| Coccidiosis in chicken is caused by one or a variety of the coccidia that belonging to Eimeria parasite on chicken intestinal epithelial cells, which is a worldwide distribution protozoosis. can result in huge losses to the poultry industry. This trial successfully isolated a strain Eimeria coccidia by using cellophane and capsule for the single oocyst separation, and finally identified it as Eimeria tenella Anhui Feixi strain (AHFX) using morphological and molecular methods; then infected chicks with the obtained oocysts that sporulated, different stages of serum biochemical changes were studied of the infected chickens.Firstly, collected the cecal contents from the chickens that had a typical symptoms of cecal coccidiosis from a chicken farm in Feixi County, Anhui; filtered by40mesh copper sieve and260mesh nylon sieve after mixing with saline, then collected the precipitate after centrifugating the filtrate10minutes at3000r/min. Adding an appropriate amount of2.5%potassium dichromate to the precipitate, cultured in28℃incubator for sporulation until more than85%of the oocysts sporulated, sporulated oocysts stored at4℃refrigerator. Single oocyst separation was carried out with the cellophane and empty capsules as the carrier, diluted the oocyst liquid to each drop of oocysts solution containing only1or2oocysts; dampened with a drop to the cellophane that in the size of0.5cm×0.5cm, observed the morphological characteristics of the oocysts, and then placed in the empty capsules, inoculated to206-day-old chicks orally, started checking oocysts from the fecal after inoculating5days to determine if the chicken had infected.Subsequently, the offspring of the single oocyst (first generation) were inoculated to14-day-old chicks in the dose of1×104oocysts per chicken, observed the mental status, appetite availability and stool changes of the chickens daily.115hours after inoculation, checked the faces every2hours and recorded the time of first oocysts appeared and the potential period; collected the oocysts (second generation) and recorded the minimum sporulation time. Randomly measured50oocysts of the first generation and second generation, tested the size difference (F test).Thereafter, extracted the coccidian genomic DNA according to a conventional method after rinsing and concentrating the oocysts solution. Designed primers with reference to Yao-Chi Su:ETI:5’-CGCTGCTGGTTTTACAGGTT-3’. ET2:5’-GCTGAAGCAAAGTTCCAAGC-31for PCR amplification, sequencing and analyzing cluster of PCR products.Finally,8021-day-old chicks were inoculated with the oocysts of Eimeria tenella Anhui Feixi (AHFX) strain in the dose of5×104oocysts per chicken; after inoculation0day,5days,9days and13days, took cardiac blood and measured average weight index of immune organ, and serum biochemical indicators.The results show that:(1) Five chickens were successfully infected by the single oocyst separation technology, the successful rate was25%.(2) Proliferated the offspring of the single oocyst separation, the size of the second-generation oocysts is17.0～23.6μm×15.8～20.3μm, with an average size of21.1μm×18.0μm, ovate index of1.17, the potential period of140hours. Tested (F test) the significant difference between the oocysts of single oocyst separation (first generation) and the oocysts of proliferation (second generation), the results showed no significant difference (P>0.05), indicating that the second-generation coccidia oocysts are in the same size with the oocysts of first generation, and other various morphological features are in line with the characteristics of Eimeria tenella. Preliminary identified the obtained coccidia as Eimeria tenella, tentatively named Eimeria tenella Anhui Feixi (AHFX) strain.(3) Identified the obtained coocidia by applying molecular biology method, successfully amplified one463bp fragment by PCR; the sequence analysis showed that the amplified fragment have been reported. The homology in ITS-1was98.2%between the isolated strain and E.tenella (GQ856310); they were very close in phylogeny. The obtained sequence was uploaded to GenBank with the accession number of JX477100.(4) Animal experiments show that:compared with the control group, after infected5days, average weight, content of Na, TP and ALB in serum was significantly lower(P<0.01), content or activity of Ca, GOT, LDH-L and IgA in serum was significantly higher (P<0.01), content or activity of K, GPT and NO in serum was significantly increased(P<0.05), activity of CAT in serum was significantly reduced (P<0.05); after inoculated9days, average weight and content of ALB in serum extreme significantly reduced (P<0.01), level of IgG in serum extreme significantly increased (P<0.01), activity or content of GSH-Px, T-SOD and TP in serum significantly reduced (P<0.05); after infection13days, the average weight and activity of T-SOD in serum significantly reduced (P<0.01), thymus index, activity or content of CAT, TP in serum significantly reduced (P<0.05), content of Ca in serum was significantly higher (P<0.05); during the entire experiment, the spleen index, bursa of Fabricius index, the content or activity of P. Mg, ALP and IgM in serum showed none significant changes (P>0.05). In summary, this study using single oocyst separation technology successfully isolated an Eimeria coccidia, and identified it as Eimeria tenella AHFX strain by morphological and molecular biological methods, initially proved the changes of serum biochemical parameters in different stages after chickens infected the Eimeria tenella AHFX strain. The isolation, identification of the Eimeria tenella AHFX strain and the findings of serum biochemical parameters changes of chickens infected the coccidian provided basic theory for coccidiosis prevention for Anhui Feixi area. |
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