden, 49% (95%CI: 39-59%) in Germany, 63% (95%CI: 57-69%) in Spain and 76% (95%CI: 67-84%) in The Netherlands and for beef herds 41% (95%CI: 31-50%) in Germany, 46% (95%CI: 41-51%) in Spain and 61% (95%CI: 50-72%) in The Netherlands. No beef herds were examined in Sweden. The lowest animal true prevalence was estimated in dairy cattle in Sweden (0.5% (95%CI: 0.1-0.8%)) while the highest animal true prevalence was estimated for dairy cattle in Spain (16.2% (95%CI: 14.9-17.5%)). Within-herd prevalences varied greatly, with very few farms in Sweden having more than 10% seropositive animals while in Spain more than 10% of the herds had within-herd prevalences between 50 and 100%. Seropositivity was significantly associated with herdtype (beef versus dairy), age, breed and region within countries. The results of this supranational comparative study showed that the importance of N. caninum infection varied greatly within in Europe. Estimates of prevalence can be used to calculate the economic impact of N. caninum infection as well as to evaluate the effect of prevention and control strategies over time.

Intra-class correlation attributable to claw trimmers scoring common hind-claw disorders in Dutch dairy herds.

Data of a cross-sectional study on prevalence of seven common bovine-claw disorders were used to calculate the intra-class correlation coefficients (ICCs) for claw trimmers. Fifteen professional claw trimmers were trained in diagnosing claw disorders uniformly. During regular trimming of all dairy cows in a herd, they recorded the presence (yes/no) of hind-claw disorders for digital dermatitis (DD), chronic laminitis (CL), sole ulcer (SU), white-line disease (WLD) and interdigital hyperplasia (HYP). For the claw disorders interdigital dermatitis/heel-horn erosion (IDHE) and sole haemorrhage (SH), claw trimmers also recorded (using graduated scores 1-3) the extent of the lesion. Complete information of 21,153 animals from 361 herds in The Netherlands was recorded between July 2002 and December 2003. To assure objective recording of claw disorders, ideally there would not be any correlation between observations. However, quantified ICCs from claw-trimmers observations ranged from 4.9% for DD to 38.0% for CL, while ICCs for herd ranged from 7.9% for SU to 26.7% for DD. Changes in the cut-off values for the diagnosis of IDHE and SH did not result in any relevant changes of the ICCs from claw-trimmers observations and these remained larger than the ICC explained by the herd. Based on these results, we conclude that for estimating measures of frequencies for different claw disorders, uniformity in recording certain diagnoses becomes an issue to consider when involving more than one observer.

Herd- and cow-level prevalence of digital dermatitis in the Netherlands and associated risk factors.

The objective of this cross-sectional study was to reevaluate the prevalence of digital dermatitis (DD) and associated risk factors in The Netherlands. Between May 2002 and December 2003, information about the presence or absence of DD lesions and other claw disorders on the hind claws of dairy cows in The Netherlands was collected by 20 hoof trimmers during trimming of all dairy cows in 383 herds. A questionnaire was used to acquire information regarding management and housing. Additional information, such as parity, breed, and stage of lactation of the cows, was provided by the Dutch Herd Book Organization. Digital dermatitis was present in 21.2% (SE = 0.3) of the study population (n = 22,454 cows). The herd prevalence varied from 0% (9.1% of the herds) to 83.0%. Most common was herd prevalence between 5 and 10%. The Holstein-Friesian breed was at higher risk for DD [odds ratio (OR) = 1.7] than was the Meuse Rhine IJssel breed (dual purpose breed). The risk for DD decreased with increasing parity. Cows at the peak of their lactation (30 to 60 d in milk) and in the third parity had higher odds for DD in comparison with cows that were later than 60 DIM. The presence of other claw disorders, such as interdigital dermatitis/heel horn erosion (IDHE), interdigital hyperplasia (HYP), and interdigital phlegmon, appeared to be predisposing for DD. Based on estimation of the population-attributable fraction, it was concluded that if IDHE, HYP, and interdigital phlegmon were not present among the study population, respectively, 32.2, 9.0, and 1.1% of the DD cases could have been prevented. The risk for DD slightly decreased when cows affected by IDHE had access to pasture. Because of the interrelation between infectious claw disorders, an effective intervention strategy against DD should focus on an integrated approach to the control of all infectious claw diseases. Cows trimmed >12 mo before the study (during regular trimming of the entire herd) were at lower risk for DD than were cows that were trimmed at shorter intervals. Animals that had >8 h of access to pasture were at higher risk for DD (OR = 1.6) compared with no access to pasture. Finally, cows in small-sized herds (<45 cows) affected with HYP were at lower risk (OR = 0.6) for DD than were cows affected with HYP in medium- and large-sized herds (60 to 85 cows per herd).

Evaluation of three enzyme-linked immunosorbent assays for detection of antibodies to Neospora caninum in bulk milk.

