Parasitol Res (2009) 105:113116 DOI 10.

1007/s00436-009-1369-1

ORIGINAL PAPER

Susceptibility of different turkey lines to Histomonas meleagridis after experimental infection

Lotfi AbdulRahman & Hafez Mohamed Hafez

Received: 8 December 2008 / Accepted: 2 February 2009 / Published online: 17 February 2009 # Springer-Verlag 2009

Abstract In the present investigation, three turkey lines, namely wild Canadian turkeys (WCT), British United turkey (BUT-Big6) and KellyBronze turkeys (KBT) were compared for their susceptibility to infection with Histomonas meleagridis. All birds were kept on wood shaving as litter from day 1 on during the entire observation period. On day 28, 1820 birds per turkey line were infected with H. meleagridis intracloacally. All birds were observed for 4 weeks after infection. The mortality rate was 95% in WCT, 78% in BUT-Big6 and 75% in KBT. In WCT, the first deaths occurred at day 6 and ended at day 13 postinfection, whilst for BUT-Big6 and KBT, birds died from days 10 to day 20. In KBT group, the mortality started at day 10 and lasted until day 17 after infection. At necropsy, all birds that died showed lesions typical for histomoniasis in the caeca and liver. The obtained results demonstrate that all tested turkey lines are susceptible to infection; however, the mortality rate for the wild Canadian turkey is statistically significantly higher compared to the other tested two lines.

Introduction Blackhead or histomoniasis is a protozoan disease primarily of turkeys. However, several other species like chickens, quail and peafowl are also susceptible. In the past, histomoniasis has been controlled very well with
L. AbdulRahman : H. M. Hafez (*) Institute of Poultry Diseases, Faculty of Veterinary Medicine, Free University Berlin, Knigsweg. 63, 14163 Berlin, Germany e-mail: hafez@vetmed.fu-berlin.de

Dimetridazole as a treatment and/or Nifursol, which was used as a feed additive. In the European Union, the last therapeutic drug against histomoniasis was banned in 1995 and the last prophylactic drug in 2003. Since then, several outbreaks of histomoniasis in turkey flocks caused considerable economic losses. Cases in chickens and game birds have occurred less frequently (Hafez et al. 2005a). In turkeys, the disease is characterised by listlessness, anorexia, droopy wings and yellow-sulfur-coloured faeces. Infections with blackhead in turkeys are often fatal, with high morbidity in survivors. It is the most fatal parasitic disease of turkeys capable of causing mortality up to 100%. Histomonas as parasite depends on complicated interactions with caecal worms, earthworms, intestinal microflora and coccidia to spread from flock to flock and to develop full pathogenicity (McDougald 2003). Currently, the control of histomoniasis is based on good hygienic measures at farm level, on using drugs that reduce the presence of caecal worms and other herbal products with variable results. The genetics of resistance to disease is an area of great interest in several animal species and poultry. Selective breeding for resistance to pathogens in animals has demonstrated that resistance and susceptibility to pathogens are controlled by both genetic and environmental factors. Genetic resistance to disease is a widespread phenomenon in all animal species due to the genetic control of the immune system. Especially in poultry, it is often possible to find individual differences in susceptibility to most diseases (Bumstead et al. 1991). Most of the investigations on genetic resistance to disease in poultry have been carried out in chickens and to less extent in turkeys. They focused primarily on viral and/or bacterial diseases. However, some studies have been on the genetic mechanisms involved in resistance to coccidial infections and to Ascaridia galli in chickens (Pinard-Van der Laan et al. 1998; Permin and

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Ranvig 2001). In addition, Curtice (1907) showed that chickens are less susceptible to Histomoniasis than turkeys, but more important in disseminating Histomonas meleagridis and Heterakis gallinarum. Furthermore, Al-Khateeb and Hansen (1974) showed between chicken breeds some differences in the susceptibility to histomoniasis after intracloacal experimental infection. Recently, Reynaud et al. (2005) compared the susceptibility of an industrial heavyweight turkey strain with a traditional lightweight strain to H. meleagridis infection. The response to infection of the heavyweight strain was in every way comparable to that of the traditional lightweight strain, with exception in the course of the infection. The 100% mortality was attained 7 days later in the industrial heavyweight turkey strain compared to traditional light turkey strain. The present investigation was carried out to compare the pathogenicity of H. meleagridis in three different turkey lines.

Results In WCT, the deaths started by day 6 and ended by day 13 post-infection, whilst in BUT-Big 6, the mortality started on day 10 and the last deaths observed at day 20 postinfection. In KBT, the deaths took place from day 10 to day 17 (Table 1). The mortality rate was 95% in WCT, 78% in BUT-Big 6 and 75% in KBT. The mortality rate of the wild Canadian turkey was statistically significantly higher compared to the other tested two lines (Table 1): WCT vs. BUT-Big 6 (P =0.001); WCT vs. KBT (P <0.001) and KBT vs. BUT (P =0.837).

