Morphometric Analysis of the Intestine in Experimental Coccidiosis in Broilers Treated with Anticoccidial Drugs

Background: Coccidiosis causes morphologic alteration in intestinal mucosa resulting in reduction of absorptive surface. Anticoccidials used as feed additives may induce changes in the intestinal mucosa. This study was designed to assess intestinal morphometry in broilers infected with Eimeria under different anticoccidial treatments. Methods: To evaluate the effect of salinomycin and amprolium+ethopabate on intestinal morphometry in broilers experimental coccidiosis, in Tehran, Iran in May 2015, fifty-four Ross 308 birds were randomly divided into two challenged and unchallenged groups at the age of 12 days. The birds were challenged with Eimeria field isolate at day 14. Different growth and parasitological parameters including weight gain, feed consumption, FCR, macroscopic lesion score and oocyst score were recorded 7 d post-inoculation. Histological sections from four main parts of intestine (anterior, middle, lower intestines and cecum) were prepared and analyzed. Villus width and length and total mucosal thickness were measured microscopically. Results: Amprolium+ethopabate and salinomycin significantly reduced coccidiosis gross lesions in infected birds. Microscopically anticoccidial administration in the presence of infection has significantly increased the villus length while the presence of amprolium+ethopabate in the absence of infection has greatly increased the mucosal thickness and villi height in comparison to the control group. Conclusion: Anticoccidials may induce some histological changes in the mucosa when there is no parasite to be affected. Some of these effects may be advantageous for the intestinal epithelium integrity and hence the birds’ performance

Immunoinformatics and Similarity Analysis of House Dust Mite Tropomyosin

Background: Dermatophagoides farinae and Dermatophagoides pteronyssinus are house dust mites (HDM) that they cause severe asthma and allergic symptoms. Tropomyosin protein plays an important role in mentioned immune and allergic reactions to HDMs. Here, tropomyosin protein from Dermatophagoides spp. was comprehensively screened in silico for its allergenicity, antigenicity and similarity/conservation.Materials and Methods: The amino acid sequences of D. farinae tropomyosin, D. pteronyssinus and other mites were retrieved. We included alignments and evaluated conserved/ variable regions along sequences, constructed their phylogenetic tree and estimated overall mean distances. Then, followed by with prediction of linear B-cell epitope based on different approaches, and besides in-silico evaluation of IgE epitopes allergenicity (by SVMc, IgE epitope, ARPs BLAST, MAST and hybrid method). Finally, comparative analysis of results by different approaches was made.Results: Alignment results revealed near complete identity between D. farina and D. pteronyssinus members, and also there was close similarity among Dermatophagoides spp. Most of the variations among mites' tropomyosin were approximately located at amino acids 23 to 80, 108 to 120, 142 to 153 and 220 to 230. Topology of tree showed close relationships among mites in tropomyosin protein sequence, although their sequences in D. farina, D. pteronyssinus and Psoroptes ovis are more similar to each other and clustered. Dermanyssus gallinae (AC: Q2WBI0) has less relationship to other mites, being located in a separate branch. Hydrophilicity and flexibility plots revealed that many parts of this protein have potential to be hydrophilic and flexible. Surface accessibility represented 7 different epitopes. Beta-turns in this protein are with high probability in the middle part and its two terminals. Kolaskar and Tongaonkar method analysis represented 11 immunogenic epitopes between amino acids 7-16. From comparative analysis of predicted probable consensus epitope regions by machine learning approaches these epitopes were gained: AA23-48, AA59-80, AA91-110, AA114-143, AA154-168, AA182-200, AA208-225, and AA254-272. Prediction of allergenic proteins by AlgPred server showed 10 matches for IgE epitope, and prediction by hybrid approach showed that IgE epitope is undoubtedly the major allergen.Conclusion: Immunoinformatic approaches in allergenic protein analysis are now reliable tools for explanation/interpretation of clinically observed complexities. Results of present study, would help in HDM immunotherapy against several species of parasites as a wide range epitopic desensitization or prevention (vaccine) regime

Comprehensive phylogenetic, similarity and allergenicity analysis of Boophilus genus tick Tropomyosin protein

