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Research in Veterinary Science 82 (2007) 225–231

Experimental coccidiosis provoked by Eimeria acervulina inchicks simultaneously fed on ochratoxin A contaminated diet

V. Koynarski a, S. Stoev b,*, N. Grozeva b, T. Mirtcheva c, H. Daskalov d,J. Mitev e, P. Mantle f

a Department of Parasitology, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgariab Department of General and Clinical pathology, Faculty of Veterinary Medicine, Trakia University, Stara Zagora 6000, Bulgaria

c Department of Biochemistry, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgariad Department of Veterinary Hygiene, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria

e Department of Ecology and Zoochygiene, Faculty of Agriculture, Trakia University, Stara Zagora, Bulgariaf Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK

Accepted 5 July 2006

Abstract

The progression of coccidiosis provoked by Eimeria acervulina was followed in chicks fed on OTA-contaminated as well as on OTA-free diets. More heavy progress of duodenal coccidiosis, including mortality, occurred in OTA-treated chicks as can be seen from thehigher value of lesion (3.50) and oocyst (31.65) indices. A stronger decrease of serum total protein was found in OTA-treated chicks(22.80 g/l) than in chicks infected with E. acervulina(24.20 g/l), but that decrease was strongest in chicks treated with OTA and simul-taneously infected with E. acervulina (19.71 g/l). The serum concentration of uric acid was significantly increased in all chicks exposed toOTA, most notably in those additionally infected with E. acervulina (1020.6 (l mol/L), whereas the serum enzyme activity of AST wasincreased only in chicks infected with E. acervulina and highest in those fed OTA contaminated diet (122.2 U/L). OTA induced degen-erative changes in kidneys, liver and heart as well as a depletion of lymphoid tissue in the lymphoid organs and a decrease of body weight.Coccidiosis induced only a slight growth depression and duodenal hemorrhages in addition to characteristic duodenal damages. Theimpairment of kidney function, histopathological changes and general growth depression were stronger when chicks infected with E.

acervulina were also given OTA.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Ochratoxicosis; Ochratoxin A; Coccidiosis; E. acervulina; Pathology

1. Introduction

Ochratoxin A (OTA) is a widespread mycotoxin in lotsof components of animal feed. Therefore, the significanceof this mycotoxin for animal health has been comprehen-sively reviewed (Kuiper-Goodman and Scot, 1989; Marqu-ardt and Frolich, 1992). The most important economicproblems reported when OTA-contaminated feed wasgiven in chicks were the growth depression and increasedmortality, although some other specific damages on many

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doi:10.1016/j.rvsc.2006.07.004

* Corresponding author. Fax: +359 42 670624.E-mail address: stoev@uni-sz.bg (S. Stoev).

tissues were also noted (Huff et al., 1974; Dwivedi andBurns, 1984, 1986; Stoev et al., 2000a, 2002b,c, 2004).The increased mortality among chicks or pigs fed onOTA-contaminated diet was seen to be due to OTA-pro-voked susceptibility to secondary bacterial infections(Stoev et al., 2000b) or to a heavy progression of someoften encountered parasitic diseases (Stoev et al., 2002b),because of the suppression in both humoral and cell-med-iated immune response in such animals as an aspect of och-ratoxicosis (Burns and Dwivedi, 1986; Stoev et al., 2000a,b,2002c). Our observations on naturally encountered chicknephropathy in Bulgaria have identified a rather high inci-dence of mortality which can only partly be attributed to

Table 1Experimental design

Group OTA in feed betweendays 1 and 14 (ppm – mg/kg)

Inoculation with E. acervulina

– day 7 (4 · 105 oocystsper chick per os)

I None E. acervulina

II 4 NoneIII 4 E. acervulina

Control None None

Feed levels of ochratoxin A (given between days 1 and 14 of the experi-ment) and inoculation with E. acervulina (at day 7 of the experiment) ofvarious experimental groups of chicks.

