Abstract
Histomoniasis is a serious disease in poultry. All chemotherapeutics with known efficacy against its causative agent, Histomonas meleagridis, have been banned from use as prophylactic or therapeutic use in production animals. In a search for possible alternatives, the in vivo effects of the herbal products Enteroguard™ and Protophyt™ were examined. Two-week-old turkeys allocated into 13 groups of 18 birds were either sham inoculated (negative control group) or were inoculated with 100, 3162 or 200 000 histomonads per bird. Control groups (no feed additives, dimetridazole, or Histostat-50™) were included in the study. No morbidity or mortality was observed in the negative control group or in the groups inoculated with 100 histomonads per bird. Mortality was 100% in the groups inoculated with 200 000 histomonads per bird and either untreated (positive control group) or receiving Protophyt SP™, Protophyt™ SP and Protophyt™ B, Enteroguard™, or Histostat-50™. Mortality was 17% in the dimetridazole-treated group. In the groups inoculated with 3162 histomonads per bird, mortality was 100% for the positive control group and the group receiving Enteroguard™, and was 94% in the group receiving Protophyt™ SP. In the present study, Enteroguard™ or Protophyt™ was not found to be effective against histomoniasis.
Introduction
Histomoniasis (infectious enterohepatitis, blackhead disease) is a disease of galliforms and other species of birds, mainly affecting the liver and caecae. The severity of the disease varies over the different species. In turkey flocks, for example, mortality can be very high, whereas in chickens the symptoms are generally less severe (McDougald, 1997). The causative agent of histomoniasis, Histomonas meleagridis, is transmitted between flocks through the vector Heterakis gallinarum (Graybill & Smith, 1920). Within a flock, direct lateral transmission of H. meleagridis occurs between birds (Hu & McDougald, 2003).
Effective chemotherapeutics such as nifursol and nitroimidazoles are no longer allowed in both the United States and the European Union (McDougald, 2005). In addition, the European Union also banned arsenical compounds (Byrne, 2001). Therefore, there is an urgent need for alternative antihistomonal products (Hu & McDougald, 2004). In the field, herbal products are used to prevent outbreaks of histomoniasis (Hafez & Hauck, 2006). However, there are only a few scientific in vivo studies backing up the efficacy of such products against H. meleagridis. Duffy et al. (2005) observed a positive effect of Natustat™, a yeast-derived mannonoligosaccharide combined with organic mineral nutrients and plant extracts on caecal and liver lesions scores but not on mortality. In a recent study (Hafez & Hauck, 2006), Protophyt™ SP (feed additive) and Protophyt™ B (drinking water additive) were found to reduce mortality following experimental inoculation with H. meleagridis from 50% (untreated group) to 20% (additives), with no mortality in the control group.
In the present study the antihistomonal effects of Enteroguard™ and Protophyt™ are examined on turkey poults that were experimentally inoculated with three different inoculation doses of histomonads. Enteroguard™ is a product based on garlic and cinnamon, with allicin and cinnemaldehyde as possibly active compounds. Protophyt™ is based on volatile oils extracted from garlic, cinnamon, rosemary and lemon.
