Kinetics of specific immunoglobulin A, M and G production in the duodenal and caecal mucosa of chickens infected with Eimeria acervulina or Eimeria tenella

Pergamon

Eimeria tenda F. GIRARD,*

G. FORT,* P. YVOR6*

and P. QtJeRl?f$

*Laboratoire

de Protozoologie, Station de Pathologie Aviaire et de Parasitologic, lnstitut National de la Recherche Agronomique, 37380 Nouzi/i-v, France TLaboratoire de Viroiogie Aviaire et d’Oncologie, Station de Pathologic Aviaire et de Parasitologic, Institut National de la Recherche Agronomique, 3?380 Nouzilly, France (Received

3 July 1996: accepted

19 February

19971

Abstract-Girard

F., Fort G., Yvorh P. & Q&r6 P. 1997. Kinetics of speciRc AVMaadG t rind caed macosa of ddckeas it&Wed with Ekeriu ocewnh or E&n#!fa feweffa. h&tofogy 27z 803-g@. The development and ~~~~ati~GBl~~e~~~y~~~~~~~~~ ) or Eimerfa h?nf& (tot#ecama).neiocal memast#e antibody levels in tbe sapernatants of 16 II at 41°C, 5% CO*, 95% air. Speellk l&M was de&c&d 1 week al&r E. and the sped& IgA and IgG contents of the d aruIaecllmwere (P < O&01) after 2 weeks. The int&nl specillc IgG ConteQt was rakseai. E. ie#le&¶ in&&ion in the daodenum artd caemm, lxtqmtively, 1 and 2 weeks pi. wss always significantly bigher in the , dnodemun for E. tene/hz). This ex uiuo c&We aSSay of

hnmurtere+nrseofintestinalareassboweda levels of IgG were a& measWed.Theroleoftl& remains to be determined. 0 19!V Australian Society for Parasitology. PaWshed by Elsevier Scieaee Ltd.

Keyword.7:

l?iwteriu:

chicken:intestinalimmune responses

INTRODUCTION

Chicken coccidiosis is caused by intestinal protozoan parasites of the genus Eimeria. The diseasecan lead to reduced productivity and/or major lossesof livestock (Biggs, 1982). The 7 speciesof Eimeriu each parasitize different areas of the intestine. For example, E. ucervu&a is restricted to the duodenum and E. tenella is located in the caeca. The drive to develop vaccines against avian coccidiosis, in particular using recom$To 2 4142

whom correspondenceshould be addressed.Fax: 33 17 74. 803

binant proteins (Crane ef al., 1991), has revealed the need for a better understanding of the intestinal and systemic immune response triggered durmg the mfection which results in a state of acquired resistance (Rose, 1987). Cellular immune mechanisms are thought to play an essentialrole in chicken coccidiosis.Severalstudies, including experiments with thymectomy (Rose & Long, 1970), cyclosporin A treatment to block T-cell immune response(Lihehoj, 1987) or adoptive transfer of spleen cells or peripheral blood lymphocytes from Eimeria-infected chickens to uninfeted chickens (Rose & Hesketh+ 1982) have shown the importance

