Veterinary Microbiology, 9 (1984) 121--129 121 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

INFLUENCE OF RETICULOENDOTHELIOSIS ON THE SEVERITY OF EIMERIA TENELLA INFECTION IN BROILER CHICKENS

M.X.J. MOTHA and J.R. EGERTON

Department of Veterinary Clinical Studies, The University of Sydney, Camden, N.S. W. 2570 (Australia)

(Accepted 20 September 1983)

ABSTRACT

Motha, M.X.J. and Egerton, J.R., 1984. Influence of reticuloendotheliosis on the severity of Eimeria tenella infection in broiler chickens. Vet. Microbiol., 9: 121--129.

Broiler chickens free of maternal immunity to reticuloendotheliosis virus (REV) were used in the experiment. Two groups of 25 chickens were inoculated with REV at one day of age. One of these groups and another group of 25 chickens were inoculated with Eimeria tenella sporulated oocysts at 7 days of age. Chickens inoculated with E. tenella showed bloody 'diarrhoea from 12 to 14 days of age. Six out of 25 chickens died (P < 0.05) at 13 and 14 days of age in the dual infected group. At 14 days of age, when chickens were killed, the lesion score in the combined infection group, was statistically different from that in the chickens inoculated with E. tenella alone. Also the weights of the bursa of Fabricius and thymus were lower in the two REV infected groups than in the controls. Although REV infection alone adversely affected the weight gain and feed conversion, with combined infection this effect was much greater. Following REV inoculation most of the chickens showed feathering defects and all the examined chickens were viraemic at 21 days of age. At the same age, all but one chicken failed to show pre- cipitating antigenaemia and about one-half of these chickens showed a very low serum neutralisation titre. None of these chickens showed precipitating antibodies.

INTRODUCTION

The economic importance of multiple infections in chickens was high- lighted by Bagust and Dennett (1979), as the major individual poultry dis- eases continue to be brought under control. The problems of multiple infec- tions are worse when birds are affected with immunosuppressive infections such as infectious bursal disease (Cheville, 1967), Marek's disease (Purchase et al., 1968) and reticuloendotheliosis (yon Bulow, 1977).

Recent investigations have shown that chickens infected with reticulo- endotheliosis virus (REV) are more susceptible to viral (Witter et al, 1979; Yoshida et al., 1981; Motha, 1982) and bacterial (Howell et al., 1982; Motha and Egerton, 1983) infections. No work has been reported to show the effect of REV on the susceptibility of chickens to protozoan infections.

0378-1135/84/$03.00 © 1984 Elsevier Science Publishers B.V.

1 2 2

Chickens infected with REV at Day 1 and housed in conventional deep litter pens showed mortal i ty due to coccidiosis (M.X.J. Motha, 1980, 1981, personal observation). In view of this observation this s tudy was undertaken to determine the effect o f REV on the development and severity of Eirneria tenella infection in broiler chicken.

MATERIAL AND METHODS

Chickens

One hundred one<lay-old broiler cockerels free of REV maternal anti- bodies (MAb) were donated by a poultry breeding organisation.

R E V

REV isolate Q 1/77 (Ratnamohan et al., 1980) was used at the fifth pas- sage level for the inoculation of chickens in the serum neutralisation (SN) and agar gel precipitation (AGP) tests. Preparation of cell cultures and virus stock and storage o f the virus have been described previously (Motha, 1982).

Eimeria tenella

A strain of E. tenella isolated from a chicken was supplied by Mr. P. Groves, Lilly Industries Pty. Ltd., West Ryde, N.S.W., Australia. Sporulated oocysts were prepared from caecal contents in potassium dichromate from donor chickens 7--8 days after oral inoculation and stored at 4 ° C.

SN test for R E V antibodies

The detect ion of neutralising antibodies (Ab) was carried out as described previously (Motha and Egerton, 1983). For end-point ti tration the serum samples were further diluted in serial two-fold steps and the titre was ex- pressed as the 50% endpoint {Reed and Muench, 1938).