     Boophilus genus ticks are responsible for transferring some pathogens and reducing production factors in cattle. Tropomysin (TPM) protein has actin regulator activity and playing important role in immune and allergic reactions. The main goal is to determine different aspects of phylogenetic, similarity, homology, structure and allergenicity of TPM protein. In prior study, we identified TPM by using Mass-spectrometry in Boophilus anulatus larva proteins extraction. Analysis by NCBI and Mascot software showed complete similarity of this protein with Boophilus microplus. TPM Blasting, invertebrates TPM sequences retrieval, aligning and analyzing of conserved and variable regions along sequences were next steps. Also, construction the phylogenetic tree, overall mean distances estimation, homology protein secondary structure, allergencity analysis was achieved. The most similar sequences to Boophilus genus TPM are Haemaphysalis sp., Scolopendra sp. and etc., respectively. The multiple sequence alignment showed that conserved and variable regions stretched in different part of TPM. The close relationships in Phylogenetic tree between Ticks and Mites were seen, although the TPM sequences in ticks are more similar to each other than to mites and assume as the nearest relatives. Insects TPM like worms, located in two separated clades, and Trichinella spiralis in worm clades are more related taxa to members of ticks and mites groups. Furthermore, overall mean distances over sequence pairs reflects TPM conservation during speciation. TPM has high homology in different species and has two domain of α-helix that cannot form disulfide bonds. Finally, allergenicity analysis by separated and hybrid approach showed it undoubted is allergen and candidates some peptides as responsible for allergenicity of TPM. The comprehensive analysis of TPM has never been easy, especially when we attempt to make statements from different aspects about this protein.  Our study revealed the some unique and valuable aspects of TPM protein of Boophilus genus, and will help to further studies on mentioned protein

Immunization of Cattle with Tick Salivary Gland Extracts

Background: Rhipicephalus (Boophilus) annulatus tick is one of the most important ectoparasite of cattle. Re­cently, several laboratories in the world have been concentrated on immunizing cattle against tick using various types of tissue extracts of ticks. The aim of this study was to evaluate the effect of immunization of cattle with tick salivary gland extract on biological parameters of ticks and humoral immune responses of cattle. Methods: Fourteen more dominant protein bands identified as immunogenic by Western-blot analysis were eluted from polyacrylamide gel. Test and control groups were injected three times with eluted proteins and sterile PBS (pH= 7.2) respectively with equivalent amount of adjuvant. After four weeks a tick challenge was performed. Fi­nally, biological parameters of collected engorged female ticks were recorded and humoral immune responses to immunization measured by ELISA. Results: The results indicated immunization of cattle resulted in reduction in mean tick counts, attachment, en­gorgement weights, feeding index, egg mass weight, hatchability and fertility index (respectively 63.1%, 62.6%, 30.2%, 36.4%, 40%, 78.7% and 13.3%) and increased duration of feeding, pre-oviposition and incubation period of eggs (respectively 8.6%, 45 and 31.34%). All changes were statistically significant (P< 0.05). Results showed an increase in antibody production of test group from the first week after immunization. The antibody level was boosted following tick infestation. Conclusion: This investigation indicates that immunization of cattle with these antigens could induce a protective immune response against Rh. (B.) annulatus tick that would be expected to provide a safe non-chemical means of tick control

Designing of potential vaccine candidates of fused cathepsin L and tropomyosin genes of Rhipicephalus (Boophilus) annulatus tick larva

Introduction: Regarding emerging tick resistance against acaricides, researches have been shifted toward alternative approaches such as immunologic methods. Vaccine preparation is an alternative way in which choosing appropriate protein with high immune induction potency is a prerequisite. In addition according to studies, using more than one protein could better enhance the immune induction and antibody production. Choosing immunogenic epitopes from selected proteins and adjoining them with a suitable linker is one of the novel approaches in vaccine design. Materials and methods: Based on the fact that both cathepsin and tropomyosin proteins of Rhipicephalus tick were previously recognized as potent immunogenic antigens, we predicted the immunogenic epitopes of these proteins by immunoinformatic methods. Among studied epitopes, those that were met by multiple bioinformatics tools were used. Results: Finally, the polytopic construction was designed by assembling the selected epitopes and connecting them with linkers. Conclusion: Using immunoinformatic tools, we predicted the characteristics of two genes of Rhipicephalus annulatus tick larva as fused potent vaccine candidates namely, cathepsin and tropomyosin

Molecular and morphological description of <i>Isospora</i> sp. from the common mynah (<i>Acridotheres tristis</i>) and a preliminary survey of two anticoccidial drugs in natural infection