804

F. Girard e/ al.

of T-cells for inducing a protective immune response which limits oocyst production in primary and subsequent infections. The intestinal epithelium is the primary target tissue for coccidia, but little information is yet available on the intestinal T-cell immune system in chickens (in contrast to the mammalian system). Studies on T-lymphocyte subsets during an Eimeria infection and subsequent challenge in chickens by FACS analysis after intestinal lymphocyte isolation or by immunohistology have shown drastic changes in T-lymphocytes at the intestinal site of parasite development. After an E. maxima infection, CD4+ and CD8+ cells participated in the response, but the contribution of CD8+ cells was the highest (Rothwell et al., 1995). After a second infection with E. acervzdina, a significant increasein the duodenum of CD8+ cells occurred in a strain of chickens that is relatively resistant to primary infection (Lillehoj & Bacon, 1991; Lillehoj, 1994). CD8+ cells are also involved in E. acervulina sporozoite transport to the site of development (Trout & Lillehoj, 1995; Lillehoj & Trout, 1996). It is rather difficult at present to draw a general picture of which modifications of the immune system appear in the intestine as a result of infection with avian coccidia. The literature shows discrepancies between studies due to the different techniques used, and also in the kind of chickens, their origin and age, and the dose and speciesof coccidia used. The role of antibodies in protection against coccidiosis in chickens is more controversial. The results of bursectomy experiments do not suggesta protective role for circulating antibodies in a primary infection (Rose & Long, 1970; Lillehoj, 1987) and the passive transfer of specific serum antibodies protects chickens against coccidiosis only in some cases(JVallach et al., 1994). But antibodies produced locally in the intestine after an Eimeria infection may play a part. In chickens, secretory IgA has indeed been identified (LebacqVerheyden et ul., 1972; Leslie & Martin, 1973). Davis et al. (1978) showed by immunohistology that the number of intestinal cells synthesizing IgA increases after an E. tenella infection. Furthermore, IgA-rich post-infection caecal contents were shown to inhibit sporozoite invasion and their development in cell cultures. This suggests that secretory IgA is part of the protective immune response to Eimeriu. This is supported by work showing that a high IgA concentration in the cystic bile is accompanied by low numbers of sporozoites in the intestinal lumina of immunized chickens (Mockett & Rose, 1986; Rose & Hesketh, 1987). But there is still no indication as to where in the intestine these specific antibodies are produced during an Eimeria infection. We have, therefore, monitored the production of specificantibodies within infected and uninfected sites of the intestine after E.

acervulina or E. tenella infection. Zigterman et al. (1993) used an ex viva culture assayof intestinal fragments lOdays after an infection with E. tenella and found increases in specific antibodies, mainly in the caeca. We have used the same assay to analyse the distribution and the relative amounts of specific local IgM, IgG and IgA antibody production in the intestine over 3 weeks following infection with E. acervuiina or E. tenella. MATERIALS

AND

METHODS

Parasites. E. acervulina and E. tenella weremaintainedby passagethrough coccidia-freechickens.Unsporulated oocystswereobtainedon day 6 p.i. from faecesfor E. acervuZina and on day 7 p.i. from the caecalcontentsfor E. ?enella. They werepurified and allowedto sporulateusingstandard procedures(Bontemps& Yvorb, 1974).The solublesporozoiteantigenwasobtainedfrom purified sporulatedoocysts, broken Gith glassbeads, sonicited at 0% in phospgatebufferedsaline(PBS).nH 7.2 and centrifugedat 10000~for

10 min. The proiein &&ent in the superna&t was mea&red with a Bicinchoninic acid protein assay (Smith et al., 1985) (Pierce, Rockford, IL, U.S.A.) and diluted for ELISA. Animals and experimental design. Histocompatible GBl white leghorn chickens were reared coccidia-free until 8 weeks of age. Birds were then orally infected with 20000 sporulated oocysts of E. acervulina or with 2000 sporulated oocysts of E. tenella. Seven, 14 and 21 days p.i., 6 control chickens were compared with 8 E. acerz&na-infected chickens and 4 control chickens with 8 E. tenelZa-infected chickens. In the case of specific antibodiesagainstcaecalE. tenella, 4 controls were compared with 10 infected chickens.

