Association between proliferation status of infected and non-infected mononuclear cells with tissue lesions in acute bovine theileriosis.

Tropical or Mediterranean theileriosis in dairy cattle is widely distributed in many tropical regions of the world. The purpose of this study was to evaluate the proliferation status of mononuclear cells infected with Theileria annulata schizonts in different tissues and its relationship with the pathogenesis of the parasite in cattle by histopathology, immuno-histochemistry and polymerase chain reaction (PCR). Blood and tissue samples of eight Holstein cattle that had been lost due to theileriosis and eight healthy slaughtered cattle of the same breed were collected as a control group after necropsy. The piroplasms in the blood smears and the schizonts in the cytoplasm of the lymphocytes and macrophages of the lymph nodes were microscopically detected. Histopathologically, the proliferation of macrophages, lymphocytes, and plasma cells in lymph nodes and the heart, congestion, and bleeding in the red pulp of the spleen, portal tracts of the liver, interstitial tissue of the kidneys, multifocal necrosis and ulceration in the abomasum together with hyperemia and hemorrhages and lymphoblastic infiltration in the submucosa and lamina propria adjacent to these lesions and emphysema with ecchymotic hemorrhage in the lungs were evident. Immunohistochemistry identified the proliferated cells as mostly Cluster of Differentiation 3- Positive T lymphocytes and macrophage marker antibody 387- positive macrophages. Positive results of PCR for the Tams1 30.00 kDa gene were observed in lymph nodes, liver, lung and abomasum. It was concluded that the pathological changes were the result of schizont-infected macrophage proliferation leading to severe uncontrolled proliferation of uninfected T lymphocytes.

The Protective Effect of Artemisia spicigera Ethanolic Extract against Cryptosporidium parvum Infection in Immunosuppressed Mice.

BACKGROUND: A specific treatment has not yet developed for cryptosporidiosis, and some of the used drugs had side effects in immunodeficient patients. The goal of an appropriate remedy is to remove symptoms and improve immune responses in hosts. The current study was designed to evaluate the therapeutic efficacy of Artemisia spicigera ethanolic extract in experimentally infected immunosuppressed mice. METHODS: Thirty six NMRI mice, 4-6 wk old, were randomly divided into six equal groups. C1: uninfected, treated control; C2: infected, untreated control; T1, T2, T3, and P: infected, treated with 0.2, 2, and 20 mg/ml extract, and 5mg/ml paromomycin, respectively. Mice were experimentally infected by oral administration of 104 oocysts/animal of Cryptosporidium parvum and treated orally for eight days per 12h, starting 12h before experimental infection. The presence of oocyst shedding, weight gain/loss, and the histopathology of ileum sections were examined. RESULTS: Results revealed that oocyst shedding was significantly (P<0.05) reduced in treatment groups. There was no significant difference between the mean of weight gain/loss in the infected control and treated groups. Histopathological analysis of ileum sections further supported the parasitological findings. CONCLUSION: Artemisia spicigera had acceptable efficacy as a therapeutic agent for cryptosporidiosis.

Vaccination with rEGVac elicits immunoprotection against different stages of Echinococcus granulosus life cycle: A pilot study.

Vaccination against dog-sheep transmission cycle is necessary to control cystic echinococcosis (CE) infection. A multi-epitope multi-antigenic recombinant vaccine was developed-comprising the three putative vaccine antigens EG95, Eg14-3-3 and EgEnolase-was cloned and expressed. In a pilot experiment, the multi-antigen vaccine was assessed in 15 dogs and 15 sheep (five experimental groups and three animals in each group) by two subcutaneous doses 28 days apart. To evaluate the efficacy of the vaccine candidate first immunological analysis were done comprising IgG and IgE antibodies and the cytokine IL-4 in sera of the immunized dogs and sheep. Serum IgG, IgE, and IL-4, in particular in the dogs, were increased after the two rounds of vaccine candidate injection, while the total number of hydatid cysts was reduced (~85.43%). This pilot trial indicated significant immune protection efficacy against E. granulosus especially in dogs, while its efficacy in sheep was not as high as dogs. The multi-antigenic candidate vaccine is proposed as a protective vaccine modality in both dogs and sheep.

