Development and Evaluation of Selected Chemoprophylaxis Candidates and a Candidate Live-Attenuated Vaccine for Prevention of Histomoniasis in Turkeys

Histomoniasis, commonly known as blackhead disease, has increased in prevalence due to the regulatory ban of prophylactics and therapeutics within the past 30 years. The objective of this thesis was to evaluate the efficacy of selected dietary chemoprophylaxis candidates as well as an in vitro live-attenuated vaccine candidate Histomonas meleagridis for prevention of histomoniasis. Chapter one addresses deoxycholic acid and a biliogenic diet intended to endogenously increase production of this secondary bile acid. Deoxycholic acid was effective in vitro but failed to prevent histomoniasis when evaluated during the in vivo experimental disease trial with turkeys. The biliogenic diet did not reduce disease prevalence. Chapter two addresses dietary inclusion of 0.2% boric acid to prevent disease. The selected concentration of boric acid was unsuccessful in disease prevention. Chapter three addresses the experiments conducted to evaluate select ages, doses, and routes of a candidate live-attenuated vaccine. The live-attenuated vaccine candidate has exhibited slight reduction in histomoniasis severity when administered intracloacally on d14. Although the practicality of this current experimental vaccine administration approach may be limited, further research must be conducted in order to further elucidate conferred immune response and investigate the viability of this vaccine

Effects of Cyclic Chronic Heat Stress on the Expression of Nutrient Transporters in the Jejunum of Modern Broilers and Their Ancestor Wild Jungle Fowl

snibaThe mechanisms associated between growth rate, gut integrity and heat stress (HS) responses are not known. The current study aimed to evaluate the effect of chronic HS on jejunal nutrient transport in slow- (ACRB from 1950), moderate- (95RAN from 1995), rapid-(modern broilers, MRB) growing birds, and their ancestor wild jungle fowl (JF). One-day male chicks (n=150/line) were placed by line in environmentally controlled chambers and kept under the same environmental conditions until d28. On d29, an 8-h daily cyclic HS (36ºC) was applied to half of the chambers, which lasts until d55, while keeping the rest under thermal neutral conditions (TN, 24°C). Jejunum tissues were collected for morphology assessment and molecular analysis of carbohydrate-, amino acid- and fatty acid- transporters. MRB exhibited the highest BW followed by 95RAN under both conditions. HS decreased FI in MRB and 95RAN, which results in lower BW compared to their TN counterparts, however no effect was observed in ACRB and JF. MRB showed greater villus height to crypt depth ratio under both environmental conditions. Molecular analyses showed that GLUT2, 5, 10, and 11 were upregulated in MRB compared to some of the other populations under TN conditions. HS down regulated GLUT2, 10, 11, and 12 in MRB while it increased the expression of GLUT1, 5, 10, and 11 in JF. GLUT2 protein expression was higher in JF compared to ACRB and MRB under TN conditions. It also showed an increase in ACRB but no effect on 95RAN and MRB under HS conditions. ACRB exhibited greater expression of EAAT3 gene as compared to the rest of populations maintained under TN conditions. HS exposure did not alter the gene expression of amino acid transporters in MRB. Gene expression of CD36 and FABP2 was up-regulated in HS JF birds. Protein expression of CD36 was down-regulated in HS JF while no effect was observed in ACRB, 95RAN and MRB. Taken together, these data are the first to show the effect of HS on jejunal expression of nutrient transporters in three broiler populations known to represent 70 years of genetic progress in the poultry industr

In vitro and in vivo evaluation of chlorhexidine salts as potential alternatives to potassium dichromate for Eimeria maxima M6 oocyst preservation

IntroductionCoccidiosis caused by the Eimeria spp., an Apicomplexan protozoon, is a major intestinal disease that affects the poultry industry. Although most cases of coccidiosis are subclinical, Eimeria infections impair bird health and decrease overall performance, which can result in compromised welfare and major economic losses. Viable sporulated Eimeria oocysts are required for challenge studies and live coccidiosis vaccines. Potassium dichromate (PDC) is typically used as a preservative for these stocks during storage. Although effective and inexpensive, PDC is also toxic and carcinogenic. Chlorhexidine (CHX) salts may be a possible alternative, as this is a widely used disinfectant with less toxicity and no known carcinogenic associationsMethodsIn vitro testing of CHX gluconate and CHX digluconate exhibited comparable oocyst integrity and viability maintenance with equivalent bacteriostatic and bactericidal activity to PDC. Subsequent use of CHX gluconate or digluconate-preserved Eimeria oocysts, cold-stored at 4°C for 5 months, as the inoculum also resulted in similar oocyst shedding and recovery rates when compared to PDC-preserved oocysts.Results and discussionThese data show that using 0.20% CHX gluconate could be a suitable replacement for PDC. Additionally, autofluorescence was used as a method to evaluate oocyst viability. Administration of artificially aged oocysts exhibiting >99% autofluorescence from each preserved treatment resulted in no oocyst output for CHX salt groups