Ex vivo culture of intestinal tissue. The method of Zigterman et al. (1993) was used. Briefly, the duodenum and caeca were taken from killed chickens and immersed in lOm1 Hank’s balanced salt solution (HBSS, Gibco, manufactured by Life Technologies Ltd, Scotland) containing 500 IU ml-’ penicillin (Diamant, France) and 5OOpgmlV’ streptomycin (Diamant, Fran=). Intestinal fluids were removed by flushing the mucosal surfaces with HBSS. The duodenal and caecal tissues were cut into 1 g pieces (3 or 4g for one tissue) and each was cut into small pieces (about 2-3mm) and washed 3 times with HBSS penicillin+treptomycin (lOmin, 3OOg) and resuspended in 5 ml of RPM1 1640 Dutch modification (Gibco) supplemented with 1OO~gmlV gentamycin (Sigma, St Louis, MO, U.S.A.), 40 mM HEPES (Gibco) and 2mM L-glutamine (G&o). The suspensions were centrifuged (5 min, 300g) and 750 ~1 aliquots of supematant (t =0) were taken. The tissue samples were resuspended and incubated at 4l“C, 5% COJ95% air in 25 cm* tissue culture flasks (Nunc, Rostilde, Denmark) for 16 h. The samples were then centrifuged and 750~1 aliquots were taken (t= 16). Specific IgA, IgM and IgG isotypes in aliquots (t=O, t = 16) were determined by ELISA. Sera. Blood samples were obtained from chickens by cardiac puncture, allowed to clot for 4 h and centrifuged (10 min, 3000g). The sera were aliquoted in 1 ml vials and kept at - 2O’C until used. ELZSA. Microtitre plates (Nunc) were coated by incubation overnight at 37’C with lOpgml-’ soluble E. acervuha or E. tenella antigen in carbonate buffer (pH 9.6) (100~1

Intestinal antibody response to avian coccidiosis well-‘). Two per cent bovine serum albumin (Boehringer, Mannheim, Germany) (ZOO~1 well-‘) was then added for I h. The’wells were washed and samples (100~1 well-‘) diluted in PBS (pH 7.2) were incubated for 1 h at 37C. The wells were then washed 3 times and 100~1 of I of the following serum polyclonal antibodies was added: horseradish peroxidase (HRPO) conjugated goat anti-chicken IgM p chain (1:2000; Bethyl Laboratories, Montgomery, TX, U.S.A.), HRPOconjugated goat anti-chicken IgA a chain (1:2000; Bethyl Labomtories), or alkaline phosphatase-conjugated rabbit anti-chicken IgG Fc fragment specific (1: 10 000; Jackson IR Laboratories, PA, U.S.A.). Finally, depending on the secondary antibody used, colour was developed using 100 ~1 of a 0.1 pg ml- ’ solution of 2-2’-amino-bis(3-ethylbenzthiazohne 6-sulfonic acid) diammonium (Sigma) in citrate buffer (pH 4) (IgA and IgM), or 100 ~1 of a 1pg ml-’ solution of p-nitrophenylphosphate (Amresco, OH, U.S.A.) in diethanolamine (pH 9.8) (IgG) to each well and incubation for 1 h at 37C. Bound isotypes in intestinal supernatant and sera were quantified by the absorbance (,4) at 505nm. The antibody released by the intestinal fragments during culture was calculated by subtracting the ,4505values off = 16 aliquots from A 505 nm

s

those oft = 0 ahquots. .For each chicken, the specitic caecal or duodenal antibody production was expressed as the /lTg8. mean value of 3 or 4 intestinal pieces of 1 g. S~afistical anah&. Data for each group (see Anrmais and Experimental design) were analysed by Student’s r-test

RESULTS E. acervulina

bzfectiotl

There were significant amounts of specific IgA in the parasitized duodenum (P c 0.001) and the caeca (P < 0.001) from the second week after infection (Fig. 1). The specific IgA content of the caeca was lower than that of the duodenum. Production did not increase 3 weeksp.i. and was not significantly diRerent from that of control chickens. The background measured for specific IgA was higher in the duodenum A505

0.6

0.6

0.5 0.4 0.3 0.2 0.1

0.5 0.4

nm

w

0 I

A505nm

14

1

21 Days p.i.

&M&l-

A 505 nm

14

21 Days p.i

7

14

21 Days p.i

I

14

21 Days p.i.

m

0.21

;

1.4

2.1 Days

A505

"m

p.i.

w

2 1.5

1

14

21 Days p.i.

Fig. 1. ELISA determination of specific IgA (1: 1), IgG (1:4) and IgM (1: 1) antibodies against .!?.acer&inu in the duodenum (left) and caeca (right). Days post-infection are indicated along the x axis and absorbance (&) is on they axis. I&K& bars, infected birds; open bars, control birds. The statistical signillcance of differences between control and infzeted birds by Student’s f-test is indicated by: *P < 0.05, **P -=I 0.01, ***P .:. 0.001.

