Identification of expressed protein profiles and antigenic determination are some of the most challenging aspects of proteomics. Two-dimensional gel electrophoresis (2-DE) combined with immunoblot analysis were employed to study the N. caninum proteome. Protein sample preparation was carried out by first conducting sonication, followed by adding lysis buffer containing 7M urea plus 2M thiourea to the purified tachyzoites in order to complete disruption. A total of 335 differentially expressed protein spots were detected using pH 4-7 IPG strip (7 cm) that were run in a 56 kVh isoelectric focusing (IEF) system. Of the spots analyzed, 64 were identified as antigenic spots on immunoblot profile. Major antigenic spots appeared at 65 kDa (pI 5.2-5.3), 51 kDa (pI 5.5), 38 kDa (pI 5.1), 33 kDa (pI 4.4), 29 kDa (pI 5.6) and 15.5 kDa (pI 5.0) were observed to be significantly distinct compared to the rest of the antigenic spots. The results indicate that combination of 2-DE and immunoblotting methods were thought as very useful tools in defining both proteins and antigens of N. caninum tachyzoites. Additionally, present 2-DE profiles may be valuable in further proteomic approaches and study of the pathogen.
Animals ; Antibody Formation ; Antigens, Protozoan/*analysis/isolation&purification ; Electrophoresis, Gel, Two-Dimensional/methods ; Electrophoresis, Polyacrylamide Gel ; Epitopes/analysis ; Image Processing, Computer-Assisted ; Immunoblotting/methods ; Isoelectric Focusing ; Neospora/*chemistry/immunology ; Proteome/analysis ; Proteomics ; Protozoan Proteins/*analysis/isolation&purification

Cryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and beta,-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.
Animals ; Cryptosporidium/*isolation & purification ; Cytoskeletal Proteins/genetics ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Genes, rRNA ; Giardia lamblia/*isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Portugal ; Protozoan Proteins/genetics ; RNA, Protozoan/genetics ; RNA, Ribosomal, 18S/genetics ; Risk Assessment ; Sequence Analysis, DNA ; Water/*parasitology

The present study was designed to investigate polymorphism in Duffy binding protein (DBP) gene of Plasmodium vivax isolates of Korea. Thirty samples were obtained from P. vivax patients in Yonchon-gun, Kyonggi-do in 1998. The PCR products of the samples were subjected to sequencing and hybridization analyses of the regions II and IV of P. vivax DBP gene. Two genotypes, SK-1 and SK-2, were identified on the basis of amino acid substitution and deletion. The genotype of 10 isolates was SK-1 and that of 20 isolates was SK-2. Most of the predicted amino acids in the region II of DBP gene were conserved between the Korean isolates and Belem strain except for 4-5 amino acid substitutions. In the region IV of DBP, a 6-bp insert that was shown in the Sal-1 allele type was found in SK-1, and a 27-bp insert that was shown in the Papua New Guinea allele type was found in SK-2. In conclusion, the present findings suggest that two genotypes of P. vivax coexist in the endemic area of Korea.
Amino Acid Sequence ; Animals ; *Antigens, Protozoan ; Base Sequence ; Carrier Proteins/*analysis/chemistry/*genetics ; DNA, Protozoan/genetics ; Genotype ; Human ; Korea ; Malaria, Vivax/parasitology ; Molecular Sequence Data ; Plasmodium vivax/*genetics/isolation & purification ; Polymerase Chain Reaction ; *Polymorphism (Genetics) ; *Protozoan Proteins ; Receptors, Cell Surface/*analysis/chemistry/*genetics ; Support, Non-U.S. Gov't

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5'and 3'ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.
Animals ; Antigens, Protozoan/genetics ; Blood/*parasitology ; Cat Diseases/*parasitology ; Cats ; Cluster Analysis ; DNA Fingerprinting/methods ; DNA, Protozoan/genetics/isolation & purification ; Genotype ; Korea ; Polymerase Chain Reaction/methods ; Polymorphism, Restriction Fragment Length ; Protozoan Proteins/genetics ; Toxoplasma/*classification/*genetics/isolation & purification ; Toxoplasmosis, Animal/*parasitology

Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1.
Amino Acid Sequence ; Animals ; Antigens, Protozoan/chemistry/*genetics ; Base Sequence ; Cloning, Molecular ; DNA, Protozoan/chemistry/genetics ; Goat Diseases/parasitology ; Goats ; Indonesia ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; Protozoan Proteins/chemistry/*genetics ; Sequence Alignment ; Sequence Analysis, DNA ; Toxoplasma/*genetics/*immunology/isolation&purification ; Toxoplasmosis/parasitology ; Zoonoses/parasitology

