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

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

[Molecular study on Ehrlichia canis in thrombocytopenic dogs]

Infection with Ehrlichia canis, a gram negative obligatory intracellular bacterium, causes canine monocytic ehrlichiosis which is the worldwide disease in dogs. The objective of this study was to investigate the prevalence of E. canis in thrombocytopenic dogs using nested PCR and diagnostic role of thrombocytopenia in the infection. Blood samples collected from 40 dogs attended in Teaching small animal hospital of Tehran University were classified as group A [platelet counts below 101.000/microL, thrombocytopenic, n=11], B [101.000-200.000/microL, thrombocytopenic, n=15] and C [platelet counts more than 201.000/microL, non-thrombocytopenic, n=14] according to their platelet counts. 16S rRNA was analyzed by nested PCR using specific primers. 16S rRNA gene fragment of E. canis were detected in five samples of group A [45.5%], three samples of group B [20%], and one sample of group C [7.1%]. Prevalence rate of infection was statistically higher in group A than the other groups [p=0.02]. In total, approximately one third of thrombocytopenic dogs had demonstrable E. canis infection [30.7%]. While thrombocytopenia cannot be considered as specific marker for detection of E. canis infection, it can be used as a surveillance test prior to other diagnostic methods

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

Evaluation of benzimidazole resistance in Haemonchus contortus using comparative PCR-RFLP methods

In order to deworm the ruminants especially of sheep in Iran, consumption of benzimidazoles has more than 2 decades history and today farmers are using imidazothiazoles, macrocyclic lactones and mostly benzimidazole compounds [BZs] to treat infected farm animals. It has been demonstrated that the most common molecular mechanism leading to BZ sresistance in Haemonchus contortus is a single mutation at amino acid 200 [phenylalanine to tyrosine] of the isotype 1 of beta tubulin gene. According to the report of such mutations in Iranian Teladorsagia circumcincta isolates with Restriction Site Created PCR-RFLP, we decided to evaluate the frequency of such mutations in H. contortus in three different geographical areas of Iran. A total of 102 collected adult male H. contortus were evaluated with PCR-RFLP [using PSP1406I as restriction enzyme]. By means of a second step to compare function of different methods and to increase sensitivity of detection mechanism, a third of samples were examined by another PCR-RFLP method [using TaaI as restriction enzyme] and finally beta tubulin gene of two samples was sequenced. All of samples were detected as BZss homozygote. Finally, beta tubulin gene sequencing of two samples showed no point mutation at codon 200. It seems that BZ resistance of H. contortus in Iran is not a serious problem as anticipated before

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

Nucleotide sequence analysis of the second internal transcribed spacer [ITS2] in hyalomma anatolicum anatolicum in Iran

Ticks are important acarina that infest animals. They are obligatory blood sucker arthropods which economically impact cattle industry by reducing weight gain and production. Moreover, they are important vectors of viral, bacterial, rickettsial and parasitic pathogens infecting humans and animals. In view of the importance of Hyalomma anatolicum anatolicum in pathogen transmission, including Theileria lestoquardi in Iran, the accurate identification of this tick is critical. Although many keys are available as aids, morphological identification of tick species such as Hyalomma anatolicum anatolicum [Koch, 1844; Hoogstral and Kaiser, 1959] is difficult and expert knowledge is required. False morphological identification at the level of species and subspecies is common, particularly for Hyalomma excavatum complex members which are prevalent in Iran. For example, the high similarity between Hyalomma anatolicum anatolicum and Hyalomma anatolicum excavatum is the cause of confusion in the identification of these species. In this study, polymerase chain reaction [PCR] techniques were used for identification of Hyalomma anatolicum anatolicum based on analysis of the gene sequence of the Second Internal Transcribed Spacer [ITS2] of this tick. The ITS2 nucleotide sequence of Hyalomma anatolicum anatolicum was 963 base pairs [bp] in length and exhibited 93% homo logy with other GenBank registered ITS2 sequences of this subspecies [accession no: FJ593700.1]. The complete ITS2 region sequence was identified in this study and registered in GenBank under accession number HQ123320