F. Girard

806 Table

lLSeruma

Days

pi.

IgA,

14 21

&A Control Infected Control Infected Control Infected

al.

IgM and IgG antibodies to E. following infection Serum

I

et

0.046~0.001b 0.094 2 0.009* 0.058 k 0.002 0.207~0.013*** 0.077kO.026 0.071 L-o.013

antibodies

acervdina antigen against W

0.117&0.002 0.329&0.025*** 0.085~O.Ol9 0.183~0.013*** 0.111~0.011 0.188~0.012**

‘Serum diluted 1:160 for IgM, 1:40 for IgA and 1:640 for IgG. ‘MeankS.E.M. *P < 0.05, **P < 0.01, ***P < 0.001, statistical ences between control vs infected, by Student’s z-test.

than in the caeca, despite identical treatment of tissues in the culture assay. The duodenal supernatant of parasitized birds contained a significant amount of specific IgM (P -C0.001) by the first week p.i. The unparasitized site (caecum) contained less specific IgM, but it was still above the control level (Fig. 1). This local production of specific anti-E. acervulina IgM remained rather low, but was significant during the 3 weeks after infection, although it decreased in the second (P -C0.05) and third weeks (P < 0.001). Specific anti-E. acervdina IgG was produced later than specific IgM or IgA, with significant amounts in the duodenum and caeca 2 weeks after infection (Fig. 1). This production was significantly greater in the duodenum of infected chickens than in controls during the third week (P -C0.05) but not in the caeca. Infection with E. acervulina triggered a rapid serum antibody response (Table 1). Specific circulating IgM (P c 0.001) and IgA (P c 0.05) were first detected during the first week after infection, in parallel with the appearance of local specific IgM. The serum titre of specific IgG was high in the second week (P c 0.001) and decreased further in the third week (P -C0.001) p.i. Serum specific IgA concentrations were significantly different from those of control chickens during the first week p.i. (P c O.OS),although local production was not yet significantly different. The serum specific IgA rose during the second week (P < 0.001). E. tenella infection Specific IgA (Fig. 2) was significantly elevated from controls 2 weeks after infection, but only in the parasitized caeca (P c 0.05). The duodenal and caecal supernatants of parasitized birds contained significant amounts of specific anti-E. tenella IgM in the first week p.i. (P c 0.001) (Fig. 2). The specificproduction decreased during the third week, but was still sig-

during

the 3 weeks

E. acerdina kxG 0.076 k 0.026 0.090~0.016 0.120~0.061 1.994kO.O06*** 0.206~0.016 1.184+0.121***

significance

of differ-

nificantly higher in the parasitized chickens during the second week pi. (P x 0.001). The amount of specific IgM was greater in the parasitized caeca than the duodenal samples. Specificanti-E. tenella IgG was produced later than specificIgM or IgA, appearing during the second week p.i. in the parasitized caeca (P c 0.001) and in the non-parasitized duodenum (P c 0.05). Significant amounts (P c 0.001) were still present during the third week p.i. The amount of specific IgG was greater in the parasitized caecum than in the unparasitized duodenum. Specific serum IgM (P c 0.01) IgA (P -C0.01) and IgG (P -C 0.05) were detected at day 7 (Table 2). IgM, IgA and IgG increased during the second week p.i. Only significant amounts of IgG (P -C0.001) were detected during the third week after infection.

