babesiosis is one of the most common infections of free - living animals worldwide and is gaining increasing interest as an emerging zoonosis in humans . canine babesiosis is important tick - borne protozoal disease of dogs with a worldwide distribution . the parasites replicate in the red blood cells and initiate a mechanism of antibody - mediated cytotoxic destruction of circulating erythrocytes ( 1 ) . the severity of babesiosis in dogs ranges from subclinical infection , the development of mild anemia to widespread organ failure and death . the diagnosis of infection with babesia is usually based on the detection of pathogens in peripheral blood under a microscope . all large babesia ( 3 - 5 m ) were designated b. canis , whereas all small babesia were thought to be b. gibsoni ( 0.5 - 2.5 m ) ( 2 ) . large b. canis was divided into three different species , namely b. canis vogeli , b. canis canis and b. canis rossi ( 3 ) . b. canis vogeli is the most widespread canine piroplasm which found in tropical , subtropical and mediterranean regions . b. gibsoni occurred in asia , north american , and northern and eastern africa ( 4 ) . canine babesiosis has varying clinical signs which depend on babesia species , host immunity , age and concurrent diseases . the most common found in canine babesiosis are fever , anemia , jaundice , hemoglobinuria , depression , and weight loss ( 5 ) . to provide a point of reference to study the epidemiology and clinical expression of canine babesiosis in china , we evaluated 30 cases of canine babesiosis by mean of clinical history , physical examination , hematological , restriction fragment length polymorphism of pcr products ( pcr - rflp ) and sequencing analysis . thirty dogs of different breeds , ages naturally infected with canine babesia were collected from the animal hospital of nanjing agricultural university between september 2012 and september 2013 . a routine physical examination performed beforehand . basic information on the breed , age , gender , tick infestation history and access to the outdoors were provided by the owners . edta - anticoagulated blood was collected for complete blood counts , smear observations and pcr analysis . complete blood count ( cbc ) was assessed with an automatic cell counter ( abx micros 60 , france ) . the following parameters were assessed : red blood cell count ( rbc ) , hemoglobin concentration ( hgb ) , hematocrit ( hct ) , mean corpuscular volume ( mcv ) , mean corpuscular hemoglobin ( mch ) , mean corpuscular hemoglobin concentration ( mchc ) , red distribution width ( rdw ) , plt count , white blood cell count ( wbc ) , wbc differential count including neutrophils , lymphocytes , monocytes . genomic dna was isolated from 200 l aliquots of edta - anticoagulated blood using tianamp genomic dna kit ( tiangen , china ) , according to the manufacturer s instruction . the concentration of dna was estimated using optical density ( o.d . ) readings at 260 nm , and the purity of dna was checked by calculating the ratio of the o.d . readings at 260 nm and 280 nm , measured using a spectrophotometer ( eppendorf , germany ) . pcr was conducted with a set of primers ( forward primer b.com 339-f : 5-gtcttgtaattg gaatgatggtgac-3 , reverse primer b.com 339-r : 5-atgcccccaaccgttcctatta-3 ) that amplified 340 bp fragment of the 18s rrna genes from b. canins and b. givsoni but not mammalian dna ( 6 ) . amplification of the full length 18s rrna genes ( 1700 bp ) were performed using universal primers : the forward primer b.com 1700-f : 5- aacctggttg atcctgccagtagtcat-3 and the reverse primer b.com 1700-f 5- gaatgatccttccgca ggttcacctac-3 ( 7 ) . pcr reaction mixtures contained the following components : 1 l genomic dna template ( 0.5 g/l ) , 12.5 l of premix ex taq ( takara , dalian ) , 0.5 l of each primer ( 10 mol / l ) , and 10.5 l water . touchdown pcr amplification was performed in veriti 96-well thermal cycler ( applied biosystems , usa ) . the initial touch down cycle was denaturation at 96 c for 30 s , annealing at 65 c for 30 s , and extension at 72 c for 30 s ( 90 s for full length 18s rrna genes ) . during the touchdown phase , the annealing temperature was decreased at the rate of 1 c for every cycle of the amplification reaction . after 10 touchdown cycles , 25 standard pcr cycles were performed under the following conditions : denaturation at 96 c for 30 s , annealing at 55 c for 30 s , extension at 72 c for 30 s ( 90 s for full length 18s rrna genes ) , and a final extension at 72 c for an additional 5 min . pcr products were resolved by electrophoresis through a 2% agarose gel for about 30 min at 120 v in tris - acetate - edta buffer . pcr products were purified using the tiangel midi purification kit ( tiangen , china ) . the 339 bp pcr product was subjected to restriction enzyme digestion using hinf i according to manufacturer s instructions ( takara , dalian ) and visualized by the 12% denaturing polyacrylamide gel electrophoresis . the 1700 bp pcr product was cloned in pmd18-t vector ( takara , dalian ) according to the manufacturer s instructions . the sequencing of positive plasmid was conducted by invitrogen , using both the universal forward and