Departamento de Medicina Veterinária Preventiva, Centro de Ciências Agrárias, Universidade Estadual de Londrina UEL

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1 Rev. Bas. aasitol. Vet., Jaboticabal, v. 19, n. 1, p. 1-6, jan.-ma ISSN X (impesso) / ISSN (eletônico) Full Aticle ost-challenge hematological evaluation with viulent stain of Eimeia tenella in boiles immunized with attenuated stain o spoozoite poteins fom homologous stain Avaliação hematológica pós-desafio com cepa viulenta de Eimeia tenella em fangos de cote imunizados com cepa atenuada ou poteínas de espoozoítos de cepa homóloga. Alexey Leon Gomel Bogado 1,2 *; João Luis Gacia 1 ; atícia Fenandes Nunes da Silva 3 ; Maa Regina Stipp Balain 1 ; José da Silva Guimaães Junio 1 1 Depatamento de Medicina Veteináia eventiva, Cento de Ciências Agáias, Univesidade Estadual de Londina UEL 2 ogama de ós-gaduação em Ciência Animal, Univesidade Estadual de Londina UEL e Bolsista CNq 3 ogama de ós-gaduação em Ciência Animal, Univesidade Estadual de Londina UEL e Bolsista CNq Received July 8, 2009 Accepted Septembe 10, 2009 Abstact Hematological paametes wee evaluated in boiles immunized and challenged with Eimeia tenella. Boile chickens of Hubbad stain, females, coccidian-fee, wee kept in wie cages and inoculated on the thid day. The expeiment was designed to include five sots of teatment with thee eplicates each. T1 was the negative contol goup, T2 eceived 500 attenuated spoulated oocysts by gavage, T3 was the positive contol, T4 eceived 50 µg of spoozoite potein + Quil A vaccine, and T5 eceived Quil A without spoozoite potein + BS, the last two though nasal oute on days 0, 7, and 21. On the 31 st day, all teatments wee challenged with homologous viulent stain of E. tenella in the dose of oocysts, with the exception of T1. One week late, blood sampling, lesion scoes, and cecal oocyst count wee caied out. The paasitological paametes showed statistical significance (p < 0.05) and thee was no damage to the hematological paametes of bids (p > 0.05) by ANOVA test. The coelations suggest that the blood paametes wee impaied by effects of the paasite on tissue, showing levels of hemohage and/o hydation. Keywods: Eimeia tenella, hematology, attenuated stain, spoozoite potein. Resumo Foam avaliados os paâmetos hematológicos em fangos de cote imunizados e desafiados com Eimeia tenella. intos de cote fêmeas da linhagem Hubbad, lives de coccídios, foam mantidos em bateias metálicas e inoculados no teceio dia. O expeimento foi delineado po cinco tatamentos com tês epetições cada, sendo: T1 contole negativo, T2 ecebeu 500 oocistos espoulados atenuados via oal, T3 contole positivo, T4 ecebeu vacina contendo 50 μg de poteínas de espoozoítos + Quil A e T5 ecebeu Quil A + BS, sendo os dois últimos po via nasal nos dias 0, 7 e 21. No dia 31, todos os tatamentos foam desafiados com cepa viulenta homóloga de E. tenella na dose de 8, oocistos, exceto T1. Uma semana depois, foi ealizada amostagem de sangue, escoe de lesão e contagem de oocistos cecais. Os paâmetos paasitológicos apesentaam significância estatística (p < 0,05), sem que causassem pejuízos aos paâmetos hematológicos das aves (p > 0,05), pelo teste ANOVA. As coelações sugeem que os paâmetos sanguíneos foam afetados pelos efeitos do paasita no tecido, apesentando níveis de hemoagia e/ou hidatação. alavas-chave: Eimeia tenella, hematologia, cepa atenuada, poteínas de espoozoítos. *Coesponding autho: Alexey Leon Gomel Bogado Depatamento de Medicina Veteináia eventiva, Cento de Ciências Agáias, Univesidade Estadual de Londina UEL, Campus Univesitáio, Rod. Celso Gacia Cid, R 445, km 380, C 6001, CE Londina - R, Basil; alexey@yahoo.com.b Suppoted by: CNq.

