The Epidemiological Investigation, Pathogenicity Experiments, Pharmic Prevention And Treatment Of Isospora Suis In Henan

The coccidiosis in pigs is commonly parasitism protozoal disease, which can cause extensive spread and prevalence in the world. To piglets, this disease mainly causes diarrhea, decreased food appetite, loss of body weight, and some relative clinical asymptoms. The severious infected pigs can become death. However, the adult pigs are mostly the parasite hosts, with no clinical signs are seen, however, which are the common source of infections. Coccidiosis severiously restrictes the development of pigs industry, which can cause more large economic loss. According to a report, the cost used for preventing and curing coccidiosis in pigs in European and American countries was more than 100 millions U.S. dollar per year. However, due to the strict host specificity, animal model only limited in pigs, the selfish characteristics of parasite and the relatively large difficulty of experimental management, and the low reproductive rate of Coccidia and the uncertainty of artifical infection, thus factors mentioned above should cause the minor studies on pig Coccidia. In this paper, we conducted the biological characterizations and phylogenetic relationship study of Isospora suis in pigs.1. To understand the prevalence and distribution of Coccidium spp. in pigs in Henan Province, a total of 24 scale pigs farms and 12 specialized households of pigs breeding from 17 cityies/counties (such as Shangqiu, Luoyang, Luohe, Hebi, Zhengzhou, et al.) in Henan Province were investigated by saturated brine flotation technique between May 2005 and August 2008. The results showed 34 farms or households were found Coccidium spp. infection, with 94.44% of postive rate. Thereinto, the infection rates ofⅠ. suis and Eimeria spp. were 83.33% and 80.56%, respectively. TheⅠ. suis-positive rate in scale pigs farms reached to 91.67%, which was significantly higher than that of specialized households (66.67%). However, the Eimeria spp.-positive rate in specialized households (83.33%) was higher than that in scale farms (79.19%). A total of 2,388 fecal samples were examined, with the overall infection rate of Coccidium spp. was 13.11%. The infection rates of Coccidium spp. in scale farms and specialized households were 12.83% and 14.00%, respectively. There was not significant difference between theⅠ. suis infection rate and the Eimeria spp. infection (7.96% and 5.15%, respectively). However, the infection rate ofⅠ. suis in scale farms (8.90%) was significantly higher than that in specialized households (4.85%). In contrast, there was a reverse result in the Eimeria spp. infection rate in scale farms and specialized households (3.39% and 9.16%, respectively). There was significant difference between the infection rate of Coccidium spp. and different age groups in different scale pigs farms. In scale farms, the prevalence of Coccidium spp. gradually increased before 20 to 30-day-old piglets, whereas the infection rate decreased significantly after the age groups. However, in specialized households, the highest infection rate (25.71%) was seen in 11-20 days of age and the lowest prevalence (3.67%) was observed in 21-30-day-old piglets, then the infection rate again increased to 18.12% in 31-40 days of age. The infection rates ofⅠ. suis in scale farms, specialized households, and the overall infection rate in different scale farms were 16.43%,20%, and 17.13%, respectively. Moreover, the highest prevalence rate all concentrated in 11-20 days of age, and the prevalence rate gradually increased and decreased before and afer the age group, respectively. In addition, the ages of 190Ⅰ. suis-positive piglets were mainly concentrated in 11-20 days, with 28.33% and 51.85% of infection rate in scale farms and specialized households. NoⅠ. suis-positive piglets was seen in more than 41 days of age in specialized households. The prevalence ofⅠ. suis ranged from 1.25% to 50% in different scale farms and 1.79%-15.28% in specialized households. With the increasing of age, the overall infection rate of Eimeria spp. gradually increased in scale farms, the highest prevalence rate (7.48%) was seen in more than 41-day-old piglets. In specialized households, the infection rates of Eimeria spp. in 31-40 days and more than 40 days of age were 15.22% and 12.17%, respectively. Which was significantly higher than those in lower than 30 days of ages and the all age groups of pigs in scale farms. In contrast, in scale farms, the infeciton rates of Eimeria spp. were relatively high in 21-30 days and more than 41 days of age, with 5.50% and 5.86% of prevalence, respectively. However, there was significant difference in the infection rate of Eimeria spp. in different farms or specialized households, with the highest infection rate reached to 60%. The difference of Eimeria spp. infection in different in different scale farms was significant, with the infection rate ranging from 0.84% to 19.15%. Whereas the infection rate of Eimeria spp. in different age groups in different specialized households ranged from 2.78% to 23.26%. The ages of 123 Eimeria-positive piglets were mainly concentrated in more than 41 days, with the highest infection rate was 