Abstract
The effect of attenuated Histomonas meleagridis on pullets was investigated and the protection of vaccinated adult laying hens against a severe challenge was studied in the same experimental setting. Four groups of 25 pullets were set up at 18 weeks of life and birds in two groups were vaccinated with in vitro-attenuated H. meleagridis. Chickens in two groups (vaccinated and non-vaccinated) were challenged 5 weeks later with virulent histomonads, while the remaining groups were retained until termination of the study 11 weeks post vaccination. Vaccination of pullets did not have any impact on their subsequent performance. Egg production of non-vaccinated but challenged birds dropped significantly (P≤0.05) between 2 and 4 weeks post challenge (p.c.) to 58.7%, compared with 90% in control chickens. At 4 weeks p.c., the drop in egg production in vaccinated and challenged birds was significantly lower (P=0.02) than in non-protected layers. Pathological changes were found only in challenged birds 2 and 6 weeks p.c. Several non-vaccinated birds showed severe lesions in the caeca with sporadic involvement of the liver and atrophy of the reproductive tract. Vaccination prior to challenge reduced the incidence of pathological findings. For the first time, vaccination of pullets with in vitro-attenuated histomonads could be shown to be an effective and safe prophylactic tool to prevent a severe drop in egg production of commercial layers following experimental infection.
Introduction
Histomonosis (syn. histomoniasis) is a parasitic disease of gallinaceous birds causing inflammation of the caecum and the liver. The disease can be severe and may result in high mortality in turkey flocks, from which it was first reported (Cushman, 1893). Fatal histomonosis occurs also in chickens, although the disease may not induce overt clinical signs in this host (McDougald, 2005). The subclinical status of chickens after infection with a virulent mono-eukaryotic culture of Histomonas meleagridis has been confirmed under experimental conditions (Hess et al., 2006). Post-mortem investigations have revealed severe lesions in the caecum and sporadic involvement of the liver of infected chickens killed at specific time points after infection (Liebhart et al., 2011; Zahoor et al., 2011). Despite the absence of clinical signs, necropsy clearly demonstrated the high virulence of histomonads propagated in vitro for a few passages whereas long-term cultivation results in parasites that are unable to induce lesions.
To date, an adverse effect of H. meleagridis on the performance of layers has only been reported in the context of natural infections (Gerth et al., 1985; Esquenet et al., 2003), and even here no detailed experimental data are available. Nevertheless, the problem is of major importance as recent data from serological surveys confirm the spread of H. meleagridis in chickens, particularly those kept in free-range flocks (Grafl et al., 2011; van der Heijden & Landman, 2011).
At present, most developed countries have non-existent or at best limited possibilities for prevention and treatment of the disease because national regulations have banned effective chemotherapeutics in order to satisfy the requirements of consumer protection. As a consequence there is an urgent demand for new approaches to protect or treat poultry against histomonosis. It was recently shown that vaccination effectively protects turkeys against a severe challenge (Hess et al., 2008) and has no negative impact on young turkeys and chickens (Liebhart et al., 2011). However, possible side effects of the candidate vaccine on the laying performance of adult chickens and the protective potential of the prototype vaccine in adult chickens have not yet been investigated.
The work described here aimed to investigate the influence of virulent histomonads on the laying performance of chickens, the possible side effects of a recently developed vaccine against histomonosis, and the protection of vaccinated chickens against a severe challenge.
Materials and Methods
Layers
One hundred Lohmann brown-classic hens (Lohmann Tierzucht GmbH, Cuxhaven, Germany) were obtained from a commercial breeder at week 18 of life. The birds were divided into four groups of 25 (Table 1) and housed in separate rooms under negative pressure on deep litter. Each room was equipped with six nesting boxes. The pullets were individually marked by Swiftack™ (Heartland Animal Health, Inc., Fair Play, MO, USA). Feed and water were provided ad libitum during the experiment, except for a feed withdrawal for 5 h after vaccination and challenge to minimize the impact of feed intake on infection. The experiment was discussed and approved by the institutional ethics committee and was licensed by the Austrian government (licence number 68.205/0026-II/10b/2008).