2 2 Bogado, A.L.G. et al. Rev. Bas. aasitol. Vet. Intoduction The poulty coccidiosis is one of the most impotant paasitic diseases of chickens and the main species commonly found in field ae Eimeia acevulina, E. maxima, and E. tenella, each one infecting a specific egion of the intestine (LILLEHOJ et al., 2004). Eimeia tenella, the most pathogenic of the thee species mentioned above, inhabits the chicken s caeca and may cause hemohage that accompanies the emegence of the second stage schizont development (ALLEN, 1997). Accoding to Allen (1997), duing infection the po-inflammatoy cytokines can induce many cells to poduce nitic oxide (NO) by induction of NO synthase (inos), which shows toxicity to both coccidian paasites and the host cells, and the vasodilatation action is associated with hemohage poduced duing infection. It is known that the pathogenic effects caused by E. tenella species impai othe systems in the chicken s body. A numbe of studies have evaluated the effects on blood cells. The studies evaluating the hematological paametes in bids infected with Eimeia spp. ae geneally esticted to the evaluation of the globula volume and some constituents of plasma, such as caotenoids, lipids, and total potein (CONWAY, 1993; ALLEN et al., 1996; 2004; ALLEN, 1997; FETTERER; ALLEN, 2001). It is impotant to know the population of blood cells ciculating afte challenge with coccidiosis in susceptible and immunized bids to bette chaacteize the immunity geneated and associated with the pathogenic effects caused by this potozoan. This study aimed to measue the hematological paametes of two goups, one composed of chickens immunized with E. tenella attenuated stain and anothe immunized with spoozoite poteins associated with adjuvant and challenged with homologous viulent stain. Mateial and Methods 1. Expeiment place The expeiment was caied out in the Laboatoy of aasitology and aasitay Disease (LD), Laboatoy of Clinical athology and Veteinaian Hospital Isolation Secto, petaining to the Depatment of eventive Veteinay Medicine of Univesidade Estadual de Londina (UEL), located in the noth of the State of aaná. 2. Animals Boile chickens of Hubbad stain, females, coccidian-fee, wee obtained fom a commecial hatchey at the day of hatch and wee kept in wie cages with wate and food ad libitum. Fo the multiplication of viulent and attenuated E. tenella stains, 80 bids wee povided with feed containing anticoccidial dug until 10 days old; fom the 11 th day of age onwads the feed was fee of anticoccidial dugs. The bids wee inoculated on the 15 th day, afte fasting fo 6 hous. In the immunogenicity evaluation, 180 chicks wee housed in wie cages and wee given manufactued feed in accodance with poulty industy specifications (state, gowe, and finishe), without anticoccidial dugs. All pocedues involving animals wee conducted in accodance with the pinciples of Ethical Committee fo Expeimental Animals of the Univesidade Estadual de Londina (CEEA/UEL), gant numbe 52/ Attenuated stain vaccine, spoozoite potein vaccine, and viulent stain (challenge) Used in this study was a viulent stain of Eimeia tenella, obtained in the field afte necopsies of infected chickens and maintained in the LD though passages in chickens. Attenuation of viulent stain was used accoding to the technique descibed by Jeffes (1975), which showed a educed pepatent peiod (pp) fom 167 hous fo the oiginal paent stain to 137 hous ove 20 passages (unpublished data). Oocyst puification was collected by salt flotation fom thei pathological site at the end of the pepatent time accoding to the pocedue descibed by Long (1971). Afte excystation and puification of the spoozoite of E. tenella by density gadient centifugation in ecoll, accoding to the technique descibed by Dulski and Tune (1988), poteins of the spoozoite wee obtained by sonication, and then wee associated with adjuvant Quil A. 4. Expeimental