Three ELISAs for the detection of antibodies against Neospora caninum in bulk milk were evaluated in 162 Dutch dairy herds. The first ELISA was the Dutch Animal Health Service (AHS) in-house ELISA, developed from the routine in-house serum ELISA. The other two ELISAs were commercial milk ELISAs from IDEXX and LSI. Blood samples of all lactating cows in 162 dairy herds were tested using the AHS in-house serum ELISA. Based on previous studies in the Netherlands a within-herd N. caninum seroprevalence of 15% was associated with increased risk for reproductive losses. This percentage was therefore used as positive seroprevalence cut-off value. Repeatability of the ELISAs was evaluated by testing on three different days. The AHS in-house ELISA lacked specificity, probably due to use of a different batch of antigen on the second and third test-day. Cut-off values were determined using misclassification costs term calculations. At cut-off values 0.6 for the IDEXX and 0.2 for the LSI, a herd sensitivity of 61% (95% CI: 49--73%) and 47% (95% CI: 35--60%) was estimated. Herd specificity at these cut-off values was 92% (95% CI: 87--98%) for the IDEXX and 94% (95% CI: 90--99%) for the LSI ELISA. The positive and negative predictive values were 84% (95% CI: 68--100%) and 86% (95% CI: 79--94%) for the IDEXX ELISA, and 85% (95% CI: 67--100%) and 82% (95% CI: 74--90%) for the LSI ELISA. The agreement between all possible combinations of test-days was expressed by kappa values. These were found to be slightly higher for the IDEXX than for the LSI ELISA. It is concluded that both commercial ELISAs performed satisfactorily to detect a within-herd seroprevalence of N. caninum in lactating cows of at least 15%.

Assessing the repeatability and reproducibility of the Leg Score: a Dutch Claw Health Scoring System for dairy cattle.

The optimal moment for trimming the claws of all dairy cows in a herd was investigated by assessing the external rotation of the hind claws of individual cows relative to the spinal column. This leg score consisted of three independent descriptors: 1 (good/normal), 2 (moderately deviant), and 3 (severely deviant). This study assessed the repeatability and the reproducibility of the leg score system, and the consistency of the advice given subsequently about trimming of the hind claws of all cows in the herd. Repeatability was assessed for 52 cows that were scored twice on the same day by 11 observers; the kappa value ranged from 0.17 to 0.66 (mean: 0.36). The probability of the same result for both assessments ranged from 0.49 to 0.80 (mean: 0.61). Claw trimming was advised if at least 20% of the cows had a leg score of 3. On the basis of the scores, 3 observers consistently advised trimming of the hind claws of all the cows in the herd, and 6 observers consistently advised against the need for trimming in the short term; 2 observers had an inconsistent advice. The reproducibility of the scoring system was assessed in two dairy herds (62 and 50 cows). Eight observers evaluated the leg score of the cows of both herds on the same day. The mean kappa value of the leg score for all pairs of different observers (A-B, A-C etcetera) was 0.24 [-0.08-0.49]. In conclusion, the leg score is not a reliable method for determining the optimal moment for claw trimming in dairy cattle. The reasons for the inconsistent observations require further investigation.

Risk factors for infection with Foot-and-Mouth Disease virus in a cattle population vaccinated with a non-purified vaccine in Iran.

In this study, we estimated the level of Foot-and-Mouth (FMD) virus infection in a cattle-dense north-western province of Islamic Republic of Iran and analyzed putative risk factors for FMD infection. Calves (6-24 months of age) from all 17 districts of West Azerbaijan were tested for antibodies against non-structural proteins (NSP-Ab) of FMD virus. A proportional stratification with a minimum of 30 epi-units was applied for 3 different husbandry systems: villages, dairy and mixed farms. Within an epi-unit, 30 calves were sampled. For the interpretation of ELISA test results, we used the 50% inhibition (50PI) cut-off as per producer's instructions and created one at 75% inhibition (75PI) based on the lowest point of the histogram of PI results. This approach resulted in three categories of outcomes; negative (N), low-positive (LP) and high-positive (HP). A generalized mixed-effect model for binary outcomes was used for analysing putative risk factors and was run for both cut-off values. A total of 8378 calves from 202 villages, 51 dairy farms and 28 mixed farms were eligible for analysis. The percentage of calves testing positive (LP+HP) was 53.7% (95% Confidence interval (CI): 52.6%-54.8%), with 39.6% (95% CI: 38.6-40.7%) testing HP (n=3309) while 14.1% (95% CI: 13.5-15.0%) of calves tested LP (n=1188). Of 281 epi-units sampled, all calves sampled tested negative in only 2 epi-units (0.7% (95% CI: 0.1-2.5%)) and more than 25 calves tested positive in 29 epi-units (10.3% (95% CI: 7.0-14.5%)). Outcomes of regression modelling using the 50 PI cut-off indicated that, for each month increase in age, the odds of testing positive increased 1.01 times (95% CI: 1.00-1.03). The odds of calves testing NSP-positive increased 1.46 times (95% CI: 1.22-1.77) for calves residing in epi-units that had experienced clinical FMD in the 12 months preceding this study. The odds of calves owned by livestock owners who traded livestock testing positive were 1.4-1.6 times higher than those owned by persons not engaged in trading while the odds for calves testing positive in dairy herds was 1.62 (95% CI: 1.10-2.35) times higher compared with calves in villages. The results of the model using the 75 PI cut-off value resulted in comparable estimates, with the age-effect becoming more evident. These results have confirmed widespread FMD infection and were used in developing a risk-based control strategy on FMD, in line with Stage 1 of the Progressive Control Pathway for FMD (PCP-FMD).