A multi-method and structure-based in silico vaccine designing against Echinococcus granulosus through investigating enolase protein.

Introduction: Hydatid disease is a ubiquitous parasitic zoonotic disease, which causes different medical, economic and serious public health problems in some parts of the world. The causal organism is a multi-stage parasite named Echinococcus granulosus whose life cycle is dependent on two types of mammalian hosts viz definitive and intermediate hosts. Methods: In this study, enolase, as a key functional enzyme in the metabolism of E. granulosus (EgEnolase), was targeted through a comprehensive in silico modeling analysis and designing a host-specific multi-epitope vaccine. Three-dimensional (3D) structure of enolase was modeled using MODELLER v9.18 software. The B-cell epitopes (BEs) were predicted based on the multi-method approach and via some authentic online predictors. ClusPro v2.0 server was used for docking-based T-helper epitope prediction. The 3D structure of the vaccine was modeled using the RaptorX server. The designed vaccine was evaluated for its immunogenicity, physicochemical properties, and allergenicity. The codon optimization of the vaccine sequence was performed based on the codon usage table of E. coli K12. Finally, the energy minimization and molecular docking were implemented for simulating the vaccine binding affinity to the TLR-2 and TLR-4 and the complex stability. Results: The designed multi-epitope vaccine was found to induce anti-EgEnolase immunity which may have the potential to prevent the survival and proliferation of E. granulosus into the definitive host. Conclusion: Based on the results, this step-by-step immunoinformatics approach could be considered as a rational platform for designing vaccines against such multi-stage parasites. Furthermore, it is proposed that this multi-epitope vaccine is served as a promising preventive anti-echinococcosis agent.

Comparative survey on scolicidal effects of selenium and silver nanoparticles on protoscolices of hydatid cyst.

Hydatidosis is a zoonotic diseases which affects human and livestock animals. The surgical treatment of hydatid diseases in man is still the most effective approach, but spillage of protoscolices is the risk of surgery. Therefore, it is essential to develop alternative approaches. Injection of protoscolicidal agents into the cysts preoperatively is an alternative method.The aim of the present study was the determination and comparison of lethal effects of silver nanoparticles (Ag-NPs) and selenium nanoparticles (Se-NPs) on protoscolices of hydatid cyst. Livers and lungs of sheep, which was naturally infected by hydatid cyst were collected from Tabriz slaughterhouse and transferred to the parasitology laboratory. Initially, the surface of cysts was disinfected by 70% Alcohol. Then, 25 ml of each cyst fluid were aspirated by sterile syringe and transferred to Erlenmeyer flask. After 30 minutes, the supernatant was discarded and protoscolices were washed with phosphate-buffered saline and tested by 1% eosin to assess their viability. Next, the samples of protoscolices, which were over 90%, were selected and the effect of 50, 125, 250 and 500 (µg/ml) of Se-NPs and Ag-NPs in the exposure times of 10, 20, 30 and 60 min were evaluated. Data were analysed by SAS software and analyzed by Duncan's comparison test. The significant differences were considered to be p<0.0001.The difference between the scolicidal effects of Se-NPs was statistically significant for all examined concentrations and exposure times compared to the control group (p<0.0001). On the other hand, the scolicidal effect of Ag-NPs in 10 and 20 min exposer was not significant in comparison to the control group. Overall scolicidal effect of Se-NPs was higher than Ag-NPs and the difference was significant (p<0.0001).The results of the present research indicated that it is possible to use Se-NPs as an effective scolicidal treatment. It is suggested the study of employing this compound in vivo or in combination with other procedures in treating hydatid cyst in further studies.

A novel B- and helper T-cell epitopes-based prophylactic vaccine against Echinococcus granulosus.