44.44%, followed by 23.16% in 21-30 days old piglets. Whereas in specialized households, the higher infection rates were observed in more than 41 days and 31-40 days of ages, with 45.10% and 41.18% of prevalence rate, respectively. A total of eight species from two genus Coccidium were discovered, includingⅠ. suis, E. suis, E. porci, E. neodebliecki, E. debliecki, E. perminuta, E. scabra, and E. yanglingensis sp. The overall OPG value was 9313 (200-46400), and 7827 (200-46400) forⅠ. suis in scale farms. Whereas in specialized households, the overall OPG value was 10818 (800-24600), and 8300 (2400-22600) forⅠ. suis.4-6 mixed infection species of Coccidium were seen in each age group in the Coccidium-positive farms or specialized households. Thereinto, theⅠ. suis was the predominant species and found in all age groups of piglets.2. To understand the biological characterization ofⅠ. suis, the single oocyst isolation method was used to obtian the pure oocysts ofⅠ. suis. Then 30 sporulatedⅠ. suis oocysts were respectively inoculated orally to six 3-day-old piglets. The passaged oocysts were identified pureⅠ. suis by morphological characteristics and were collected for further amplification. The passaged oocysts were respectively inoculated orally to six 3-day-old and 22 5-day-old piglets, with 2,000 oocysts for each piglet, thus the large number of pure oocysts ofⅠ. suis were obtained. The present results indicated that there was some assiciation between the shape of oocysts and the time of oocysts shedding. And the sporulation time of oocysts was related to different temperature, especially between the thin-wall-type oocysts and the thick-wall-type oocysts.3. To understand the regulation of oocysts shedding of piglets infectedⅠ. suis, ten different dosages of oocysts were used (30,0.2×104, 1×104,5×104,15×104,20×104, 22×104,30×104,50×104,and 100×104) to inoculate orally the Coccidia-free piglets, with 2 mL oocysts solution for each piglet. The results showed that the prepatent period was 9 days for the group of 30 oocysts, whereas the prepatent periods of the other 9 groups were 4-6d and major 5 days. The patent periods had large difference, the shortest time was 4 days and the longest time was 15 days, however, the majority ranged from 5 days to 9 days. The peak period of oocysts shedding concentrated in 7-11 days post inoculation (DPI). Moreover, the second peak period of oocysts shedding was 4-6 days after 1-2 days ended the first peak period of oocysts shedding. The present results suggested that there were associations between the prepatent period, patent period, infection intensity and the infection dosage. Higher inoculated dosage, the prepatent period and the patent period shorter, OPG values greater; smaller inoculated dosage, the prepatent period and the patent period longer, OPG values smaller too.4. In order to reveal the dynamic changes of the blood physiological index and serum biochemical values of piglets infected withⅠ. suis, nine six-day-old piglets were inoculated withⅠ. suis sporulated occysts. The blood physiological index and serum biochemical values were checked on by Sysmex sf-3000 cytoanalyze and 7060 automatic biochemistry analyzer (Hitachi).The checked-time are before and 1,3,6,9,11 days post-infection(DPI), respectively. Results show that white blood cell raised on 1 DPI and reached peak value on 11DPI. Red blood cell reduced on 3 DPI and came to the lowest point on 9 DPI, which was refection on 11 DPI. Alanine aminotransferase activity rised gradually and reached peak value on 11 DPI. Aspartate aminotransferase became the peak value on 6 DPI. Alkaline phosphatase descented on 1 DPI, and came to the lowest point on 11 DPI. Lactate dehydrogenase,γ-glutamyltransferase and a-hydroxybutyrate dehydrogenase were reduced but had no significant change. Albumin reduced on 1 DPI, which was refection on 11 DPI. Globulin decreased, but has no significant change. Glucose rised but has no significant change. Blood urea nitrogen depressed on 1-3 DPI, then rised gradually up to 11 DPI. Uric acid rised on 1-3 DPI, then deduced until 11 DPI. Total cholesterol depressed. Triglyeride rised on 1-9 DPI and returned pre-infection level on 11 DPI. K+ content reduced on post infection but the difference was no obviously. However leucocyte neutrophil, lymphomonocyte, hemoglobin, haematocrit, total protein, Na+, Cl-, Ga2+ and Mg2+ had no significant change.5. In order to reveal the dynamic changes of the T lymphocyte subgroups in piglets, nine six-day-old piglets were inoculated withⅠ. suis sporulated occysts. CD3+, CD4+, CD8+ T cells were checked on before and post the day inoculation 1,3,6,9,11 (DPI), respectively. Results showed that the amounts of CD3+, CD4+T cells increased on DPI 3 and then decreased, but the diversity was not significant (p>0.05); however, CD8+ T cell raised on DPI 6 and reached peak value on DPI 9 (P＜0.05). The ratio of CD4+/CD8+began to descend on DPI 6, and came to the lowest point on DPI 11. Those indicated that the immunological functions of the piglets infected withⅠ. suis were reinforced on DPI 3, however, which were suppressed after DPI 8.6. To understand the endogenous development ofⅠ. suis and the pathogenicity to piglets, nine different dosages (5.0×104,1.0×105,1.0×104,5.0×104,1.0×105,1.5×105,2.0×10',5.0×105,1.0×106 sporulated oocysts ofⅠ. suis) were used to inoculate orally Coccidia-free 3-day-old piglets. The results showed that two distinct types of meronts were seen in tissue sections. They were binucleate type I meronts, type I merozoites, and multinucleate typeⅡmeronts, typeⅡmerozoites. The endogenous development ofⅠ. suis mainly developed in parasitophorous vacuole of epithelium mucosae cells and intestine crypt in the middle and posterior segment of jejunum and ileum. Macrogamonts lack the highly eosinophilic wall-forming bodies, and the oocyst wall is usually inconspicuous. Two types of oocysts were formed, including thin-wall-type oocysts and the thick-wall-type oocysts. The thin-wall-type oocysts often atrophy or fracture during sporulation. The clinical signs were related to different infection dosages of oocysts, however, all infected piglets showed the symptom of diarrhea. Increased with the infection dosages, clinical symptoms worsened, fecal scores recorded and mortality of infection piglets increased. Liveweight gain of infection piglets with larger dosages oocysts was decreased significantly. The patent of infection piglets with lower dosages oocysts occured after diarrhea whereas The patent and diarrhea of infection piglets with higher dosages oocysts almost occurred at the same times. Thus, these results indicated theⅠ. suis had a higher pathogenicity to piglets.7. A total of 5 18S rRNA sequences from the genus Isospora (Ⅰ. suis ISU97523,Ⅰ. beill DQ060658,Ⅰ. ohioensis AF029303,Ⅰ.felis L76471,Ⅰ. robini AF080612) were downloaded from the Genbank database, the sequences were aligned by the software of OMIGA 2.0 and produced a consence sequence. Then the primers for nested-PCR were designed according to the consence sequence obtained using Primer 5.0 software. The relative conditions including annealing temperature, Mg2- level, dNTP quantity, template, and the concentration of the primers were optimizated. The specificity study showed that the designed primers could amplify excellent aim strap of Isospora spp. and Eimeria spp. In addition, the primers also could amplify Toxoplasma gondii. However, the Cryptosporidium spp. and some common intestinal bacteria (such as Escherichia coli, Clostridieum welchii, and Chain coccus) were not successfully amplified by designed primers. The results of sensitivity study indicated that the primers could successfully amplify the DNA sample correspondent to 0.5 oocysts. The results of simulation field study showed that the detection rate ofⅠ. suis oocysts by established nested-PCR method was more higher than that using MacMaster's method. Moreover, The established systems could amplify the DNA sample from 135 oocysts in feces. The 4Ⅰ. suis isolates from different areas and a isolate from passaged oocysts were conducted phylogenetical relationship study based on the present nested-PCR methods and by the software of MEGA 4.0. The results suggested that there were no sequence difference between the five isolates in Henan Province, with a 100% of similarity. In the phylogenetical tree, the five isolates consisted a single taxon. In conclusion, the established nested-PCR method in this study could use for molecular epidemiological investigation of Isospora suis in pigs and the phylogenetical ralationship research.8. To evaluate the effectiveness of Toltrazuril against Isospora suis,7-day-old piglets infected naturally withⅠ. suis were given orally Toltrazuril for 4 times. The piglets were divided into 3 groups, with 5 piglets for per group. The dosage of Toltrazuril was 15 mg per piglet for the first group,12.5 mg per piglet for the second group. Piglets not given Toltrazuril but infected withⅠ. suis were as control group. The average values of anticoccidial index (ACI) were 1.387 and 1.322, respectively. Oocysts disappeared in feaces on the third day and the forth day post administration, respectively. The cure rate was one hundrend percent. The present results indicated Toltrazuril was effective againstⅠ. suis infection.9. To evaluate the preventive and treating effectiveness of Toltrazuril against Isospora suis,3-day-old piglets infected naturally withⅠ. suis were given orally Toltrazuril for one time,20 mg to 25 mg per kilogram bodyweight. The piglets of chemoprophylaxis were not infected withⅠ. suis and their bodyweight were significantly gained. The symptoms of sick piglets disappeared at 3 day to 4 day after treatment, negative rate of oocysts was 100%, weight gain increased 11.63% to 13.99% to compared with the control group and ACI were 1.357 to 1.380. The present results indicated Toltrazuril was effective againstⅠ. suis infection. Based on the characters of isosporosis's epidemiology and a series researched results, preventive and controlling measures of intensive pig farm were established. The methods were put into practice Zhengzhou, Luoyang and Kaifeng in Henan. The resulte showed that the infection rate ofⅠ. suis decreased from 30.84% before implementation of the program to 5.09% to 8.80%, OPG was significantly lower and isosporosis of piglets was controlled well. Enormous economic and social benef have been produced and pig industry has improved in Henan Provence after the application.