Materials and Methods
Medicated feed and drinking water
Non-medicated turkey starter feed (no. 94040; Arkervaart-Twente, Nijkerk, The Netherlands) and non-medicated turkey feed (no. 94041; Arkervaart-Twente) were mixed with the different antihistomonal products: either 200 parts/106 dimetridazole (1,2 dimethyl-5-nitroimidazole, D4025; Sigma, Zwijndrecht, The Netherlands) or 375 parts/106 Histostat-50™ (no. 560101; Alpharma Inc., Fort Lee, USA), or 3000 parts/106 Protophyt SP™ (Phytosynthèse, Riom, France) or 500 parts/106 Enteroguard™ (Orffa, Giessen, The Netherlands). To ensure an adequate distribution of the product in the feed, the following feed mixing procedure was used. A maximum quantity of 25 kg feed was mixed per run. After accurate weighing of the product it was transferred to a plastic bag. Approximately 500 g feed was added and thoroughly mixed. Subsequently, the contents of the bag were transferred to a larger plastic bag that contained approximately 2.5 kg feed, followed by thorough mixing. During the next mixing step another 2.5 kg feed was added and mixed again. Thereafter, the contents of the bag were mixed with the remainder of the 25 kg feed for approximately 15 min using a blender (no. 19091; Naturamix, Haarlem, The Netherlands). The medicated feed was collected in a labelled paper bag. All of the medicated feed needed for the whole experimental period, except for the dimetridazole feed, was produced within 2 days and was stored at room temperature until use. The dimetridazole feed was freshly mixed every week and stored at 4°C in the dark, as recommended by the manufacturer. The blender was thoroughly cleaned between products using a brush, followed by operation of the blender with 5 kg non-medicated feed that was subsequently discarded.
Drinking water with a herbal additive (Protophyt™ B) was freshly prepared every day by adding 2 ml Protophyt B™ per litre of water.
Assessment of feed mixing procedure
Samples were taken from the feeds with dimetridazole and Histostat-50™ and were analysed for dimetridazole and arsenic content to assess the mixing procedure.
Dimetridazole was quantified by high-performance liquid chromatography after extraction with acetonitrile/methanol. Briefly, 5 g feed was moistened with 15.0 ml water. After 5 min, 35.0 ml of a mixture (1:1) of acetonitrile (Fisher Scientific, Loughborough, UK) and methanol (Biosolve, Valkenswaard, The Netherlands) was added. The suspension was shaken for 30 min at room temperature. The extract was filtered through a paper filter (Schleicher, Dassel, Germany) and eluted over 4 g neutral alumoniumoxide (Fisher Scientific) packed in a glass column (25 ml). The first 2 ml eluted filtrate was not used. The extract was diluted 10 times with mobile phase. The mobile phase consisted of 170 ml sodium-acetate buffer (0.01 mol/l pH 6.0) with 30 ml acetonitrile. The high-performance liquid chromatography column was a 3.0 mm C18 Chromspher packed with 40 µm reversed phase material (Varian, Middelburg, The Netherlands). Dimetridazole was quantified against standard material (Rhône-Poulenc, Amstelveen, The Netherlands) at 320 nm. All handling was done under yellow light to protect degradation of dimetridazole. The detection limit was 0.8 mg/kg. The recovery of the method is between 90% and 95% in the range of 2 to 500 mg/kg.
Arsene was determined by Inductively Coupled Plasma (ICP). Briefly, 1 g feed to which 6 ml of 70% HNO3 (Baker Chemicals, Deventer, The Netherlands) had been added was destructed in a magnetron digestion unit (Milestone Inc., Shelton, USA) until a clear destruate remained. After complete solubilization, 50 ml water was added. Arsene was measured with an ICP analyser (Optima 3300 DV; Perkin Elmer, Waltham, USA).
Experimental set-up
In two separate rooms (6.85×4.48 m2), room A and room B, 13 stainless steel wire pens (1.3×1 m2) were constructed. On the left side of both rooms four pens (groups 1 to 4, and groups 8 to 11, respectively) were positioned at a distance of 40 cm, while on the right side of the rooms three pens (groups 5 to 7) or two pens (groups 12 and 13) were placed, also at a distance of 40 cm. The side walls of all pens were covered with plastic to minimize the risk of cross-infections between pens. Approximately 8 cm wood shavings was used as bedding litter. The experimental rooms were only accessible for qualified personnel, and involved changing of footwear and overall clothing.
One-day-old BUT Big 6 poults (120 females and 120 males) were kept separately in two pens for 1 week, with non-medicated drinking water and feed (turkey starter) ad libitum. The lighting program was 2 h of darkness and 22 h of light.