Identification and purification of a specific and immunogenic antigen of the laminated layer of the hydatid cyst and production of an antigen-specific monoclonal antibody

Cystic echinococcosis [CE] is an infection caused by the larval stage of Echinococcus granulosus. This is widely distributed through Iran, where a variety of animals act as intermediate host. The immunogenic antigens [Ag] of different compartments of the hydatid cyst have been already determined. One of these compartments is the laminated layer [LL]. We have extracted a protein with the MW of 24 kDa from a lysate prepared from the LL and produced a monoclonal antibody [mAb] against this protein. Five mAb named P[3]F[6s], P[2]Hp[4s], P[1], A[6s], P[1],C[3s], and P[1],F[7s] have been produced. The isotype analysis showed that P[3]F[6s] is IgG[1], and the rest are IgM. P[3]F[6s] was purified from the ascitic fluid of mice injected with P[3]F[6s] hybridoma intraperitoneally. Western blot and LLISA analysis showed that this mAb could recognize the purified 24 kDa prepared from lysate of LL. Since a 24 kDa protein has been shown to be an immunogenic Ag, this protein can be used as a candidate for the development of diagnostic tests and vaccine strategies. For these aims, P[3]F[6s] can be used for the purification of this 24 kDa protein

Serological study of Neospora caninum in pregnant dairy cattle in Tehran, Iran

Seven hundred and sixty-eight blood samples of pregnant cattle from four Holstein dairy herds that are farmed in the vicinity of Tehran were used to evaluate the seroprevalence of Neospora caninum infection by enzyme-linked immunosorbent assay [ELISA]. Two hundred and ninety-eight of the 768 blood samples [38.8%] were positive for this infection. The prevalence of infection in the herds varied from 18.7% to 65.1%. The abortion rate in seropositive and seronegative animals was 20.67% and 10.11%, respectively. Thus, the risk of abortion was approximately double the rate in seropositive cows [p<0.0005]. There was a high correlation between the infection rate and the age in one herd. In other three herds, no significant correlation was found between infection rate and age. This is the first extended study with regards to the rate of Neospora caninum infection in pregnant cattle in the vicinity of Tehran

[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

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

Molecular and pathological study of bovine aborted fetuses and placenta from Neospora caninum infected dairy cattle

The objective of the study was to evaluate the presence of Neospora caninum organisms in the brain of aborted fetuses and placentas of full-term calves born of seropositive cows. During 2006-2007, 12 brains of aborted calves from Neospora seropositive cattle and 7 placentas from seropositive dams giving birth to full-term calves, from four dairy cattle farms located around Tehran province, Iran were examined by Nested-PCR and histopathology techniques. The Nested-PCR demonstrated that all of 12 aborted fetal brain samples and 5 of 7 placentas were infected by N. caninum. Mild to severe placentitis was observed in 5 placentas. Severe hyperemia and perivascular and perineuronal edema revealed in all fetal brain. In 3 out of 12 brains, scattered foci of hemorrhages, neuropilar necrosis and gliosis were present. In addition, nonpurulent encephalitis with severe lymphohistiocytic perivascular cuffing in one case and a small tissue cyst like Neospora caninum cyst in other calf were observed. Our results confirmed the molecular and histopathologic findings of other studies about Neospora caninum infection and it seems to support the hypothesis that Neospora infection is associated with bovine abortion in Iran

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

[ study of rhipicephalus species in Iran]

