Rüdiger Hauck

Comparison of the Specificity and Sensitivity of PCR, Nested PCR, and Real-Time PCR for the Diagnosis of Histomoniasis

Abstract Blackhead, also known as enterohepatitis, is caused by a protozoan parasite called Histomonas meleagridis. Clinical symptoms are nonspecific. Until now, diagnosis has been mainly based on postmortem lesions and microscopical and histopathological examination. In many cases, especially in layer flocks, these conventional methods are not sufficient, as the lesions are sometimes not clear. The technique for isolation of histomonads in vitro offers many advantages, but the confirmation of histomonads growing in culture may require a time-consuming procedure of rectal inoculation of culture material into chickens or turkeys. The aim of our investigation was to establish a conventional polymerase chain reaction (PCR), a nested PCR, and a real-time PCR, and to examine their specificity as well as sensitivity in the diagnosis of histomoniasis. The obtained results have shown that the conventional PCR is more sensitive than the real-time PCR. Furthermore, the sensitivity of the PCR can be increased by adding the nested PCR. However, the real-time PCR is more specific.

Efficacy of a herbal product against Histomonas meleagridis after experimental infection of turkey poults

Abstract Histomoniasis (infectious enterohepatitis, blackhead) is caused by the protozoan parasite Histomonas meleagridis (H. meleagridis). After the ban of all prophylactic and therapeutic drugs in the European Union, histomoniasis is increasingly responsible for considerable economic problems to the poultry industry. The aim of this study was to investigate the effect of a herbal product with extracts from cinnamon, garlic, lemon, and rosemary on H. meleagridis in turkey poults in vivo. For this purpose, 60 two-week-old poults were divided into three groups. Group 1 received the herbal product in the feed six days before infection and in water three days before infection, then in feed and drinking water until the end of the experiment. Groups 2 and 3 were left untreated. At week 3 of age, Groups 1 and 2 were infected intracloacally with H. meleagridis. Three weeks after infection the surviving birds were euthanized and examined for pathological lesions. Mortality was 20% in Group 1 and 50% in Group 2. There were no deaths in Group 3. DNA of histomonads was detected in all examined caeca and livers of the dead birds, but was not detected in any examined organ of the surviving birds of all groups. There was no noticeable difference in the lesion scores of the dead birds between the groups. The surviving birds of all groups did not show lesions post mortem. Since all effective prophylactic and therapeutic drugs against histomoniasis were banned in the EU, under given conditions the investigated herbal product seems to be an effective alternative for the reduction of mortality in turkeys caused by histomoniasis.

Detection of DNA of Histomonas meleagridis and Tetratrichomonas gallinarum in German Poultry Flocks Between 2004 and 2008

Abstract Between 2004 and 2008, 338 samples from 156 German turkey, chicken, and peacock flocks with suspected histomonosis (histomoniasis) were sent to the Institute for Poultry Diseases of the Free University Berlin. Most samples were from ceca or livers; the other samples were organ pools or were taken from other organs or the environment. In 108 samples from 65 flocks, histomonal DNA was detected by polymerase chain reaction (PCR). Tetratrichomonas gallinarum DNA was found in 5.3% of investigated samples from flocks infected with Histomonas meleagridis and in 27.4% of investigated samples from flocks that were not infected with H. meleagridis. For subtyping of the strains, the C-profiling method, a method used to analyze the internal transcribed spacer-1 (ITS-1) of the rRNA gene, was modified to be more specific for H. meleagridis. Results showed the presence of more than the three subtypes described so far. There was no clear correlation between the subtype and the host. By C-profiling the clonal cultures, heterogeneous ITS-1 sequences were shown to probably result from intragenomic differences between rRNA genes.

Protective oral vaccination against infectious bursal disease virus using the major viral antigenic protein VP2 produced in Pichia pastoris.