A GP test

Precipitating Ab and antigenaemia were examined by the method de- scribed by Ianconesduiand Aharonovici (1978). A precipitin line between the test serum samples and the REV antigen was considered to be evidence of REV Ab and a precipitin line appearing between the reference serum and the test serum sample was considered to be REV antigenaemia. The fusion of the above precipitin lines with the lines appearing between the REV antigen and the reference serum helped to ascertain the specificity of this test.

123

Virus isolation

REV viraemia was detected by the DEF plaque test as described by Motha (1982). In this experiment 0.2 mi of serum was used for virus isolation. Chicken embryo fibroblast (CEF) growth medium was prepared using Medi- um 199 wi thout foetal calf serum and the petri dishes were inoculated with the serum samples at the time of seeding. On the third day of inoculation this culture medium was changed to CEF maintenance medium.

Caecal lesion and faecal scores

Caecal lesion scoring was carried out as described by Johnson and Reid (1970) on a scale of increasing severity from 0 to 4. Scoring of droppings for haemorrhage induced by E. tenella was performed at 12--14 days of age (Morehouse and Baron, 1970).

Management

Each group of chickens was housed in a different deck of a Petersime electric bat tery brooder (Petersime Incubator Co., Gettysburg, OH, U.S.A.) and fed on experimental unmedicated broiler starter crumbles. Feed and water were made available ad libitum.

Experimental procedure

Four groups (A--D) of 25 chickens each (Table I) were used in this experi- ment. On Day 1 Groups A and C were inoculated subcutaneously wi th 0.4 × 10 s tissue culture infective dose 50% (TCIDs0) of REV. At Day 7 Groups B and C were inoculated into the crop using 10 s sporulated E. tenella oocysts per bird. Group D comprised uninoculated controls.

All chickens were weighed individually at 7, 14 and 21 days of age and be- tween 7 and 21 days of age the feed consumption of each group was re- corded. The chickens in Group D were bled at 3 days and group A were bled at 21 days of age for REV serology. At 14 days of age 5 chickens from each group and all dead chickens were necropsied and examined for caecal lesions. The bursa of Fabricius and the lobes of thymus glands on the right side of each bird were weighed. All chickens were examined for feathering defects ( "nakanuke" Koyama et al., 1976) at 21 days of age. The chicken droppings were examined for blood.

Statistical analysis

Student 's t test was applied to analyse differences between weights, and ×2 test was applied to analyse differences between mortalities and caecal lesion scores. Yates ' correction was applied since the expected values were small for the X 2 test.

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125

RESULTS

Chickens inoculated with E. tenella alone (Group B), and E. tenella and REV (Group C) showed b loody diarrhoea from 12 to 14 days of age. There was no difference in the degree of faecal scoring between these two groups (Table I). However, 6 ou t of 25 chickens died at 13 and 14 days of age in Group C whereas there were no mortalities in the other 3 groups. The differ- ences in mortal i ty between Group C and Group D were statistically signifi- cant (0.01 < P < 0.05). At necropsy, caeca were distended with a large amount of clot ted and unclot ted blood and mucosal debris.

When 5 chickens from each group were killed at 14 days of age, chickens from Group C had a higher lesion score than those in Group B. This differ- ence was statistically significant (0.01 < P < 0.05). Chickens in Groups A and D did not show any caecal lesions. The mean weights of thymuses and bursae of Fabricius are shown in Table II. Although Groups A and C showed a lower mean weight in each organ, only the mean weight of the thymuses in Group A was statistically different from that of Group D (P < 0.025).

Uninfected chickens grew faster than those in Groups A, B and C. The dif- ferences were statistically significant at 7, 14 and 21 days except in the case of 7-day-old chickens in Group C. Also, the weight gains of birds in Groups A and B were statistically different from those of Group C at 14 and 21 days of age. There were no differences in weight gain between Group A and Group B chickens at the same ages. Feed conversions in Group A, B and D were very similar and in Group C was substantially higher than those of Group D. Almost all the chickens infected with REV (Groups A and C) showed feathering defects at 21 days of age (Table I).