Mortality attributable to bluetongue virus serotype 8 infection in Dutch dairy cows.

In 2007, bluetongue virus serotype 8 (BTV-8) re-emerged in the Netherlands and a large number of farmers notified morbidity and mortality associated with BTV-8 to the authorities. All dead cows in the Netherlands are registered in one of the three age classes: newborn calves <3 days, calves 3 days to 1 year, and cows >1 year. These registrations result in a complete data set of dead cattle per herd per day from 2003 until 2007. In this study, the mortality associated with BTV-8 for the Dutch dairy industry was estimated, based on this census data. Default, mortality associated with BTV-8 was estimated for the confirmed notification herds. Moreover, an additional analysis was performed to determine if mortality associated with BTV-8 infection occurred in non-notification herds located in BTV-8 infected compartments. A multivariable population-averaged model with a log link function was used for analyses. Separate analyses were conducted for the three different age groups. Confirmed notification herds had an increased cow mortality rate ratio (MRR) (1.4 (95% CI: 1.2-1.6)); calf MRR (1.3 (95% CI: 1.1-1.4)); and newborn calf MRR (1.2 (95% CI: 1.1-1.3)). Furthermore, in non-notification herds in BTV-8 infected compartments, mortality significantly increased 1.1 times (95% CI: 1.1-1.1) in cows, 1.2 times (95% CI: 1.2-1.2) in calves and 1.1 times (1.1-1.1) in newborn calves compared with BTV-8 non-infected months. Using objective census data over a 5-year period, the MRRs indicated increased mortality associated with BTV-8 infection not only in herds of which the farmer notified clinical signs but also in non-notification herds in infected compartments.

The increase in seroprevalence of bluetongue virus (BTV) serotype 8 infections and associated risk factors in Dutch dairy herds, in 2007.

Bluetongue virus serotype 8 (BTV-8) emerged in the Netherlands in August 2006 for the first time. In the winter of 2006/2007, during the transmission free period, the government decided to establish a sentinel network to monitor the re-emergence of BTV-8 in 2007. Between June and December 2007, a sentinel network of 275 dairy herds with 8901 seronegative cows at start, was in place for BTV-8 testing in milk samples. Besides estimates of the monthly BTV-8 within-herd prevalence per region (south, central and north), this sentinel was used to determine BTV-8 associated risk factors. Information on management and housing practices that were hypothesized to be related to the increase in BTV-8 prevalence (risk factors) were used. Complete information on BTV-8 testing and management was obtained for 234 herds. At herd level, the increase in seroprevalence was defined as the total increase in seroprevalence amongst sentinel cows per herd during the sampling period (in months) divided by the number of sampling months in which the spread of BTV-8 occurred within the particular compartment in which the herd was located. This parameter was used as dependent variable in the linear regression analysis. The final model revealed four risk factors remained in the final model. Herds in the central and southern region of the Netherlands had a higher monthly increase in seroprevalence 6.4% (95% CI: 3.1-9.9) and 10.1% (95% CI: 6.2-14.3), respectively compared to herds in the northern region. Furthermore, there was a strong association with grazing. The monthly increase in seroprevalence in cattle pastured a few hours per day or throughout the day was 5.6% (95% CI: 1.4-10.2) to 11.4% (95% CI: 6.0-17.3) higher, relative to that for cattle kept indoors. For cattle that grazed outdoors throughout the day and the night, the monthly increase in seroprevalence was 13.6% (95% CI: 7.2-20.8). In addition, an association was found between the monthly increase in seroprevalence and some factors relating to stable design. Keeping the stable doors closed during the day was linked to a higher seroprevalence rate compared to that in stables with the door left open (3.6% (95% CI: 0.3-7.1)). Furthermore, a horizontal ventilation opening (>30 cm) along the walls of the stable, and with a windbreak curtain, appeared to offer some protection (-3.0% per month (95% CI: -6.0 to 0.2)) as compared to stables that had no or, only a small, ventilation opening (<30 cm). By the second half of 2007, bluetongue had spread over throughout the Netherlands. Our study indicated that there were some management factors that may help limit exposure to BTV-8 and its consequences.