<p>Coccidian parasites, especially <i>Isospora</i>, are prevalent parasites in Passeriformes. Isosporan oocysts from common mynahs (<i>Acridotheres tristis</i>) are incompletely described. Detailed knowledge on biology, prevalence, pathogenesis and treatment of avian isosporiasis is scant. In this study, isosporan oocysts isolated from common mynahs were morphologically and molecularly characterized. The medication efficiencies of diclazuril and sulfadiazine-trimethoprim in isosporiasis in naturally infected mynahs were evaluated. Isosporan oocysts from common mynahs were described morphologically by microscopic imaging. The 18S rRNA and COI genes were amplified using PCR and the resultant products were sequenced and analysed phylogenetically. To evaluate the efficiencies of diclazuril and sulfadiazine-trimethoprim, two experimental treatment groups and a null control were assigned. General health status, weight and oocysts per gram of faeces were evaluated. Oocysts from all birds contained isosporan oocysts that were morphologically and dimensionally similar (<i>P</i> < 0.05). The oocysts were spherical; with no oocyst residuum, micropyle or polar granules. At both loci, phylogenetic analyses placed the <i>Isospora</i> isolate in the same clade with <i>Isospora</i> spp. from other Passeriformes. Both of the anticoccidials were well tolerated by the birds, a rapid reduction in oocyst excretion was noted at the commencement of treatment and 72 h after drug administration, oocyst excretion zeroed in all treated birds. Based on morphological and molecular data, this isolate does not resemble any previously described isosporas, hence <i>Isospora tristum</i> n. sp. is proposed for the current species. Both evaluated anticoccidials seemed to be efficient in reduction of oocyst production and can be recommended for the treatment of mynah isosporiasis.</p

Molecular Epidemiology and Phylogeny of Crimean-Congo Haemorrhagic Fever (CCHF) Virus of Ixodid Ticks in Khorasan Razavi Province of Iran

Background: Crimean–Congo hemorrhagic fever (CCHF) is a fatal disease caused by Nairovirus classified within the Bunyaviridae family. The virus is transmitted to humans through the bites of infected ticks or direct contact with viremic animals or humans. The current study aimed to detect the virus genome in ticks from Khorasan Razavi Province. Methods: One hundred hard ticks were collected randomly from 100 sheep in four different areas of the province. Collected ticks were kept alive and identified. All the ticks were analyzed for the presence of CCHF virus genome using reverse transcriptase polymerase chain reactions (RT-PCR). Results: The identified ticks were belonging to Hyalomma marginatum (16% female and 6% male), Rhipicephalus turanicus (52% female and 25% male), and Dermacentor raskemensis (1%). The CCHF virus genome was found in Hyalomma marginatum (5% male from Taibad and Sabzevar region and 1% female from Taibad). Genetic analysis of the virus genome isolated from two regions (Sabzevar and Taibad) showed 100% identity. Conclusion: This study indicated that CCHF should be regarded as a risk-borne infection in this province. Therefore, special health management is needed to control this disease

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatuslarvae

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatusis an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines

Transmission of Leishmania infantum by Rhipicephalus sanguineus (Acari: Ixodidae) in Dogs

Background: Rhipicephalus sanguineus is the most widely distributed tick in the world, which is partly due to its biological flexibility and the global distribution of its major host, the domestic dog. In Mediterranean region it could be principal reservoir host for Leishmania infantum, usually transmitted by the phlebotomine sand flies. In this study, we evaluated the vector potential of R. sanguineus in transmitting L. infantum to uninfected dogs. Methods: During 2014, five dogs with clinical manifestations of canine visceral leishmaniasis (CVL), high anti-Leishmania antibody titers and tick infestation, were selected from CVL endemic areas (Tehran and Alborz provinces). At least, twenty live ticks were removed from each infected dog. After morphological identification, the ticks were divided into two groups; ticks belonging to the first group were dissected for parasitological examinations and semi-nested PCR assay, and those of the second group were selected for the transmission of CVL caused by L. infantum to uninfected dogs. Following tick infestation, all uninfected dogs were kept for 9 months and examined monthly for clinical and serological tests. Results: Nearly, 67% of ticks were infected by L. infantum using the semi-nested PCR. All other parasitological tests of ticks were negative. Clinical examinations and serological tests of the investigated dogs revealed negative results. Nested-PCR test results performed on splenic biopsy samples of dogs were also negative. Conclusion: L. infantum-positive R. sanguineus ticks were unable to transfer L. infantum from infected dogs to healthy ones. The detection of L. infantum DNA in ticks collected from naturally infected dogs by semi-nested PCR does not prove their vectorial competence