Study On A Suspected Pathogenic Ciliophora In Eupolyphaga

Objective: The background is that a patient who ate a living Eupolyphaga causes lung infections and brain lesions and eventually dies.After excluding other causes,Eupolyphaga was dissected and found to be parasitic with a variety of nematodes and protozoa.Parasitic protozoa include three different forms of Ciliophora and Amoeba.In this paper,studies were carried out on suspected Balantidium with high infection rate and highest infection rate in Eupolyphaga.Metheods: Eupolyphaga was dissected,Ciliophora was isolated and purified,and its morphological characteristics were observed under unstained and stained state.Primers were designed by reviewing the literature.After extracting the DNA of the worm,PCR amplification was carried out,and the target band was observed by electrophoresis.Twenty-four KM mice were randomly divided into the normal immunization group(group A)and the immunosuppressed group(group B).The two groups of mice were randomly divided into 4 groups(A1A2/A3/A4 or B1/B2/B3/B4),in which A1 and B1 groups were intraperitoneal injection with 0.2ml worm suspension containing about 100 live Ciliophora,and A2 and B2 groups were gavage.A3 and B3 were intraperitoneally injected with 0.2 ml of normal saline,and A4 and B4 were gavage with 0.2 ml of normal saline.The behavioral changes of the mice in each group were observed after infection,and the serum,lung,liver and brain organs of each group were collected at the 8th week.The anti-Ciliophora antibody levels in mice serum were detected by ELISA,and the differences among the groups were compared.Visually and microscopically compare the differences in lung,liver and brain lesions,and find out if there are Ciliophora in the tissues.Results: 1.Morphological observation:There are two periods of trophozoitesand cysts of the Ciliophora.The trophozoites are asymmetrically elliptical,which the front end is thinner and more blunt at the back end,and the cilia are arranged closely and neatly outside the plasma membrane.The cell mouth is wider at the front end,and the pharyngeal length extends to the ventral body and the length of the body is 3/5,and the cytoplast is in the posterior part.The cytoplasm is colorless and translucent,containing granules and food vacuoles or stretch vesicles.An elliptical or longgiant nucleus is located in the body,and even small nucleus are visible.The average body length is 68.64±14.15?m(47.40?m-98.60 ?m),the average body width is 53.50±9.67 ?m(38.70 ?m-73.50 ?m);the giant nucleus length is 28.12±6.28 ?m(14.60 ?m-37.70 ?m)and the width is 9.93±5.65 ?m(3.00?m-19.60 ?m);The cilia length is 4.17±0.88?m(2.30?m-6.10?m).The morphological characteristics of cysts are circular,with cyst wall,the size is(54.50±10.67)?m×(54.50±10.67)?m and the nuclear size is(18.12±6.28)?m×(17.43±5.65)?m.The cells are rich in granules,which are visible in food bubbles and round giant nucleus.Compared with the morphological data,it conforms to the basic characteristics of the Balantidium.2.Molecular biology analysis:After reviewing the literature,three pairs of primers related to the 18 S rRNA conserved sequence of the Balantidium were designed.DNA was extracted from the Ciliophora for PCR amplification,and the target gene of about 1500 bp was observed by electrophoresis to be consistent with the theoretical value.3.Infectious and pathogenicity:a.There was no significant difference in body weight changes between mice with different immune status and different infection pathways(P>0.05);b.Anti-Ciliophora antibodies were detected in the serum of mice with different immune status after oral and intra-abdominal infection(compared with uninfected mice,the OD values ofeach group were >2.1);c.There was no significant difference in the serum level of Anti-Ciliophora antibodies between the two mice with different immune status(C>0.05).d.The difference of anti-Ciliophora antibodies in serum of mice infected with Ciliophora in two different ways was no statistical significance(P>0.05);e.Both normal and immunosuppressed mice can cause severe inflammatory hemorrhagic lesions in the lungs after infection with Ciliophora.A few liver lesions can be seen.Individual brain lesions can be seen,but no typical pathogens are found in the tissues.Conclusions: 1.The Ciliophora,which has a high infection rate in the hindgut of Eupolyphaga,is similar to but different from the morphology of the Balantidium species.2.The Ciliophora can cause a variety of organ lesions after infection in mice.