DISCUSSION

Little information is available on local antibody production by the intestine of chickens infected with Eimeria. Our results showed that a primary E. acervulina infection triggers a significant specificantibody immune response, both in the parasitized (duodenum) and unparasitized (caeca) sites, with IgM appearing in the tirst week p.i., and IgA and IgG during the second week. The IgA response was of short duration and the specific IgM response decreasedin subsequent weeks. The amplitude of the production of specific immunoglobulins was nevertheless always greater in the parasitized area (duodenum) than elsewhere for IgA and IgM. The same relative amounts of specific IgG were observed in the duodenum and in the caeca. The patterns for each isotype in the serum were identical, except for the quick initial rise of IgA during the first week pi. A similar pattern of local and systemic IgM antibody response was noted after an E. tenella infection, but specific circulating IgG was detected as

Intestinal antibody response to avian coccidiosis

i

1-4

14

7

21

A505m

21

Days p.i

Daysp.i

v

A 505 ain

0.25 0.2

w

.

0.25 0.2 0.15

0.15 0.1

0.1 0.03

O.O§ 0

0

i

1.4

I

2.1

14

A 505 nm

21

Days p.i

Days p.i

A 505 nm

-

1.5

1.5 1

-r

-8.

1

0.5

l t

0.50 i-&2@

0

14

7

21

7

14

Days p.i

21

Days p.i

Fig. 2. ELISA determination of speciIic IgA (l:l), IgG (1:4) and IgM (1: 1) antibodies against .I?.~eneila in the duodenum (left) and caeca (right). Days post-infection are indicated along the x axis and absorbance (&J is on they axis. Wck bars. infected birds; open bars, control birds. The statistical significance of differences between control and infected birds by Student’s c-test is indicated by: *P < 0.05, **P c 0.01, ***P < 01001.

early as the first week p.i. and specific intestinal IgA was not detected during the first week p.i. The cx uiuo culture of intestinal tissuescoupled to Table 2-Serum’

IgA, 1gM and IgG antibodies to E. ceneiiu antigen during the 3 weeks following infection

Days pi. I 14 21

an ELBA ii a useful method of analysing the development of an antibody response in distinct areas of the intestine (Zigterman er ul., 1993). As specific

bc.4 Control Infected Control Infected Control Infected

Serum antibodies against E. feneila W

0.028 k 0.004” 0.066~0.010** 0.027 & 0.004 0.150*0.019*** 0.001 ~0.001 0.036kO.016

0.076&0.011 0.200&0.032** 0.091~0.014 0.252 k 0.03Y 0.003 * 0.002 o&41* 0.002

kG

a.334ko.o4o a.750*0.129* a.337ko.033

2.905~0.0+?1~** 0218~0.043 2.046*0.148***

%rnm diluted I:160 for IgA and IgG and I:320 for IgM. ‘Mean&S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001, statistical significance of differences between control vs infected, by Student’s z-test.

