Molecular characterization of Rhipicephalus [Boophilus] annulatus from Iran by sequences of cytochrome c oxidase sub-unit I [COI] and the second internal transcribed spacer [ITS2

Traditionally, morphological features of Rhipicephalus [Boophilus] annulatus from closely-related ticks have been considered for their identification and differentiation. However, it is difficult and requires expertise in order to accurately identify and differentiate engorged female ticks and some developmental stages such as larva and nymph from other similar ticks. Hence, molecular markers may be a suitable alternative. Mitochondrial cytochrome c oxidase subunit I [COI] gene and the second internal transcribed spacer [ITS2] fragments of Rh. [Bo.] annulatus were sequenced to assess the use of molecular techniques for identifications and phylogenetic studies of these ticks. Polymerase chain reaction [PCR] technique was performed based on the analyses of COI and ITS2 sequences of ticks collected from two different regions in Iran [Golestan and Mazandaran]. The length of COI and ITS2 sequences were 1539 and 1158bp, respectively. The nucleotide similarity of COI gene was 91.3% between the ticks examined from the two different regions. The deduced amino acid sequences from COI showed 98.6% similarity between the ticks studied and showed 98.2 and 99.6% similarity with the only complete sequence of Rh. [Bo.] annulatus [AGH19677] registered in GenBank. The obtained complete nucleotide sequences of ITS2 from Rh. [Bo.] annulatus from Golestan and Mazandaran revealed 99.9% similarity, while the other ticks registered in GenBank 95 to 99% similarity [KC503267, AF271270, AF271272, JQ412126]. It seems that COI and ITS2 sequences could provide suitable genetic markers for discrimination and genetic characterization of Rhipicephalus [Boophilus] annulatus

[ study of oocyst shedding pattern and weight changes in neonatal calves experimentally infected with Cryptosporidium parvum]

Background: Cryptosporidium parvum is a protozoan parasite which belongs to apicomplexa phylum. The parasite infects both wild and domesticated animals and human beings as wellOBJECTIVES: The purpose of the present study was to detect oocyst shedding and diarrhea pattern in experimental cryptosporidiosis and their correlation with weight loss in neonatal calves

Methods: Twelve Holstein calves of both sexes were obtained at birth from dairy farm and randomly divided into two groups of 6 calves. Six calves were orally infected with 10[7] C.Parvum oocysts at the 12h post parturition. The control group was not infected. Clinical signs were examined and fecal samples were collected by the rectal examination twice a day. All calves were weighed from day 0 to day 30 with 3 days intervals to determine effects of cryptosporidiosis on weight gain

Results: All infected calves were noticeably depressed and had a decreased appetite from 3 days post inoculation [DPI] while they received colostrum. Subsequently, watery diarrhea with clumps of mucus and yellow or pale changes of feces color were observed. The infected calves have had diarrhea for 5-8 days that remarkably had got dehydrated. The most severity of diarrhea was 4-6 DPI. Oocyst excretion started 4 DPI, peaked at 6 DPI [60.48×10[6] +/- 9.03oocysts/g feces] and continued until 11 DPI. Control calves had no diarrhea and other clinical signs during the whole period of the trial. The mean weight gain of control group was significantly higher than inoculated group during experiment [p<0.001]. The Weight of the infected calves was retarded until 9 days old and then risen subsequently

Conclusions: Present study showed the role of C.Parvum as the primary cause of diarrhea and weight loss among neonatal calves

[Implementation and designing of line-detection system based on electroencephalography [EEG]]

In Brain-based lie detection systems which have been recently introduced as substitutes or classic lie detection systems, the procedure for recognition of guilty and innocent subjects is done by inspection of brain signals which are acquired during the specific Polygraph test. With the aim of increasing the performance, this paper presents a powerful method for detection of Guilty persons in lie detection systems using brain signals. It was an experimental study. The employed method is based on the extraction of P300 components from brain signals. In this way, the test protocol was designed based on Odd-ball method, firstly. This test was done on 16 people and their brain signals were acquired. After preprocessing, p300 amplitude was extracted for each person from brain signals, and finally Guilty and Innocent persons were classified by comparing amplitude through Bootstrapped Amplitude Difference [BAD] method. The obtained results show that the proposed method has detected correctly 7 out of 8 guilty persons and 8 out of 8 innocent persons. Also, the validated results show the promise of the proposed approach in discrimination of guilty subjects from innocent subjects by the accuracy of 93.75%. Knowing the existence of precious information in brain signals and their relation with brain's cognitive activities and also considering the performance of the proposed method, there are enough reasons use the proposed approach for detection of guilty persons from innocent ones. Further, in comparison with previous methods, the impact of man ability to control brain signal parameters and creating incorrect feelings been reduced through the proposed method

mRNA expression study on small amount of Theileria annulata lymph node biopsy sample using SMART-cDNA technology