Table 1. Experimental design to investigate the impact of vaccination and/or challenge with histomonads on the performance of layers.
Experimental design
Pullets in group V (vaccinated only) and group V+C (vaccinated and challenged) were vaccinated at 18 weeks of life with attenuated parasites of the clonal culture H. meleagridis/Turkey/Austria/2922-C6/04. The histomonads were preserved at −150°C after attenuation by long-term in vitro cultivation (Hess et al., 2008). Following thawing and propagation of the attenuated parasites (passage 295), histomonads were diluted and the cell number was determined using a Neubauer cell counting chamber (Sigma-Aldrich, Vienna, Austria). The vaccine was administered cloacally using a conventional pipette and orally via a crop tube placed on a syringe (Omnifix F Solo 1 ml; B. Braun Melsungen AG, Melsungen, Germany). Individual doses of 104 histomonads in 300 µl culture medium consisting of Medium 199 with Earle's salts, l-glutamine, 25 mM HEPES and l-amino acids (Gibco, Invitrogen, Lofer, Austria), 15% foetal calf serum (Gibco) and 0.66 mg rice starch (Sigma-Aldrich) were applied. In accordance with a recently established infection model for histomonosis (Hess et al., 2006), birds in group C (challenged only) and group V+C were challenged 5 weeks post vaccination (p.v.) with highly virulent histomonads. The in vitro propagation of the clonal culture was limited to 21 passages to maintain the parasites’ virulence. The preparation and application of the challenge inoculum was performed as described for the vaccination. Chickens in group NC (negative control) were kept as control birds without vaccination or challenge. The birds were observed over a period of 11 weeks, apart from five chickens per group that were sacrificed 2 weeks post challenge (p.c.).
Examination of birds and sampling procedures
The chickens were examined daily for adverse clinical signs. At weekly intervals the body weight was recorded and blood samples taken for serological investigations (see below). At the same time, cloacal swabs were collected to identify birds excreting histomonads by re-isolation as previously described (Hess et al., 2006).
Laying performance
Eggs were collected daily from the nest boxes and the litter of each pen and the egg count of every group was noted. In addition, the weight of each egg was taken and the eggs were examined for malformation. The contents of several eggs of each group were examined visually for abnormalities.
Serology
Blood samples were collected shortly before vaccination, on the day before challenge and weekly p.c. Sera were obtained from blood samples by centrifugation (Rotunda 460; Hettich, Tuttlingen, Germany) at 3300×g for 12 min and stored at −20°C. Antibodies against H. meleagridis were detected by means of an established enzyme-linked immunosorbent assay protocol (Windisch & Hess, 2009). As in previous work, a cut-off level at an optical density of 0.54 was selected to differentiate between positive and negative results.
Post-mortem examination
Birds were anaesthetized by intravenous application of thiopental (Sandoz, Kundl, Austria) before bleeding to death, and necropsy was performed to observe lesions caused by H. meleagridis. The severity of lesions found in the caecum or the liver was evaluated according to an established scoring system (Windisch & Hess, 2009; Zahoor et al., 2011). Another focus during necropsy was the examination of the reproductive activity of the hens. Tissue samples of the caeca were taken and frozen at −20°C for further examination.
Polymerase chain reaction
DNA of the caeca was processed using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions. Pure DNA was added to the HotstarTaq Master Mix (Qiagen) and the partial 18S rRNA gene of H. meleagridis was amplified by means of established procedures (Grabensteiner & Hess, 2006).
Statistical analysis
The weight of birds and the number of eggs from each group were analysed statistically. Calculations were performed with one-way analysis of variance followed by t tests (average body weight) or by Fisher's least significant difference as a post hoc procedure (number of eggs) using the statistical software package SPSS® Version 17 (IBM® SPSS® Statistics; IBM Corporation, Somer, New York, USA). P≤0.05 was considered significant.
