Genetic Characterization of Hydatid Cysts of Different Intermediate Hosts.

Cystic echinococcosis is an important cosmopolitan parasitic zoonosis that causes public health and economic problems in Egypt. The present study was undertaken to identify genotypes of hydatid cyst (HC) DNA isolated from different animal isolates and to identify the genotype of secondary hydatid cysts (HCs) developed in rabbits experimentally infected with camel HC for detection of any genetic mutation. In the present study, we extracted DNA from the germinal layers of 8 HCs collected from 3 camels, 1 cattle, 1 sheep and 3 donkeys in addition to 3 secondary HCs collected from rabbits experimentally infected with camel HC. PCR amplification of the ITS1 gene of all examined samples showed an amplified DNA band at 1115 bp. The partial nucleotide sequences of the ITS1 gene of all isolates were aligned and compared with the reference sequences of the genotypes G1-G8 in GenBank. The camel and rabbit samples were identified as Echinococcus canadensis genotype 6 (G6), while the cattle and sheep samples belonged to E. granulosus sensu stricto (G1). The donkey isolates belonged to E. equines (G4). Alignment of the ITS1 partial nucleotide sequences of the camel HCs and rabbit secondary HCs isolates with the G6 partial nucleotide sequence in GenBank was performed. Both camel HCs and rabbit secondary HCs isolates exhibited the same sequence identity matrix, which indicated the absence of mutation in the rabbit secondary HCs. It can be concluded that camel and rabbit samples were identified as E. canadensis (G6), the cattle and sheep samples belonged to E. granulosus sensu stricto (G1) and donkey isolates belonged to E. equines (G4). No mutation occurred during HCs transmission from camel to rabbit.

Molecular Characterization and Phylogenetic Analysis of Toxocara Species in Dogs, Cattle and Buffalo in Egypt.

Toxocara canis of dogs and Toxocara vitulorum of cattle and buffalo are nematode parasites that cause serious economic and public health problems all over the world. This study aims to provide molecular data to identify and distinguish between Toxocara spp. from dogs, cattle and buffalo in Egypt. Moreover, constructing a phylogeny and phylogenetic relationships among these Toxocara spp. were performed through an analytic study of ATPase-6, a mitochondrial gene; 12S, small subunit ribosomal RNA gene and ITS-2, the second internal transcribed spacer nuclear ribosomal gene. T. vitulorum from cattle and buffalo were found to be almost identical. The ATPase- 6 and 12S regions showed 87.78 % and 90.38 % nucleotide similarity between T. canis and T. vitulorum, while for the ITS-2 region, only 78.38 % was found. Analysis of the three studied genes revealed that each Toxocara spp. has distinct molecular characteristics. Moreover, it was revealed that these genes, especially the ITS-2 gene, are useful and sensitive molecular markers for classifying and studying the phylogenetic analysis and relationships among closely related Toxocara spp. All sequences obtained in this study were registered in the GenBank under the accession numbers: MG214149 -MG214157.

Synergistic haemolytic activity and its correlation to phospholipase D productivity by Corynebacteruim pseudotuberculosis Egyptian isolates from sheep and buffaloes

Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).

Synergistic haemolytic activity and its correlation to phospholipase D productivity by Corynebacteruim pseudotuberculosis Egyptian isolates from sheep and buffaloes.

Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).

Evaluation of cercarial antigen for the serodiagnosis of fasciolosis in experimentally and naturally infected sheep.

The value of cercarial antigen for diagnosis of experimental and natural sheep fasciolosis was studied by enzyme linked immunosorbent assay (ELISA) and enzyme linked immunotransfer blot (EITB). In ELISA, the antibody levels of experimentally infected sheep with Fasciola gigantica appeared at 2 weeks post infection (PI), gradually increased till 7 weeks PI and nearly remained at the same level from 7 to 13 weeks PI (the end of experiment). Also, the sensitivity and specificity of cercarial antigen for diagnosis of naturally sheep fasciolosis were 100 and 90%, respectively. In EITB, in the sheep experimentally infected with F. gigantica, the band of 32.5kDa molecular weight polypeptide appeared at 2 weeks PI and continued till the end of experiment. Also, the cercarial antigen recognized 32.5kDa molecular weight band with all sera from naturally infected sheep with fasciolosis (n = 25). This band did not cross-react when tested with sera from infected sheep with Cysticercus tenuicollus (n = 20). This study suggests that, the 32.5kDa molecular weight polypeptide could be used as sensitive and specific epitope for the serodiagnosis of sheep fasciolosis.