A major issue in many gene expression studies utilizing small amount of biological materials is the limited quantity of RNApurified from clinical samples, which is often used for RT-PCR or standard Northern blot analysis. The SMART cDNA synthesis method and subsequent SMART-cDNA-PCR technique was used to analyse 3 genes in macroschizonts of Theileria annulata in small lymph node biopsy material. The SMART-cDNA of TaSp gene was cloned in pTZ57R/T-vector and sequenced. We focused on genes encoding surface proteins TaSp, TaD and HSP70. Our results showed that SMART cDNA dependably reproduces the expression profile found in messenger RNA. The RT-SMART-PCR showed the amplification of the processed mRNAs. The sequencing analysis showed that the amplified cDNA was coded for TaSp protein in Theileria annulata. It was concluded that the SMART PCR technique is practical for amplification of complete sequence of mRNAs in the form of cDNAs, and therefore for gene expression studies if only small amounts of starting material are available

Identification of Gyrodactylus gurleyi in Carassius auratus using morphometric and molecular characterization

Gyrodactylus is a small monogenean ectoparasite that lives on the skin and fins of most of the world's fish species. Gyrodactylus appears to be one of the most prevalent parasites found in ornamental fish, especially in Cyprinids. Goldfish [Carassius auratus] are a popular ornamental fish that are highly contaminated by Gyrodcatylus. The present study is aimed to identify morphological and molecular characteristics of the Gyrodactylus parasite on gold fish. The morphological identification of Gyrodactylus specimens was performed using the measurements and drawings of opisthaptoral hard parts of the parasites. The molecular species description was based on a polymerase chain reaction [PCR] of partial sequence of the 5.8S region of ribosomal RNA, and a partial sequence of the internal transcribed spacer 2 [ITS2] of ribosomal RNA. The nucleotide sequences of the PCR products were compared with corresponding sequencing registered in GenBank. Based on the morphometric analysis and sequencing, the Gyrodactylus specimens were described as Gyrodactylus gurleyi. A combination of molecular techniques with morphological analysis seems to be the best approach for the identification of Gyrodactylus speices

Cloning, and molecular characterization of polymorphic Iranian isolate theileria annulata surface protein [TaSp]

Because of the strong immunologic responses of surface protein TaSp in Theileria annulata infected host, we tried to characterize this protein in a T. annulata isolate from Iran. The RNA prepared from T. annulata infected cells was used to produce SMART-DS-cDNA. The Double strand cDNA was then amplified with primers derived from TaSp mRNA sequences. The PCR product was cloned in pTZ57R/T vector, sequenced and registered under accession no. JQ003240 in GenBank. The sequence analysis showed 90%-94% nucleotide sequence identity and 68%-94% amino acid homology to the corresponding sequences of TaSp gene by T. annulata, T. sp. china I, T. sp. china and T. lestoquardi and three T. annulata reported from Iran respectively. Interestingly, the sequence analysis also showed small nucleotide sequence region near the 5` end in which the presented TaSp protein differed very strongly from the other known TaSp sequences. For the preparation of the recombinant protein, the cDNA was cloned in pQE-32 vector, the recombinant protein was prepared and assayed by Theileria infected bovine serum. The polymorphism in TaSp gene could be detected in intra- as well as inter species. The different characterized TaSp proteins had a common identic region, which may be helpful for development of broad band vaccine based on the recombinant proteins. The polymorphism in this gene, make this protein also interesting for the diagnostic purposes

Molecular study of sheep malignant theileriosis at Barka region in the Sultanate of Oman

We used the PCR technique based on the abovementioned primer pair and sequencing to demonstrate the Theileria infection in the sheep samples collected from Sultanate of Oman. According to the frame work of "integrated control of ticks and tick borne diseases in globalized world managed by EU-ICTTD-3 project, the samples from blood, liver, spleen, lymph node and lung were sent to the laboratory of Iranian Research Center for Ticks and Tick-borne Diseases [IRCTTD]. Samples from blood smear and impression smears from liver, spleen, lymph node, and lung were analyzed by Geimsa staining. The DNA was extracted from the abovementioned samples and analyzed by PCR technique using specific primers derived from the nucleotide sequences of 18S rRNA gene of T. lestoquardi, which can amplify the common region in other Theileria and Babesia spp. Subsequently the amplified DNA was sequenced. The analysis of blood smears of the sheep was negative for piroplasmosis performed through the Giemsa staining. The impression smears prepared from liver, spleen, lymph node, and lung showed suspicious structures mimicking Theileria schizonts in some cells. The results showed an expected PCR product of 428 bp in length, which is specific for Theileria spp. The PCR products were subsequently sequenced. The corresponding nucleotide sequence is registered under accession number JF309152 in GenBank. The sequence alignment in Gen Bank showed that the PCR products had 99% homology to the known T. lestoquardi registered under accession number AF081135 in the GenBank. Oman sheep are highly susceptible for Theileria infection and the infected sheep mostly die before the microschizonts or erythrocytic form of Theileria appears in the nucleated or erytrocytic cells respectively

Detection of single dactylogyrus spp. in DMA extracted from infected gill tissue of fishes using polymerase chain reaction