reverse primers of puc plasmid . sequence alignments were performed using blastn , which is available on the ncbi server ( http://www.ncbi.nlm.nih.gov ) . thirty dogs of different breeds , ages naturally infected with canine babesia were collected from the animal hospital of nanjing agricultural university between september 2012 and september 2013 . a routine physical examination performed beforehand . basic information on the breed , age , gender , tick infestation history and access to the outdoors were provided by the owners . edta - anticoagulated blood was collected for complete blood counts , smear observations and pcr analysis . complete blood count ( cbc ) was assessed with an automatic cell counter ( abx micros 60 , france ) . the following parameters were assessed : red blood cell count ( rbc ) , hemoglobin concentration ( hgb ) , hematocrit ( hct ) , mean corpuscular volume ( mcv ) , mean corpuscular hemoglobin ( mch ) , mean corpuscular hemoglobin concentration ( mchc ) , red distribution width ( rdw ) , plt count , white blood cell count ( wbc ) , wbc differential count including neutrophils , lymphocytes , monocytes . genomic dna was isolated from 200 l aliquots of edta - anticoagulated blood using tianamp genomic dna kit ( tiangen , china ) , according to the manufacturer s instruction . the concentration of dna was estimated using optical density ( o.d . ) readings at 260 nm , and the purity of dna was checked by calculating the ratio of the o.d . readings at 260 nm and 280 nm , measured using a spectrophotometer ( eppendorf , germany ) . pcr was conducted with a set of primers ( forward primer b.com 339-f : 5-gtcttgtaattg gaatgatggtgac-3 , reverse primer b.com 339-r : 5-atgcccccaaccgttcctatta-3 ) that amplified 340 bp fragment of the 18s rrna genes from b. canins and b. givsoni but not mammalian dna ( 6 ) . amplification of the full length 18s rrna genes ( 1700 bp ) were performed using universal primers : the forward primer b.com 1700-f : 5- aacctggttg atcctgccagtagtcat-3 and the reverse primer b.com 1700-f 5- gaatgatccttccgca ggttcacctac-3 ( 7 ) . pcr reaction mixtures contained the following components : 1 l genomic dna template ( 0.5 g/l ) , 12.5 l of premix ex taq ( takara , dalian ) , 0.5 l of each primer ( 10 mol / l ) , and 10.5 l water . touchdown pcr amplification was performed in veriti 96-well thermal cycler ( applied biosystems , usa ) . the initial touch down cycle was denaturation at 96 c for 30 s , annealing at 65 c for 30 s , and extension at 72 c for 30 s ( 90 s for full length 18s rrna genes ) . during the touchdown phase , the annealing temperature was decreased at the rate of 1 c for every cycle of the amplification reaction . after 10 touchdown cycles , 25 standard pcr cycles were performed under the following conditions : denaturation at 96 c for 30 s , annealing at 55 c for 30 s , extension at 72 c for 30 s ( 90 s for full length 18s rrna genes ) , and a final extension at 72 c for an additional 5 min . pcr products were resolved by electrophoresis through a 2% agarose gel for about 30 min at 120 v in tris - acetate - edta buffer . pcr products were purified using the tiangel midi purification kit ( tiangen , china ) . the 339 bp pcr product was subjected to restriction enzyme digestion using hinf i according to manufacturer s instructions ( takara , dalian ) and visualized by the 12% denaturing polyacrylamide gel electrophoresis . the 1700 bp pcr product was cloned in pmd18-t vector ( takara , dalian ) according to the manufacturer s instructions . the sequencing of positive plasmid was conducted by invitrogen , using both the universal forward and reverse primers of puc plasmid . sequence alignments were performed using blastn , which is available on the ncbi server ( http://www.ncbi.nlm.nih.gov ) . a total of 30 dogs infected with canine babesiosis were diagnosed by clinical examination and observation of intra - erythrocytic inclusions within blood smears . giemsa - stained blood smears showed the presence of small pear - shaped parasites ( fig . 1 ) . of the 30 dogs , males ( 80% ) were more affected than females ( 20% ) . the most prevalent clinical abnormalities were lethargy ( 100% ) , anorexia ( 100% ) , pale or icteric mucous membranes ( 80% ) , fever ( 70% ) and dark urine ( 70% ) . thirty dogs ( 100% ) had the rbc , hgb and hct values below the reference values . twenty - night dogs had the plt values below the reference values , while only one case ( 3.3% ) presented values of plt within the normal values . the mean values of the other parameters were normal in dogs infected by canine babesiosis . 