Results
Clinical signs and laying performance
The droppings of two birds in group C and one in group V+C appeared yellowish at 2 weeks p.c. and one bird in group C had to be euthanized 20 days after challenge due to injuries caused by pecking by its pen mates. Except for differences in egg production, the vaccinated and/or challenged birds did not show clinical signs or a significant loss of body weight (Figure 1). None of the eggs showed any abnormalities and the egg weights increased continuously, reaching a final mean weight of 58.9 g, with a maximum deviation of 0.8 g. The weekly laying performance of each group represents the total number of eggs per hen and is shown in Figure 2. In the first week of the experiment, the number of eggs was below 10% in every group consistent with the age of the hens. The laying performance of chickens in groups C and NC increased during the following 4 weeks until it reached above 85% at the day of challenge, when it resembled the values of the vaccinated groups V+C (84.6%) and V (87.4%). The first significant drop in egg production of birds in group C was observed 2 weeks after challenge, when the production was down to 65.7%, a reduction of nearly 30% compared with the control group (93.7%). The decrease in numbers of eggs in group C continued over the following 2 weeks to 58.7% and 60.2%, respectively. Vaccinated and challenged birds showed an egg production of 75.7% after challenge, slightly below the values recorded from groups NC or V, which were between 79.3% and 96.4% in the final weeks of the experiment. This implies a maximum deviation of the weekly egg production before and after challenge within group V+C of up to 9.43% compared with 28.73% in group C. Statistical analyses proved significant differences between birds in group C and birds in groups NC or V at weeks 2, 3 and 4 p.c. The egg production of vaccinated and challenged birds (group V+C) hardly decreased p.v., except for 2 weeks after challenge when a significant reduction was noticed compared with birds that were only vaccinated or not treated. However, layers in group V+C produced a significantly higher number of eggs than non-vaccinated but challenged hens at week 4 p.c.
Published online:
07 February 2013Figure 1. Average body weight of challenged (group C), vaccinated and challenged (group V+C), vaccinated only (group V) and control birds (group NC) collected each week. *Significant difference between group NC and group C (P≤0.05).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2013/cavp20.v042.i01/03079457.2012.760841/20130207/images/medium/cavp_a_760841_o_f0001g.gif"})
Figure 1. Average body weight of challenged (group C), vaccinated and challenged (group V+C), vaccinated only (group V) and control birds (group NC) collected each week. *Significant difference between group NC and group C (P≤0.05).
Published online:
07 February 2013Figure 2. Laying performance of challenged (group C), vaccinated and challenged (group V+C), vaccinated only (group V) and control birds (group NC) calculated per week. Vaccination of the birds was performed at 18 weeks of life, followed by challenge 5 weeks later. Each group consisted of 25 chickens at the time of vaccination, of which five birds were killed 2 weeks p.c. In group C, one bird had to be euthanized 20 days p.c. due to severe injuries resulting from pecking by other chickens. *Significant difference between group NC and group C or group V+C (P≤0.05). #Significant difference between group V+C and group C (P≤0.05).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2013/cavp20.v042.i01/03079457.2012.760841/20130207/images/medium/cavp_a_760841_o_f0002g.gif"})
Figure 2. Laying performance of challenged (group C), vaccinated and challenged (group V+C), vaccinated only (group V) and control birds (group NC) calculated per week. Vaccination of the birds was performed at 18 weeks of life, followed by challenge 5 weeks later. Each group consisted of 25 chickens at the time of vaccination, of which five birds were killed 2 weeks p.c. In group C, one bird had to be euthanized 20 days p.c. due to severe injuries resulting from pecking by other chickens. *Significant difference between group NC and group C or group V+C (P≤0.05). #Significant difference between group V+C and group C (P≤0.05).
Re-isolation
Recovery of live histomonads from cloacal swabs was intermittent over the course of the experiment (Table 2). Only five of 50 vaccinated hens (groups V and V+C) tested positive before the challenge. Following challenge, eight hens in group V+C and six non-vaccinated birds in group C excreted histomonads until termination of the trial. Within the same period, hens in groups V and NC did not excrete H. meleagridis. Additionally, the flagellate Tetratrichomonas gallinarum was detected in cloacal swabs of 23 birds, including chickens from all groups.
