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The effect of BCG, zymosan and Coxiella burnetti extract on Eimeria infections

Article  in  Immunology and Cell Biology · September 1996

DOI: 10.1038/icb.1996.61 · Source: PubMed

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Immunology^ and Cell Biology (1996) 74, 346-348

Brief Communication

The effect of BCG, zymosan and Coxiella biirnettiextract on Eimeria infections

NC SMITH" and KS OVINGTON^

^ Institut fur Parasitologie, Universitdt Zurich. Zurich. Switzerland and'^Division of Biochemistry and MolecularBiology. School of Life Sciences. Australian National University. Australian Capital Territory, Australia

Summar>' Infection of animals with species of Eimeria induces a hyper-reactivity to endotoxin as manifestby a greatly increased capacity of infected animals to produce TNF in response to LPS in vivo compared withuninfected animals. This finding indicates priming for hyperactivation of macrophages by Eimeria infectionand raises the possibility that non-specific triggering of macrophages by agents such as Bacille Calmette-Guerin (BCG), zymosan or Coxiella burnetti extract may be a simple means of control for coccidiosis.However, all of these agents enhanced oocyst excretion in mice, rats or chickens infected with Eimeriavermiformis. Eimeria nieschulzi or Eimeria tenella. respectively, without affecting the patent period.

Key words: Bacille Calmette-Guerin (BCG), chickens, Coxiella burnetli, Eimeria spp., mice, rats, TNF,zymosan.

Introduction

Non-specific resistance to many parasites can be inducedin experimental animals by prior treatment with immuno-stimulants such as Bacille Calmettc-Gucrin (BCG).Corynehacterium parvum. Coxiella hurnetli. cord factor,COAM, zymosan, glucan. Salmonella and Listeria. Thishas been shown for trypanosomes, Plasmodium. Babesia.Leishmania and various helminths.' Particularly con-vincing studies demonstrated that, aside from an abilityto induce resistance to Babesia microti in mice, all theseagents are capable of inducing anti-tumour activity andhyper-reactivity to endotoxin.^""^ In the same studies, itwas shown that other immunomodulators such as Staphy-lococcus. Streptococcus, thioglycollate and colloidal car-bon did not induce anti-tumour activity or hyper-reactivity to endotoxin, nor did they induce resistance toB. microti. Thus, the effective agents appear to possess aparticular ability and it is probably significant that most ofthem are demonstrated inducers or enhancers of gamma-intcrferon production.'

Gamma-interferon is known to play an important rolein resistance to many parasites including Eimeria spp. Inparticular, the ability of inbred strains of mice to resistprimary infection with Eimeria vermiformis is related totheir ability to produce gamma-interfcron in response tothe parasite.^"^ Additionally, the ability of inbred miceto control primary infection is correlated with their abilityto generate free radicals in response to either parasiteextracts or zymosan,^ indicating a non-specific com-

Correspondence: Dr NC Smith, Queensland Institute of Med-ical Research, The Bancroft Centre, PO Royal Brisbane Hospital,Brisbane, Qld 4029, Australia.

Received 21 December 1995; accepted 6 February 1996.

ponent in effector cell activation. Our aims were,, there-fore, to: (i) test the hypothesis that infection with Eimeriainduces a hyper-reactivity to endotoxin; and (ii) subse-quently, test whether agents such as BCG, zymosan and C.burnetti extract could confer resistance to these parasites.

Materials and methods

Administration of LPS to rodents infected with Eimeriaand quantitation ofTNEin their sera

Eight-week-old female BALB/c mice (Australian National Uni-versity animal care facility) or SIV rats (University of Zurichanimal care faeility) were infected with 2000 sporulated E. vermi-formis oocysts or 50 000 sporulated Eimeria nieschulzi oocysts,respectively, these being doses where pathological manifestationsindicative of TNF production (eg, anorexia and cachexia) be-come obvious. Eight days after infection, the animals were in-jected i.p. with LPS (Sigma, St Louis. USA; phenol-extractedfrom Esclwrichia coli serotype O128:B12 : 30 pg for mice andI mg for rats). Serum samples were collected 90 min later fromthe animals and TNF assayed as previously described.^

Administration of BCG. zymosan andC. burnettiextract and quantilation of parasite load after challengewith Eimeria