After 1 week all birds were tagged (Swifttack), individually weighed and divided into six weight classes per sex. All birds of each weight class were then randomly distributed over the 13 pens. Each group consisted of nine female and nine male poults. Until the termination of the experiment at the age of 6 weeks, light was provided for 16 h per day.
The experimental groups 1 to 7 (Table 1) in room A were given a “standard” inoculation dose of 200 000 histomonads per bird, whereas the birds in room B received either a low inoculation dose of 3162 histomonads per bird (groups 8, 10, and 12) or a very low inoculation dose of 100 histomonads per bird (groups 9, 11, and 13). The inoculations were done during two consecutive days in room A, and room B, respectively. The negative control group (group 2, no treatment and no inoculation) was directly positioned next to the positive control group (group 1, inoculated but not treated). During handling of the animals throughout the experiment (feed supply, removal of dead birds from the pens, etc.) the negative control group was always handled last in order to detect possible cross-infection.
Table 1. Experimental groups and inoculation dose
At the age of 7 days (i.e. at transfer of the animals to the experimental groups), the starter feed with additives was supplied ad libitum. However, non-medicated feed and drinking water was continued in group 7 until 2 days before inoculation, when starter feed with Protophyt SP™ and drinking water with Protophyt B™ was supplied to the birds.
At the time of inoculation at 2 weeks of age, all groups changed from turkey starter feed to turkey feed with the appropriate feed additive for the remainder of the experimental period (until 6 weeks of age).
After inoculation, the groups were inspected three times a day and dead birds removed from the pens. When birds were very sick and did not eat for two consecutive inspections, these were euthanized by injection with T61.
H. meleagridis strain
Strain /Deventer/NL/AL327-type I/03 (Van der Heijden et al., 2006), a Dutch field strain that was successfully propagated in culture (Van der Heijden et al., 2005), was used for inoculation. On two consecutive days, the isolate was resuscitated, cultured in Dwyer's medium (Dwyer, 1970), and subcultured once in modified Dwyer's medium (Van der Heijden et al., 2007) consisting of Medium 199 with Hanks salts (Gibco-BRL, Grand Island, USA) with 10% heat-inactivated horse serum (Gibco-BRL) and 0.096% w/v white rice powder (Arrowhead Mills Inc., Hereford, USA). After pooling of the contents of four tissue culture flasks, a 1:5 pre-dilution was made in pre-warmed Medium 199 with Hanks salts and counted using a Bürker-Türk haemocytometer and phase contrast microscopy at 200x magnification. Subsequently, the suspensions were further diluted in M199 medium to obtain a concentration of 200 000 histomonads/ml and were counted before and after the inoculation on both inoculation days. On the second inoculation day, the 200 000 histomonads/ml suspension was further diluted in order to obtain the desired inoculation doses (Table 1). The inoculation doses were aliquoted for each experimental group by pipetting 25 ml into pre-warmed 50 ml tubes that were kept warm until use.
Inoculation
The turkeys were essentially inoculated by the method of Chappel (1975). The birds were inoculated intracloacally with 1 ml histomonad suspension using a 10 ml syringe (Pharma-Plast) with a blunt tip. After inoculation a finger was placed over the cloaca for 30 sec to prevent voiding of the inoculum. Subsequently the birds were suspended in inverted position for a maximum of 5 min but at least for the time that the cloaca was observed pulsating (usually for approximately 1 min after stimulation). In only three out of 234 cases was voiding of the inoculum seen (birds in groups 3, 7 and 12), after which these turkeys received a new inoculum. No birds were seen discharging after return to their pens. The negative control group was inoculated with 1 ml M199 medium instead of histomonads.
Postmortem examination
Thorough postmortem examination was performed on all experimental birds either during the experiment (in case of mortality or severe disease) or at the end of the study at the age of 6 weeks. Birds that died during the experiment were kept at 4°C before being subjected to postmortem examination within 1 or 2 days.