Table 2. Results of re-isolation, PCR and post-mortem investigations of chickens vaccinated at 18 weeks of life with attenuated histomonads and/or challenged with virulent histomonads 5 weeks later.
Serology
The mean value of circulating antibodies against H. meleagridis in birds of each group is shown in Figure 3. Prior to vaccination at 18 weeks of life, the mean titre of every group was below the cut-off value. Positive sera from vaccinated birds in group V were first noted 8 weeks p.v. and the antibody titres of these birds remained constantly above the cut-off until the end of the experiment. The mean titre of serum samples from layers that were only challenged rose steeply from 2 weeks p.c. until termination of the experiment. The birds in group V+C showed a slight increase of antibodies against the parasite starting prior to challenge. However, after 1 week p.c. the titres in the sera of these birds increased sharply and levels of antibodies over the following weeks were higher than in birds of all other groups. The mean titre of hens in group NC remained below the cut-off value until the end of the study.
Published online:
07 February 2013Figure 3. Comparison of antibody titres against H. meleagridis in serum samples from chickens at 18 weeks of life (prior to vaccination), 5 weeks later before the challenge and at weekly intervals after challenge. Values above the optical density of 0.54 were considered positive.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2013/cavp20.v042.i01/03079457.2012.760841/20130207/images/medium/cavp_a_760841_o_f0003g.gif"})
Figure 3. Comparison of antibody titres against H. meleagridis in serum samples from chickens at 18 weeks of life (prior to vaccination), 5 weeks later before the challenge and at weekly intervals after challenge. Values above the optical density of 0.54 were considered positive.
Post-mortem examination
Pathological findings are summarized in Table 2. Two weeks p.c. five birds per group were killed, of which three hens in group C and two birds in group V+C showed lesions in the caeca typical of histomonosis. At the same time after challenge, spots of necrosis in the liver were found in two birds of group C and in one bird of group V+C. Another finding in chickens killed 2 weeks p.c. was an atrophy of the ovary and the oviduct of three birds in group C and one bird in group V+C (Figure 4). Severe lesions in the caeca and the liver were also observed in the bird in group C that had to be euthanized 20 days p.c. Necropsies at the end of the experiment, 6 weeks p.c., revealed lesions consistent with histomonosis in six of 19 birds in group C and in three of 20 birds in group V+C. Pathological changes of the affected caeca were consistently recorded, with a maximum lesion score of 4 during necropsies at weeks 2 and 6 p.c. Milder lesions were observed in the livers (lesion scores 1 to 3) and were only found in hens that contracted caecal lesions, with the exception of a single bird in group C. With the exception of two birds, one in group C and one in group V+C, all remaining hens were in production at the end of the trial. Vaccinated birds that were not challenged with virulent histomonads in group V and chickens in group NC did not show any pathological aberrations (lesion scores=0).
Published online:
07 February 2013Figure 4. Experimentally induced lesions in the caeca (4a) and the liver (4b) of a chicken killed 2 weeks post challenge (Bird 1 in group C). 4c: The bird's ovary and the oviduct were found to be atrophied.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2013/cavp20.v042.i01/03079457.2012.760841/20130207/images/medium/cavp_a_760841_o_f0004g.jpg"})
Figure 4. Experimentally induced lesions in the caeca (4a) and the liver (4b) of a chicken killed 2 weeks post challenge (Bird 1 in group C). 4c: The bird's ovary and the oviduct were found to be atrophied.
Polymerase chain reaction
Of the five birds per group killed 2 weeks p.c., only two in group C and three in group V+C had H. meleagridis DNA in the caeca (Table 2). At the end of the experiment, 6 weeks after challenge, the caeca of all birds in group V+C were found positive by polymerase chain reaction (PCR). From groups C and V, eight and six caeca, respectively, were recorded positive.
