Tetratrichomonas gallinarum granuloma disease in a flock of free range layers.

Granuloma disease in a flock of free range productive layers in the Netherlands in 2017 is described. The disease resembled granuloma outbreaks in layers caused by Tetratrichomonas gallinarum in 2013 and occurred in the same area in which the rearing farm considered as the source of the 2013 outbreaks was located. Between 55 and 84 weeks of age mortality was 20.3% (breeder's norm 3.9%). All dead hens examined (n = 20) showed granulomas especially in liver and ceca. Nine hens with or without liver and/or ceca granulomas were examined for trichomonads in mentioned organs by in situ hybridization (ISH), nested PCR, and cloning and sequencing. Ceca were also examined by culture. T. gallinarum ISH was positive in all livers and ceca with granulomas and negative in case granulomas were absent. T. gallinarum strain 13/16632, which caused the 2013 outbreaks was found in 4/8 hens with granulomas. Moreover, other trichomonads were detected: a T. gallinarum strain GPO-like and a Simplicimonas sp. strain GABC1-like. Mixed infections also occurred. Infectious causes of granuloma disease other than the afore-mentioned trichomonads could be excluded. Trichomonad DNA was not detected in environmental samples and wild ducks originating from the farm of concern, except for one duck in which the same Simplicimonas sp. as in hens was detected, leaving the source of the T. gallinarum infection in hens unknown. It is concluded that the herein described granuloma disease likely was caused by T. gallinarum strain 13/16632. However, the pathogenicity of the other trichomonads found remains to be clarified.

Granuloma disease in flocks of productive layers caused by Tetratrichomonas gallinarum.

In 2013, seven outbreaks of granuloma disease occurred in Dutch flocks of productive layers housed on different farms. These outbreaks were characterized by increased mortality and high incidence of granulomas, mainly in caeca (340/408 hens = 83%) and livers (69/408 hens = 17%). Mortality started to increase between 21 and 35 weeks of age and reached 3.7% to 11.0% exceeding the breeder's norm in periods ranging from 9 to 48 weeks. Some flocks also showed decreased egg production and/or loss of mean egg weight. All affected flocks were linked to one rearing farm, which therefore seemed to be the source of the disease. However, no signs of disease had been observed at this rearing farm. Sentinel hens placed in one of the affected flocks to determine whether the disease had an infectious nature developed granulomas identical to those seen in the outbreaks. Next, by fulfilling Koch's postulates it was shown that Tetratrichomonas gallinarum was the aetiological agent of the granuloma disease. The condition was reproduced in mature specified pathogen free White Leghorn hens (GD - Animal Health, Deventer, the Netherlands) by inoculation via both an artificial and a natural route with a well-defined axenic T. gallinarum isolate obtained from one of the affected flocks. Other causes of granuloma disease were excluded.

Quantification of parasite shedding and horizontal transmission parameters in Histomonas meleagridis-infected turkeys determined by real-time quantitative PCR.

To gain more insight into the within flock transmission of Histomonas meleagridis, the shedding of parasites was quantified by a newly developed real-time quantitative (q)PCR and the basic reproduction number (R0) and the mean number of secondary infections per infectious bird per day in a susceptible population (ß) of H. meleagridis in the absence of heterakis were assessed. Forty turkeys were divided into two groups of 10 and 30 birds at 14 days of age. Birds of the first group were inoculated with 200,000 histomonads each, the second group served as a susceptible contact group. Cloacal swabs were taken at -1, 1, 4, 7, 9, 11, 14, 18 and 21 days post inoculation (p.i.) to assess the shedding of the parasite by the qPCR (detection limit 330 histomonads/ml droppings). The experiment ended at 28 days p.i. Mortality was 100% in the inoculated birds and started at day 12 p.i., while in the contacts, it was 83% and started at 16 days p.i. Shedding started 1 day after the inoculation in both groups. The mean shedding levels (and 95% CI) expressed as parasite equivalents per gram cloacal content on a log10 scale in the inoculated, contact birds that died and contact birds alive were 2.0 (1.6-2.4), 1.6 (1.4-1.9) and 1.2 (0.5-2.0), respectively. Birds that died shed histomonas more often and were infectious for 13.4 days; in contrast, those that recovered were infectious for 5.7 days. R0 was estimated to be 8.4 and ß 0.70. Simulations made with the parameters obtained were in agreement with the experimental results, confirming their validity.

Schmallenberg virus epidemic in the Netherlands: spatiotemporal introduction in 2011 and seroprevalence in ruminants.

