Performing Department
Poultry Science
Non Technical Summary
Blackhead disease, caused by the protozoan parasite Histomonas meleagridis, has become common in some poultry production units often resulting in a 30% mortality rate in chickens and up to 90% mortality rates in turkeys. Nitarsone (4-nitrophenylarsonic acid), the only approved product available as a Blackhead disease preventative, was recently withdrawn due to growing consumer concern over its arsenical content. Consequently, as of January 1st, 2016 there are no approved drugs for the prevention and treatment of Blackhead disease. Although important research has been accomplished, significant gaps in knowledge regarding the treatment and epidemiology of Blackhead disease remain. More information is desperately needed regarding disease reservoirs, vectors, mechanisms of transfer, husbandry practices that may increase or reduce the susceptibility of the flock and therapeutic approaches. Previous research has mostly focused directly on H. meleagridis. This parasite is an anaerobic microorganism and cannot survive outside of a host for an extended period of time. Thus, when studying the epidemiology of H. meleagridis, research should also concentrate on the cecal worm Heterakis gallinarum, which is an indispensable host for H. meleagridis and vectors that transmit the worm eggs.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Histomonas meleagridis is an anaerobic protozoan and the causative agent of blackhead disease. H. meleagridis can cause up to 100% mortality in turkey flocks and up to 30% mortality in chicken flocks. It is believed that turkeys are highly susceptible to the disease because they fail to mount an effective immune response to the parasite. With the withdrawal of the approval of nitarsone by the EU and USA, there are currently no drugs on the market to prevent or treat blackhead disease. The loss of antiprotozoal treatments is also leading to the emergence of other protozoal diseases such as Tetratrichomonas gallinae and Cochlosoma anatis.Identify feed additives such as essential oils, a phytogenic compound, organic acids, and a yeast derivative that may boost the bird's immune response or inhibit H. meleagridis growth and prevent blackhead disease.Determine if a vaccination strategy can be used to prevent blackhead.Determine if litter amendments can reduce the time H. meleagridis is alive and infective in the litter and determine if this inhibits transmission of blackhead in turkeys.Identify the genetic basis of blackhead disease resistance and sensitivity in chickens and turkeys respectively.As an anaerobic microorganism, H. meleagridis cannot survive outside of a host for an extended period of time. Thus, when studying the epidemiology of H. meleagridis, research should also concentrate on the cecal worm Heterakis gallinarum, which is an indispensable host for H. meleagridis. H. gallinarum is a nematode parasite that lives in the cucum of chickens and other galliform birds. It is mildly pathogenic, but eggs from the organism can carry the protozoan parasite H. meleagridis and thus are a key component to studying blackhead disease.Develop a molecular diagnostic test for H. gallinarum.Identify reservoirs of H. gallinarumIdentification of compounds that destroy H. gallinarum eggs in the environment.Determine if worming agents against H. gallinarum can prevent turkeys from developing blackhead disease when infected with worm eggs.
Project Methods
Laboratory Trials: All laboratory trials will be approved by the North Carolina State University Institutional Biosafety Committee.In vitro screening of compounds that affect H. meleagridis growthStrain. A H. meleagridis strain was isolated from a field outbreak in Buford, GA and is used in all animal trials. An attenuated version of this line (BMA) was produced by multiple passages and will be used for laboratory experiments.Treatments and counting. Compounds are prepared fresh before inoculation into Dwyer's media with H. meleagridis cells. Dimetridazole (12.5 ppm) is used as positive control, while water (or solution used to suspend the compound) is used as a negative control. Flasks treated with different compounds are incubated at 40° C and counted using a Neubauer hemocytometer at different time points (from 8 to 48 hrs. There are 3 replicates per treatment, with each flask being counted 6 times. Molecular Diagnostics Test for Heterakis gallinarum Sequencing of H. gallinarum. Samples of the H. gallinarum worm will be obtained and processed using a genomic DNA extraction kit for pure H. gallinarum genomic DNA. This will allow for the sample to be sequenced using a shotgun approach.Heterakis gallinarum primer design. Multiple sets of conventional PCR primers will be developed based on the sequence of the candidate loci. Two types of candidate genes will be chosen: those that are highly conserved will be used to diagnose H. gallinarum, and those which cover a variable region, and along with sequence analysis will allow us to determine if different strains of H. gallinarum exist and tract their movement. These primers will be tested using the H. gallinarum DNA isolated from multiple sources other nematodes relevant to poultry.Validation of qPCR test for H. gallinarum using various strains. Using the H. gallinarum DNA sequence obtained from the previous aim, sets of qPCR primers will be designed in order to develop an assay. The H. gallinarum worms obtained from the previous aim will be incubated to elicit egg production. To test the sensitivity of this assay, eggs will be separated into tubes with differing amounts of eggs. qPCR will be