| As a worldwide protozoan parasite,Giardia lamblia parasitizes in the small intestines of many mammals includ ing humans,dogs,and cats.It can cause giardiasis with diarrhea as the main symptom.In this study,Giardiastrains from stray dogs and cats in Guangdongweregenotypiedby PCR amplification based on bg and tpi genes,and Giardia infections between different age,gender,district and their main genotypes were investigated.In the meantime,Tm-shift genotyping method for detection of cat-derived G.lambliawas established based on bg gene in order to provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G.lamblia.Firstly,a total of 527 canine faecal samp les from stray dogs of pet shelters in Shantou,Foshan,Shenzhen and Shaoguan,Guangdong province of China were examined by light microscopy.The tpi and bg genes of theirisolates were amplifiedby using normal or nested PCR,cloned and sequenced.The results showed that total infective rate was10.8%?57/527?and amplicated tpi and bg fragments were 334bp and 511bp,respectively.Based on the phylogenetic analysis,25 isolates?24 assemblages AI and 1assemblage D?in canine isolates were typed by tpi gene and 57 isolates?26assemblages AI,18 assemblage C and 13 assemblages D?in canine isolates were typed by bg gene.The dominant genotypes of young dogs??1 year?and adult dogs?>1 year?were the assemblage AI and C,respectively,and mixed infection were only found in the young dogs.The dominant genotypes of both male and female dogs were the assemblage C and AI,respectively,and mixed infection were found in both male and female dogs.Dogs infected with assemblage AI,Cand D were found in threedistricts?Shantou,Shenzhen and Shaoguan?,which only two assemblages?AI and D?were found in Foshan.In addition to Shaoguan area,mixed infection were found in other districts.Secondly,a total of 104feline faecal samples from stray cats of pet shelters in Shantou,Zhongshan,and Shaoguan,Guangdong province of C hina were examined by light microscopy.The tpi and bg genes of theirisolates were amplifiedby using normal or nested PC R,cloned and sequenced..The results showed that total infective rate was5.8%?6/104?and amplicated tpi and bg fragments were 334bp and 511bp,respectively.Based on the phylogenetic analysis,6 isolates?6 assemblages AI?in feline isolates were typed by tpi gene and 6 isolates?3 assemblages AI and 3 assemblages F?in feline isolates were typed by bg gene.Mixed infection was found in different age and gender group.In addition to Shaoguan,mixed infection were found in other districts.Finally,to develop Tm-shift genotyping method for detection of cat-derived Giardia lamblia,two sets of primers with two GC-rich tails of unequal length attached to their5'-end were designed according to two SNPs?BG434 and BG170?of?-giardin?bg?gene,and specific PC R products were identified by inspection of a melting curve on real-time PCR thermocycler.A series of experiments on the stability,sensitivity,and accuracy of Tm-shift method was tested,and clinical samples were also detected.The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F.Coefficient of variation of Tm values of assemblage A and F was 0.14 and 0.07%in BG434 and 0.10 and 0.11%in BG170,respectively.The lowest detection concentration was 4.52×10-5 and 4.88×10-5 ng/?L samples of assemblage A and F standard plasmids.The Tm-shift genotyping results of ten DNA samples from the cat-derived G.lamblia were consistent with their known genotypes.The detection rate of clinical samples by Tm-shift was higher than that by microscopy,and their genotyping results were in complete accordance with sequencing results.The results indicatedthat there is a zoonotic potential of G.lambliafrom stray dogs and cats in Guangdong provinve,and it is necessary to pay attention and to take the necessary prevention and control measures to pro tect human health.The Tm-shift genotyping method is rapid,specific,and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G.lamblia. |
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