This study aimed at estimating the Schmallenberg virus (SBV) seroprevalence in dairy heifers, non-dairy adult cattle, sheep and goats in the Netherlands after cessation of SBV transmission at the end of 2011. Archived serum samples from ruminants submitted to the GD Animal Health Service for monitoring purposes between November 2011 and March 2012 were selected and tested for presence of SBV-specific antibodies using an in-house ELISA. Animal seroprevalences were estimated at 63.4% in dairy heifers, 98.5% in adult non-dairy cattle, 89.0% in sheep and 50.8% in goats. Multivariable analyses were carried out to describe the relationship between potential risk factors and the ELISA outcome S/P%. The overall SBV seroprevalence in ruminants and ruminant herds in the Netherlands at the end of 2011 was high, with considerable differences between species and farm types. No gradient spatial pattern in final seroprevalence could be detected and therefore no suggestions about the site of introduction and spread of SBV in the Netherlands in 2011 could be made. In dairy heifers, it was shown that S/P% increased with age. In sheep, S/P% was lower in animals located in the coastal area. Whether herds were located near the German border did not affect the S/P% in sheep nor in dairy heifers. An attempt was made to gain insight in the spatiotemporal introduction of SBV in the Netherlands in 2011, by testing sheep serum samples from 2011. A seroprevalence of about 2% was found in samples from April, June and July 2011, but the ELISA positive samples could not be confirmed in a virus neutralization test. A clear increase in seroprevalence started at August 2011. From mid-August 2011 onwards, seropositive samples were confirmed positive by virus neutralization testing. This indicated the start of the epidemic, but without a clear spatial pattern.

Development and validation of an indirect Enzyme-linked immunosorbent assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants.

To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3-100.0%). The ELISA showed a high repeatability (cv=6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats.

Prevalence of exposure and infection of Lawsonia intracellularis among slaughter-age pigs.

The extent of clinical or subclinical infection associated with Lawsonia intracellularis within Dutch pig herds was uncertain. A case-control study of slaughter age pigs was used to study natural infection within Dutch herds and to compare diagnostic methods. From six case herds where clinical disease had been identified recently, and six disease-free herds, 40 pigs of slaughter-age were examined postmortem. The diagnostic methods used were: serology, gross examination, Haematoxylin and Eosin stain (HE), Warthin-Starry silver stain, Lawsonia-specific indirect immunoperoxidase of the ileum, and PCR of ileum mucosa and colon contents. There were 59% seropositive pigs in case herds and 26% seropositive pigs in control herds. Using immunohistochemistry, 57% of case herds and 46% of control herds were bacteria positive in the ileum mucosa. It was concluded that a majority of Dutch herds contain L. intracellularis infected finisher pigs. In some herds this is associated with clinical outbreaks of acute haemorrhagic enteropathy but in other herds no clinical disease is apparent. Many seropositive pigs in herds without clinical disease had evidence of Lawsonia antigen in sites other than the apical cytoplasm of proliferating epithelial cells, particularly the supranuclear region. It was uncertain whether to classify these pigs as having "recovered" from an infection or whether they have a sub-clinical or chronic form of the disease. We concluded that PCR examination of faeces and serology probably provide more specific results than gross examinations at slaughter, and that a monoclonal antibody-based examination of ileum mucosa should be the accepted screening method for this infection.

First international ring trial of ELISAs for Salmonella-antibody detection in swine.

An international ring trial for Salmonella-ELISAs for swine serology was organized. Twelve laboratories participated and used "in-house" ELISAs or commercially available kits. In total 47 well-defined sera from various sources, including inoculation studies with Salmonella-strains from sero-groups B, C1, C2, D, and E1, were tested blindfold. The specificity of most ELISAs was satisfactory, but relatively large differences were found between the sensitivities of the tests. It is concluded that international reference samples should be made available to guarantee a minimum level of sensitivity.

A longitudinal study of Salmonella enterica infections in high-and low-seroprevalence finishing swine herds in The Netherlands.

The purpose of this investigation was to study the incidence and course of Salmonella infections in finishing pig herds in order to asses the stability of a given Salmonella herd status. Five low- and 7 high-seroprevalence herds were followed for seven sampling rounds. Each round, blood and faecal samples were tested in an indirect ELISA and by bacteriological culturing, respectively. In high-seroprevalence herds a positive Salmonella status was an indication of a long-term problem and the status was relatively stable over time. The herds experiencing clinical salmonellosis were not necessarily the herds with the highest seroprevalence. It is possible to deliver sero-negative finishers to the slaughterhouse, even though these pigs were seropositive as growers. In three out of five low-prevalence herds, major infection incidents occurred, indicating that changes in the Salmonella status should be anticipated. Low-prevalence herds can remain negative over a longer period of time as a result feeding a complete liquid feed containing fermented by-products.

