Evaluation of live-attenuated Histomonas meleagridis isolates as vaccine candidates against wild-type challenge

Abstract

Repeated serial in vitro passage of Histomonas meleagridis, the etiological agent of histomoniasis (blackhead) of turkeys, was demonstrated to markedly achieve attenuation and reduction of virulence as compared to the original wild-type isolate. Four experiments were performed to evaluate the route (oral vs. intracloacal) and age (day-of-hatch vs. d 14) for administration of attenuated H. meleagridis isolates as vaccine candidates against homologous or heterologous wild-type challenge. Attenuated H. meleagridis were developed from 2 different strains (Buford strain originating in Georgia; PHL2017 strain originating in Northwest Arkansas). Buford P80a (passage 80, assigned as isolate lineage "a" following repeated passage) was selected as the primary vaccine candidate and was evaluated in Experiments 1-3. Experiment 4 evaluated selected candidates of attenuated PHL2017 (P67, P129) and Buford (P80a, P200a, P138b, P198c) strains against Buford wild-type challenge. As has been demonstrated previously, wild-type H. meleagridis cultures administered orally after 1 day of age were not infective in the current studies, but infection with wild-type cultures could be induced orally at day-of-hatch. Infection was effectively achieved via the intracloacal route at day-of-hatch and in older turkeys (d 21, d 28-29, d 35). Intracloacal inoculation of turkeys with the attenuated passaged isolates as vaccine candidates at d 14 was shown to produce significant (P < 0.05) protection from mortality, reduction in body weight gain, as well as reduction in hepatic and cecal lesions in these experiments following challenge with either the homologous wild-type isolate or from a wild-type strain obtained years later from a geographically disparate area of the United States. Inoculation with the attenuated H. meleagridis isolates at day-of-hatch, either orally or cloacally, did not produce significant protection against subsequent wild-type challenge. While offering significant protection with minimal vaccine-related negative effects, the protection from cloacal vaccine administration was neither significantly robust nor encouraging for industry application using the methods evaluated in the present manuscript since mortalities and lesions were not completely reduced which could thereby potentially allow transmission from residual infection and shedding within a flock.

Keywords: Histomonas meleagridis; histomoniasis; live-attenuated; turkey; vaccination.

Figure 1

Experimental timelines for administration of live-attenuated vaccine candidate (Vacc) Histomonas meleagridis and subsequent challenge with Buford strain wild-type H. meleagridis (WTH). The Buford strain was isolated from infected chickens in Georgia; PHL2017 strain was isolated from infected turkeys in Arkansas. Vacc passage number and isolate indicator are included in each group name, where applicable. Abbreviations: C-PC, Challenge Phase positive-challenged control; NC, non-challenged control; V-PC, Vaccination Phase positive-challenged control.

Figure 2

Experiment 1 frequency of lesion scores during Challenge Phase for (A) liver and (B) cecae. Numbers within columns indicate the number of turkeys per evaluated lesion score. Numbers at the top of each column indicate the lesion score mean ± SE for that group with different superscripts denoting significance (P < 0.05). Lesion scores were based on a scale of “0” to “3” and were analyzed using the Proc Mixed Procedure in SAS 9.4 software. Challenge Phase began on d 29 when the Vacc and C-PC group were intracloacally challenged with 2 × 10⁵ Buford WTH cells/turkey. Abbreviations: C-PC, Challenge Phase positive-challenged control; NC, non-challenged control; Vacc, live-attenuated Histomonas meleagridis; WTH, wild-type H. meleagridis.

Figure 3

Experiment 2 frequency of lesion scores during Vaccination Phase for (A) liver and (B) cecae and during Challenge Phase for (C) liver and (D) cecae. Numbers within columns indicate the number of turkeys per evaluated lesion score. Numbers at the top of each column indicate the lesion score mean ± SE for that group with different superscripts denoting significance (P < 0.05). Lesion scores were based on a scale of “0” to “3” and were analyzed using the Proc Mixed Procedure in SAS 9.4 software. Vaccination Phase began on d 0 with administration of respective dose and route of either Vacc Buford P80a or Buford WTH cells/turkey; Challenge Phase began on d 21 with the intracloacal administration of 2 × 10⁵ Buford WTH cells/turkey to all groups, except for C-PC Oral 200k which received the dose orally. Turkeys from the NC group were redistributed to form the new C-PC groups for the Challenge Phase. Abbreviations: C-PC, Challenge Phase positive-challenged control; V-PC, Vaccination Phase positive-challenged control; Vacc, live-attenuated Histomonas meleagridis; WTH, wild-type H. meleagridis.

Figure 4

Experiment 3 frequency of lesion scores during Vaccination Phase 1 for (A) liver and (B) cecae; Vaccination Phase 2 for (C) liver and (D) cecae; Challenge Phase for (E) liver and (F) cecae. Numbers within columns indicate the number of turkeys per evaluated lesion score. Numbers at the top of each column indicate the lesion score mean ± SE for that group with different superscripts denoting significance (P < 0.05). Lesion scores were based on a scale of “0” to “3” and were analyzed using the Proc Mixed Procedure in SAS 9.4 software. Vaccination Phase 1 began on d 0 with administration of 2 × 10⁵ cells/turkey of either Vacc Buford P80a or Buford WTH cells/turkey via respective route; Vaccination Phase 2 began on d 14 with the introduction of a d 14 Vacc group and new PC (formed from subsets of the NC) which received intracloacal administration of 2 × 10⁵ either Vacc Buford P80a or Buford WTH cells/turkey, respectively; Challenge Phase began on d 28 with the intracloacal administration of 2 × 10⁵ Buford WTH cells/turkey. Abbreviations: C-PC, Challenge Phase positive-challenged control; NC, non-challenged control; V1-PC, Vaccination Phase 1 positive-challenged control; V2-PC = Vaccination Phase 2 positive-challenged control; Vacc, live-attenuated Histomonas meleagridis; WTH, wild-type H. meleagridis.

Figure 5

Experiment 4 frequency of lesion scores during Vaccination Phase for (A) liver and (B) cecae on d 28; Vaccination Phase for (C) liver and (D) cecae on d 35; Challenge Phase for (E) liver and (F) cecae. Numbers within columns indicate the number of turkeys per evaluated lesion score. Numbers at the top of each column indicate the lesion score mean ± SE for that group with different superscripts denoting significance (P < 0.05). Lesion scores were based on a scale of “0” to “3” and were analyzed using the Proc Mixed Procedure in SAS 9.4 software. Vaccination Phase began on d 14 with the intracloacal administration of 2 × 10⁵ cells/turkey of the respective Vacc isolate while the PC Buford received the same dose of Buford WTH; Challenge Phase began on d 35 with the intracloacal administration of 2 × 10⁵ Buford WTH cells/turkey. Abbreviations: C-PC, Challenge Phase positive-challenged control; NC, non-challenged control; V-PC, Vaccination Phase positive-challenged control; Vacc, live-attenuated Histomonas meleagridis; WTH, wild-type H. meleagridis.