Table 1 Course of mortalities in WCT, BUT-Big 6 and KBT Days p.i. No. of dead birds per day per group WCT BUT-Big6 0 0 0 0 0 0 0 0 0 4 2 3 0 1 1 1 0 0 1 1 0 0 0 0 0 0 0 0 14 77.8 18 KBT 0 0 0 0 0 0 0 0 0 3 0 2 5 0 2 1 2 0 0 0 0 0 0 0 0 0 0 0 15 75.0 20

Materials and methods Three turkey lines, namely British United turkey (BUTBig6), KellyBronze turkeys (KBT) and wild Canadian turkeys (WCT), were used. WCT were bred in captivity for about 30 inbred generations and not intentionally selected. Eighteen to 20 birds per turkey line were used. The birds were kept from the first day on wood shave as litter. On day 28, the birds were infected with H. meleagridis strain Turkey/Germany/GB 551/04, which was isolated from naturally infected meat turkey flock and confirmed using real-time polymerase chain reaction (PCR; Hafez et al. 2005b). The strain was proven to be free from Tetratrichomonas gallinarum and Blastocytis sp. using PCR as described by Grabensteiner and Hess (2006). Each bird received 1 ml modified Dwyers medium (Van der Heijden et al. 2005) containing 300,000 histomonads intracloacally. All birds were observed for 4 weeks after infection and daily mortality was recorded. Postmortem was conducted on all birds that died. After 4 weeks, the surviving birds were euthanised and examined for pathological changes. Lesions in caeca and liver were scored according to McDougald and Hu (2001). From all turkeys which died in the course of the study and survivals that were euthanised at the end of the trial, samples were taken and tested using real-time PCR. Statistical methods The KaplanMeier method was used to construct survival curves, which were then compared by the log-rank test, with correction for multiple comparisons. A value of P < 0.05 was considered significant.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Total deaths Percentage No. of birds/group 0 0 0 0 0 1 1 3 4 5 1 2 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 18 94.7 19

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At necropsy, all birds that died showed lesions typical for histomoniasis in the caeca and liver. No further gross lesions could be observed in any other organs. The mean lesions score of died wild Canadian Turkey was 4 for caeca and 3 for livers, and in BUT-Big 6 and KellyBronze turkeys, the score was 4 for both caeca and liver. The surviving birds that had been euthanatised did not show neither clinical signs nor gross lesions, and histomonad DNA could not be detected in their caeca and livers using real-time PCR.

Discussion In addition to management practises, like separation of turkeys and chickens and indoor rearing, histomoniasis has been very well controlled with a variety of therapeutic and prophylactic drugs in the last 35 years. Since the ban of all therapeutic and prophylactic drugs in the European Union, several outbreaks of histomoniasis in turkey flocks causing considerable economic losses were observed (Hafez et al. 2005b). It was shown that in chickens, some antibiotics possess a limited efficacy against H. meleagridis, whilst tested anticoccidials had almost no effect after experimental infection with H. meleagridis and caecal coccidia (Hu and McDougald 2002, 2004). As the chance to register another nitroimidazole for use in turkeys seems to be limited, new interventions toward prophylaxis and therapy are required and other approaches have to be found. Considerable interest has been focused on alternative plant-derived products. Several investigations were carried out in vitro as well as in vivo and showed variable results (Chossat 2002; Zenner et al. 2003; Duffy et al. 2005; Hafez and Hauck 2006; Grabensteiner et al. 2007; Hauck and Hafez 2007). Concerning the genetic resistance or susceptibility of different poultry species to infection with H. meleagridis, only few literatures are available. Curtice (1907) showed that chickens are less susceptible to histomoniasis than turkeys, but more important in disseminating H. meleagridis and H. gallinarum. In addition, Al-Khateeb and Hansen (1974) showed that Babcock chickens were more susceptible to histomoniasis than were Cornish Rocks after intracloacal experimental infection. Similar results were obtained by Chute et al. (1976) by comparing the responses of White Leghorn, New Hampshire chickens and Beltsville small white turkeys to experimental infections with H. meleagridis and H. gallinarum. The infection in Beltsville small white turkeys caused liver lesions and high mortality, whilst in chickens, only caecal lesions without mortality. On the other hand, the incidence of the infection was higher in White Leghorn than in the New Hampshire.

Reynaud et al. (2005) compared the susceptibility to H. meleagridis infection of two turkey lines, an industrial heavyweight strain (H.W.) and a traditional lightweight strain; without given additional background information about the examined turkey strains, both strains were highly susceptible to the infection. The 100% mortality, however, was attained 7 days later in the H.W. strain. At necropsy, there was no difference in the severity of gross and histological lesions between the two different turkey lines. The obtained results in the present investigation demonstrate that all tested turkey lines are susceptible to infection; however, the mortality rate of the wild Canadian turkey was statistically significantly higher compared to the other tested two lines. The mortality rate within the three lines varied between 75% and 95%. Van der Heijden and Landman (2008) showed that using lower infectious dosage (3,162 histomonads/bird) can induce 100% mortality in BUT-Big 6 turkey poults after experimental infection at second week of age. They speculated that the relative high virulence of the strain used might explain the fact that mortality was high despite a relatively low inoculation dose. In general, the mortality rate after experimental infection could be varied in accordance to several factors such as age and genetic origin of the birds, the infectious dose and isolates used (Lund 1955; Hu et al. 2004; Hauck et al. 2006; Hess et al. 2006). The liver lesion scores obtained in BUT-Big 6 and KBT were higher than those obtained in WTC, even though the mortality was much less in those two lines. One possible explanation is that WTC are less tolerant, not to the parasite itself but to the lesions induced by it. Therefore, a milder degree of liver destruction might cause mortality. The current lack of effective tools to treat histomoniasis in turkey flocks under field conditions and the high susceptibility of the all tested turkey lines to the experimental infection show paramount importance in finding new effective drugs.

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