Six weeks after inoculation the remaining turkeys were transported in boxes to the postmortem room and euthanized using carbon dioxide. At necropsy, liver and caecal lesions were scored on a scale ranging from 0 (no lesions) to 4 (McDougald & Hu, 2001).
Statistical analysis
The time of survival in the groups with different treatments was analysed using one-way analysis of variance. Group means were adjusted using Bonferroni's method. The residuals were assessed for being normally distributed. The liver and caecum lesion scores between groups were analysed using the non-parametric Kruskal–Wallis comparison test. Statistical analysis was performed using Statistix 8 (Analytical Software, Tallahassee, USA).
Results
Assessment of the feed mixing procedure
The average dimitridazole level of the feed mixed with dimetridazole was 182 parts/106 (desired dose 200 parts/106), while the average arsenic level of the feed mixed with Histostat-50™ was 68 parts/106 (desired dose 63 parts/106), which corresponds to 405 parts/106 Histostat-50™ (desired dose 375 parts/106).
H. meleagridis inoculation
Not a single turkey died in the negative control group (no treatment, sham inoculated) and at postmortem the livers and caecae of all birds did not show abnormalities (all were scored 0). In the experimental groups inoculated with the standard dose of 200 000 histomonads per animal (Figure 1), mortality was high in most of the groups. The first turkeys died from histomoniasis around day 10 post-inoculation (p.i.). In the positive control group (no feed additive) and in the groups treated with either Protophyt SP™ or Enteroguard™ at day 13 p.i. approximately 50% of the animals had died, while 50% mortality was (slightly) delayed by 1 or 2 days in the experimental group with combined therapy with Protophyt SP™ and Protophyt B™ and in the group treated with Histostat-50™. At days 16 to 18 p.i. almost all animals had died in the positive control group and in the groups treated with either Protophyt™ or Enteroguard™, while 100% mortality was delayed for 3 days in the group medicated with Histostat-50™. In the experimental group that received dimetridazole, the first two turkeys died at 17 days p.i. Nevertheless, mortality in this group was limited to 17% at 30 days p.i. when the experiment was terminated. Surprisingly, 13 of the 15 surviving animals in this group presented typical lesions in the caecae and liver at necropsy.
Published online:
16 January 2008Figure 1. Survival of turkeys in experimental groups following inoculation with 200 000 histomonads per turkey.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2008/cavp20.v037.i01/03079450701784883/20160222/images/medium/cavp_a_278514_o_f0001g.gif"})
Figure 1. Survival of turkeys in experimental groups following inoculation with 200 000 histomonads per turkey.
The different treatments of the groups in which the turkeys were inoculated with 200 000 histomonads per bird had a significant effect (one-way analysis of variance, P<0.05) on the time of survival—and also when the dimetridazole group was left out of the analyses (Table 2). Only in the groups receiving dimetridazole or Histostat-50™ did birds die significantly later than in the positive control group (Bonferroni's pair-wise comparisons test, P<0.05). Among these groups the effect of treatment was significant for the liver lesion scores (Kruskal–Wallis, P<0.05) but not for caecal lesion scores (P>0.05). Pair-wise comparisons revealed no differences between the groups in liver lesion scores.
Table 2. Comparison of mortality day and caeca and liver lesions scores of turkeys that died following inoculation with H. meleagridis
The results in the three groups inoculated with the low dose of 3162 histomonads per animal (Figure 2) were very similar. The first turkeys died in the positive control group (no feed additive) at 7 days p.i., even prior to the positive control group inoculated with 200 000 histomonads per animal. In the other two groups treated with Protophyt SP™ or Enteroguard™, mortality was first seen at 9 or 12 days p.i., respectively. In all three groups 50% mortality occurred at 15 days p.i., which was 2 days later than the groups inoculated with 200 000 histomonads per animal. Finally, maximum mortality occurred at 19 days p.i., also slightly delayed compared with the groups inoculated with 200 000 histomonads per bird. The single turkey in the Protophyt SP™ group that survived throughout the experiment had no lesions in the liver or caecae upon necropsy.