Investigation of Histomoniasis Prevention in Poultry

Histomonas meleagridis is the etiological agent of histomoniasis, also commonly known as blackhead disease. This protozoal disease of poultry is detrimental to turkeys with flock mortalities often reaching 80-100%, although other gallinaceous birds are susceptible. Since the voluntary removal of nitarsone in 2015, the poultry industry is suffering with no approved prophylactics, therapeutics, or vaccines for this disease. The objectives of this dissertation were to evaluate multiple methods for prevention or control of histomoniasis, including dietary chemoprophylaxis and vaccination. Specifically, this research evaluated quinine as a chemoprophylactic candidate (Chapter 3) or live-attenuated H. meleagridis as vaccine candidates (Chapter 4) in an experimental challenge model. Quinine is an antiprotozoal phytochemical that has previously shown efficacy against Plasmodium spp., the etiological agent of poultry malaria; therefore, quinine was hypothesized to confer antihistomonal properties. Quinine effectively reduced (P \u3c 0.05) viable histomonads in vitro but did not mitigate histomoniasis when evaluated in vivo, as indicated by similar (P \u3e 0.05) post-challenge body weight gain (BWG), mortalities, and lesion scores (LS) as compared to the positive-challenged control (PC) group. Taken together, these data suggested that phytochemicals should be further evaluated against histomoniasis but both in vitro and in vivo studies should be conducted against multiple isolates. Four experiments were conducted to evaluate age, route, and administration dose of selected live-attenuated H. meleagridis isolates as vaccine candidates (Vacc) against homologous or heterologous wild-type H. meleagridis (WTH) challenge in turkeys. Day-of-hatch administration of Vacc did not confer protection against subsequent WTH-challenge; however, WTH-challenge at day-of-hatch via the oral route induced histomoniasis whereas oral challenge at d21 did not induce disease. When administered intracloacally at d14 at a dose of 2 x 105 cells/turkey, the Vacc isolates generally resulted in reduced (P \u3c 0.05) disease severity during Challenge Phase, as indicated by lower mortalities, lower LS, and improved BWG as compared to the PC group. During Vaccination Phases, the Vacc groups resulted in lower LS and mortalities (P \u3c 0.05) as compared to the PC group and were not different (P \u3e 0.05) as compared to the non-challenged control group, indicating the reduced virulence and apparent safety of Vacc isolates for application to turkeys. Furthermore, d14 intracloacal vaccination offered some protection against homologous and heterologous WTH-challenge. Overall, this research demonstrated vaccination against histomoniasis to be possible although protection was not robust

Effect of Morinda citrifolia (Noni)-Enriched Diet on Hepatic Heat Shock Protein and Lipid Metabolism-Related Genes in Heat Stressed Broiler Chickens

Heat stress (HS) has been reported to alter fat deposition in broilers, however the underlying molecular mechanisms are not well-defined. The objectives of the current study were, therefore: (1) to determine the effects of acute (2 h) and chronic (3 weeks) HS on the expression of key molecular signatures involved in hepatic lipogenic and lipolytic programs, and (2) to assess if diet supplementation with dried Noni medicinal plant (0.2% of the diet) modulates these effects. Broilers (480 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to two environmental conditions (heat stress, HS, 35°C vs. thermoneutral condition, TN, 24°C) and fed two diets (control vs. Noni) in a 2 × 2 factorial design. Feed intake and body weights were recorded, and blood and liver samples were collected at 2 h and 3 weeks post-heat exposure. HS depressed feed intake, reduced body weight, and up regulated the hepatic expression of heat shock protein HSP60, HSP70, HSP90 as well as key lipogenic proteins (fatty acid synthase, FASN; acetyl co-A carboxylase alpha, ACCα and ATP citrate lyase, ACLY). HS down regulated the hepatic expression of lipoprotein lipase (LPL) and hepatic triacylglycerol lipase (LIPC), but up-regulated ATGL. Although it did not affect growth performance, Noni supplementation regulated the hepatic expression of lipogenic proteins in a time- and gene-specific manner. Prior to HS, Noni increased ACLY and FASN in the acute and chronic experimental conditions, respectively. During acute HS, Noni increased ACCα, but reduced FASN and ACLY expression. Under chronic HS, Noni up regulated ACCα and FASN but it down regulated ACLY. In vitro studies, using chicken hepatocyte cell lines, showed that HS down-regulated the expression of ACCα, FASN, and ACLY. Treatment with quercetin, one bioactive ingredient in Noni, up-regulated the expression of ACCα, FASN, and ACLY under TN conditions, but it appeared to down-regulate ACCα and increase ACLY levels under HS exposure. In conclusion, our findings indicate that HS induces hepatic lipogenesis in chickens and this effect is probably mediated via HSPs. The modulation of hepatic HSP expression suggest also that Noni might be involved in modulating the stress response in chicken liver