Epidemiological Investigation Of Animal Toxoplasmosis And Analysis On Pathogenicity And MicroRNA Expression Profile In Swine Infected By Type Chinese ? Isolate Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasitic protozoan with worldwide distribution that causes serious parasitic zoonosis.Infection of T.gondii is very prevalent in animals.The feline is the only final host and plays a vital role in the life cycle of T.gondii and in the spread of toxoplasmosis.T.gondii is commonly inapparent infection in most animals,however,can cause disease and death in pigs.The pork infected with T.gondii is an important source of human toxoplasmosis infection.In China,toxoplasmosis in pigs was first reported in the 1970s,and caused serious economic losses to the pig industry.Although recently the reports on clinical cases of swine toxoplasmosis were rather sparse,however,the epidemiological survey showed that T.gondii infection was still high prevalence in slaughter pigs and live pigs in farms.T.gondii is the only recognized species in the genus Toxoplasma.However,genetic polymorphisms exist among T.gondii strains.The distribution of T.gondii genotypes varies among different geographic regions.And in China,Chinese I is the dominant genotype of T.gondii in human and animals.In this present study,the epidemiological investigation of toxoplasmosis was conducted in sick pigs and stray cats.The piglets were infected with a type Chinese I isolate of T.gondii from a dead pig using three ways.After infection the clinical symptoms and immunopathological characteristics of pigs were observed,and the interactions between T.gondii and host were analyzed at the level of microRNA based on high throughput sequencing.1.Molecular epidemiological investigation and genetic diversity of T.gondii in animalsFrom September 2011 to August 2016,the tissue samples from 139 sick pigs,and tissue and serum samples from 64 stray cats,were collected.The T.gondii DNA or the specific antibodies aganist T.gondii in the samples was detected by PCR and ELISA.The genotypes for 10 genetic markers of T.gondii were identified by PCR-RFLP.The results showed that positive rate for T.gondii was 46.04%(64/139)in the sick pigs.The geographical locations and ages were the risk factors inducing swine toxoplasmosis,while no direct correlation occurs between the T.gondii prevalence and seasonal climate for sick pigs.The total rate of T.gondii infection was 29.84%in the 610 tissue samples,in which the prevalence was significantly higher(P<0.05)in the lung and heart samples.The total prevalence of toxoplasmosis in stray cats was 34.38%(22/64),in which 17.19%(11/64)by PCR and 25.00%(16/64)by ELISA,respectively.The six distinct genotypes including ToxoDB#9(Chinese I),ToxoDB#10(Type I),ToxoDB#213,ToxoDB#89,and two new genotypes(New 1 and New 2)were identified from the twenty-two samples that were successfully genotyped.ToxoDB#9 genotype was the predominant genotype in pigs and stray cats in Jiangsu Province,and it was the first time that ToxoDB#89 and two new genotypes were found in China.These data indicated that Toxoplasma gondii isolates from animals in the region had limited genetic diversity.2.Genotyping and virulence analysis of T.gondii isolates from human and dead pigsAfter the method purifying T.gondii tachyzoites were established,a large number of purified tachyzoites of T.gondii isolates from human(KS strain)and pigs(YZ-1,YZ-2)were acquired,respectively.The genotypes for 10 genetic markers of T.gondii isolates were identified by PCR-RFLP.Then,groups of ICR mice(SPF grade)were respectively intraperitoneally inoculated with different doses of tachyzoites,ranging from 104 to 100 of each isolate.The virulence of the three isolates was determined based on the mortality and the average of survival time of infected mice.Subsequently,virulence-related factors were cloned and sequenced.The rensults showed that KS isolate was Type I(ToxoDB#10),and isolates YZ-1 and YZ-2 belonged to ToxoDB#9(Chinese?),and these three isolates were virulent to mice,similar to the reference strain RH.Analysis of T.gondii virulence-related factors showed that the deletions of 15 bp and 3 bp fragments were found in the GRA6 gene of YZ-1 and YZ-2 isolates compared with KS isolates and RH strains.All isolates contained a Pvu ? restriction site in the KHB fragment and five tandem repeats in the 5'UTR region of SAG1,both of which were associated with T.gondii virulence.However,the five loci in the KHB fragment could not be used to distinguish between virulent and non-virulent strains.These data suggest that the molecular basis of the virulence in type Chinese ? isolates may be different from type ? strains,and type Chinese ? isolates have their own characteristics in virulence-related genes.3.Pathological characteristics of piglets infected with type Chinese ? isolate of T.gondiiThe healthy piglets were infected with type Chinese ? isolate of T.gondii(YZ-1 isolate)using three ways including injection intravenously,injection intraperitoneally and oral delivery of mice infected with T.gondii.Meanwhile,the uninfected piglets were set as a control group.After infection,the clinical manifestations were observed and the rectal temperature was measured daily.At 10,25,50 and 80 days after infection,the piglets in each group were randomly selected to autopsy for observing the gross lesions.Then the tissues of the piglets were taken to make sections for observing the histopathological