226 V. Koynarski et al. / Research in Veterinary Science 82 (2007) 225–231

OTA-contaminated feed (Stoev et al., 2002a). The descrip-tions of pathology of spontaneous chick nephropathy varyconsiderably, especially if another secondary disease arisesspontaneously along with OTA-induced nephropathy inchicks (Stoev et al., 2002b) that can occur frequentlybecause of the immunosuppression accompanying thisnephropathy (Stoev et al., 2000a,b). Therefore, it is impor-tant to clarify the complex clinicomorphological findings insuch cases, which derive from both diseases as well as tofollow the progression of coccidiosis in the same cases.At present, there are very scarce and contradictious dataon progression of coccidiosis in chicks fed on OTA-con-taminated diet. For example, Huff and Ruff (1982) reportedan increase of serum uric acid and an increase of relativeweight of liver in chicks fed OTA and infected with E.

tenella, which was confirmed by Stoev et al. (2002b), butboth the relative weight of liver and the uric acid concen-tration in serum were decreased in chicks fed OTA andinfected with E. acervulina (Huff and Ruff, 1982). That’swhy, we decided to extend our recent study on progressionof coccidiosis provoked by E. tenella in chicks fed on OTA-contaminated diet (Stoev et al., 2002b) and included E.acervulina in our further investigations in this area.

2. Materials and methods

2.1. OTA production

Aspergillus ochraceus (isolate D2306, as used by Tapiaand Seawright, 1984; and Stoev et al., 2000b, 2002b) wasgrown on sterilized shredded wheat (40 g) in 500 ml conicalflasks, moistened by a 40% (w/v) addition of sterile waterand incubated on a rotary shaker at 27 �C for 2 weeks(Harris and Mantle, 2001). The brown granular product,which bore no obvious sign of fungal growth or sporula-tion, was sterilized at 80 �C for 1 h and stored at �20 �C.A sample was analyzed for ochratoxins; batches typicallycontained OTA (about 2 mg/g) and relatively small compo-nent of the biologically-inactive alkaloid ochratoxin B (des-chloro-ochratoxin A). No other mycotoxins were producedin this solid substrate fermentation process and the neces-sary dilution by approximately 103 when homogenized intochick ration made only a minimal addition of other compo-nents of the molded shredded wheat substrate (Stoev et al.,2000b, 2002b).

2.2. Experimental design

Specific pathogen-free chicks (Plymouth Rock) werepurchased at a week of age and housed in wire floor cageswith continuous infra-red lighting at a temperature suitablefor their age. Commercial complete standard feed withoutany medication (Smesler, Stara Zagora, Bulgaria) with orwithout added OTA was available ad libitum. The chickswere housed in 3 experimental and 1 control groups (12birds in each) and treated either or both with OTA andE. acervulina (Table 1).

2.3. Measurements

The chicks were weighed at the beginning of the exper-iment and after one and two weeks. For following thepathomorphological changes in various internal organs aswell as oocyst and lesion indices the chicks from eachgroup were slaughtered two weeks after the beginning ofthe experiment or one week after the infection with E. acer-

vulina, as the first mortalities occurred in group III in thattime (Table 1).

2.4. Histological examination

Tissues for histological examination were taken fromkidneys, liver, lung, heart, thymus, bursa of Fabricius,spleen, intestine, cerebellum, brain and medulla and fixedin 10% neutral buffered formalin. The fixed tissues wereprocessed for paraffin embedding, sectioned at 6 lm andstained with haematoxylin–eosin.

2.5. Parasitological examination

The required quantity of oocysts was obtained from nat-urally susceptible chicks (two-weeks of age) inoculatedexperimentally with oocysts of E. acervulina according toLozanov (1980). The oocysts were preserved in 2.5% solu-tion of potassium bichromate (K2Cr4O7) at 4 �C until usedfor inoculation (Table 1). The oocyst index in slaughteredchicks was examined according to Cucler (1959), whereasthe lesion index in the same chicks was evaluated accordingto Johnson and Reid (1970) (Table 2).

2.6. Hematological and biochemical examination

Blood samples were taken at slaughter at the end of the14th day of the experiment and examined for various bio-chemical parameters within 1–2 h of collection, immedi-ately after separation of serum. The serum total proteinwas measured by Bio-La-Test (Lachema Diagnostica,Brno, Czech Republic) and uric acid by EnzUric-FT-test(Labordiagnostica, Gopecke, Germany). The serumenzyme activity of AST (asparate-aminotransferase) wasmeasured by Cormay test (Smolenskiego, Warsaw,Poland).

Table 2Mean values of lesion and oocyst indices in slaughtered chicks (7 daysafter inoculation with E. acervulina).