Published online:
16 January 2008Figure 2. Survival of turkeys in experimental groups following inoculation with 3162 histomonads per turkey.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2008/cavp20.v037.i01/03079450701784883/20160222/images/medium/cavp_a_278514_o_f0002g.gif"})
Figure 2. Survival of turkeys in experimental groups following inoculation with 3162 histomonads per turkey.
No significant effect of treatment in the groups inoculated with 3162 histomonads per bird was found, either for number of days of survival (one-way analysis of variance, P>0.05) or for caecal or liver lesion scores (Kruskal–Wallis, P > 0.05).
In the three experimental groups inoculated with 100 histomonads per animal and either non-treated (positive control, no feed additive) or treated with Enteroguard™ or Protophyt SP™, no mortality was seen and the livers and caecae were normal (score 0) at postmortem.
No differences were seen in mortality of female or male turkeys after experimental inoculation with H. meleagridis (Figure 3). Additionally, there was no significant relation (regression analysis, P>0.05) between the weight of the poults at the start of the experiment and the number of days the bird survived inoculation, even if female and males results were analysed separately. In the groups inoculated with 100 histomonads per bird (i.e. no mortality), the overall consumption of feed with either no additive, Protophyt™ SP or Enteroguard was 32.5, 33.1, or 32.3 kg/group, respectively.
Published online:
16 January 2008Figure 3. Comparison of survival of male and female turkeys among all groups after experimental inoculation with H. meleagridis.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2008/cavp20.v037.i01/03079450701784883/20160222/images/medium/cavp_a_278514_o_f0003g.gif"})
Figure 3. Comparison of survival of male and female turkeys among all groups after experimental inoculation with H. meleagridis.
Discussion
The ban of effective chemotherapeutics against H. meleagridis in commercial poultry (Byrne, 2001) propelled the search for alternative antihistomonal products. In the present study, two herbal products were tested for a possible anti-histomonal effect. However, both Protophyt™ and Enteroguard™ had no effect on mortality following inoculation of turkey poults with 200 000 histomonads per bird. In the positive control group, in the groups receiving either Enteroguard™ or Protophyt™ SP as a feed additive, and in the group of turkeys that received Protophyt™ SP as feed additive and Protophyt™ B in drinking water, mortality was 100%. The latter finding is in agreement with the results of a field study in turkeys (Chossat, 2002), but in contrast with a recent in vivo experiment (Hafez & Hauck, 2006). In the latter study turkeys provided with Protophyt™ SP in the feed 6 days before inoculation and Protophyt™ B in the drinking water 3 days before inoculation showed 20% mortality, instead of 50% mortality in the positive control group, after experimental inoculation with 147 500 histomonads per bird. This difference was only just significant (chi-square, P=0.047). Moreover, their birds were housed in cages, making re-infections through direct lateral transfer as under practical conditions less likely. The only positive effect of the Protophyt™ SP and Protophyt™ B treatment in the present study was a slight delay in mortality of 1 or 2 days in comparison with the other groups. Here, both Protophyt™ SP and Protophyt™ B were supplied only 2 days before inoculation. Possibly more time is needed for the products to partly protect birds against histomoniasis following an inoculation with H. meleagridis.