changes.On the other hand,the tissues were used for mouse bioassay and PCR detection.The results showed that all the piglets infected using three methods were successfully infected and displayed the different clinical symptoms,such as fever,weight loss,cough,cyanosis,asthma,and acute death,etc.The gross lesions mainly occurred in lung,liver,spleen and lymph node during the acute infection.But these gross lesions were not obvious in the chronic infection.During the acute infection the histopathological changes mainly occurred in the lung,spleen,liver,lymph nodes and intestinal mucosa,including bleeding and necrosis.During the chronic infection,however,the histopathological changes were mainly meningitis and alveolar emphysema.T.gondii tissue cycsts were found in the brain at 50 days after infection.But T.gondii tissue cycsts in the brain tissue disappeared at 80 days after infection,and PCR detection and mouse bioassay also showed that T.gondii were negative in the brain tissues.These foundings show that type Chinese ? strains can form cysts in the brain of piglets,which can not set up the chronic infection in mice,however,the cysts can not exist for a long time.4.Changes of immune-related factors in piglets infected with T.gondiiAfter piglets infected with T.gondii YZ-1,the blood samples of the piglets were collected at the frequency of 4 times during the 1st week,following by 2 times weekly during the 2nd-4th weeks after infection,and then once a week until the end of the experiment.A fluorescence quantitative PCR detection method was established to test the whole blood DNA of infected piglets,and the changes in cellular immunity and humoral immunity levels of piglets were monitored at different time points after infection with T.gondii by ELISA.The results showed that the method of fluorescence quantitative PCR with high sensitivity was suitable for the clinical diagnosis.It was found that the number and the duration of parasites in the blood were different among infected groups.Thl-type cytokines,including IFN-y and IL-12,of pigs in the intravenous and intraperitoneal injection groups was significantly higher than those in the control group in the early infection stage(P<0.05),in which IL-12 plays a role firstly,and so as pigs in feeding mice infection group.Th2-type cytokines(IL-10,IL-4),the concentration changes of two cytokines were different in serum.And the concentration of IL-10 was significant increase earlier in pigs of feeding mice infection group.However,the changes of IL-4 in serum were relatively flat.IgM antibody appeared earlier,while the concentrations of IgG antibody were significantly higher in 7-14 days after infection(P<0.05)and continued for a relatively long time.These results indicate that Thl-type cytokines are up-regulated to activate the cellular immune response after the infection with T.gondii,and then the Th2-type immunoregulatory cytokines play a role in preventing the immune injury and stimulating the antibodies against T.gondii.5.Effects on miRNA expression in the spleen of pig during the acute and chronic infection phases by T.gondiiThe healthy piglets were infected by T.gondii tachyzoites of YZ-1 strain with injection intravenously.And the spleens were collected from the piglets during acute(at 10 days after infection)and chronic(at 25 days after infection)infection phases and then were used to extract the total RNA,respectively.The small RNA libraries were created and high-throughput sequenced after quality check qualified.After a series of data screening,genome comparison,classification annotation,and analysis of differentially expressed miRNAs and their target genes though GO and KEGG enrichment.The results indicated that there were 38 differentially expressed miRNAs significantly in spleens of between infected and uninfected piglets(P<0.05).In the acute infection phase,there were 35 miRNAs showing significant difference,among which 22 were up-regulated and 13 down-regulated,while there were only 6 differentially expressed miRNAs during the chronic infection period,among which 4 were up-regulated and 2 miRNAs were down-regulated.Moreover,our foundings revealed that the differentially expressed miRNAs mainly affected the immune status and level of host following T.gondii infection though the expression regulation of Toll-like receptor pathway-related gene,cytokine receptor and STAT family members.6.Effects on miRNA expression in the brain of pig during the acute and chronic infection phases by T.gondiiThe healthy piglets were infected by T.gondii tachyzoites of YZ-1 strain with injection intravenously.The brains were collected from the pigs during acute(at 10 days after infection)and chronic(at 25 days after infection)T.gondii infection phases and then were used to extract the total RNA.The changes of miRNA expression in brain tissue were analyzed according to the methods used to study on spleen above.The results showed that 89 miRNAs,containing 51 up-regulated and 38 down-regulated,were significantly different in expression between infected and uninfected pigs during acute infection phase,while the expression of 69 miRNAs,including 32 miRNAs up-regulated and 37 down-regulated,was significantly different at the chronic infection phase.And the differentally expressed miRNAs were mainly associated with immune during the acute infection phase,and were mainly involved in tumor mechanism and neuro developmental regulation in the chronic phase.