Group Number ofexamined chicks

Lesion indexb Oocyst indexc

I (E.ac.) 12 2.41 ± 0.31a 26.31 ± 0.96a

II (OTA) 12 0 0III (OTA + E.ac.) 8 3.50 ± 0.26 31.65 ± 0.52

Control 12 0 0

± SEM (standard error of the mean).a Significant difference compared to group III (p < 0.05).b Lesion index (the maximum is 4) was examined using the following

scheme: assessment 1 – scattered white plaque-like lesions confined to theduodenal mucosa (maximum 5 lesions per square centimeter); assessment2 – lesions are much closer together, but not coalescent; the intestinal wallshows no thickening; assessment 3 – lesions are numerous enough to causecoalescence in the lesion size, giving the duodenal mucosa a coatedappearance; the intestinal wall is thickened and the content is watery ;assessment 4 – coalescing of the lesions is much more complete and nodistinctive lesions may appear on the duodenal mucosa; the intestinal wallis considerably thickened.

c Oocyst index (the maximum is 40) was examined using the followingscheme: assessment 0 – from 0 up to 0.1 million oocysts in the intestine forchick; assessment 1 – from 0.1 up to 1 million oocysts; assessment 10 –from 1 up to 5 millions oocysts; assessment 20 – from 5 up to 10 millionsoocysts; assessment 40 – more than 10 millions oocysts.

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2.7. Statistical methods

Non-parametric Mann–Whitney in addition to Stu-dent’s t-test was used to estimate significant differencesbetween the mean values of various parameters in differentgroups of chicks.

3. Results

3.1. Clinical observation

Diarrhoea was the main clinical sign seen first in chicksgiven OTA (groups II and III) on day 3 from the beginningof the experiment, but the same was also seen in chicks ofgroup I, 5 or 6 days after inoculation with E. acervulina andwas most pronounced in chicks infected with E. acervulinaand simultaneously given OTA (group III) at that time.Other clinical signs, depression, weakness and dullness, ruf-fled feathers, reduction of feed intake and growth depres-sion occurred in all experimental groups though mainlyin groups II and III. One chick from group III dead at

Table 3Mean values of body weight (b.w.) in chicks of various experimental groups at

Group Day 1 Day 7

b.w. (g) Number of chicks b.w. (g)

I (E.ac.) 85 ± 4.0 12 152.9a ± 7.7II (OTA) 83 ± 3.5 12 103.1* ± 5.5III (OTA + E.ac.) 86 ± 2.8 12 95.4* ± 6.0

Control 84 ± 3.6 12 160.4a ± 6.6

± SEM (standard error of the mean).a Significant difference compared to group III (p < 0.05).* Significant difference compared to controls (p < 0.05).

day 13 and three more dead at day 14 of the experimentas was expected from the higher values of lesion and oocystindices (Table 2) and the significant decrease of bodyweight (Table 3) in slaughtered chicks of the same OTA-treated group.

3.2. Body weight

A statistically significant decrease of body weight wasobserved in all experimental groups compared to the con-trol group, but was most marked in groups given OTA.A significant decrease of body weight was seen in chicksgiven OTA and simultaneously infected with E. acervulina

compared to chicks given only OTA (Table 3).

3.3. Biochemical and hematological findings

Total protein in serum was significantly decreased inchicks of all experimental groups, most notably in thosetreated with OTA and especially in group treated withOTA and E. acervulina simultaneously (Table 4).

A significant increase was observed in serum uric acid inall OTA-treated groups, but was highest in chicks addition-ally infected with E. acervulina (Table 4).

Serum enzyme activity of AST was somewhat increasedin all experimental groups, but that increase was confidentonly in groups infected with E. acervulina (Table 4).

3.4. Gross pathology and parasitological findings

There were a few subcutaneous muscular hemorrhagesin chicks of OTA-treated groups II and III. The kidneysand liver in these chicks were congested and enlarged.Their gall bladder was also distended by bile. The mucosalsurface of small intestine was hyperemic and covered withmucous fluid. The pancreas was pink-coloured. The menin-ges were slightly hyperemic.

In chicks infected with E. acervulina (groups I and III)there were various white plaque-like lesions confined tothe duodenal mucosa, which sometimes coalesced, mostnotably in OTA-treated chicks, giving the intestinalmucosa a coated appearance. Also, the mucosal surfaceof duodenum was hyperemic and coated with mucous fluid.In addition, some red spots (2–3 mm diameter) were seenunder duodenal serosa. The intestinal wall of the duode-

the beginning and at end of the first and second weeks of the experiment

Day 14

Number of chicks b.w. (g) Number of chicks

12 243.7a,* ± 11.3 1212 155.4a,* ± 12.7 1212 110.0* ± 9.5 8

12 314.6a ± 16.8 12

Table 4Mean serum values of total protein, AST (asparate-aminotransferase) and uric acid in chicks of various experimental groups at day 14 of the experiment.