808

F. Girard et al.

immunoglobulins are detected after incubation of washed intestinal tissue for 16 h, this test detects the releaseof existing antibodies and any new production by the B cells during the incubation, as shown by Zigterman et c& (1993) using protein synthesisinhibitors such as cycloheximide or cytochalasin A, which efficiently blocked IgA release. The authors used these techniques lOdays after infecting chickens with E. tenella and demonstrated the production of specific IgM, IgA and IgG antibodies in the caecum, where E. tenelIa replicates. Earlier studies used precipitating and neutralization tests to reveal an increasein specific IgA in the caecal contents by 7 days pi., with a peak lOdays later which was associated with recovery (Davis et a/., 1978; Davis & Porter, 1979). To the best of our knowledge, no other kinetic studies of the antiE. acervulina intestinal immunoglobulin response have been performed. The secreted IgA has not yet been shown to have any protective action in chickens during coccidiosis. But extracts of the caecal contents of chickens infected with E. tenella that are rich in IgA can damage the sporozoites, causing most of them to lose the ability to differentiate, even when they can still invade the enterocytes (Davis et af., 1978). Others have used immunofluorescence and agglutination assays to show that specific IgA antibodies in the caecum and bile after an E. tenella infection can bind to the sporozoite membrane (Trees et al., 1989). Our results show that the medium from cultured duodenum and caecafrom E. acervulina- or E. tenellainfected chickens contain considerable amounts of specific IgG. Production does not begin until the second week of infection, while specific IgM production is highest in the first week of infection. The IgA response is restricted to the second week pi. This seems to contradict the immunohistological data of Davis et af. (1978) who found almost no specific IgG+ cells in the submucosa of the caecum of chickens infected twice with E. tenella, but a large number of specific IgA+ cells. Jeurissen et al. (1989) used monoclonal antibodies against chicken IgM and IgA, and found mostly IgM+ and IgA+ plasma cells in the lamina propria of the villi and IgM and IgAantibodies in germinal centres of the submucosa 3 weeks after 2 infections with E. tenella. No count of IgG+cells was reported. More recently, Rothwell et al. (1995) detected IgM+ B cells in the intestinal epithelium of the jejunum, where the parasite multiplies, after an E. maxima infection,. There were 2 peaks of proliferation, the first 1 day after infection and a second 6 days later. They detected IgA+ cells in the epithelium after the second challenge with oocysts 15 days after the prepatent period. The importance and function of IgGs in the intestinal contents is still a matter of speculation. IgGs

have been found in the intestine of mammals, but their origin has not been well identified. Some authors think IgGs transude from the serum (Wernet ef al., 1971), but others favour local production (Dahlgren et al., 1987). Our experiment suggests that these IgGs are secreted locally, because they were not detected in the culture supernatant at the beginning of the culture, but were present after 16h, after extensive washing of the intestinal mucosa. Leslie et al. (1971) described IgG as secretory immunoglobulins in the fowl. Others have shown that IgA are the predominant isotype in the gut contents, although IgG is found occasionally (Lebacq-Verheyden et al., 1974; Trees et al, 1989). But IgGs are fragile and easily destroyed in intestinal fluids. Differences in the techniques used could explain why they are not usually detected. IgGs have been detected in the gut of white leghorn chickens when pilocarpine was used to induce secretion (Porter & Hoh, 1992). Our results show that the amplitude of the specific antibody production, especially of the IgM and IgA isotypes, depends on the intestinal area, with greatest production in the parasitized duodenum for E. acervulina. But significant, although lower, production of specific IgM and IgA also occurred in the caeca. Specific IgG productions in the duodenum and caecum 2weeks post-infection were essentially the same. The response to E. tenella likewise involved significant specific production of IgA, IgM and IgG isotypes in the duodenum, which is not colonized. Weisz-Carrington et al. (1979) suggest that lymphocytes primed by parasitic antigens in the intestinal mucosal circulate throughout the intestine and reach intestinal sites where Eimeria does not develop. Our results suggest that this could take place very quickly after infection. Another possibility is that membrane antigens are released into the intestine, and that these trigger an immune response in distinct areas of the intestine. Corroborating these observations, Jeurissen et al. (1989) found, using imrnunohistology, that the immunity of chickens immunized against E. tenella seemed to inhibit the penetration of the sporozoites not only in the caeca but also partially in the small intestine. In conclusion, an ex vivo intestinal culture assay was used to follow the mucosal immune response in 2 areas of the intestine after E. acervuhza or E. tenella infection. Antibody responses appeared quickly both at the site of parasite development and also significantly in the other intestinal area. Specific IgA production was indeed triggered, but the response was transient and small. But specific IgG production was high, suggesting that it is involved in local protection against Eimeria in chickens. Acknowledgements-F.

Girard was supported by a grant

Intestinal antibody response to avian coccidiosis from Region Centre (France). The English text was checked by Dr Owen Parkes.

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