design The expeiment was designed to include five teatments with thee eplicates each and 12 bids pe eplicate, in a completely andomized model. The teatments wee: T1 was the unvaccinated, unchallenged contol, T2 eceived 500 attenuated oocysts by gavage and was challenged, T3 was the unvaccinated, challenged contol, T4 eceived 50 µg of spoozoite potein + Quil A vaccine by nasal oute and was challenged, and T5 eceived Quil A without spoozoite potein + BS by nasal oute and was challenged. The fist day of immunization of the thee-day-old chickens was consideed as day zeo fo the puposes of oganization and intepetation of the data. The teatments T4 and T5 wee boosted by the same oute 7 and 21 days afte the pimay immunization, and teatment T2 was kept in litte pens afte immunization. On the 31 st day, the challenge was caied out with spoulated oocysts of homologous viulent stain of the species E. tenella in the dose of oocysts, with the exception of T1. 5. Blood samples, body weight measuements, and oocyst-counting (OoG) The weighing of food and bids of each eplicate wee caied out on the challenge day and one week afte the challenge to calculate the weight gain (WG) and feed convesion (FC). The lesion scoe of the teatment (six animals in each teatment) was assessed seven days afte eithe vaccination o challenge, accoding to the pocedue descibed by Johnson and Reid (1970). The emaining animals wee euthanized on day 45. The oocyst-counting of caecal contents was pefomed daily fo a week in all teatments afte the challenge, using the euthanized animals. The hemogam was pefomed one week afte the challenge. Biefly, the blood samples wee dawn by cutaneous ulna vein punctue of thee animals of

3 v. 19, n. 1, jan.-ma ost-challenge hematological evaluation with viulent stain of Eimeia tenella in boiles 3 each teatment and stoed with EDTA. Hematological paametes wee evaluated: plasma potein, globula volume, hemoglobin, total eythocyte and leukocyte count, diffeential leukocyte count, and detemination of eythocyte indexes (mean copuscula volume, mean copuscula hemoglobin concentation, and mean copuscula hemoglobin), as epoted by Campbell (2007). The hemoglobin measuement was detemined by the cianometahemoglobin method with a modification: the eagent/blood mixtue was centifuged befoe the optical density eading to avoid oveestimation of hemoglobin concentation. The eythocyte and leukocyte counts wee pefomed in a 1:200 dilution of blood in Natt and Heick (1952) solution. Afte mixing, the diluted blood was placed on the hemocytomete, emaining at est fo five minutes. The ed cells wee counted in fou lage cone squaes and in cental squaes, and the numbe of cells counted was multiplied by 10,000 to calculate the total numbe of cells pe cubic millimete. The total leukocyte count was caied out accoding to the methodology descibed by Campbell (2007), though the sum of all leukocytes (LEU) pesent in the nine lage squaes of the hemocytomete gid aea, accoding to the Equation 1: LEU µl = ( Total of nine squae cells lage + 10% ) 200 (1) The diffeential counting of leukocytes was pefomed by pepaation of blood smeas stained with Giemsa and obseved in the 1000 objective (immesion). Additionally the total plasma potein concentation was detemined in a photometic coloimetic test using the Biuet method. 