Group Total protein (g/l) AST (U/L) Uric acid (lmol/L) Number of chicks

I (E.ac.) 24.20a,* ± 0.89 114.8* ± 4.7 165.5a ± 32.6 12II (OTA) 22.80* ± 1.74 99.6a ± 4.8 872.6* ± 96.7 12III (OTA + E.ac.) 19.71* ± 0.81 122.2* ± 7.1 1020.6* ± 212.4 8

Control 35.40a ± 0.87 79.0a ± 8.6 214.6a ± 20.9 12

± SEM (standard error of the mean).a Significant difference compared to group III (p < 0.05).* Significant difference compared to controls (p < 0.05).

Fig. 2. Photomicrograph of kidney in chick fed on 4 ppm OTA in diet.Necrotic debris and hyaline drops in the lumen of some tubules. H/E, scalebar = 33.3 lm.

228 V. Koynarski et al. / Research in Veterinary Science 82 (2007) 225–231

num was often thickened and the content was waterymainly in OTA-treated chicks as can be seen from thelesion indices (Table 2).

The number of oocysts in the intestine and the values ofthe oocyst indices were also clearly higher in chicks givenOTA in diet (Table 2).

3.5. Histopathology

No histopathological changes were found in internalorgans of control chicks.

In kidneys of OTA-treated groups (II and III) peritubu-lar capillaries were congested. Degenerative changes (gran-ular degeneration or cloudy swelling as well askaryopycnosis or karyolysis) in epithelial cells of the prox-imal convoluted tubules were observed (Fig. 1) as well asfocal mononuclear cells infiltration was seen in the renalinterstice. Some of the tubules contained necrotic debris,granular or hyaline casts in the lumen (Fig. 2). A limitedproliferation of connective tissue and activation of capil-lary endothelium were also noticed in the same chicks.These damages were stronger in chicks given OTA and E.

acervulina (group III) than in those given OTA alone(group II). In chicks treated with E. acervulina alone (groupI) only a slight hyperemia of peritubular capillaries andscarce granular degeneration in epithelial cells of the prox-imal convoluted tubules were seen sometimes.

Cloudy swelling, granular or vacuolar degenerationwere seen in the livers of OTA-treated groups, but the

Fig. 1. Photomicrograph of kidney in chick fed on 4 ppm OTA in diet andinfected with E. acervulina. Granular degeneration and karyopycnosis inepithelial cells of the proximal tubules. H/E, scale bar = 33.3 lm.

same were more marked in group III, infected also byE. acervulina(Fig. 3). Also, there was an activation of cap-illary endothelium and Kupffer’s cells, hyperemia and per-icapillary edema, in addition to perivascular infiltration ofmononuclear cells. Only a slight hyperemia of capillariesand scarce granular degeneration in hepatocytes wereoccasionally seen in chicks of group I, treated with E.

acervulina alone.Degenerative changes in lymphoid organs were only

seen in chicks of groups II and III. There was a pro-nounced depletion of lymphoid cells in the cortical zone

Fig. 3. Photomicrograph of liver in chick fed on 4 ppm OTA in diet andinfected with E. acervulina. Granular and vacuolar degeneration, karyo-pycnosis and karyolysis in hepatocites H/E, scale bar = 33.3 lm.

Fig. 6. Photomicrograph of duodenal mucosa in chick fed on 4 ppm OTAin diet and infected with E. acervulina. Presence of many preliminaryforms of oocysts (schizonts) in intestinal mucosa. Damaged distal part ofthe villi. H/E, scale bar = 50 lm.

V. Koynarski et al. / Research in Veterinary Science 82 (2007) 225–231 229

of the thymus and sometimes the region between medullaand cortex was not well defined or the cortex became verythin. Similar depletion of lymphoid cells (Fig. 4) anddegenerative changes (karyopyknosis and karyorrhexis) inthe lymph follicles were seen in the bursa of Fabricius. Aslight cellular depletion or degenerative changes were alsoobserved in the white pulp of germinal centres in the spleenof chicks in the same groups. No histopathological changeswere seen in the lymphoid organs in chicks of group I,excepting the slight hyperplasia of the white pulp of thespleen.