Introduction: In this study, we targeted the worm stage of Echinococcus granulosus to design a novel multi-epitope B- and helper T-cell based vaccine construct for immunization of dogs against this multi-host parasite. Methods: The vaccine was designed based on the local Eg14-3-3 antigen (Ag). DNA samples were extracted from the protoscoleces of the infected sheep's liver, and then subjected to the polymerase chain reaction (PCR) with 14-3-3 specific forward and reverse primers. For the vaccine designing, several in silico steps were undertaken. Three-dimensional (3D) structure of the local Eg14-3-3 Ag was modeled by EasyModeller software. The protein modeling accuracy was then analyzed via various validation assays. Potential transmembrane helix, signal peptide, post-translational modifications and allergenicity of Eg14-3-3 were evaluated as the preliminary measures of B-cell epitopes (BEs ) prediction. Having used many web-servers, a well-designed process was carried out for improved prediction of BEs. High ranked linear and conformational BEs were utilized for engineering the final vaccine construct. Possible T-helper epitopes (TEs) were identified by the molecular docking between 13-mer fragments of the Eg14-3-3 Ag and two high frequent dog class II MHC alleles (i.e., DLA-DRB1*01101 and DRB1*01501). The epitopes coverage was evaluated by Shannon's variability plot. Results: The final designed construct was analyzed based on different physicochemical properties, which was then codon optimized for high-level expression in Escherichia coli k12. This minigene construct is the first dog-specific epitopic vaccine construct that is established based on TEs with high-binding affinity to canine MHC alleles. Conclusion: This in silico study is the first part of a multi-antigenic vaccine designing work that represents as a novel dog-specific vaccine against E. granulosus. Here, we present key data on the step-by-step methodologies used for designing this de novo vaccine, which is under comprehensive in vivo investigations.

A novel in silico minigene vaccine based on CD4+ T-helper and B-cell epitopes of EG95 isolates for vaccination against cystic echinococcosis.

EG95 oncospheral antigen plays a crucial role in Echinococcus granulosus pathogenicity. Considering the diversity of antigen among different EG95 isolates, it seems to be an ideal antigen for designing a universal multivalent minigene vaccine, so-called multi-epitope vaccine. This is the first in silico study to design a construct for the development of global EG95-based hydatid vaccine against E. granulosus in intermediate hosts. After antigen sequence selection, the three-dimensional structure of EG95 was modeled and multilaterally validated. The preliminary parameters for B-cell epitope prediction were implemented such as the possible transmembrane helix, signal peptide, post-translational modifications and allergenicity. The high ranked linear and conformational B-cell epitopes derived from several online web-servers (e.g., ElliPro, BepiPred v1.0, BcePred, ABCpred, SVMTrip, IEDB algorithms, SEPPA v2.0 and Discotope v2.0) were utilized for multiple sequence alignment and then for engineering the vaccine construct. T-helper based epitopes were predicted by molecular docking between the high frequent ovar class II allele (Ovar-DRB1*1202) and hexadecamer fragments of the EG95 protein. Having used the immune-informatics tools, we formulated the first EG95-based minigene vaccine based on T-helper epitope with high-binding affinity to the ovar MHC allele. This designed construct was analyzed for different physicochemical properties. It was also codon-optimized for high-level expression in Escherichia coli k12. Taken all, we propose the present in silico vaccine constructs as a promising platform for the generation of broadly protective vaccines for species and genus-specific immunization of the natural hosts of the parasite.

Survey of hydatidosis infection in slaughtered camel (Camelus dromedarius) in Tabriz area, Northwest Iran.

Hydatid Disease is the name given to the condition caused by the zoonotic tapeworm Echinococcus granulosus. The tapeworm spends most of its adult life in the intestine of its definitive host, namely canids and in particular the dog. The tapeworm eggs become voided in the canids' faeces and as a result of ingesting the eggs, infection passes to the intermediate host, commonly herbivores while grazing. However, humans can become accidentally infected and hydatid cysts may develop throughout the body. During April 2010-February 2014, a total 198 camels, which had been sent to the abattoir, the daily number of hydatid infected livers and lungs of camels slaughtered at Tabriz abattoir were recorded. To be sure about the validity of recorded data, observed data were collected daily. Approximately 29 (14.64 %) of camels were infected according to this survey. Age wise, the prevalence of infection in young animals (under the age of 5 years) was 4 (2.02 %), whereas in animals between 5 and 10 years and over, the prevalence of infection was 11 (5.55 %) and 14 (7.07 %) respectively. Sex wise, female animals had a higher prevalence with 17 (19.76 %) cases in camels, whereas in the males, there were 12 (10.71 %) cases in camels. There was a notable difference found in our study between male and female animals (P < 0.05). Infections were recorded in two visceral organs with the Lungs having the highest prevalence in camels, and the liver had low infected in camels. The results of this study suggest that infection of camels with hydatid cyst is common in Tabriz, Iran and that this may constitute economic and health problems in the meat industry.