6. Statistical analysis The esults wee submitted to analysis of vaiance (ANOVA) and when significant (p < 0.05), the means of vaiables WG, FC, and hematological paametes wee compaed by Tukey s test. The student t-test was used to establish diffeences between the aveages of the post-challenge cecal OoG. The lesion scoes (LS) wee analyzed by the Kuskal Wallis non-paametic test and Dunn s multiple compaisons test. A value of 0.05 was consideed significant. Data not nomalized wee tansfomed pio to analyses using the Johnson tansfomation within Minitab. Statistical analyses fo all data wee pefomed using Minitab vesion 14 (Minitab Inc., State College, USA). Results and Discussion 1. ost-challenge clinical and paasitological paametes Afte challenge with viulent homologous stain, the WG and FC showed no statistical significance (p > 0.05), as shown in Table 1. The lesion scoe showed significance (p < 0.001) in the Kuskal-Wallis test and Dunn multiple compaison test. Two goups wee fomed: the T3 and T5 teatments (3.75 and 3.33, espectively) with highe lesion scoes; and the T1 and T2 teatments (0.00 and 0.17, espectively) with a mino lesion scoe. Teatment T4 (1.92) showed intemediate lesion in elation to the two goups above (Table 1). Table 1. Immunogenicity test caied out seven days afte the challenge, with feed convesion, weight gain, and lesion scoe of teatments Teatment Immunogenicity (day 38) Ooc 10 5 ± SD FC ± SD WG (g) ± SD LS Day 38 T ± ± b ± b T ± ± b ± b T ± ± a ± a T ± ± ab ± a T ± ± a ± a value The indexes ( a ) and ( b ) of lesion scoe and oocyst numbe, showed the diffeence of the means detemined by nonpaametic Kuskal Wallis test followed by Dunn s multiple compaison (p < 0.05) and the diffeence of means detemined by t-student test (p < 0.05). FC = Feed convesion; WG = Weight gain; LS = Lesion scoe; SD = Standad deviation; g = Gams. Teatments wee: T1 = Negative contol; T2 = Received 500 attenuated spoulated oocysts by gavage; T3 = ositive contol; T4 = Received 50 µg of spoozoite potein + Quil A vaccine; T5 Received Quil A without spoozoite potein + BS. The numbe of oocysts pe gam of cecal contents was significantly deceased (p < 0.01) in the T1 and T2 teatments in compaison to X goup, while the T3, T4, and T5 teatments showed high oocyst counts. The challenge with oocysts pe bid was not enough to cause significant diffeences between the WG and FC paametes. To induce clinical o subclinical coccidiosis to evaluate hematological data it might be necessay to give a highe challenge dose. Howeve, the paasitological paametes showed a pathogenic effect in the positive contol teatment, caused by the challenge and shown by thei high lesion scoes and the highest oocyst poduction, which we believe wee sufficient fo the hematological evaluation. Biefly, we evaluated the bid hematological changes with a challenge capable of causing the clinical coccidiasis fom. 2. Hematological paametes post-challenge The analysis of vaiance showed no significant diffeences (p > 0.05) among teatments fo plasma potein, globula volume (GV), hemoglobin (HB), total eythocytes (TE), leukocytes (LEU), lymphocytes (LYMH), heteophils (HET), eosinophils (EOS), monocytes (MON), and eythocyte indexes (mean copuscula hemoglobin, mean copuscula hemoglobin concentation, and mean copuscula volume), as shown at Tables 2 and 3. Only little infomation is available in the liteatue concening the evaluation of hematologic alteations in bids infected with E. tenella. The few studies which have analyzed hematological paametes ae esticted to the leukocyte pofile and type of immune esponse to infection caused by the paasite. Conway et al. (1993) assessed levels of infection by E. tenella and othe species and obseved a gadual decease of the globula volume, plasma total potein, weight gain, and inceased feed convesion to the numbe of inoculated oocysts. These esults wee not epoduced in this expeiment because of the challenge