Surprisingly, the mortality in the group medicated with Histostat-50™ was 100%. Nitarsone (4-nitrophenyl-arsonic acid) is the active compound of this product, and has been reported to be highly effective for preventive use in feed, although it is probably histomonastatic since a relapse has been described after withdrawal of medication (McGuire & Morehouse, 1952). In addition, some mortality was seen in the experimental group medicated with dimetridazole, especially toward the end of the experiment at 4 weeks after inoculation. Moreover, a large proportion of the surviving turkeys showed severe lesions at necropsy and it is probable that more turkeys would have died had the experiment been continued. Since dimetridazole is considered highly effective (McDougald, 2005) this result was unexpected. It is unlikely that this was caused by a slightly lower dose of dimetridazole (182 parts/106 instead of 200 parts/106) in the feed since the 50% protective dose of dimetridazole was found to be 38 parts/106 (Lucas, 1961), and with 100 parts/106 (McGuire et al., 1963) or 125 parts/106 (Lucas & Goose, 1965) close to 100% protection against histomoniasis was obtained. Also, the recovery of the dimetridazole test was determined during the validation of the assay and found to be 87% in the range of 5 to 100 parts/106. It is expected that for higher concentrations up to 200 parts/106 the recovery is approximately the same. Therefore, the actual dimetridazole concentration in the feed was probably close to the desired 200 parts/106.
A dose–response relation between the size of the infective dose and mortality was reported by Lund (1955). After inoculation with 105, 104, 103, 102, or 10 histomonads per turkey, mortality was 100%, 82%, 25%, 5%, and 5%, respectively. But the infective dose in itself probably also does not explain the results of the present experiment. Most inoculation studies used infective doses between 105 and 106 histomonads per animal (McDougald, 2005). In addition, in the present experiment a lower (3162) and a much lower (100) inoculation dose were examined, although only in positive control groups and in groups of birds having either Protophyt SP™ or Enteroguard™ as a feed additive. In the groups inoculated with 3162 histomonads per bird, again all birds of the positive control group and the group treated with Enteroguard™ died. In the group of turkeys that was treated with Protophyt SP™ only a single bird survived the experiment. Thus, also at lower inoculation doses, the herbal products had no effect or hardly any effect on mortality. In all three groups inoculated with 100 histomonads per bird, however, not a single bird died during the experiment or showed lesions at necropsy.
The relative high virulence of the strain used might explain the fact that mortality was high, despite a relatively low inoculation dose. This might also be the cause of the considerable morbidity in the dimetridazole-treated group towards the end of the experiment. This is speculative, however, since there have been no reports on the effect of chemotherapeutics on strains of different virulence, and it needs further investigation.
The inoculation procedure (Chappel, 1975) was probably very effective as none of the experimental poults (except for a few birds that were re-inoculated) showed discharge of the inoculum after returning to their pens. This may also have contributed to the higher mortality found compared with other in vivo studies using a different intracloacal inoculation method. On the other hand, infected birds are known to shed H. meleagridis as early as 2 days after inoculation (McDougald, 2005; Hess et al., 2006). Since the experimental turkeys were housed on litter direct lateral transmission of the parasite (Hu & McDougald, 2003), causing re-infections could have contributed to the higher mortality.
A possible explanation for the high mortality in the Histostat-50™-medicated group of birds may be that the administration of this feed additive started just 1 week before inoculation. This may have been too short for prophylactic treatment. Nitarsone, the active compound in Histostat-50™, is a prophylactic rather than a curative drug (Hu & McDougald, 2004).
Cross-contamination between groups, potentially masking an antihistomonal effect of a certain product, was highly unlikely. The negative control group (no medication, no inoculation) that was placed directly next to the positive control group with an inoculation dose of 200 000 histomonads per turkey remained free from clinical disease throughout the experiment and the birds showed no lesions at necropsy. The same was true in the experimental groups inoculated with 100 histomonads per bird as these were placed next to the groups inoculated with 3162 histomonads per bird and also remained free of disease, while almost every bird in the neighbouring pen died of histomoniasis.