The Efficacy of Ultraviolet Irradiation on Trichophyton Species Isolated From Nails.

BACKGROUND: The treatment of onychomycosis is a challenge and infections are typically more severe and difficult to treat in toenails than in fingernails. OBJECTIVES: The current study aimed to investigate the fungicidal effect of ultraviolet radiation on the growth of dermatophytes isolated from nails. PATIENTS AND METHODS: Samples from patients with clinical manifestations of onychomycosis were inoculated onto Sabouraud dextrose agar and incubated at 30°C for 14 days. Isolated species were identified by specific laboratory examinations; UV-A, UV-B, and UV-C light were used to irradiate two strains of Trichophyton mentagrophytes and T. rubrum. Colony count, size and growth rate of the isolated fungi were evaluated under laboratory conditions. RESULTS: Trichophyton rubrum type 1 was less sensitive to UV-A and UV-C, and more sensitive to UV-B than type 2. T. mentagrophytes type 2 was slightly responsive to UV-A therapy, although no decrease in colony count was observed. Increased doses of UV-B and UV-C irradiation decreased the counts. The effect of radiation on colony size was dependent on the dose and type of irradiation. CONCLUSIONS: UV-A, UV-B, and UV-C light seem to be effective in decreasing colony growth of the most prevalent fungi, which caused onychomycosis in the current study samples. Further studies are needed to determine the efficacy of ultraviolet light therapy, identify possible side effects, and establish appropriate dosages for the antifungal effect of this therapy.

Occurrence of Linguatula serrata nymphs in cattle slaughtered in Tabriz, Iran.

The Linguatula serrata is a tongue-shaped parasite that infects carnivores or insectivorous reptile as final and herbivores as intermediate host. The aim of this study was to evaluate the prevalence of nymphal stages of L. serrata in mesenteric nodes (MLN) and mediastinal lymph nodes of cattle slaughtered in Tabriz slaughterhouse, North West of Iran. Mesenteric and mediastinal lymph nodes of 640 cattle of different sex and age were inspected. A digestion method was applied for investigation of samples revealing an infection prevalence of 18.9 %. The MLNs in 97 cattle out of 640 (15.1 %) and the mediastinal lymph nodes in 47 cattle out of 640 (7.3 %) were infected by L. serrata nymphs. The results showed infection rate of mesenteric lymph nodes higher than mediastinal mesenteric lymph nodes (P < 0.05). The infection rate increased with age (P < 0.05). Although a significant difference seen in the infection rate between male and female but it was not significant at the same age groups of male and female (P > 0.05). In addition, there was a significant difference in the infection rate of different seasons (P < 0.05). Linguatulosis occurs as an endemic zoonosis in the northwest of Iran and has an active transmission life cycle.

Fecal egg counts for gastrointestinal nematodes are associated with a polymorphism in the MHC-DRB1 gene in the Iranian Ghezel sheep breed.

