Detection, typing and control of Histomonas meleagridis

Abstract

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.