No difference was found between female and male birds regarding the susceptibility to an experimental inoculation with H. meleagridis.
| Experimental room | Group | Feed additive | Dose (histomonads/bird) |
|---|---|---|---|
| A | 1 | No (positive control) | 200 000 |
| 2 | No (negative control) | 0 | |
| 3 | Dimetridazole | 200 000 | |
| 4 | Histostat-50™ | 200 000 | |
| 5 | Protophyt SP™ | 200 000 | |
| 6 | Enteroguard™ | 200 000 | |
| 7 | Protophyt SP/B™ | 200 000 | |
| B | 8 | No (positive control) | 3162 |
| 9 | No (positive control) | 100 | |
| 10 | Protophyt SP™ | 3162 | |
| 11 | Protophyt SP™ | 100 | |
| 12 | Enteroguard™ | 3162 | |
| 13 | Enteroguard™ | 100 |
| Mortality day | Caeca lesions | Liver lesions | ||||||
|---|---|---|---|---|---|---|---|---|
| Dose (histomonads/bird) | Feed additive | n a | Mean | Standard deviation | Mean | Standard deviation | Mean | Standard deviation |
| 0 | No additive | 0 | – | – | – | – | – | – |
| 100 | No additive | 0 | – | – | – | – | – | – |
| Protophyt SP™ | 0 | – | – | – | – | – | – | |
| Enteroguard™ | 0 | – | – | – | – | – | – | |
| 3162 | No additive | 18 | 15.1 | 2.68 | 3.9 | 0.24 | 3.1 | 0.90 |
| Protophyt SP™ | 17 | 15.1 | 2.30 | 3.9 | 0.24 | 3.0 | 0.79 | |
| Enteroguard™ | 18 | 15.5 | 1.69 | 4.0 | 0.00 | 3.3 | 0.49 | |
| 200 000 | Dimetridazole™ | 3 | 25.7A | 7.25 | 3.3 | 0.58 | 3.3 | 0.58 |
| Histostat-50™ | 18 | 16.2B | 3.25 | 3.9 | 0.32 | 3.0 | 0.59 | |
| Protophyt SP™/B™ | 18 | 14.5BC | 1.74 | 3.9 | 0.32 | 3.4 | 0.51 | |
| No additive | 18 | 13.7C | 1.15 | 3.9 | 0.24 | 3.3 | 0.46 | |
| Protophyt SP™ | 18 | 13.3C | 1.45 | 3.9 | 0.24 | 3.6 | 0.50 | |
| Enteroguard™ | 18 | 13.5C | 1.82 | 4.0 | 0.00 | 3.4 | 0.51 | |
| Means with different superscript uppercase letters are significantly (P<0.05) different among the groups with the same infective dose. aNumber of birds that died during the experiment. | ||||||||
Acknowledgements
This study was funded by the commodity of the Board for Poultry and Eggs. The authors thank Orffa Additives (the Netherlands) and Biosynthèse (France) for providing Enteroguard™ and Protophyt™, respectively. They also thank Johan Welner and colleagues for their assistance in the biotechnological handling of the experimental birds.
References
- Byrne, D. 2001. Commission Regulation EC No. 2205/2001, amending Council Directive 70/524/EEC concerning additives in feeding stuffs as regards withdrawal of the authorization of certain additives. Official Journal of the European Communities, L297: 3–4. [Google Scholar]
- Chappel, L.R. 1975. Using the HmL1 strain of Histomonas meleagridis in evaluating chemotherapeutants in chicks. Avian Diseases, 19: 679–682. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Chossat , L. (2002) . L'Histomonose en production A.O.C. “Dinde fermiere de Bresse” essai de prevention par phytotherapie . Doctoral thesis, ÉCole Nationale Veterinaire D'Alfort Lyon, 20 Feburary . [Google Scholar]
- Duffy, C.F., Sims, M.D. and Power, R.F. 2005. Evaluation of dietary Natustat™ for control of Histomonas meleagridis in male turkeys on infected litter. Avian Diseases, 49: 423–425. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Dwyer, D.M. 1970. An improved medium for cultivating Histomonas meleagridis. Journal of Parasitology, 56: 191–192. [Crossref], [PubMed], [Google Scholar]
- Graybill, H.W. and Smith, T. 1920. Production of fatal blackhead in turkeys by feeding embryonated eggs of Heterakis Papillosa. Journal of Experimental Medicine, 31: 647–655. [Crossref], [PubMed], [Google Scholar]