Dactylogyrus spp. are monogenean worms found mostly as ectoparasites on the gills of several fish species, including carp and goldfish. These parasites are commonly detected by microscopic analysis of the gill lamellae, but this is time-consuming and technically difficult. In contrast to this conventional method, molecular techniques provide specific, sensitive and safe detection of parasites. In the present study, polymerase chain reaction [PCR] and subsequent DNA sequencing were used to detect Dactylogyrus spp. Specific common primers were designed to amplify the ITS-1 region of the rRNA gene of Dactylogyrus spp. Dactylogyrus worms were collected from 100 goldfishes and identified using a dissection microscope. Then, single worms were used for DNA extraction. To evaluate the PCR, a single parasite was added to a parasite-free gill, which then had its DNA extracted. Subsequently, the PCR products were purified and sequenced. Comparison of the nucleotide sequences of the PCR products with GenBank sequences showed that there was 100% homology with sequences from two Dactylogyrus spp., namely Dactylogyrus vastator and Dactylogyrus dulkeiti [registered under accession numbers AJ 564159 and AJ 564126, respectively]. The results obtained from sequence analyses were consistent with species identification by microscopy. Therefore, the results show that it is possible to develop a sensitive and precise PCR method for the detection of Dactylogyrus-mfected fish using DNA extracted from the whole gill

Recombinant Cryptosporidium parvum p23 as a candidate vaccine for cryptosporidiosis

Cryptosporidium parvum is a ubiquitous protozoan, which develops within the microvillous membrane of enterocytes in a wide variety of vertebrates, including man. Cryptosporidiosis is an important parasite causing severe diseases in the immunodeficient people especially AIDS patients. Cryptosporidiosis has been also reported as a com-mon serious primary cause of outbreaks of diarrhea in newborn calves. The aim of this study was to confirm that P23 was an immunogenic antigen in domestic isolates of C. parvum. We isolated cryptosporidial oocysts from the naturally infected calves. The oocysts were then purified and characterized as C. parvum by nested PCR. To obtain the recombinant P23 protein, we isolated the mRNA from oocyst of C. parvum, and synthesized the cDNA. The cDNA was then amplified using specific primers for P23 gene. Sequencing of PCR product showed 100% homology to the known P23 sequences in GenBank. The double strand P23-cDNA was then cloned in pGEX-5X-2 expression vector and P23-recombinant protein was prepared. West-ern blot analysis of recombinant P23 showed that it could be recognized by the positive C. parvum serum. Furthermore, serum from immunized goat with the recombinant P23 protein also recognized a protein band with approximately 23 kDa in lysates prepared from the oocytes. Since P23 is an immunodominant surface glycoprotein expressed in the early phase of infection and the immunogenic epitopes are found in its residual chain of amino acid sequence, the recombinant P23 could be recom-mended as a favorable candidate for vaccination against C. parvum infection

Specific egg yolk antibody against recombinant cryptosporidium parvum P23 protein

Cryptosporidium parvum is a parasitic protozoan that functions as important causative agent of diarrhea in human and animals. The host's immune response to surface antigens of C. parvum has been previously demonstrated. In this respect, the role of humoral immunity in the development of host protective immunity against this protozoon has been well demonstrated. The effect of specific chicken egg yolk antibody [IgY] against recombinant C. parvum P23 was examined. IgY sample was prepared from eggs of chickens immunized with recombinant C. parvum protein p23 and analyzed with C. parvum lysate and recombinant P23. The anti P23 specific IgY was recognized a protein band with approximately 23 kDa in lysates prepared from the C. parvum oocysts. Also dot blot analysis of recombinant P23 showed that it could be recognized by the anti P23 specific IgY up to 1/1000 dilution of antibody. But the best antibody dilution for immunological studies was determined as 1:200. Since P23 is an immunodominant surface glycoprotein expressed in the early phase of infection, specific IgY against recombinant p23 could be recommended as a favorable candidate for passive immunization against C. parvum infection in human and animals

[Identification of cryptosporidium parvumby PCR and its determination of protein pattern and immunogenic antigens]

Cryptosporidium parvum is an apicomplexan protozoan parasite which causes diarrhea in both human and wide range of animals. Since this protozoa causes remarkable economic losses in cattke industry in Iran, the molecular determination of porotozoa and characterization of its protein pattern and immunogenic antigend are the aim of this study. In this study, diarheatic fecal samples of calves suspected for cryptosporidiosis were collected and identified. Purification and concentration of cryptosporidial oocysts from fecal samples was performed. Oocysts were confirmed as Cryptosporidium parvum by semi-nested PCR using specific primers designed from 18srRNA gene of Cryptosporidium parvum. Acalf with negative antibodies against Cryptosporidium parvunm was infected with 5 x10[6] oocysts. 5 days after inoculation, oocysts were isolated and purified. Soluble proteins from sporozoites were prepared and analyzed by SDS-PAGE and western blotting. There was an intense recognition of some10 to 100 kDa, ten low molecular weight proteins were recognized between 20-40 kDa, six separated protein bands was recognized between 40-70 kDa, immunoreactive proteins were present at different molecular weights between 17-260 kDa. Three antigens of apparent molecular weights 20-30 kDa, three antigen bands between 40-60 kDa and 2 bands 70-75 kDa were identified. Antibody responses to cryptosporidial antigens at high molecular weights were successfully diagnosed with apparent molecular weights 130, 170, 216 and 257kDa