4 4 Bogado, A.L.G. et al. Rev. Bas. aasitol. Vet. Table 2. Haematological means values ± standad deviation of plasma potein (), globula volume (GV), hemoglobin (HB), total eythocytes (TE), mean copuscula volume (MCV), mean copuscula hemoglobin concentation (MCHC), and hemoglobin copuscula media (HCM) Teatment (g.dl 1 ) GV (%) HB (g.dl 1 ) TE ( 10 6.mL 1 ) HCM (pg) MCV (fl) CHCM(g.dL 1 ) T1 4.2 ± ± ± ± ± ± ± 1.3 T2 4.3 ± ± ± ± ± ± ± 3.2 T3 4.2 ± ± ± ± ± ± ± 0.6 T4 4.0 ± ± ± ± ± ± ± 2.3 T5 4.0 ± ± ± ± ± ± ± 1.2 value The data set was analyzed by ANOVA and when statistically significant (p < 0.05), the mean of the teatments wee compaed by Tukey s test. Table 3. Haematological mean values ± standad deviation of leukocytes, the absolute and elative values of lymphocytes, heteophils, monocytes, and eosinophils Teatment Leukocytes Lymphocytes Heteophils Monocytes Eosinophils ( 10 3.μL 1 ) ( 10 3.μL 1 ) (%) ( 10 3.μL 1 ) (%) ( 10 3.μL 1 ) (%) ( 10 3.μL 1 ) (%) T ± ± ± ± ± ± ± ± ± 3.8 T ± ± ± ± ± ± ± ± ± 5.2 T ± ± ± ± ± ± ± ± ± 5.8 T ± ± ± ± ± ± ± ± ± 0.0 T ± ± ± ± ± ± ± ± ± 1.4 value The data set was analyzed by ANOVA and when statistically significant (p < 0.05), the mean of the teatments wee compaed by Tukey s test. not being able to cause clinical o sub-clinical disease and due to the time of blood sampling afte the challenge. Changes in peipheal blood leukocytes fom chickens affected by E. tenella wee detected only in pimay infections in a study conducted by Natt (1959), whee sevee bleeding was detected. Rose et al. (1979) obseved that fist infection with E. maxima in the chicken leads to an incease in the ciculating leukocytes numbe (heteophils, lymphocytes, and monocytes) in two phases, one lymphocytosis befoe and anothe afte the peak poduction of oocysts. In the peiod when the oocyst poduction eached a peak, infected bids had lymphopenia, so that the numbe of ciculating leukocytes did not diffe statistically fom the contol goup. Challenge with homologous stain in the immunized bids showed a apid and shot immune esponse, etuning to nomal levels on the second day post-challenge. In assessing the leukocyte esponse of immunized and challenged bids, Rose et al. (1984) obseved a significant decease in the peipheal leukocytes numbe one hou afte oocyst inoculation, due to ciculating lymphocytes depletion. Then thee was a gadual incease of the leukocytes, and afte seven hous the cells of the immunized goup wee above the total count of the contol goup. The leukocytosis was late found to be elated to the inceased numbe of ciculating heteophils. These facts could explain the absence of leukocyte changes in this study. Accoding to the authos above, changes in the cell numbe do not occu duing the peak poduction of oocysts. Anothe possibility would be that the challenge was not pathogenic enough to cause cellula changes, due to non-significance of the weight gain and feed convesion paametes. The numbe of eosinophils in this study was simila between infected and contol animals, in ageement with the study by Rose et al. (1979) with E. maxima species. Unlike in mammals, the ole of eosinophil in bids is not fully known and studies have shown its paticipation in eactions of type IV hypesensitivity (delayed), as occus with mammalian eosinophils (CAMBELL, 2007). 3. Coelations between evaluated paametes Significant coelations (p < 0.05) wee detected between WG and TE ( = 0.74); MON and FC ( = 0.57); lesion scoes and the OG (=0.69); lesion scoe and TE ( = 0.56); and lesion scoe and MON ( = 0.76). Thee wee significant coelations between the hematological paametes: GV and HB ( = 0.65); GV and plasma potein ( = 0.63); GV and HET ( = 0.57); GV and MON (=0.60); HB and plasma potein ( = 0.60); LYMH and HET ( = 0.94); and HET and MON ( = 0.55). The coelations of all paametes used in this immunogenicity study ae listed in Table 4. Based on these esults, some hypotheses on the pathogenesis of the disease have been poposed to explain the inteactions between the paametes unde the conditions in which the expeiment was caied out. The hematological paametes TE, GV, and HB ae geneally associated with hydation levels and/o hemohage. The coelation between the WG TE ( = 0.74 and p = 0.004) is invesely popotional, suggesting that if a decease in weight gain occus, it may be accompanied by an eythocyte concentation in challenged bids. The othe coelations ae as follows: TE lesion scoe ( = and p = 0.048); HB plasma potein ( = and p = 0.029); GV plasma potein ( = and p = 0.021); and HB GV ( = and p = 0.016) indicate a diect elationship between the vaiables. All coelations above ae linked to dehydation due to diahea, inceased intestinal

