Adapting Eimeria tenella to Grow in Primary Chicken

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Adapting Eimeria tenella to Grow in Primary Chicken Kidney Cells Following Repeated Passages between Cell Culture and Chickens Author(s): Jianfei Zhang, Eric Wilson, Shiguang Yang and Mark C. Healey Source: Avian Diseases, Vol. 41, No. 1 (Jan. - Mar., 1997), pp. 111-116 Published by: American Association of Avian Pathologists Stable URL: http://www.jstor.org/stable/1592450 Accessed: 18-08-2015 06:44 UTC REFERENCES Linked references are available on JSTOR for this article: http://www.jstor.org/stable/1592450?seq=1&cid=pdf-reference#references_tab_contents You may need to log in to JSTOR to access the linked references.

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AVIAN DISEASES 41:111-116,

1997

Adapting Eimeria tenella to Grow in Primary Chicken Kidney Cells Following Repeated Passages Between Cell Culture and Chickens Jianfei Zhang,A Eric Wilson,B Shiguang Yang,c and Mark C. HealeyCD Medicine Institute of GuangdongAcademy of AgriculturalScience, AVeterinary Wushan, Guangzhou, People'sRepublic of China 510640 MolecularBiology Department, Montana State University,Bozeman, Montana 59717 BVeterinary CDepartmentof Animal, Dairy, and VeterinarySciences, Utah State University,Logan, Utah 84322-5600 Received29 March 1996 SUMMARY. The present study was undertakento adapt a field isolate (FO)of Eimeria tenella to grow in primary chicken kidney cells (PCKCs) by selecting for characteristicsof the parasite rather than modifying the culture and/or environmentalconditions. Fourteen generations (F1 to F14) of E. tenella were produced following repeatedpassagesbetween PCKCs and chickens.Although Fl yielded only a 28% increasein oocysts in PCKCs compared with FO, F2 to F5 produced from 259% to 277% more oocysts, respectively.There was no significantincreasein the percentageof oocysts produced in PCKCs by F6 to F14 compared with F5. GenerationsFl to F14 demonstrateda greaterpropensityfor multiple infections within the same host cell than did FO. For example, it was not uncommon to observetwo, three, and occasionallyfour oocystswithin a single PCKC. Chickensinoculated with FO oocysts generallyexperiencedgreaterpathogenesisby day 7 postinoculation than chickens inoculatedwith F14 oocysts as measuredby decreasedbody weights, increasedcecal lesions, and a higher mortalityrate. RESUMEN. Adaptacion de Eimeria tenella para su crecimientoen celulas primariasde rinon de pollo, despues de pasajesrepetidosentre cultivo celulary pollos. Este estudio se hizo paraadaptaruna cepa de campo de Eimeriatenellaa creceren celulas primariasde rifi6n mediante la seleccion por sus caracteristicasparasiticasen vez de modificarlapor cultivo o condiciones ambientales.Se produjeron14 generaciones(F1 a F14) de E. tenelladespues de pasajesrepetidosen celulasde rifi6n y pollos. Aunque la generaci6nF1 produjo solo un aumento del 28% de oocistos en las celulas de rifi6n comparadocon las generacion FO, las generocionesF2 a F5 produjeron259% a 277% mas oocistos, respectivamente. No hubo un aumento significanteen el porcentajede oocistos producidos en las celulas de rifi6n en las generacionesF6 a F14, comparadocon la generaci6nF5. Las generaciones F1 a F14 demostraronuna mayor tendenciaparacausarinfeccionesmultiplesdentro de la misma celula huesped que la generaci6nFO.Por ejemplo, fue comuinobservardos, tres y ocasionalamentecuatro oocistos dentro de una sola celula renal. Los pollos inoculadoscon oocistos de la generaci6nFOgeneralmentemostraronmayor patogenicidadal dia 7 despues de la inoculaci6n que los pollos inoculados con oocistos de la generacionF14, medido por la disminuci6n de los pesos corporales,aumento en las lesiones cecalesy mayor porcentaje de mortalidad. Key words: Eimeriatenella,field isolate, primarychicken kidney cells Abbreviations:FBS = fetal bovine serum;HBSS = Hanks' balancedsalt solution;PCKCs =primarychicken kidney cells

Improved techniques for growing Eimeria tenella in cell culture have been instrumental in DCorrespondingauthor.

understanding the biochemistry of this parasite and allowing for more economical and efficient testing of anticoccidial compounds (18). Patton (11) first described the cultivation of the asex-

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J. Zhanget al.