Degenerative and slight necrotic changes as well as adesquamation of surface- or glandular mucosal epitheliumof duodenum and jejunum were seen in chicks of groups II(Fig. 5) and III, but the degenerative changes in the duode-num were stronger in the chicks of group III. Small pete-chial hemorrhages, hyperemia of vessels, slightmononuclear infiltration in the lamina propria, lots ofoocysts and third generation of schizonts, in addition tolocal degenerative changes of surface- or glandular epithe-lium were also seen in the mucosa of duodenum in chicks

Fig. 4. Photomicrograph of bursa of Fabricius in chick fed on 4 ppmOTA in diet and infected with E. acervulina. Depletion of lymphoid cells inthe central part of the lymph follicles. H/E, scale bar = 50 lm.

Fig. 5. Photomicrograph of duodenal mucosa in chick fed on 4 ppm OTAin diet. Degenerative changes and desquamation of glandular- and surfacemucosal epithelium, including totally damaged villi. H/E, scalebar = 50 lm.

Fig. 7. Photomicrograph of brain in chick fed on 4 ppm OTA in diet andinfected with E. acervulina.A pericellular and pericapillary edema. H/E,scale bar = 50 lm.

infected with E. acervulina (groups I and III), most notablyin group III (Fig. 6). The distal part of the villi of duodenalmucosa was often with desquamated epithelium or totallydamaged in the chicks of the same groups, most notablyin group III.

A perivascular edema, lytic changes and irregular stain-ing, as a result of the increased eosinophilia of some myo-fibrils were seen in the heart of chicks of groups II and III.Slight granular degeneration of myofibrils and activationof capillary endothelium were also observed in these chicks.In chicks treated with E. acervulina alone (group I) only ahyperemia of vessels, slight perivascular mononuclear infil-tration or scarce granular degeneration of myofibrils wererarely seen.

In the lung, there was only a perivascular or peribron-chial mononuclear cell infiltration and slight edematouschanges in the chicks of OTA-treated groups.

In the brain, a pericellular or pericapillary edema wasseen in chicks of groups II and III (Fig. 7). In neuronsand glia cells of the brain in the same chicks there were

Fig. 8. Photomicrograph of cerebellum in chick fed on 4 ppm OTA in dietand infected with E. acervulina. Edema in the region of the Purkinje’s cellsand slightly in the granular layer. Lytic and pycnotic changes in Purkinje’scells. H/E, scale bar = 50 lm.

230 V. Koynarski et al. / Research in Veterinary Science 82 (2007) 225–231

slight lytic changes. In the cerebellum of these chicks,edema and slight degenerative changes were observed inthe region of the Purkinje’s cells and rarely in molecularor granular layers (Fig. 8). Slight edematous changes inthe white substance of the cerebellum were occasionallyseen in the chicks of groups II and III. In the lumbosacralregion of the medulla, slight lytic changes in neurons werealso noticed in some chicks of the same groups. Only aslight hyperemia of capillaries and scarce lytic changes inneurons were occasionally seen in chicks of group I, treatedwith E. acervulina alone.

4. Discussion

It was found that intensity of clinical signs, macroscopicand histopathological changes, the growth depression aswell as impairment of kidney function, expressed by serumlevels of uric acid, were stronger when chicks infected withE. acervulina were also treated with OTA. Moreover, thecoccidiosis progresses in a heavy way and rapidly inOTA-treated chicks, as perceived from the changes inlesion and oocyst indices and especially from the chickmortality. That could be explained by OTA-provokedimmunosuppression and following sensitivity to variousinfectious and parasitic diseases (Stoev et al., 2000a,b,2002b).

The growth depression in OTA-treated chicks, accord-ing to some authors (Mohiudin et al., 1993), may be dueto the impairment of protein synthesis by OTA.

The histopathological changes in OTA-treated chicks,especially those found in kidneys, corresponded well tothe deviation of some biochemical parameters, as theincrease in the serum concentration of uric acid, whichshowed impairment in kidney function was evident. Thedegenerative changes in the epithelial cells of kidneys andliver probably are due to the route of elimination ofOTA via kidneys and partly via liver, due to enterohepatic

recirculation and hepatobiliary way of excretion of OTA(Fuchs, 1988), exerting direct toxic effect of OTA on theseorgans (Dwivedi and Burns, 1984; Stoev et al., 2000a). Theslight granular degeneration in hepatocytes in chicks trea-ted with E. acervulina alone could be due to the resorptionof toxic products as a consequence of decomposition ofnecrotic epithelial debris in the intestinal content (Lozanov,1983).