5 v. 19, n. 1, jan.-ma ost-challenge hematological evaluation with viulent stain of Eimeia tenella in boiles 5 tansit, deceased wate absoption in the caecum, and plasma exudation caused diectly by the inteaction between the paasite and the affected ogan (WILLIAMS, 2005). These esults ae in ageement with those obtained by Fukata et al. (1997), who obtained a eduction in levels of plasma poteins (p < 0.01) on the fouth day post-infection by E. tenella. On the seventh day post-infection, the numbe of ed blood cells and globula volume also showed a decease compaed to the negative contol (p < 0.001). Thee was a stong invese coelation between the LYMH and HET in the plasma ( = and p < 0.001), indicating that the pevalence of these cells changes accoding to the degee of bid susceptibility. The same was obseved with the MON and HET numbe, but at lowe intensity ( = and p = 0.044). The pevalence of these cells in the cecal tissue of the diffeent teatments was not assessed. Biefly, these coelations suggest a patial eplacement between lymphocytes and heteophils in the development of immune esponse against Eimeia tenella. The coelation between the lesion scoes and OG ( = and p = 0.004) indicates indiectly that a lage amount of paasites in the ceacum causes a highe lesion scoe. Rose et al. (1979) suggested that a geate polifeation of monocytes may be highly associated with emoval of the consideable amount of debis and may seve to assist in the defense against paasites in the affected tissue. Based on the infomation above, we found an inceased numbe of monocytes when the lesion was moe sevee, as is shown in the esult of lesion scoe MON ( = and p = 0.002). In the Rose et al. (1979) study, an incease of the monocyte numbe was moe ponounced at the end of the cycle of the paasite, afte the peak poduction of oocysts. The coelation between FC and MON ( = and p = 0.042) suggests that an incease in feed convesion is associated with lage amounts of monocytes. The set of factos that eflect in the bid pefomance is complex, involving many vaiables in host-paasite elationship. Johnson (1997) cites that the poinflammatoy cytokines, poduced by seveal cell types, among which ae the monocytes, ae esponsible fo low gowth in immunologically challenged animals. Although this esult appeas to give evidence of a pathogenic effect associated with this type of cell, unde conditions in which this expeiment was conducted, we do not have enough suppot to sustain this assetion. The assessment of goups with diffeent susceptibility levels to a single challenge facing the coelations showed GV HET ( = and p = 0.04) and GV MON ( = and p = 0.030), suggesting that levels of hydation and hemohage caused by the challenge suffe changes accoding to the pedominance of heteophils o monocytes. Conway et al. (1993) obseved that the geate the challenge of susceptible bids, the lowe the globula volume. Table 4. Coelation coefficient () and values between the paametes used to evaluate the immunity in bids afte challenge with homologous viulent stain of E. tenella on the 38 th day aamete aamete WG FC LS OG GV HB TE LEU HET EOS LYM FC LS OG HT HB TE LEU HET EOS LYMH MON WG = Weight gain; FC = Feed convesion; LS = Lesion scoe; OG = Numbe of oocysts pe gam of cecal contents; GV = Globula volume; HB = Hemoglobin; TE = Total eythocyte; LEU = Leukocyte; = lasma potein; HET = Heteophils; EOS = Eosinophils; LYMH = Lymphocytes; MON = Monocytes