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.
Animals ; Cat Diseases/*parasitology ; Cats ; China ; Cluster Analysis ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Feces/parasitology ; Giardia lamblia/*classification/cytology/genetics/*isolation & purification ; Giardiasis/parasitology/*veterinary ; Microscopy ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/genetics ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.
Animals ; Cat Diseases/*parasitology ; Cats ; China ; Cluster Analysis ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Feces/parasitology ; Giardia lamblia/*classification/cytology/genetics/*isolation & purification ; Giardiasis/parasitology/*veterinary ; Microscopy ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/genetics ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA

Resistance of Plasmodium spp. to anti-malarial drugs is the primary obstacle in the fight against malaria, and molecular markers for the drug resistance have been applied as an adjunct in the surveillance of the resistance. In this study, we investigated the prevalence of mutations in pvmdr1, pvcrt-o, pvdhfr, and pvdhps genes in temperate-zone P. vivax parasites from central China. A total of 26 isolates were selected, including 8 which were previously shown to have a lower susceptibility to chloroquine in vitro. For pvmdr1, pvcrt-o, and pvdhps genes, no resistance-conferring mutations were discovered. However, a highly prevalent (69.2%), single-point mutation (S117N) was found in pvdhfr gene. In addition, tandem repeat polymorphisms existed in pvdhfr and pvdhps genes, which warranted further studies in relation to the parasite resistance to antifolate drugs. The study further suggests that P. vivax populations in central China may still be relatively susceptible to chloroquine and sulfadoxine-pyrimethamine.
Antimalarials/*pharmacology ; China ; Chloroquine/pharmacology ; DNA, Protozoan/chemistry/genetics ; Drug Resistance/*genetics ; Folic Acid Antagonists/pharmacology ; Genotype ; Humans ; Malaria, Vivax/epidemiology/*parasitology ; Plasmodium vivax/drug effects/*genetics/isolation & purification ; Point Mutation ; Polymorphism, Single Nucleotide/*genetics ; Prevalence ; Protozoan Proteins/genetics ; Sequence Analysis, DNA ; Tandem Repeat Sequences/*genetics

A 12-year-old spayed female mixed-bred dog presented with nasal bleeding of 2 days duration and a skin nodule in the left flank. No abnormalities were found in coagulation profiles and blood pressure. Cytological evaluation of the nodule revealed numerous characteristic round organisms having a nucleus and a bar within macrophages and in the background, consistent with leishmaniasis. In vitro culture was unsuccessful but PCR of the nodular aspirate identified the organisms as Leishmania infantum, and the final diagnosis was canine leishmaniasis. No history of travel to endemic countries was noted. Because the dog had received a blood transfusion 2 years before the illness, serological screening tests were performed in all donor dogs of the commercial blood bank using the commercial Leishmania ELISA test kit, and there were no positive results. Additional 113 dogs with hyperglobulinemia from Seoul were also screened with the same kits but no positive results were obtained. To the best of the author's knowledge this is the first autochthonous case of canine leishmaniasis in Korea.
Animals ; Base Sequence ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Dog Diseases/*diagnosis/epidemiology ; Dogs ; Enzyme-Linked Immunosorbent Assay/veterinary ; Female ; Giant Cells/pathology ; Leishmania infantum/genetics/immunology/*isolation & purification ; Leishmaniasis, Visceral/diagnosis/*veterinary ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; Protozoan Proteins/genetics ; Republic of Korea ; Sequence Analysis, DNA/veterinary ; Serologic Tests/veterinary

This study aimed to identify the assemblages (or subassemblages) of Giardia duodenalis by using normal or nested PCR based on 4 genetic loci: glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal DNA (18S rRNA) genes. For this work, a total of 216 dogs' fecal samples were collected in Guangdong, China. The phylogenetic trees were constructed with MEGA5.2 by using the neighbor-joining method. Results showed that 9.7% (21/216) samples were found to be positive; moreover, 10 samples were single infection (7 isolates assemblage A, 2 isolates assemblage C, and 1 isolate assemblage D) and 11 samples were mixed infections where assemblage A was predominant, which was potentially zoonotic. These findings showed that most of the dogs in Guangdong were infected or mixed-infected with assemblage A, and multi-locus sequence typing could be the best selection for the genotype analysis of dog-derived Giardia isolates.
Animals ; China ; Cluster Analysis ; Coinfection/parasitology/veterinary ; Cytoskeletal Proteins/genetics ; DNA, Protozoan/chemistry/genetics ; Dog Diseases/parasitology ; Dogs ; Genotype ; Giardia lamblia/*classification/*genetics/isolation & purification ; Giardiasis/parasitology/*veterinary ; Glutamate Dehydrogenase/genetics ; Molecular Sequence Data ; *Multilocus Sequence Typing ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 18S/genetics ; Triose-Phosphate Isomerase/genetics