Uitroeiing van schurft op varkensbedrijven

De Projectgroep “Vrijwaring Schurft” heeft van het Programmateam “Diergezondheid in Beweging” de opdracht gekregen om, indien mogelijk, een haalbaar en controleerbaar schurftvrij-programma voor de Nederlandse varkenshouderij te ontwikkelen. De volgende onderzoeksvragen zijn hierbij beantwoord: 1 - Is het mogelijk een ELISA te ontwikkelen voor het aantonen van antilichamen in het bloed tegen de schurftmijt van het varken (Sarcoptes scabiei var. suis), die op het laboratorium routinematig kan worden toegepast? 2- Is het mogelijk om met schurft besmette varkensbedrijven door middel van een tweemalige ivermectine-behandeling van alle aanwezige varkens, aangevuld met hygiënische maatregelen maar zonder ontschurfting van de omgeving, vrij te krijgen van schurft? 3 - Kan een bedrijf dat eenmaal schurftvrij is door middel van uitsluitend hygiënische maatregelen gedurende tenminste één jaar schurftvrij blijven? 4 - Wat is het ingeschatte economische effect van uitroeiing van schurft op praktijkbedrijven

Detection, typing and control of Histomonas meleagridis

Histomonosis (blackhead) is a disease of galliform birds caused by the flagellated protozoan Histomonas meleagridis. Its primary target organs are the ceca and the liver. Especially in turkeys, mortality can be very high (up to 100%). In the 1960’s and 1970’s several effective antihistomonal compounds like nitarsone and dimetridazole were developed and the disease was brought under control. After these antihistomonals were banned from use in production animals due to possible toxic and carcinogenic properties, the disease re-emerged. The aim of the studies in this thesis was to optimize and develop methods for the detection and typing of H. meleagridis and to examine new possibilities to control the disease. Histomonas culture was optimized by adding more (100 mg instead of 12 mg) rice powder to the culture medium (Dwyer medium). This resulted in a 10-fold higher yield of parasites and the possibility to prolong cultures by addition of rice powder. Also it was found that a complex constituent, chicken embryo extract, was redundant. Another constituent, horse serum, proved essential. Several new possible antihistomonal products were examined by using in vitro (based on the imrpoved culture) and, if effective, in vivo models. Aromabioic™ and tiamulin were not effective in vitro and were not examined further. Enteroguard™ and Protophyt™ had no antihistomonal effect in vivo. Paromomycin, however, protected turkeys against an otherwise lethal challenge with H. meleagridis (full protection at 400 ppm, and partly protection at 200 ppm). A blocking ELISA was developed for the detection of antibodies in chicken and turkeys. The monoclonal antibody needed for this assay was raised against proteins extracted from H. meleagridis using a detergent (Triton X-114). While both chicken and turkeys seroconverted after a challenge with H. meleagridis, the ELISA showed no crossreactivity with Tetratrichomonas gallinarum. The ELISA is e.g. useful for seroepidemiological studies towards the distribution of H. meleagridis in chickens, which are considered a reservoir for the parasite. Finally, a novel subtyping technique (C-profiling) was developed. It is based on molecular techniques (PCR and sequening). The internal transcribed spacer 1 (ITS1) region of the rRNA gene proved sufficiently variable for subtyping. Three subtypes of H. meleagridis was found. Type III possibly is Parahistomonas meleagridis, a nonpathogenic protozoan described some decades ago. Subtyping is useful if different genotypes are associated with different pathotypes. C-profiling was applied to a closely related pathogen of humans: Dientamoeba fragilis and was found to be a promising molecular epidemiological tool for studying the transmission, geographical distribution and relationships between strains and pathogenicity of this parasite

Tetratrichomonas gallinarum granuloma disease in a flock of free range layers

International audienceGranuloma 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 ¼ 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 clarifie

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

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