Infectious bursal disease virus (IBDV) causes economically important immunosuppressive disease in young chickens. The self-assembling capsid protein (VP2) from IBDV strain IR01 was expressed in Pichia pastoris resulting in the formation of homomeric, 23-nm infectious bursal disease subviral particles (IBD-SVPs) with a yield of 76 mg/l before and 38 mg/l after purification. Anti-IBDV antibodies were detected in chickens injected with purified IBD-SVPs or fed with either purified IBD-SVPs or inactivated P. pastoris cells containing IBD-VP2 (cell-encapsulated). Challenge studies using the heterologous classical IBDV strain (MB3) showed that intramuscular vaccination with 20 µg purified IBD-SVPs conferred full protection, achieved complete virus clearance and prevented bursal damage and atrophy, compared with only 40% protection, 0–10% virus clearance accompanied by severe atrophy and substantial bursal damage in mock-vaccinated and challenge controls. The commercial IBDV vaccine also conferred full protection and achieved complete virus clearance, albeit with partial bursal atrophy. Oral administration of 500 µg purified IBD-SVPs with and without adjuvant conferred 100% protection but achieved only 60% virus clearance with adjuvant and none without it. Moderate bursal damage was observed in both cases but the inclusion of adjuvant resulted in bursal atrophy similar to that observed with live-attenuated vaccine and parenteral administration of 20 µg purified IBD-SVPs. The oral administration of 250 mg P. pastoris cells containing IBD-VP2 resulted in 100% protection with adjuvant and 60% without, accompanied by moderate bursal damage and atrophy in both groups, whereas 25 mg P. pastoris cells containing IBD-VP2 resulted in 90–100% protection with moderate bursal lesions and severe atrophy. Finally, the oral delivery of 50 µg purified IBD-SVPs achieved 40–60% protection with severe bursal lesions and atrophy. Both oral and parenteral administration of yeast-derived IBD-VP2 can therefore induce a specific and protective immune response against IBDV without affecting the growth rate of chickens.

Partial sequence of the beta-tubulin of Histomonas meleagridis and the activity of benzimidazoles against H. meleagridis in vitro.

The protozoan parasite Histomonas meleagridis is a member of the family Monocercomonadidae in the class Trichomonada. Due to food safety concerns, currently no prophylactic or therapeutic drug against the parasite is licensed in the European Union. Benzimidazoles are antiparasitic drugs, and some of them are licensed for use in food-producing animals. Benzimidazoles act on beta-tubulin, and the beta-tubulin sequence allows predictions about the efficacy of benzimidazoles. In this study, we sequenced and analyzed a part of the beta-tubulin gene of five H. meleagridis strains and of Dientamoeba fragilis. In each Histomonas strain, three to five different sequences were found. No clustering of sequences from the same strain was recognizable. A phylogenetic tree based on the amino acid sequences of trichomonal beta-tubulin genes placed the histomonal sequences on a branch with D. fragilis, separate from Monocercomonas sp. and Tritrichomonas foetus. All histomonal amino acid sequences predicted a susceptibility to benzimidazoles. However, when we tested the efficacy of five benzimidazoles, namely, albendazole, fenbendazole, flubendazole, mebendazole, and nocodazole, on H. meleagridis in vitro, all tested drugs showed no efficacy, even though the concentrations tested were higher than the concentrations found to be effective against Trichomonas vaginalis and T. foetus by other investigators.

Pilot study on the efficacy of paromomycin as a histomonostatic feed additive in turkey poults experimentally infected with Histomonas meleagridis

Paromomycin is an aminoglycoside antibiotic with activity against protozoa. Currently, paromomycin is registered for food producing animal species. In a pilot study we evaluated the efficacy of different doses of paromomycin in the feed against Histomonas meleagridis in experimentally challenged turkey poults. Groups consisting of 30 birds each were given feed with 100, 200 and 400 ppm paromomycin, respectively, starting on day 1 through to day 42. One group of 30 birds was left untreated. On day 21 all birds were infected intracloacally with H. meleagridis. Additionally, 10 birds were kept as a non-infected and non-treated control group. Before the challenge there was no significant difference between untreated and treated groups with regards to feed consumption and feed conversion rate. After the challenge, mortality was 80% in the infected non-treated birds. In the treated groups the mortality rate was 73.3%, 43.3% and 20%, respectively. No histomonal DNA was found in caeca and livers of the surviving birds. In addition, higher doses of paromomycin (200 and 400 ppm) led to reduced counts of Clostridium perfringens in the droppings. In conclusion, a prophylactic application of paromomycin as a feed additive was effective against the challenge with H. meleagridis under experimental conditions.