ual stages of this parasite in vitro. The complete

life cycle of E. tenellawas successfullymodeled in primarychicken kidney cells (PCKCs)when Doran (1) grew the parasitefrom sporozoiteto oocyst. Subsequently,a variety of cell culture manipulationshave been attempted in an effort to enhance the in vitro development of eimerian species. These have included altering the number and strain of sporozoites used to inoculate cell culture (6,8), growing parasitesin a varietyof differentcell lines (3,4,5,12), using different types and concentrations of culture medium (2), varyingvitamin and nutrient supplements (10,14,18), and changingthe ambient environment in which coccidiansare cultivated (13,17,19,20). We recentlydescribedincreasing the yield of E. tenellaoocysts in PCKCs by optimizing select growth conditions (21). Most published reports have attempted to improve the growth and reproductionof E. tenella in cell cultureby changingthe growthmedium, varying the host cell line, or manipulating specific environmental conditions. Conversely,the presentstudy describesa method for adapting E. tenella to grow in PCKCs by selecting for characteristicsof the parasiterather than modifying the cell culture. MATERIALSAND METHODS Production of oocysts. Eimeriatenella(field isolate 80) was originallyobtained from Dr. Harry D. Danforth (Animal Parasitology Institute, USDA, Beltsville,Md.). Three-week-oldwhite leghornchickens obtained from the Utah State Universitypoultry farm were housed in wire mesh cages in the Laboratory Animal Research Center on campus. Feces were collected from the ceca of these chickens7 days after they had been inoculated per os with 30,000 oocysts. The fecal debris was removed using a modificationof the gravitypan flotation techniqueas previously described (16). Oocysts were subsequently harvested from the feces and maintained at room temperaturefor 5 days in 2.5% potassium dichromate until 80% sporulationhad occurred(7) as determined microscopically.Oocysts were then decontaminated by soaking in a 40% commercialpreparation of bleach (5.25% sodium hypochlorite [w/v]) for 30 min, washed in sterile Hank's balanced salt solution (HBSS) five times, and stored at 4 C until used. Primary chicken kidney cells. Kidneys were obtained from two 2-4-wk-old white leghorn chickens and transferredto a 50-ml centrifugetube. The kidneyswere washed twice in HBSS and minced into

small pieces (approximately1 mm3). Piecesof kidney tissue were suspendedin 50 ml of 0.25% (w/v) trypsin in HBSS, stirredfor 5 min at 37 C, and allowed to sediment for approximately30 sec. After discarding the supernatant,50 ml of freshpreheatedtrypsin was added to the sedimentedkidney tissueand stirred for an additional 15 min at 37 C. The supernatant was decanted into a 50-ml tube containing 5 ml of fetal bovine serum (FBS) and centrifugedat 200 x g for 5 min. The pellet of PCKCs was resuspended by washing twice in HBSS diluted 1:10 with growth medium. Growth medium consisted of RPMI-1640 medium containing 10% heat-inactivatedFBS and 1% Hybri-Max?antibiotic/antimycoticsolution (Sigma Chemical Co., St. Louis, Mo.). Seven tenths of one milliliter of the resuspendedPCKCs was then mixed with 100 ml of growthmedium. One milliliter of the resultant suspension of PCKCs was used to seed each well of a 24-well polystyrenetissue culture plate containinga 13-mm-diameter(1.33-cm2)Thermanox? coverslip(Nunc, Inc., Naperville,Ill.). Plates were incubated at 40.5 C in an atmosphereof 7.5% CO2 for 48-60 h to allow the PCKCs to reach at least 80% confluency. Preparation of sporozoites. Approximately2 X 106 oocysts suspendedin a 3-5-ml volume of HBSS were placed in a centrifugetube containing 5 ml of glass beads and vortexed until 80% of the oocysts had ruptured(30-60 sec) as determinedmicroscopically. The glass beads were washed with 50 ml of HBSS and the sporocystswere pelleted by centrifugation for 10 minutes at 1875 x g. The supernatant was then aspiratedand the pelleted sporocystswere resuspendedin 50 ml of excystationsolution (50 ml of HBSS containing 0.375 g taurodeocycholicacid and 0.125 g trypsin). This suspension of sporocysts was incubated at 40.5 C in an atmosphereof 7.5% CO2 for approximately40 min, or until 80% excystation could be confirmed by microscopicexamination. Sporozoiteswere separatedfrom cell debrisand intact oocysts by passagethrough an 11-jumpore filter. Inoculation of PCKCsand chickens. One milliliter containing approximately40,000 purifiedsporozoites was inoculated onto individual monolayers of PCKCs growing on coverslipsplaced on the bottoms of wells in the tissue culture plates. Growth medium was replacedon days 1, 3, and 5 postinoculation by aspirating80% of the medium and adding 1 ml of fresh growth medium. Using brightfieldmicroscopy (20 x objective), the total number of oocysts producedin monolayersof PCKCs growingon each coverslipwas counted. The first generationof PCKC culture-derivedoocysts (referredto as F1) were sporulated and used to inoculate 2-wk-old chickens. The oocysts were harvestedfrom chicken manure on day 7 postinoculation, sporulated,purified, decontaminated,excysted, and the sporozoites