Administration of acidified drinking water to finishing pigs in order to prevent Salmonella infections.

The aim of the study was to test whether acidified drinking water, with two millilitres of an acid mixture per litre, was able to reduce the number of Salmonella infections in finishing pig herds. In each compartment, half of the pens were supplied with acidified water and the other pens served as negative control. In three herds the required dose was not applied to the pigs as a result of various practical problems. In another herd, all pigs remained seronegative throughout the study. Analysis of the remaining three herds showed a large and significant treatment effect in one herd (P<0.001). As a result of the small number of observations and the overall lower seroprevalence in the control groups, the other two herds only showed a statistical trend to a treatment effect (0.10

Salmonella seroprevalence at the population and herd level in pigs in The Netherlands.

The aim of this study was to provide baseline data on the population and herd Salmonella seroprevalence in sows and finishers. For the population estimates in 1996 and 1999 and the herd prevalences for sows and gilts, blood samples from swine vesicular disease (SVD) and pseudorabies monitoring programmes were used and tested in an indirect Salmonella enzyme-linked immunosorbent assay (ELISA). The herd prevalence for finishers was determined using blood samples collected at two slaughterhouses. The population prevalence for finishers in 1996 and 1999 was 23.7 and 24.5%, respectively, and for sows 40.5 and 60.4%, respectively. The prevalence in free range (FR) finishers was significantly higher (44.6%) than in intensively housed finishers in 1999, identifying a hazard group for possible extra pork and pork product contamination. Of 406 finishing herds, 9% were completely seronegative for Salmonella (cut-off OD%>10). Of these 406 finishing herds, 69.7% had Salmonella-status I (low prevalence), 21.7% status II (moderate prevalence) and 8.6% status III (high prevalence) (cut-off OD%>40). In 46 multiplying sow herds, 20 breeding sow herds and 20 matching replacement gilt herds, the average herd prevalences were 54, 44.4 and 19.3%, respectively. Two gilt herds were completely seronegative. The prevalence in the gilt herds was never higher than in the matching breeding sow herds. Agreement on methodology and calibration of ELISA tests would make these results comparable between countries and is a prerequisite for a co-ordinated and integrated program to reduce Salmonella in pork in the European Union.

Herd level husbandry factors associated with the serological Salmonella prevalence in finishing pig herds in The Netherlands.

A national program to reduce Salmonella in pork and pork products should include monitoring and intervention at farm level. To develop an adequate intervention strategy at farm level, risk factors for Salmonella infections in finishing pigs have to be determined. In this study, blood samples were collected randomly at two slaughterhouses from slaughter pigs. Samples were tested by the Dutch Salmonella ELISA, based on the O-antigens 1, 4, 5, 6, 7 and 12, using a cut-off of OD%=10. This ELISA has been calibrated against the Danish ELISA to give comparable results. Workers from herds from which at least forty blood samples had been collected, were asked to participate in a questionnaire. In total, 353 questionnaires were obtained and analysed. Significant risk factors associated with the proportion of seropositive samples were identified by multiple linear logistic regression. The feeding of a complete liquid feed containing fermented by-products and the omission of disinfection after pressure washing a compartment as part of an all-in/all-out procedure, were both associated with a lower Salmonella seroprevalence. A small to moderate herd size (<800 finishing pigs), a previous diagnosis of clinical Salmonella infection in the herd, the use of tylosin as an antimicrobial growth promoter in finishing feed, or herds which had more than 16% of the livers of their pigs condemned at the slaughterhouse as a result of white spots were associated with a higher Salmonella seroprevalence. Hypothetical intervention strategies based on these risk factors can be studied for their effect on the Salmonella seroprevalence and practical applicability in field studies.

Experimental quantification of the transmission of Sarcoptes scabiei var. suis among finishing pigs.