- Hafez, M.H. and Hauck, R. 2006. Efficacy of an herbal product against Histomonas meleagridis after experimental infection of turkey poults. Archives of Animal Nutrition, 60: 436–442. [Taylor & Francis Online], [Web of Science ®], [Google Scholar]
- Hess, M., Grabensteiner, E. and Liebhart, D. 2006. Rapid transmission of the protozoan parasite Histomonas meleagridis in turkeys and specific pathogen free chickens following cloacal infection with a mono-eukaryotic culture. Avian Pathology, 34: 280–285. [Taylor & Francis Online], [Web of Science ®], [Google Scholar]
- Hu, J. and McDougald, L.R. 2003. Direct lateral transmission of Histomonas meleagridis in turkeys. Avian Diseases, 47: 489–492. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Hu, J. and McDougald, L.R. 2004. The efficacy of some drugs with known antiprotozoal activity against Histomonas meleagridis in chickens. Veterinary Parasitology, 121: 233–238. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Lucas, J.M.S. 1961. 1,2-Dimethyl-5-nitroimidazole, 8595 R.P. Part I. Prophylactic activity against experimental histomoniasis in turkeys. Veterinary Record, 73: 465–467. [Google Scholar]
- Lucas, J.M.S. and Goose, J. 1965. Tests for activity against Histomonas meleagridis: use of the infection in chickens and a comparison of the results obtained in chickens and turkeys. British Veterinary Journal, 121: 65–75. [Crossref], [PubMed], [Google Scholar]
- Lund, E. 1955. The progress of histomoniasis (Blackhead) in turkeys as related to the size of the infective dose. Poultry Science, 34: 127–130. [Crossref], [Web of Science ®], [Google Scholar]
- McDougald, L.R. 1997. “Protozoa: other protozoan diseases of the intestinal tract”. In Diseases of Poultry, 10th edn, Edited by: Calneck, B., Barnes, H.J., Beard, C.W., McDougald, L.R. and Saif, Y.M. 890–899. Ames: Iowa State University Press. [Google Scholar]
- McDougald, L.R. 2005. Blackhead disease (histomoniasis) in poultry: a critical review. Avian Diseases, 49: 462–476. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- McDougald, L.R. and Hu, J. 2001. Blackhead disease (Histomonas meleagridis) aggravated in broiler chicks by concurrent infection with caecal Coccidiosis (Eimeria tenella). Avian Diseases, 45: 307–312. [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- McGuire, W.C. and Morehouse, N.F. 1952. Chemotherapy studies of histomoniasis. Poultry Science, 31: 603–609. [Crossref], [Web of Science ®], [Google Scholar]
- McGuire, W.C., Moeller, M.W. and Morehouse, N.F. 1963. The effect of dimetridazole on growth and the prevention of histomonosis in poultry. Poultry Science, 43: 864–871. [Crossref], [Web of Science ®], [Google Scholar]
- Van der Heijden, H.M.J.F., McDougald, L.R. and Landman, W.J.M. 2005. High yield of parasites and prolonged in vitro culture of Histomonas meleagridis. Avian Pathology, 34: 505–508. [Taylor & Francis Online], [Web of Science ®], [Google Scholar]
- Van der Heijden, H.M.J.F., Landman, W.J.M., Greve, S. and Peek, R. 2006. Genotyping of Histomonas meleagridis isolates based on internal transcribed spacer-1 sequences. Avian Pathology, 35: 330–334. [Taylor & Francis Online], [Web of Science ®], [Google Scholar]
- Van der Heijden , H.M.J.F. & Landman , W.J.M. (2007) . Modification of Dwyer's culture medium for Histomonas meleagridis . Avian Diseases , in press . [Web of Science ®], [Google Scholar]