Female C57BL/6 mice (Australian National University animalcare facility) were chosen for this part of the study because oftheir known susceptibility to E. vermiformis. characterized by anextended patent period and exacerbated oocyst excretion com-pared with other strains.'" Thus, any effect on parasite reproduc-tion by the macrophage activators should be particularly obviousin this strain. Groups of eight 5-6-week-old mice were injected

The effect of BCG. zymosan and Coxiella burnetti extract on Eimeria tnfections 347

i.p. with 10" BCG, 10' BCG, 2 mg of zymosan (Boehringer-Mannheim. Sydney, Australia). 200 )il of C burnelli extract(Commonwealth Serum Laboratories, Melbourne, Australia) orleft untreated. The selection of doses was based on the results ofClark et al.--'* Two weeks after administration of the variousagents, mice were orally infected with 10^ sporulated oocysts ofE. vermiformis. Timing of the challenge infections relative to thepretreatments with the various agents was also based on verythorough previous work, which established 2 weeks as the mini-mum interval between pretreatment and challenge for maximaleffect in a range of mouse strains,-"•* Faeces were collected dailyfrom individually caged mice and the oocysts counted as previ-ously described'" to establish total oocyst excretion per mouseand the length of the patent period (defined here as the periodover whieh oocysts are excreted).

Results

Table 2 Effect of BCG, zymosan and C burnetli extract onoocyst production and patent period ofE. vermiformis infectionsinC57BL/6mice

Agent

NonelO-* BCG10̂ BCG2 mg zymosan200 uLC burnelli

extract

Total oocvst excretion/mouse ( X 10' ̂ :

mean±s.e.m., fj = 8)

l l l ,63±9.40122,02±11.98149,21 ±26.55211.88±23.79*297.74±22.59*

Length of patentperiod (range.

w = 8, days)

7-87-87-86-87-9

Mice were treated by i.p. injection 2 weeks prior to infectionwith 10^ sporulated oocysts.

*Significantly different from controls {P<0.05) using theWilcoxon test.

Effect of LPS on TNE production in rodents infectedwith Eimeria

LPS stimulated systemic production of TNF in animalsinfected with Eimeria (Table 1). Thus, both mice infectedwith E. vermiformis and rats infected with E. niesehulzihad higher levels of TNF in their circulation if they weretreated with LPS.

Effect of BCG, zymosan andC. burnetti extract oninfections with Eimeria

None of BCG, zymosan or C burnetti extract caused areduction in oocyst excretion or the length of the patentperiod of E. vermiformis in mice (Table 2), In fact, therewas an overall tendency for increased oocyst excretion intreated mice, particularly in those mice injected with zy-mosan or C burnetli extract, without any effect on thepatent period. Similar results were obtained in rats (in-fected with E. nieschulzi) and chickens (infected withEimeria tenella) injected with 20 or 8 mg of zymosan,respectively. Thus, untreated 10-wcek-old female SIV rats(Universitat Zurich) infected with 10̂ sporulated oocystsof £", nieschulzi. excreted 99.72±38.62 (x lO*") oocysts(mean±s.e.m., n = 5) compared with 140.41 ± 13.98

Table 1 Concentrations of TNF (ng/mL) in sera taken fromBALB/c mice or SIV rats 8 days after infection with E- vermi-formis (2000 oocysts) or £, nieschulzi (50 000 oocysts). respec-tively

Injected with LPS' Not injected with LPS

Uninfected miceInfected mice

Uninfected ratsInfected rats

0.7.

4.33.

6±0.15 ±0.9*

5 ±2.05 ± 4.0*

0.4 + 0.30,5±0.2

0.1 + 0.10.3 ±0.1

Results are mean ±s,e.m.. n = 5-10 mice or 4 rats per group,'Animals were injected i.p. with 30 jig (for mice) or 1 mg (for

rats) LPS. 90 minutes prior to serum sampling,*Signif)cantly different from controls (/ '<0.05) using the

Wilcoxon test.