The degenerative changes and the depletion of lymphoidcells in the lymphoid organs in OTA-treated chicks can bein good agreement with immunosuppressive effects thatOTA can exerts in contamination levels 2–4 ppm, asreported by some authors (Holmberg et al., 1988; Harveyet al., 1992; Kozaczynski, 1994). Therefore, a heavy pro-gression of some infectious (Stoev et al., 2000a,b) and evenparasitic diseases (Stoev et al., 2002b) could be expected aswas demonstrated in the present experiment. The OTA-provoked immunosuppression could be explained by theinhibition of protein synthesis and the subsequent delayin cell division in the immune system (Harvey et al.,1992). The impaired protein synthesis in lymphocytes couldlead to the impairment in their activation, differentiationand proliferation. In addition, the decreased phagocyticactivity of natural killer cells and T-killer cells, which isprobably due to a decrease of basal interferon (Harveyet al., 1992), may additionally deteriorate the immuneresponse of OTA-treated chicks. By the present experi-ment, it was demonstrated in practice, how the knownOTA suppression of humoral and cellular immunity(NNT, 1991; Stoev et al., 2000a,b) can make worse the pro-gression of coccidiosis provoked by E. acervulina, and canalso lead to mortality among chicks at day 7 of the infec-tion that cannot be commonly provoked by E. acervulina

(Williams, 1973; Lozanov, 1983; Holdworth et al., 2004).Diarrhoea and growth depression in chicks were oftenreported as the main clinical signs in coccidiosis provokedby E. acervulina, but no mortality was seen in such chicks(Lozanov, 1983).

The edematous changes seen in various internal organs,in addition to some muscular hemorrhages, might be dueto vascular damages, provoked by OTA (Stoev et al.,2000a). Furthermore, a disturbance of blood clotting dueto a reduction in the concentration of fibrinogen in theblood and an increase in the prothrombin time, observedas a response of ochratoxicosis (Doerr et al., 1981; Priorand Sisodia, 1978), could also contribute to these muscularhemorrhages. However, the scarce petechial hemorrhagesin the duodenum are commonly observed, because of local-ization of E. acervulina in this place (Johnson and Reid,1970).

It can be supposed that the significant decrease in theserum total protein could be due to the impairment of pro-tein synthesis, provoked by OTA, as has been previouslyreported (Mohiudin et al., 1993; Stoev et al., 1999, 2000a,2002b), but the slight decrease in the serum total proteinin the chicks infected only with E. acervulina is likely a con-sequence of progression of coccidiosis and can explain the

V. Koynarski et al. / Research in Veterinary Science 82 (2007) 225–231 231

most pronounced decrease in this parameter in the chickstreated with E. acervulina and OTA simultaneously.

The strong increase in the serum level of uric acid inboth groups treated with OTA showed that the functionof kidneys was significantly impaired, which is in agree-ment with our previous investigations on OTA-toxicity(Stoev et al., 2000a, 2002b). However, the results of thepresent study contradict with the conclusion made by Huffand Ruff (1982) that the serum concentration of uric aciddecreased in chicks fed OTA and simultaneously infectedwith E. acervulina as was reported in the same paper.

Concerning the serum enzyme activity of AST, it can beconcluded that the strongest increase in this parameter inthe chicks fed OTA and simultaneously infected with E.acervulina may be due to the strongest degenerativechanges in the internal organs of the same chicks.

Finally, the present experiment was demonstrated inpractice that some often encountered parasitic diseases, ascoccidiosis provoked by E. acervulina, can progress in amore heavy way in OTA-treated chicks, and can also leadto mortality among chicks that cannot be commonly pro-voked by E. acervulina. Such mortality was only character-istic for coccidiosis provoked by E. tenella,but even in suchcases coccidiosis developed more rapidly and heavily inOTA-treated chicks as was seen from our previous experi-ment (Stoev et al., 2002b). However, our observation is incontrast with the study made by Huff and Ruff (1982), whichfound less lesions in chicks challenged with either E. acervu-

lina or E. tenella and fed OTA compared to chicks not fedOTA. The clinicomorphological finding in such cases maybe complicated, because it derives from both diseases.

Acknowledgement

This study was financially supported by the foundationof Ministry of science and education in Bulgaria.

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