Genetic variation among sheep breeds in resistance to gastrointestinal nematodes (GIN) has been demonstrated in several production environments. Relationships between the ovine major histocompatibility complex and resistance to GIN have been studied, but few studies have systematically examined this issue in less-developed and semi-arid regions. The aim of the current study was to explore associations between fecal worm egg counts (FEC) for several GIN and polymorphisms in the DRB1 gene. One hundred male lambs were selected at 4-6 months of age from weaned animals in five flocks (n = 20 per flock). Body weights were determined, FAMACHA scores based on color of the ocular mucous membranes were assigned as an indicator of anemia, and blood and fecal samples were collected twice to evaluate FEC and blood packed cell volume (PCV) and for DNA isolation. A repeated-measures analysis of variance was used to test effects of genotype on FEC. The model included fixed effects of flock, genotype, time of measurement (1 or 2), and flock × time and genoype × time interactions, and a random (repeated) effect of lamb. Two genotypes (A1A1 and A1A2) were observed following digestion of Region 1 of Ovar-DRB1 with PstI. Genotypic frequencies were 0.73 for A1A1 and 0.27 for A1A2. FEC differed between Ovar_DRB1 genotypes A1A1 and A1A2 for Marshallagia marshalli, Strongyle, and total nematode FEC. Observed FEC were 30-41% lower for genotype A1A1. Differences among genotypes were consistent across measurement times, with no effect of genotype × measurement time interaction for any parasite class (P ≥ 0.34). A significant association was observed between FAMACHA scores and lamb PCV, and the residual correlation between these two variables was -0.51 (P < 0.001). FAMACHA scores can thus be used to detect differences among lambs in PCV, and polymorphic markers of Ovar-DRB1 have potential value as an indicator of parasite resistance in applied animal breeding programs on sheep farms in this region.

External parasite infection of common carp (Cyprinus carpio) and big head (Hypophthalmichthys nobilis) in fish farms of Mashhad, northeast of Iran.

Totally 75 common carp and 100 big head fishes were caught by using net from fish farms in Mashhad, northeast of Iran. In laboratory skin, eye and fin of fishes were inspected by stereomicroscope and in second phase direct smears are prepared from probable lesions. Gills were dissected and its filaments were placed in petri dishes and fixed by Glycerin. In total 50 parasites (consist 19 protozoa and 31 metazoa) were recorded from fishes. The parasites represented in Protozoa (Ichthyophthirius multifiliis and Trichodina sp.) and Monogenea) Dactylogyrus spp.) and Copepoda (Lernea cyprinacea). During this study, infection with Dactylogyrus spp. was recorded on fish in all months. Mean intensity of Dactylogyrus spp. varied significantly among the seasons (P < 0.05). The maximum mean intensity was recorded in winter. Also infection with Lernea cyprinicea in spring was significantly higher than other seasons (P < 0.05). The results of this study together with the previously recorded prevalence of parasitic infection in fishes support that infection to external parasites (both protozoa and metazoa) is widespread cause for losses in fish farms of Iran.

Prevalence of Haemoproteus columbae and Trichomonas gallinae in pigeons (Columba domestica) in Isfahan, Iran.

The aim of this study was to determine the prevalence of Haemoproteus columbae and Trichomonas gallinae in pigeons (Columba domestica) in Isfahan, Iran. Study was done on 100 pigeons from January to June 2010 in Isfahan located in south of Iran. Oropharyngeal swabs were taken from all birds. Also blood samples were obtained from wing vein and thin smears were prepared from them. All smears were stained by Giemsa and were examined by immersion. Out of 100 Oropharyngeal swab smears 57 (57%) were positive for T. gallinae and Haemoproteus gallinae were detected in 62 pigeons (62%) from which blood smear were taken. The present study is in close agreement with other studies in Iran. But infestation rate to theses parasite in world are very different. These differences were probably linked to geographical variations and differences in the feeding habitat and abundance of vehicles of Haemoproteus.

Biochemical, hematological, and electrocardiographic changes in buffaloes naturally infected with Theileria annulata.

Changes in selected blood and serum components and electrocardiography (ECG) were investigated in 20 adults (13 females and 7 males) of water buffaloes suffering from severe theileriosis. The age of all animals used in this study ranged 1.5-5 yr. Theileriosis was diagnosed by observation of parasites in the peripheral blood and the presence of schizonts in lymphocytes that were provided from swollen lymph nodes. Statistically significant decreases were observed in the means of RBC, WBC, and packed cell volume (PCV) in blood of infected animals. The means levels of sodium, calcium, phosphorus, and potassium of infected animals were lower than healthy animals, but only the decrease of potassium was significant. The mean serum activities of aspartate transferase and alanine aminotransferase were significantly higher than in uninfected animals. Three cases had atrial premature beat, 2 cases had sinus tachycardia, 2 had sinus arrhythmia, and 1 had first degree of atrioventricular block in ECG. The present study showed that T. annulata infection in cattle is associated with hematological and biochemical, and ECG changes.