| Trichomonas gallinae is a kind of flagellate protozoa parasite that parasitizes the upper digestive tract of pigeons and causes trichomoniasis in the host.Trichomoniasis is endemic all over the world.Trichomonas gallinae has highly infectious ability,which can infect not only all kinds of pigeons but also poultry,turkey,wild birds,etc;It mainly causes necrotic ulcers and obstruction of the upper digestive tract such as the mouth,esophagus,crop,and anterior stomach of infected pigeons,resulting in difficulties in eating,drinking and breathing,slow growth and even death.It causes serious losses to the pigeon industry every year.Shandong Province is a major province of meat pigeons breeding,showing the characteristics of regional intensive breeding.The purpose of this study is to understand the infection status,gene types,cultural characteristics,and pathogenic characteristics of Trichomonas gallinae in main meat pigeon ranches in Shandong Province,to provide a scientific basis for in-depth research and clinical prevention and control of trichomoniasis.Firstly,oropharynx swabs were collected from the main meat pigeon ranches in 8regions of Shandong Province.The infection of Trichomonas gallinae was detected by culture method and microscopic examination.The isolates of Trichomonas gallinae were obtained by in vitro culture,smear,Wright Giemsa staining,and morphological identification;Secondly,three Trichomonas gallinae strains were randomly selected from each region,its Internally Transcribed Spacer gene and AP65 gene were amplified by PCR,sequenced,the gene homology was analyzed by bioinformatics software,and the phylogenetic tree was constructed to determine the genotype of protozoa strains.Thirdly,protozoa strains of different genotypes were cultured in vitro.Under conditions of different p H values（p H 1.5,p H 3.9,p H 6.3,p H 9.8,p H 12.6）,different serum concentrations（1%,5%,10%,15%,20%）and different temperatures（28 ℃,33 ℃,37 ℃,42 ℃,47 ℃）,the growth and proliferative rates of the protozoa strains were compared,and the cultural characteristics of the isolated protozoa strains were analyzed.Fourthly,the isolated strains were rejuvenated and cultured under different conditions.The artificial feeding Trichomonas-free 1-month-old squabs were orally infected,and the pathological changes were observed by tissue section staining;immunohistochemical technique,scanning electron microscope,and transmission electron microscope was used to detect the parasitic state of protozoa and analyze the pathogenic characteristics.The results showed that Trichomonas gallinae was detected in the main meat pigeon ranches in 8 regions of Shandong Province,and the individual positive rate was 82.78%（601/726）;among them,the positive rate of squabs was 81.03%（188/232）,and that of fledglings was 86.74%（229/264）;The carrying rate of nanny pigeons was 80%（184/230）.The results of genotyping of 24 strains of Trichomonas gallinae in 8 regions showed that there were 21 strains of genotype A,accounting for 87.5%,1 strain of genotype B,accounting for 4.17%,and 2 strains of genotype T,accounting for 8.33%;The homology and phylogenetic tree of AP65 gene showed that the isolated protozoa strains belonged to two branches.Genotype A strains in Jinan Tianqiao district were on the same evolutionary branch as two gene type T strains in Liaocheng pigeon ranch,and the other strains were on the same evolutionary branch.Under the condition of a basic HF medium,there was no difference between the growth and proliferation rate of genotype A and B.the growth rate of genotype A was slightly faster than that of genotype B at 12h～20h;There was no difference in doubling time between genotype A and genotype B which was 3.77 and 3.89 h,respectively;Under different p H values,the results showed that at p H 6.3,the growth and proliferation rate of the two genotypes was significantly higher than that of other p H conditions.p H < 3.9 or > 9.8 caused the proliferation of the two genotypes to slow down or stop growing.The serum concentration of 10% significantly promoted the rapid proliferation of the two genotypes and maintained the high density of the protozoa for a long time.The serum concentration of 1%and 5% also significantly promoted the rapid proliferation of protozoa in the initial stage（0h～16h）,but it was difficult to maintain the high density of the protozoa,while the two protozoa strains almost did not proliferate well at serum concentration of 15% and 20%.When the temperature was 37℃ and 42℃,the proliferation rate of the two protozoa strains was significantly faster than that of other temperature conditions.When the temperature was greater than 47℃ or less than 28℃,they almost did not proliferate.Chicken embryo allantoic cavity inoculation,duck embryo allantoic cavity inoculation,DF-1 cell inoculation,and Vero cell inoculation were used to rejuvenate Trichomonas gallinae.Among the four methods,only Vero cell inoculation failed to rejuvenate Trichomonas gallinae.The rejuvenated Trichomonas gallinae was inoculated into the allantoic cavity of chicken embryo and successfully infected artificial feeding Trichomonas-free 1-month-old white feather King squabs.The immunohistochemical results of esophageal tissue showed that Trichomonas gallinae parasitized on the surface of pigeon esophageal mucosa;No obvious pathological changes were found in esophageal pathological sections;The results of the scanning electron microscope and transmission electron microscope of pigeon esophagus and Trichomonas gallinae showed the adhesion state of Trichomonas gallinae and the shorten occur and disordered pathological damage of esophageal mucosal microvilli.In summary,Trichomonas gallinae infection is common in the main meat pigeon ranches in 8 regions of Shandong Province.There are multiple Trichomonas gallinae genotypes dominated by type A.The growth and proliferation of Trichomonas gallinae isolate stains are the best at the culture condition that is p H6.3,the serum concentration is10% and the temperature is 37℃～42℃.Trichomonas gallinae can be rejuvenated by inoculation in chicken embryo allantoic cavity,duck embryo allantoic cavity,and DF-1 cell,which is easy to be susceptible to 1-month-old squabs. |
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