Discussion
Experimental histomonosis of chickens was first investigated by Graybill & Smith (1920), who successfully reproduced the disease by feeding embryonated eggs of Heterakis papillosa that harboured H. meleagridis. We subsequently reported the establishment of an infection model for histomonosis in chickens and turkeys based on mono-eukaryotic parasites (Hess et al., 2006). In this study, no clinical signs were noted in chickens infected on their 14th day of life, similar to the present findings in which only three of 50 adult chickens showed mild yellowish diarrhoea. Lesions typical of histomonosis, mostly in the caecum of several layers during post-mortem, could be found as previously seen in chicks infected on their first day of life (Zahoor et al., 2011). There was no obvious variation in the course of histomonosis in chickens infected at different ages, similar to the situation in turkeys (Liebhart et al., 2008).
A major focus of the present study was to monitor the influence of histomonosis on egg production in laying chickens. Therefore, the birds were challenged at peak of production. For the first time, it could be shown that a significant drop in egg numbers occurred between 2 and 4 weeks after infection of laying hens with virulent parasites. The result is in agreement with reports from the field, which have mentioned a possible association of histomonosis and a reduced performance of layers (Gerth et al., 1985; Esquenet et al., 2003). However, these reports mention an increased mortality (2.4% and 6%), which was not observed in the present study. The difference presumably relates to the distinct situation in the field and a well-defined experimental setting. In the present study, atrophy of the ovary and the oviduct was found in some of the infected birds killed 2 weeks p.c., and this change most likely explains the reduced laying performance. Coincidentally, severe inflammation and necrosis of the caeca, with the sporadic involvement of the liver, were seen in several of the challenged chickens. One can hypothesize that the reduced uptake of nutrients caused atrophy of the reproductive tract. Nevertheless, egg production decreased significantly and recovered at 5 weeks p.c. with nearly all challenged birds being in production 1 week later, when the study was terminated. However, the caeca of several chickens still showed maximum lesion scores but clinical findings indicated the recovery of the birds from histomonosis.
Vaccination 5 weeks prior to challenge protected most of the chickens from the drop in egg production. Protection was demonstrated by a significantly higher number of eggs laid and by a lower incidence and severity of lesions. Some vaccinated and challenged chickens displayed lesions in the caeca and one bird had lesions in the caeca and liver, indicating that a single vaccination 5 weeks prior to challenge might be insufficient to protect all chickens against challenge. Other vaccination schedules should thus be explored in future studies.
It has been shown previously that the presence of circulating antibodies against H. meleagridis is not able to protect birds from the disease (Clarkson, 1963; Hess et al., 2008; Bleyen et al., 2009). In turkeys there was a correlation between levels of antibody and virulence of the parasite, similar to the present study (Windisch & Hess, 2009). On the day of challenge, the mean optical density value of vaccinated chickens (group V+C) was hardly above the cut-off and hens in group V did not test positive before week 8 p.v. In contrast, the mean antibody titre of vaccinated turkeys was above the cut-off by 4 weeks p.v., reflecting a difference in the immune response of chickens and turkeys (Powell et al., 2009). Possible differences with regard to the immune reactions induced by attenuated histomonads and virulent parasites need further investigation.
The effectiveness of colonization and multiplication of the parasite in the intestine of vaccinated laying hens probably correlates with protection. Live histomonads could only be isolated from a single vaccinated bird during the entire monitoring period of 11 weeks, whereas 12 vaccinated and challenged birds were found positive. From the birds that were challenged without prior vaccination, live histomonads were recovered from six birds during the 6 weeks p.c. Similarly, the number of caeca from hens that were vaccinated but not challenged and that tested positive by PCR was much lower than the corresponding number from challenged or vaccinated and challenged birds. The inability of histomonads that had been propagated for a long time to grow readily in host birds was described many years ago by Lund et al. (1967) and could be the result of a hindered or retarded re-adaption to the in vivo environment, again underlining the difference between attenuated and virulent histomonads.
The sporadic detection of T. gallinarum in the cloacal swabs of some birds from each group was unexpected but could be associated with the birds’ origin. In commercial chickens, those trichomonads have been described as common inhabitants of the caecum (McDougald, 2008; Amin et al., 2011).The possibility that T. gallinarum gave rise to adverse clinical signs or pathological changes in the present work can be excluded because chickens in the negative control group showed no negative effects from this parasite. Furthermore, the tools used to monitor infection with H. meleagridis in birds have been shown to be highly specific (Grabensteiner & Hess, 2006; Grafl et al., 2011).
