| Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis spp. are important enteric protozoa distributed in a variety of human and animal species and geographic locations. The infections caused by them are generally considered opportunistic. The ubiquitous parasites can cause transient and self-limited diarrhea or manifest as asymptomatic infections in immunocompetent individuals. Nevertheless, the infection can become intense and long-lasting in immunocompromised persons and animals, especially HIV- infected patients and neonatal animals. However, little is known about the prevalence, species/genotype distribution, and genetic features of these pathogens in dairy cattle in Heilongjiang Province in China.One objective of this study was to investigate the prevalence and genetic features of the three parasites in 526 dairy cattle from cities Harbin, Daqing, and Qiqihar, northeast China. Cryptosporidium was identified to be the most predominant among the three pathogens, with the prevalence(24.7%, 130/526; 95% CI, 0.205 to 0.290) signif icantly higher than that of E. bieneusi(5.9%, 31/526; 95% CI,0.038 to 0.080) or Blastocystis(10.3%, 54/52695% CI, 0.075 to 0.130)(p < 0.01). Five Cryptosporidium species/genotype were determined in 125 of the 130 PCR-positive specimens by DNA sequence analysis of the small subunit r RNA gene(SS U r RNA). Among them, C. parvum(87/130) and C. bovis(28/130) are the predominant species, followed by C. ryanae(5/130), C. andersoni(2/130), C. suis-like genotype(2/130), and mixed C. ryanae/C. bovis(1/130). C. parvum and C. andersoni are zoonotic species. Subtyping relying on the DNA polymorphisms of the 60 kDa glycoprotein(gp60) gene identif ied subtypes IIdA15G1(24/87) in Harbin and IIdA20G1(48/87) in Qiqihar. High prevalence of human- pathogenic C. parvum subtypes IIdA15G1 and IIdA20G1 in dairy cattle from northeast China is of great zoonotic concern. This is the first report describing C. parvum subtype IIdA20G1 in China and IIdA15G1 in Heilongjiang Province. Sequence analysis of the internal transcribed spacer(ITS) enabled identification of 11 genotypes from 31 PCR-positive specimens, including 6 known(BEB4, CS-4, EbpC, G, I, J) and 5 new genotypes(NECA1 to NECA5). The genotypes formed two genetic clusters in phylogenetic analysis, with CS-4, EbpC, G, and NECA1 to NECA5 distributed in zoonotic Group 1 and BEB4, I, and J in potentially host-adapted Group 2. All the known genotypes are zoonotic genotpes. For the detection of Blastocystis, DNA sequence analysis of the SSU r RNA gene indentified 4 known subtypes(ST4, ST5, ST10, ST14) and a new subtype(ST18) from 54 PCR-positive specimens. ST10 was the predominant genotype(41/54), followed by ST14(9/54), ST4(2/54), ST5(1/54) and ST18(1/54). ST4 and ST5 are zoonotic genotypes.In multilocus sequence typing(MLST) of C. parvum isolates, five IId A15G1 and five IIdA20G1 isolates were further typed at five genetic loci( Actin, DZ- HRGP, HSP70, ML1, and ML2). However, the isolates were successfully amplif ied only at four of the five loci. No sequence polymorphisms were observed between the isolates at individual locus. In MLST of E. bieneusi, four of another ten E. bieneusi isolates were successfully sequenced at the three microsatellite loci(MS1, MS3, MS7), while only two sequences were obtained at the minisatellite locus(MS4). Phylogenetic analysis at the four loci indicated a clustering pattern quite similar as that in the analys is at ITS locus except that several ITS Group 2 isolates of genotypes BEB4, BEB6, I, and J fall into the big group with zoonotic potential. The data confirm the increased c onceren about the zoonotic importance of ITS Group 2 genotypes that were previously considered ruminant adapted, and highlight the use of MLST in understanding the epidemiology and biology of E.bieneusi.In addition, univar iate analysis implemented in SPSS was performed to analyze the risk factors associated with the pathogen prevalence. The Odds ratios(ORs) with 95% CIs and p values were calculated to measure the infection risks. Factors associated with an increased infection risk include breeding mode(bred intensively), animal age(youth and elders), seasonality(spring and autumn), and farm history(over 10 years).In conclusion, this study described the prevalence and genetic characteristics of Cryptosporidium, E. bieneusi and Blastocystis in dairy cattle from Heilongjiang and evaluated the zoonotic potential of the species or genotypes identified. Risk factors associated with the high prevalence of Cryptosporidium infections were also analyzed for the first time in Heilongjiang Province. The findings should be helpful in understanding the epidemiology and control of cattle transmissible protozoiasis. |
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