performed using extracting DNA from the different quantities of eggs to generate a standard curve.Identify Blackhead disease reservoirs and their translocation potential Environmental samples, insects, and vermin from commercial chicken and turkey houses with a history of blackhead disease will be tested for the presence of H. gallinarum H. meleagridis.Identifying vectors of H. gallinarum. Used poultry litter will be obtained from a commercial broiler house and tested using H. gallinarum primers to determine its presence. Litter negative for H. gallinarum will be inoculated with H. gallinarum eggs and placed in a chicken isolator (30" x 30" x 30") and kept in a laboratory setting at room temperature. Darkling beetles classified as H. gallinarum-negative will be placed in the chicken isolator and allowed to roam for 24 hours. Beetles will be removed upon completion of the time period and placed in another adjacent chicken isolator with clean litter and allowed to roam for another 24 h. Beetles will be euthanized and tested for the presence of H. gallinarum in order to ascertain if beetles are viable mechanical vectors for H. gallinarum. Beetles will be separated into two groups: "outside" and "inside." Outside beetles will be placed in a sterile bag and rinsed with PBS and hand massaged in order to wash off any plausible H. gallinarum without disrupting the inside of the beetle. Inside beetles will be rinsed off with bleach thrice and then with PBS. Beetles will be placed in a bag and stomached for 1 minute to access the inner contents.Screen for Compounds that Kill Heterakis gallinarum EggsScreen Development and testing. Twenty embryonated and unembryonated H. gallinarum eggs will be suspended in 100 uL of sterile water in microcentrifuge tubes. Eggs will be tested with water, bleach, and Steriplex. Eggs will be visualized microscopically after 1 hour looking for disruption of the H. gallinarum egg surface. Treated unembryonated eggs will be placed in a 0.5% formaldehyde solution and kept at 25oC to determine if these eggs can still embryonate.Animal Trials:All the animal experimentation will be with the approval of the North Carolina State University Institutional Animal Care and Use Committee.Changes in gene expression in response to blackhead in chickens and turkeys. Cecal and liver tissue will be isolated from uninfected and H. meleagridis infected turkeys and chickens at 0, 5, and 10 days post infection. RNA will be isolated from the samples and subject to RNAseq analysis. Data from the turkey and chicken will be compared to identify differences in genes expression between the two species with a focus on known immune genes. Any differences observed will be confirmed by qPCR.Direct Challenge Model. 6 day-old turkey poults will be weighed, wing banded, and randomly transferred to cage with 6 replicates for each treatment. On day 18, birds that will be intracloacally inoculated with 1 mL of culture containing a total of 10,000-100,000 histomonads; while the control birds will be sham inoculated. Normally when histomonas infections are about 10-12 days along, signs of infection are seen in the form of diarrhea, lethargy, and a failure to gain weight. Once signs of the disease are observed in 75% of the control infected birds or treatment birds, the trial will be terminated and all birds will be weighed, euthanized, and necropsied.Lateral Transmission Trials. 20 day-old turkey poults will be weighed, wing banded, and randomly transferred to pens containing pine shaving. There will be 4 replicates per treatment or control. Treatments will consist of uninfected control birds, uninfected control birds on the treatment diet, infected control birds, infected control birds on the treatment diet. On day 18, 5 out of the 20 turkeys will be infected with 1 mL of culture containing a total of 50,000 histomonads, while the other birds will remain uninoculated, but free to interact with the infected birds. Laterally infected birds will be weighed, euthanized, and necropsied as soon as they exhibit any signs of blackhead disease. The trial will be terminate when 80% of the laterally transfected birds in the control cage or any treatment diet have been euthanized because they exhibit signs of blackhead disease.Histomonas meleagridis Vaccination trial. 10 day-old turkey poults will be weighed, wing banded, and randomly transferred to pens containing pine shaving for bedding material. There will be 4 replicates per vaccine treatment or control. On day 18 and day 25 we will inject subcutaneously 200 ug of histomonas protein suspended in 0.5 ml of phosphate buffered saline with and without 0.5 ml of Freund's Complete Adjuvant or intracloacally inoculated with 100,000 attenuated histomonads. Two weeks after the last injection or inoculation, half of the birds will be infected intracloacally with 1 mL of culture containing a total of 30,000 virulent histomonads per bird, while the other half of the birds will be infected after 4 weeks. Once signs of the disease are observed in 75% of the control infected birds or treatment birds, the trial will be terminated and all birds will be weighed, euthanized, and necropsied.Broiler Breeder Uniformity Model. At hatch, Cobb 500 by-product male chicks will be obtained from a local hatchery and immediately placed in floor pens with fresh pinewood shavings. At 10 days of age, chicks will be inoculated intracloacally with 100,000 H. meleagridis cells. Each of the experimental diets will be replicated with 4 pens each containing 20 chicks. The study will be terminated after 35 days when broilers will be weighed to calculate growth parameters.