2 , the primer set b.com 339-f and b.com 339-r successfully amplified an approximately 339 bp dna fragment from all 30 dogs infected with canine babesiosis . the babesia species can be distinguished based on the polymorphism of hinf i restriction site within the 339 bp fragment of the 18s rrna genes from b. canins ( no hinf i restriction site ) , and b. gibsoni ( hinf i restriction site ) . all the 339 bp dna fragment from30 dogs infected with canine babesiosis could be digested into two dna fragment ( 185 bp and 154bp ) with the hinf i , which indicated that the parasites were b. gibsoni . the 1700 bp pcr products amplified from 3 dogs were purified and cloned in the pmd18-t vector . the sequences of three full length 18s rrna genes had the same sequences and had been submitted to embl sequence databases ( accession no . phylogenetic analysis of the three full length 18s rrna genes showed that the sequences exhibited a high level of homology ( 99 - 100% ) with the other b. gibsoni isolated from japan ( accession no . the results of sequences analysis were shown that those dogs were infected with b. gibsoni . the primary goal of this research was to investigate the epidemiology and clinical expression of canine babesiosis in nanjing , china . in the present study , 30 cases infected with babesia gibsoni were confirmed by mean of clinical history , physical examination , hematological , restriction fragment length polymorphism of pcr products ( pcr - rflp ) and sequencing analysis . the cases of canine babesiosis reported in china have been considered to be caused by b. canis vogeli and b. gibsoni . b. gibsoni is the majority parasites infected dog in north of china , such as jiangsu , jilin , henan province . prevalence of b. canis vogeli in dogs was only reported in guangdong province , south of china ( 8) . b. gibsoni has a wide geographical distribution and has been reported from asia , africa , europe , the middle east and northern america . dogs are reservoir for b. gibsoni , specific fighting dogs in particular , and not an endemic species of tick . it is speculated that b. gibsoni will be reported eventually from all countries where dogs fighting is practiced ( 4 ) . but the similarity between species and subspecies has been a limiting factor ( 9 ) . serological test is a useful assay for detection of infected dogs , especially if combined with pcr . however , poor specificity , inability to differentiate acute from chronic infection were major limitation of serological tests ( 10 ) . pcr method represents a powerful tool not only for genotype identification but also in cases when blood smears do not provide a certain diagnosis . recent advances in molecular methodology ( pcr - based methods and subsequent dna sequencing ) have made it possible to detect and identify piroplasms with greater sensitivity and specificity than with traditional methods ( 11 ) . in this study , microscopical most of the dogs infected with b. gibsoni showed abnormalities such as lethargy , anorexia , fever , anemia , and thrombocytopenia , which were in accordance with other researches ( 5 ) . the most common abnormalities of hematological parameters were rbc , hgb and hct , which were observed in all affected dogs . the most consistent and severe change in blood picture observed in the course of canine babesiosis is thrombocytopenia ( 1 , 12 ) . these values are the most inconsistent and the less reliable in terms of characterizing the organism s reaction in canine babesiosis ( 12 ) . the morphological similarity between species and subspecies of babesia has led to much confusion over accurate diagnosis using light microscopy ( 13 ) . moreover , the detection of babesia parasites is difficult in dogs with unapparent or chronic infection since the level of parasitemia is very low ( 14 ) . pcr , in combination with restriction fragment length polymorphism ( rflp ) analysis offers an effective and rapid means of discriminating between species ( 13 , 15 ) . in this study , all 339 bp dna fragment from 30 dogs infected with canine babesiosis could be digested with the hinf i , which indicated that the parasites were b. gibsoni . sequencing of the full length 18s rrna genes provides a reliable method for both species identification and phylogentic analysis and allows new sequences to be compared with the sequences of other babesia ( 7 , 16 ) . the results of this study revealed that canine babesiosis in nanjing , china were due to infection of b. gibsoni . in the present study , the most common abnormalities detected in blood of dogs infected with b. gibsoni were anemia and thrombocytopenia . the results of pcr - rflp and sequencing analysis indicated that b. gibsoni was the main species responsible for canine babesiosis cases at the time of the study in nanjing , china .
backgroundto provide a point of reference to study the epidemiology and clinical expression of canine babesiosis in china.methodsa total of 30 dogs infected with canine babesiosis were evaluated by mean of clinical history , physical examination , hematological , restriction fragment length polymorphism of pcr products ( pcr - rflp ) and sequencing analysis.resultthe most prevalent clinical abnormalities were lethargy ( 100% ) , anorexia ( 100% ) , pale or icteric mucous membranes ( 80% ) , fever ( 70% ) and dark urine ( 70% ) . 
 hematology parameters revealed that anemia and thrombocytopenia were the major abnormalities in blood of dogs infected with canine babesia . 
 the results of pcr - rflp and sequencing analysis indicated that b. gibsoni was the main species responsible for canine babesiosis cases at the time of the study in nanjing , china.conclusionsthe results provide valuable information for better understanding of the epidemiology of canine babesiosis in china .