( X lC*) for rats treated with zymosan. The range of patentperiods was 5-6 days for both groups. Untreated 4-wcek-old female LSL chickens (Animaico, Stetten, Switzerland)infected with 10̂ sporulated oocysts of E. tenella excreted379.0 ±48.4 ( X 10'') oocysts (mean±s.e.m., « = 4) com-pared with 472.2±22.0 ( x 10 )̂ oocysts for chickens in-jected with zymosan. Again, the patent period for bothgroups of chickens was 5-6 days (treatment with the im-munostimulants, C parvum and BCG, as well as infectionwith Salmonella galinarum have also been reported tohave little effect on infections with Eimeria inchickens''),

Discussion

This study demonstrates that infection with Eimeria in-duces hyper-reactivity to endotoxin as evidenced by thegreatly enhanced systemic production of TNF in infectedmice and rats injected with LPS (Table 1). These data arecomplemented by earlier work'- showing that macro-phages from Eimeria-mfcc'iC(\ chickens have an enhancedability to produce TNF and IL-1 in viiro. indicating thatcoccidial infection may lead to macrophage activation.These series of observations suggest that non-specific im-munomodulation by agents such as BCG, zymosan and C/)Hr«('//; extract could represent a simple means of controlfor coccidiosis. However, this was shown to be clearly notthe case and, at least in mice infected with /:, vermiformis.parasite reproduction actually seemed to be significantlyenhanced by prior treatment with the immunostimuiants(Table 2).

Why treatment with agents such as BCG, zymosan andC burnetti extract should cause an increase in oocystexcretion by animals infected with Eimeria is puzzling butthere are several potential explanations based on the abil-ity of these agents to activate macrophages.--^ First, pre-treatment with the various immunomodulators may haveexhausted the macrophages prior to infection with £7mt'-ria thereby rendering the hosts more susceptible to infec-tion. This does not appear to be a ver>' likely explanation,however, since similar inoculation regimens were used by

in the successful induction of resistance to Plas-

348 NC Smith ei a\.

modium and Babesia. Second, macrophage activationmay actually lead to the production of factors, includingcytokines, which promote parasite growth.'^ Alterna-tively, macrophage-derived products may indirectly in-crease parasite reproduction by interfering with effectiveimmune responses to primary Eimeria infections. Thisproposal may also be flawed because, at least in mice,inefficient resistance to Eimeria is characterized by a pro-longed patent period reflecting extra cycles of schi-zogony;'"* this did not occur in our study since the patentperiod was unaffected by the immunomodulators, indi-cating that a similar number of asexual divisions occurredin treated and untreated animals.

A plausible explanation for the positive effect of themacrophage activators on oocyst production is that onlythe initial establishment of infection was affected. Therole of macrophages in modulating infections with Eime-ria is controversial; experiments with silica and carag-eenan designed to poison macrophages have yielded con-tradictory results but infections with E. vermiformis inmacrophage-deficient mice were greater than in controlmice and T cell-derived factors are capable of enhancingmacrophage killing off . tenella sporozoites in v/7ro.''However, it may be important that, although damagedsporozoites may be engulfed and killed by macrophages,there is scant evidence that uptake of normal, healthysporozoites by macrophages is in any way deleterious tothe parasite.'' In fact, macrophages, along with intraepi-thelial lymphocytes, have been implicated in the transportof invasive stages of Eimeria to their final infectivesite.'-'' '^ Thus, sporozoites are engulfed by monocytesvery early in the lifeeycle and, in species such as E. acer-vulina, E. maxima. E. necatrix. E. tenella. E. falciformisand E. vermiformis. this is an important step in ensuringthat the parasites are transported from the intestinal sur-face to the crypt epithelia, where their development oc-curs. Even in species where development occurs in thesurface epithelia (e.g. E. brunetii and E. praecox) uptakeby mononuclear cells occurs and a considerable portion ofsporozoites of all species may actually be transportedthrough extraintestinal sites before returning to the gutsome hours later. This phenomenon may help to explainthe increase in oocyst excretion associated with pretreat-ment with BCG, zymosan or C. htmietti extract. It isconceivable that increased activity of intestinal mono-

cytes may result in more efficient engulfment of sporozo-ites, actually resulting in more sporozoites arriving attheir preferred infective site. In this way, a greater numberof parasites would become established in the intestineand, ultimately, oocyst excretion would be increased with-out any effect on the length of the patent period.

Acknowledgements

This work was supported by the Australian ResearchCouncil and the Bundesamt fur Bildung und Wissen-schaft, Bern (Project: Coccidiosis COST-89).

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