In this study, the rate of S. scabiei var. suis transmission among finishing pigs was quantified in a contact transmission experiment. Forty piglets originating from a mange free farrow-to-finish herd were randomly allocated to three groups and one S. scabiei var. suis infested finishing pig was subsequently added to each of these groups. After 35 days, the three seeder pigs were removed from the groups and the remaining 40 pigs were re-allocated to five pens. Ear scrapings, to be examined for mites, were collected from each pig on days 1, 14, 28, 42, 56, and 84 of the experiment. Blood samples, to be tested for antibodies against S. scabiei, were collected from each pig on days 0, 14, 28, 42, 56, 70, 84 and 112 after the introduction of the seeder pigs. From the results of the ear scrapings and the blood samples the number of susceptible (not infested) and infested pigs was derived at the time of each sample collection and the number of new infestations in the intervals between the sample collections. From these data the infestation rate parameter beta (average number of new infestations per infested pig per day) was estimated by use of a Generalised Linear Model (GLM) and accordingly, beta was estimated at 0.056 (95% CI: 0.037-0.085) infestations per infested pig per day.Next, by use of beta, the transmission of S. scabiei was simulated in a population of 100 finishing pigs for 100 days after the introduction of a single infested pig. For this purpose, 500 simulations were done. The 90% confidence interval of the number of infested pigs at day 100 ranged from 12 to 88 (median: 63). It was concluded that transmission of S. scabiei among finishing pigs is slow. Due to the presumed lower contact rate between sows as compared to finishing pigs, it is anticipated that transmission of S. scabiei among sows will even be slower than among finishers These findings are of particular interest for the development of surveillance programmes for S. scabiei free herds.

Production performance and pruritic behaviour of pigs naturally infected by Sarcoptes scabiei var. suis in a contact transmission experiment.

Reports on the effects of mange on the production performance of pigs are conflicting. So far, studies have used experimental infections, by depositing encrusted lesions from chronically infected pigs into the ears of experimental pigs. However, this is a poor representation of what happens under natural field conditions. The purpose of our study was to quantify the effects of sarcoptic mange on production performance and pruritus in pigs that were infected by contact with S. scabiei var. suis-infected pigs. A total of 80 piglets were matched by sex and weight and randomly divided between experimental and control compartments. In the experimental compartment, each of three naturally S. scabiei var. suis-infested pigs were randomly allocated to three pens with 13 susceptible pigs each. From day 0 to 35, the growth performance of pigs in the experimental compartment was significantly (P=0.04) worse (35 g/d) than of pigs in the control compartment. From day 35 to 112, there was a statistical trend (P=0.10) that the growth performance of pigs in the experimental compartment was lower (50 g/d) than that of pigs in the control compartment. For the complete fattening period (0-112 or more days), the growth performance of pigs in the experimental compartment was significantly (P=0.05) worse (41 g/d) than that of pigs in the control compartment. Mean feed conversion ratio (kg feed per kg gain) was 2% higher in the experimental compartment compared with the control compartment. Pigs in the experimental compartment had a nine times (95% CI: 2 - 44) higher chance of showing pruritic behaviour than pigs in the control compartment.

Validation of ELISAs for the detection of antibodies to Sarcoptes scabiei in pigs.

An Enzyme-linked ImmunoSorbent Assay (ELISA) was developed for the detection of antibodies to Sarcoptes scabiei. This 'Animal Health Service'-ELISA (AHS-ELISA) was compared with a commercial test (Checkit(R) Sarcoptest) using experimental and field sera. The experimental study was a contact infestation experiment. Eighty piglets were randomly divided between the experimental and control group. After introduction of three Sarcoptes scabiei var. suis infested pigs in the experimental group, both groups were monitored by determining scratching indices, taking ear scrapings and blood samples in Weeks 0, 2, 4, 6, 8, 12 and 16. Four pigs in the control group were immunised with either Dermatophagoides pteronyssinus (Dp) antigens (n=2), or Acarus siro (As) antigens (n=2). In the control group all (non-immunised) pigs were negative in all tests. In the experimental group only slightly elevated scratching indices were observed, with a maximum in Week 8. After 2 weeks for the first time an ear scraping was positive (2.5%). In Week 8 the highest number of positive ear scrapings were found (25.0%). Positive results in the Sarcoptest were first obtained in Week 12 (10.5% positive), while eventually 29.0% of the finishing pigs were positive after 16 weeks. The AHS-ELISA first detected a serological response after 6 weeks (5. 0% positives), increasing until after 16 weeks a large proportion (74.2%) of the finishing pigs were seropositive, making the AHS-ELISA the most sensitive test. In the AHS-ELISA one As-immunised pig remained seronegative, but the other hyper-immunised pigs crossreacted. In the Sarcoptest, only Dp-immunised pigs had elevated Optical Densities (OD's) albeit below the cut-off level. Although hyper-immunisation is not a representation of field conditions, it cannot be excluded that the AHS-ELISA is not 100% specific.Field samples were taken from 20 sows in 30 herds, classified as mange-free, suspect, or infested. On a herd level there was high agreement among the ELISAs. Both serological tests were suitable to distinguish mange-free herds from infested herds. In one infested herd the decline of maternal antibody in piglets was studied by sampling 40 piglets from 20 different litters. The lowest average OD using the AHS-ELISA was found at 5 weeks of age, followed by a significant increase at 7 weeks. The average OD with the Sarcoptest was at a minimum level at 3 weeks, but no increase was found later. For screening of herds, interference of maternal antibodies is avoided by sampling at an age of 7 weeks or older.