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AdaptingEimeriatenellato cell culture preparedas described above. Sporozoiteswere again used to inoculate monolayersof PCKCs. Oocysts resulting from this second passagein PCKCs were referred to as F2; consecutive generations of oocysts obtained by repeatedpassageswere named in a similar numerical fashion. The process of alternating from PCKCs to chickens and back to PCKCs was repeated14 times. At the same time that F was used to inoculate chickens, FO (parent generation never before used to inoculate PCKCs) was collected from other experimentallyinfected chickens and used to inoculate additional chickens. Sporozoites excysted from FO and F1 (identical ages) were then used to concurrentlyinoculate monolayersof PCKCs growing on coverslipsin 24-well plates (12 coverslips/generation). The same process was repeatedfor FO and F2 to F5. On day 7 postinoculation,the total number of oocysts was counted for F1 to F5 and compared to FO using Student'st-test. Pathogenicity. The relativepathogenicityof FO and F14 was comparedby inoculating six groups of seven or eight chickens/groupper oswith 500 (groups 1 and 2), 10,000 (groups 3 and 4), and 50,000 (groups 5 and 6) oocysts/chicken. Mortality and body weight change for chickens in each group were recordedprior to killing the chickens on day 7 postinoculation. At the time of sacrifice,the ceca were harvestedfrom each chicken, opened lengthwisewith a scissors, and the gross pathologic changes scored accordingto the standardizedmethod of Johnsonand Reid (9). After cecal lesion scoreshad been recorded, the cores were removed and the mucosal surfaceof each cecum was vigorously scraped.The cecal cores and mucosal scrapingsfrom individualchickenswere homogenized together and the total volume adjusted to 50 ml with 40% bleach. This preparationwas stirred for 10 min at room temperatureto destroy the mucosal tissue. The total number of oocysts was then counted using a hemocytometer. RESULTS Adaptability of E. tenella to PCKCs. The adaptability of E. tenella to PCKCs was measured by the total number of oocysts produced by day 7 postinoculation for Fl to F5 compared to FO (Table 1). Generations F2 to F5, but not Fl, produced significantly more (P < 0.0001) oocysts than did FO. Increases in oocyst production ranged from 28% (F1) to 277% (F5). There was no significant increase (P > 0.05) in the percentage of oocysts produced in PCKCs by F6 to F14 compared to F5. Infectivity of PCKCs. Asexual stages of FO and F1 to F14 frequently occurred in multiples

Table 1. Adaptability of Eimeria tenella to primary chickenkidney cells following repeatedpassages between cell culture and chickens. Percent

GeneraOocystsB

increaseC

FO F1

59.98 ? 5.45 77.02 + 5.98

28

FO

29.69 ? 1.73

tionA

F2 FO F3 FO

106.56 21.43 77.88 29.44

4.88 1.29 3.53 2.40

259

F4

109.02 ? 7.05

270

FO F5

50.19 + 5.38 189.45 + 16.59

277

+ + +

263

AGenerationsof E. tenella.The parentgenerationis representedby FO(neverbeforeused to inoculateprimary chicken kidney cells [PCKCs]),whereasF1 to F5 represent cell culture-adaptedgenerations produced by repeated passages between PCKCs and chickens. BMeannumber of oocysts/cm2 of PCKCs + SE. GenerationsF2 to F5, but not Fl, produced significantly more (P < 0.0001) oocysts than did FO on day 7 postinoculation. CPercentincrease in the number of oocysts produced by F1 to F5 comparedto FO. within individual PCKCs; as many as 10 schizonts were often observed within a single PCKC. However, the gamonts and oocysts of F to F14 showed a greater propensity than FO for multiple infections within PCKCs. For example, it was not uncommon to observe two, three, and occasionally four oocysts within a PCKC (Fig. 1). When this occurred, infected PCKCs were usually surrounded either by uninfected cells, or by cells infected with only a single oocyst. Only rarely were PCKCs inoculated with FO infected with more than one oocyst. Chickens inoculated with Pathogenicity. FO oocysts generally experienced greater pathogenesis than chickens inoculated with F14 oocysts as determined by changes in body weight, cecal lesion scores, and percent mortality by day 7 postinoculation (Table 2). For instance, chickens in group 5 (50,000 FO oocysts/chicken) lost significantly more (P < 0.001) body weight than chickens in group 6 (50,000 F14 oocysts/chicken). Similarly, the cecal lesion scores for chickens in groups 1 and 3 (500 and 10,000 FO oocysts/chicken, respectively) were significantly higher (P < 0.05) than the cecal

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