This study was conducted during 3 years period [2002-2004]. Tick sampling was carried out randomly from domestic animals during seasonal activity of ticks from different parts of Iran. 2170 ticks from 151 cattle, 629 sheep, 336 goats and 33 camels were collected. The occurance of tick infestation in cattle, sheep, goats and camels was 60%, 71.4%, 53% and 46% respectively. Sampled ticks of Rhipicephalus species have been identified as; Rhipiephalus sanguineus [37.9%] Rhipiephalus bursa [49.8%] and Rhipicephalus turanicus [12.23%]. All three species adopted in ecological zone II but Rhipicephalus sanguineus was the main tick species found in four zones of Iran. The comprative tick yield obtained from animals showed that Rhipicephalus bursa was the most aboundant in zone II but Rhipicephalus sanguineus was the rarest species in zone IV. The results described here suggest that livestock had almost different pattern of tick species in any localities of Iran

Identification of tick species of dermacentor in some localities of Iran

This study conducted during 3 years period [2002-2004]. Tick sampling was randomly carried out from domestic animals during seasonal activity of ticks from six provinces of Iran. 2170 ticks from 151 cattle, 629 sheep, 336 goats and 32 camels were collected proin 30 prosseces with a mean number of 1.9 tick per animal. 209 Dermacentor ticks collected from six provinces [Kordestan, Ardebil, East Azarbaiejan, Zanjan, Khorasan and Semnan] that included 23% of collected tick population in those provinces. The diversity of Dermacentor is restricted to three species; D.niveus [50%], D.marginatus [27%] and D.raskemensis [23%]. The maximum occurance of D.raskemensis, D.niveus, D. marginatus, were occurred in provinces of Semnan, Khorasan and Kordestan, respectively. It can be concluded that livestock had almost different pattern of tick species in any localities thus distribution of D.raskemensis ,D.niveus, D. marginatus, has been confirmed this matter

[Detection of leptin and leptin receptor mRNA expression in the bovine testis]

Leptin, hormonal product of the ob gene, is known to regulate food intake, energy metabolism and reproductive functions in mammals. The mechanism by which leptin affect male reproductive system, in contrast to its well proven effects in female fertility, has been a matter of debate. Expression of leptin and its receptor in some reproductive organs suggest that leptin has both endocrine and paracrine/autocrine effects on reproduction. Various evidences have pointed to a direct role of leptin in the control of rodent testicular function such as steroidogenesis and spermatogenesis. So, detection of leptin and leptin receptor mRNA in bovine testis will be the first crucial step to an understanding of its paracrine/autocrine effect on testes in cattle. In the present study, we showed the expression of leptin mRNA as well as its functional receptor [Ob-Rb] mRNA in whole testis of Holstein cattle using reverse transcription and polymerase chain reaction [RT-PCR] analysis. To confirm the first results, RT-PCR products were amplified with Nested PCR using inner leptin primer pairs designed on different exons. Based on our results, although we could not determine the exact cell source of leptin in / testis, it suggests that besides its primary actions at the hypothalamic-pituitary level, leptin can also involved in autocrine and/or paracrine mechanisms in testicular physiology in cattle

Isolation of DNA from a single helminth using new developed kit in Iran and its PCR analysis

Nematodes are among the most common and important parasites of man and animal. DNA of a single worm can be used for several purposes, such as identification to the species, subspecies, strain and antihelmenthic resistance. DNA extraction from a single small worm using traditional methods such as phenol extraction technique faces serious problems. DNA from 20 single Haemonchus contortus was isolated using DNA isolation kit newly designed in Iran by the Research Unit of Molecular Biological System Transfer [MBST] based on the specific binding of DNA to the carrier. The genomic DNA was amplified using specific primers derived from beta-tubulin isotype 1 in PCR. The specificity of the PCR products was determined using semi-nested PCR technique. Specific PCR-product from beta-tubulin gene could be amplified with 1 ng, 100 pg and 10 pg DNA. The used DNA extraction method was safe, with high quality and quantity, fast, easy to handle and not costly for genetic analysis of even a single small worm. The Iran produced DNA extraction Kit is grounded on a selective binding of nucleic acids to a silica-based membrane and is recommended for the isolation of DNA from even small amount of biological materials