Evaluation of tests for detection of antibodies to Aujeszky's disease (pseudorabies) virus glycoprotein E in the target population.

Receiver operating characteristic (ROC) curves assess the quality of tests over the entire range of test signals. We compared the ability of an ELISA to detect antibodies to Aujeszky's disease (pseudorabies) virus gE in colostrum (test A) and in a single droplet of whole blood (test B) with the results obtained in serum (gold standard) in the target population by constructing and analyzing such curves. The area under the ROC curve, which is a quantitative measure of test performance, proved to be significantly (p < 0.01) smaller in test A than in test B or the gold standard. No significant differences in the area under the ROC curve were observed between test B and the gold standard.

Assessment of the quality of tests for the detection of antibodies to Aujeszky's disease virus glycoprotein gE in a target population by the use of receiver operating characteristic curves.

The performance of tests for the detection of antibodies to Aujeszky's disease virus glycoprotein E (gE) in a target population was evaluated by constructing and analysing receiver operating characteristic (ROC) curves. These curves assess the discriminating ability of a test over the entire range of test signals. The advantages of applying the analysis to a sample of the target population (all commercial pigs in the Netherlands), as compared to using a panel of test sera, are that the estimates of sensitivity and specificity, the comparisons between tests and the choices of the cut-off values are all relevant for the target population. The results of a gE-ELISA in colostrum (test A) and in a single droplet of whole blood (test B) were compared with the results obtained with the same ELISA in serum (gold standard). The area under the ROC curve, which is a quantitative measure of test performance, was significantly (P < 0.01) smaller with test A than test B or the gold standard, indicating that test B performed better than test A. No significant difference was observed between test B and the gold standard.

World Health Organisation--supervised interlaboratory comparison of ELISAs for the serological detection of Salmonella enterica serotype Enteritidis in chickens.

A collaborative exercise, supervised by the World Health Organisation, was set up to compare ELISAs used for the serological detection of Salmonella enteritica serotype Enteritidis in chickens. The aim was to ascertain how far agreement could be reached on the interpretation of optical density readings for high titre, intermediate titre and low titre sera. Two sets of sera were sent to 14 participants. The first set compared high, medium and low titre sera raised in specified-pathogen-free and commercial broiler breeder chickens. The second set comprised 20 sera of different antibody titres raised in commercial birds reared under laboratory conditions and sent blind. Both indirect and double-antibody sandwich blocking ELISAs were used with a number of different detecting antigens. With a few exceptions good agreement was reached on the interpretation of results obtained from high and low titre sera from the optical density obtained with a single serum dilution. Differences were observed in the interpretation of medium titre sera. The results suggested that most ELISAs produce reasonably comparable results and that practical problems may arise from interpretation of the results mainly as a result of the choice of the criteria used for differentiating sera obtained from infected and uninfected chickens. These problems are discussed.

[What possibilities does the milk-progesterone test in dairy cattle offer in clinical practice?]. / Welke mogelijkheden biedt de melk-progesterontest bij melkkoeien voor de praktijk?

A field trial was made by the Animal Health Service Foundation in Boxtel, the Netherlands, during the period from December 1984 to June 1985 for the purpose of studying the possibilities of the milk progesterone test (MPT) in dairy cattle in the field. Approximately 10,000 milk samples were taken to determine the concentration of progesterone; this was done daily using the EIA method in the laboratory of the Animal Health Service Foundation in Boxtel. The MPT offers more possibilities in the field than do the detection of oestrus and diagnosis of gestation alone. Experimental studies in eighty-six cows showed that the progesterone levels of the milk start to decrease from a few days prior to oestrus, which means that heat is predictable. The time of insemination is also more or less predictable on the basis of the MPT. It was shown that the concentration of progesterone in the milk of animals subsequently found to be pregnant was less than 1.0 ng/ml on the day of insemination. Optimum results of fertilisation are obtained after the first, second or third day on which the concentration of progesterone in the milk is less than 1.0 ng/ml.