6 6 Bogado, A.L.G. et al. Rev. Bas. aasitol. Vet. Unde the conditions in which the expeiment was caied out, we can conclude that the teatments did not diffe by ANOVA, possibly because the challenge was not as pathogenic as expected. The failue to veify changes in eythocyte and especially the peipheal leukocyte count of chickens challenged with E. tenella viulent stains may be influenced by the peiod duing which blood was collected, which coesponded to the peak of oocyst poduction. The coelations suggest that the pesence of ciculating monocytes has a diect elation with the lesion scoe incease, feed convesion, and numbe of oocyst shedding. Due to the highe numbe of paasites in the lesion site, thee is demand fo a geate quantity of phagocytes. The coelations of blood paametes GV, TE, HB, and plasma poteins with othe paametes, such as WG, OG, and lesion scoe showed levels of hemohage and/o hydation, as a consequence of effects of the paasite aggession to the tissue. Refeences ALLEN,. C. et al. Association of loweed caotenoids with potection against cecal coccidiosis by diets high in n-3 fatty acids. oulty Science, v. 75, n. 8, p , ALLEN,. C. Nitic oxide poduction duing Eimeia tenella infections in chickens. oulty Science, v. 76, n. 6, p , ALLEN,. C.; DANFORTH, H. D.; VINYARD, B. L. Development of a potective index to ank effectiveness of multiple teatments within an expeiment: application to a coss-potection study of seveal stains of Eimeia maxima and a live vaccine. Avian Disease, v. 48, n. 2, p , CAMBELL, T. W. Hematologia de mamífeos não domésticos, aves, épteis, peixes e anfíbios comuns. In: THRALL, M. A. Hematologia e Bioquímica Clínica Veteináia. 1 ed. São aulo: Editoa Roca, p CONWAY, D.. et al. Effects of diffeent levels of oocyst inocula of Eimeia acevulina, E. tenella, and E. maxima on plasma constituents, packed cell volume, lesion scoes, and pefomance in chickens. Avian Disease, v. 31, n. 7, p , DULSKI,.; TURNER, M. The puification of spoocysts and spoozoites fom Eimeia tenella oocysts using pecoll density gadients. Avian Disease, v. 32, n. 2, p , FETTERER, R. H.; ALLEN,. C. Eimeia tenella infection in chickens: effect on plasma and muscle 3-methylhistidine. oulty Science, v. 80, n. 11, p , FUKATA, T. et al. Evaluation of plasma chemisty and haematological studies on chickens infected with Eimeia tenella and E. acevulina. Veteinay Recod, v. 141, n. 2, p , JEFFERS, T. K. Attenuation of Eimeia tenella though selection fo pecociousness. Jounal of aasitology, v. 61, n. 6, p , JOHNSON, J.; REID, W. M. Anticcocidial dugs: lesion scoing techniques in battey and floo-pen expeiments with chickens. Expeimental aasitology, v. 28, n. 1, p , JOHNSON, R. W. Inhibition of gowth by po-inflammatoy cytokines: an integated view. Jounal of Animal Science, v. 75, n. 5, p , LILLEHOJ, H. S.; MIN, W.; DALLOUL, R. A. Recent pogess on the cytokine egulation of intestinal immune esponses to Eimeia. oulty Science, v. 83, n. 4, p , LONG,. L. Maintenance of intestinal potozoa in vivo with paticula efeence to Eimeia and Histomonas. In: TAYLOR, A. E. R.; MULLER, R. Isolation and Maintenance of aasites In Vivo. Oxfod: Blackwell Scientific ublication, p NATT, M.. The effect of cecal coccidiosis on the blood cells of the domestic fowl. 3. The changes in the leucocyte pictue duing the couse of the infection. Expeimental aasitology, v. 8, n. 2, p , NATT, M..; HERRICK, C. A. A new blood diluent fo counting the eythocytes and leucocytes of the chicken. oulty Science, v. 31, n. 1, p , ROSE, M. E.; HESKETH,.; OGILVIE, B. M. eipheal blood leucocyte esponse to coccidial infection: a compaison of the esponse in ats and chickens and its coelation with esistance to einfection. Immunology, v. 36, n. 1, p , ROSE, M. E.; HESKETH,.; RENNIE, M. Coccidiosis: apid depletion of ciculating lymphocytes afte challenge of immune chickens with paasite antigens. Infection and immunity, v. 45, n. 1, p , WILLIAMS, R. B. Intecuent coccidiosis and necotic enteitis of chickens: ational, integated disease management by maintenance of gut integity. Avian athology, v. 34, n. 3, p , 2005.