Effect of Coated Plant Extracts on Histomonas Meleagridis and Growth of Bacteria in Vitro

Abstract Three coated plant extracts (RepaXol® and two experimental formulations) were tested for their minimal lethal concentration for histomonads in vitro and the effect of those substances on the bacterial growth in the histomonadal culture. After 48 hr, RepaXol® and experimental formulation B were lethal to histomonads at a concentration of 1.25 µl/ml. Experimental formulation C was lethal at a concentration of 2.5 µl/ml. All products also decreased the growth of bacteria at concentrations inhibiting the growth of histomonads.

Articular Aspergillosis of Hip Joints in Turkeys

Abstract Aspergillosis is an important cause of morbidity and mortality in birds. Turkey poults are known to be particularly susceptible to fungal infection. Although the respiratory tract is the most commonly affected, dissemination can occur into virtually any organ. Here, we report an unusual outbreak of articular aspergillosis in a flock of meat turkeys with clinical signs of lameness. Between 7 and 11 weeks of age, turkeys had severe granulomatous osteoarthritis of the hip joints with necrosis of the femur head. Fungal morphology and PCR amplification and sequencing of the first ITS1-5.8S-ITS2 rDNA region identified Aspergillus fumigatus as the infectious agent. Concurrently, Staphylococcus spp. was isolated from the hip joints, which may have promoted the tropism of the fungus. Mild respiratory tract aspergillosis was observed in only one case. The findings suggest that fungal arthritis may present a specific disease entity in turkeys and should be considered as further cause of lameness in turkeys.

Detection of Histomonas meleagridis DNA in Different Organs After Natural and Experimental Infections of Meat Turkeys

Abstract Histomonas meleagridis infection of turkeys is usually accompanied by a severe disease with unspecific clinical symptoms but with distinct pathological lesions in the ceca and liver. In the literature some macro- and microscopic evidence of the spread of histomonads to the other organs has been provided. The aim of the present investigations was to use real-time polymerase chain reaction (PCR) to demonstrate the dissemination of H. meleagridis DNA to different organs after natural and experimental infection of meat turkeys. Samples from several organs were collected from a meat-turkey flock, which proved to be naturally infected with histomoniasis, and examined for histomonad DNA by real-time PCR. Histomonad DNA was detected in all investigated ceca, livers, spleens, kidneys, and pooled brain swabs. Additionally it was found in 75% of investigated samples from bursae of Fabricius, in 50% of investigated duodenums, and in 40% of investigated jejunum samples. After experimental intracloacal infection of 3-wk-old turkey poults with 147,500 histomonads, similar samples were collected from all turkeys that died. After a 3-wk observation period the surviving birds, as well as the noninfected control group, were euthanatized and samples were taken. During the entire experimental period, 10 birds out the 20 infected birds died. Histomonad DNA was detected in all investigated ceca, livers, lungs, and hearts (100%) and almost all kidneys (90%) and bursae of Fabricius (80%). On the other hand, only 30% of examined spleens and 10% of brain samples revealed positive results. Surviving infected birds were euthanatized and necropsied; histomonad DNA was found in one out of 10 livers but not in any ceca. Also, histomonad DNA could not be detected in examined cecal and lung samples from the noninfected control group.

Systematic Position of Histomonas meleagridis Based on Four Protein Genes

Abstract Phylogenetic trees based on parabasalid sequences of the small subunit rRNA placed Histomonas meleagridis in close proximity to Dientamoeba fragilis, Tritrichomonas foetus, and Monocercomonas sp. In this study, we sequenced partial genes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, and α-tubulin from 2 strains of H. meleagridis. We found 5 different GAPDH sequences, 6 different enolase sequences, and 3 α-tubulin sequences. Phylogenetic trees based on the obtained sequences showed a close relationship of H. meleagridis with T. foetus and, to some extent, Monocercomonas sp. In conclusion, our findings further corroborate the ssu rRNA–based tree. Consequently, our study also supports the hypothesis that H. meleagridis secondarily lost cytoskeletal structures.