None of the chickens in Group D showed evidence of REV neutralising or precipitating MAb at 3 days of age. In Group A, 19 out of 20 chickens showed REV precipitating antigenaemia and 9 out of 20 chickens showed REV neutralising Ab at 21 days of age. The mean Ab titre was 23.40 + 1.24. None of the chickens showed precipitating Ab at 21 days of age. All 20 chickens in Group A were viraemic at 21 days of age (Table I).

DISCUSSION

Carpenter et al. (1977) reported that chickens inoculated with REV devel- oped severe depression in T- and B-cell functions because spleen cells and peripheral blood leucocytes were suppressed in their ability to respond to phytohaemagglutinin and lipopolysaccharide. Although, in this experiment the T- and B-cell functions were not measured, the mean weights of the bursa of Fabricius and thymus were lower in the birds inoculated with REV than in the controls.

This experiment has provided other evidence of immunosuppression by REV. Thus mortal i ty in chickens with combined I~EV and E. tenella infec- tion was significantly higher than in birds with single infections or those free

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127

of both infections. Lesions attributable to coccidiosis were increased in severity when REV infection was concurrent. Further support for the view that REV increased the severity of coccidiosis is provided by weight gains and feed conversion data in the various groups of birds. Mean body weights of birds were consistently lower in the groups with the combined infection and these groups had the poorest feed conversion ratio.

Decreased resistance to E. tenella infection has also been demonstrated with infectious bursal disease (Anderson et al., 1977; Giambrone et al., 1977), hormonal (Long and Pierce, 1963) and surgically (Challey, 1962) bursectomised chickens. In our experiment, there were no differences in the faecal scores of chickens infected with E. tenella and those with the com- bined infections. Giambrone et al. (1977) had similar results in experiments with E. teneUa and infectious bursal disease virus.

The infectivity of the REV dose used was demonstrated by the virological and serological responses to the chickens in group A and clinical response ("nakanuke") observed in groups A and C. In this experiment, almost all the chickens (33 out of 34) infected with REV showed feathering defects. In a previous experiment (Motha and Egerton, 1983) when the same strain of virus was used, different proport ions of feathering defects were seen in a laying strain of chickens. The effect could be due to the breed susceptibility to the virus. Scofield et al. (1978) found differences in mortal i ty and lesion responses to REV in two different lines of chickens. A runting syndrome as- sociated with REV infection has been well documented (Witter et al., 1970; Mussman and Twiehaus, 1971; Jackson et al., 1977). In this experiment REV infection alone adversely affected weight gains and feed conversion, but not so severely as the combined infection.

The viraemia and serological results confirm some observations made by Bagust and Grimes (1979) and Witter et al. (1981). Thus, at 21 days of age all 20 surviving chickens were viraemic. Only 9 of them had Ab titres be- tween 20 and 35 and the mean Ab titre was 23.40 + 1.24. Other workers (Witter and Crittenden, 1979) consider that chickens which are viraemic and do not develop Ab are tolerant to REV infection. However, since the lowest titre measured in our test was 20 it is possible that some or all o f the remain- ing 11 birds had Ab titres below this level. Similar observations were made by Witter et al. (1981) on viraemia and Ab responses and they claimed that some chickens were partially tolerant to REV. In this experiment, the anti- genaemia detected by the AGP test gives a 95% correlation with the DEF plaque test used for virus isolation. Bagust and Grimes (1979) have reported that the virus can be readily isolated from the plasma and serum of chickens inoculated with REV at l<lay old up to 6 weeks of age, in the presence or absence of Ab.

The development of immunity to E. tenella infection results from both humoral (Long et al., 1963; Rose and Long, 1971) as well as cell-mediated mechanisms (Giambrone and Klesius, 1980; Giambrone et al., 1980). REV infects organs involved both in humoral and cell-mediated responses and this

128

expe r imen t shows tha t R E V can alter the response to E. tenella infec t ion and hence the po ten t ia l to increase losses f rom coccidiosis under field condi- t ions.

ACKNOWLEDGEMENTS

The helpful suggestions o f Mr. P. Groves, Lilly Industr ies Pty. Ltd. , West Ryde , N.S.W., Australia, and the financial suppor t f r o m the Universi ty o f S y d n e y Cancer Research F u n d and the Pos t -Graduate Research Studentsh ip are grateful ly acknowledged .

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