In conclusion, histomonosis of chickens can result in a temporary but severe drop in egg production following infection, despite the absence of additional clinical signs. This outcome is of high relevance because recent serological investigations have shown the parasite to be widely distributed in European flocks of laying hens (Grafl et al., 2011; van der Heijden der Heijden & Landman, 2011). Grafl et al. (2011) found significantly fewer seropositive birds in pullet flocks than in layer flocks, emphasizing the risk of infection during production. In a well-defined experimental setting, it could be shown that vaccination of chickens against histomonosis before adolescence significantly reduces the drop in egg production following challenge. In parallel, the number of birds showing pathological lesions is very much reduced.
| Group | Vaccinationa | Challengeb |
|---|---|---|
| (n=25) | (18 weeks of life) | (23 weeks of life) |
| C | No | Yes |
| V +C | Yes | Yes |
| V | Yes | No |
| NC | No | No |
| aVaccine: Hm/Turkey/Austria/2922-C6/04—passage 295. | ||
| bInoculum: Hm/Turkey/Austria/2922-C6/04—passage 21. | ||
| Bird number | |||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Parameter | 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 |
| Group C: birds challenged at 23 weeks of life | |||||||||||||||||||||||||
| Re-isolation prior to challenge | −a | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − |
| Re-isolation post challenge | − | − | − | +b | − | − | − | − | − | − | − | − | + | + | − | + | − | − | + | − | − | + | − | − | − |
| PCR caecum | + | − | + | − | − | − | + | − | + | + | − | − | − | + | + | + | − | − | − | − | − | + | − | − | + |
| Lesion score liver | 2 | 0 | 2 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Lesion score caecum | 4 | 0 | 4 | 4 | 0 | 0 | 4 | 0 | 0 | 0 | 4 | 0 | 0 | 0 | 4 | 0 | 4 | 0 | 4 | 0 | 0 | 0 | 0 | 0 | 4 |
| Atrophy of the reproductive tract | yc | nd | n | y | y | n | n | n | n | n | y | n | n | n | n | n | n | n | n | n | n | n | n | n | n |
| Group V: birds vaccinated at 18 weeks of life | |||||||||||||||||||||||||
| Re-isolation prior to challenge | −a | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − | − | − | − |
| Re-isolation post challenge | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − |
| PCR caecum | − | − | − | − | − | − | − | − | − | + | + | + | − | − | − | − | + | − | − | + | − | − | − | − | + |
| Lesion score liver | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Lesion score caecum | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Atrophy of the reproductive tract | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n |
| Group V+C: birds vaccinated at 18 weeks of life and challenged at 23 week of life | |||||||||||||||||||||||||
| Re-isolation prior to challenge | −a | − | − | + | − | − | − | − | − | + | − | − | − | − | − | − | − | + | − | + | − | − | − | − | − |
| Re-isolation post challenge | + | − | − | − | − | − | − | + | + | − | − | − | + | − | − | − | − | − | + | − | + | + | − | − | + |
| PCR caecum | − | + | + | + | − | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
| Lesion score liver | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Lesion score caecum | 4 | 0 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 4 | 0 | 4 | 0 |
| Atrophy of the reproductive tract | y | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | n | y | n | n | n | n | n | n |
| Parasites were detected by re-isolations from cloacal swabs taken once a week and by PCR from caecal tissue. Lesion scores of liver and caecum ranged from 0 (no lesions) to 4 (most severe changes). Birds 1 to 5 in each group were killed 2 weeks post challenge and bird 7 in group C had to be euthanized due to injuries caused by pen mates on day 20 post challenge. The remaining birds were kept until the age of 29 weeks. Negative control birds (group NC) were found negative for the detection of the parasite by re-isolation and PCR did not display pathological changes during post-mortem (data not shown). aAll re-isolations of the respective bird negative. bAt least one re-isolation of the respective bird positive. cYes. dNo. | |||||||||||||||||||||||||
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