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Arginine kinase in Toxocara canis:Exon-intron organization,functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors

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Asian Pacific Journal of Tropical Medicine 2016年第10期9卷 973-979页

作者： Susiji Wickramasinghe Lalani Yatawara Mitsuru Nagataki Takeshi Agatsuma Department of Parasitology Faculty of MedicineUniversity of Peradeniya Department of Medical Laboratory sciences Faculty of Allied Health SciencesUniversity of Peradeniya Department of Environmental Health Sciences Kochi Medical School

Objective: To determine exon/intron organization of the Toxocara canis(T. canis) AK(TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Methods: Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame(1 200 bp) of TCAK(wild type) was cloned into the BamH 1/SalI site of pM AL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pM AL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25℃ for the forward reaction(phosphagen synthesis). Results: Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K_m value of the mutant(Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K_m value of the mutant(Serine to Glycine) increased to 0.19 mM. The Km value(0.19 mM) of the double mutant(Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type(0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica(A. indica), an aminoglycoside antibiotic(aminosidine), a citrus flavonoid glycoside(rutin) and a commercially available catechin mixture against TCAK. Green and black tea(1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK. Conclusions: Arginine kinase in T. canis has a seven-exon/six-intron gene structure. However, further studies

关键词： Toxocara canis Arginine kinase Gene structure Site directed mutagenesis Inhibition kinetics

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Immunochemical characterization of antigens of Brucella canis and their use in seroprevalence study of canine brucellosis

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Asian Pacific Journal of Tropical Medicine 2011年第11期4卷 857-861页

作者： Sharma Barkha Sinha Dharmendra Kumar Singh Dhirendra Kumar Department of Epidemiology and Preventive Medicine College of Veterinary SciencePandit Deen Dayal Upadhaya Veterinary UniversityMathuraUttar PradeshPin-281001India Division of Epidemiology Indian Veterinary Research InstituteIzatnagarBareillyUttar Pradesh243122India Division of Veterinary Public Health Indian Veterinary Research InstituteIzatnagarBareillyUttar Pradesh243122 India

Objective:To explore immunochemical characterization of antigens of Brucella canis(B. canis),and the use in seroprevalence study of canine brucellosis.Methods:External hot phosphate buffer saline extract(HPBSE) and internal sonicated(SA) antigens were prepared from B.canis strain MEX 51 and imniunochemically characterized.These antigens were used to test 527 serum samples of dogs by 2-mercaptoethanol-tubc agglutination test(2 ME-TAT), agar gel immunodiffusion test(AGID).dot-ELISA and indirect enzyme-linked immunosorbent assay(I-ELISA) to assess the seroprevalence of canine brucellosis.Results:The protein content of HPBSE and SA antigens was 0.387 mg/ml.and 0.195 mg/mL,respectively,whereas carbohydrate content was 0.174 mg/mL and 0.150 mg/mL,respectively.The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(12.5%) of HPBSE and SA,revealed 6 and 8 visible peptide bands ranging from 18-80 kDa and 12-45 kDa,respectively.Western blot analysis showed immunodominant bands of MW 12.28.39 and 45 kl)a for HPBSE and 20-24 kl)a for SA. The AGII) revealed HPBSE as more specific antigen than SA but both I-ELISA and dot-ELISA indicated SA antigen to be more specific and reliable than HPBSE.The seroprevalence of canine brucellosis was 2.27%by 2ME-TAT.1.5%by AGID.3.03%by dot-ELISA and 16.12%by I-ELISA. Conclusions:On the basis of the results of present study,we concluded that HPBSE is suitable antigen for AGID,which is more specific:whereas SA antigen is suitable for I-ELISA,which is highly sensitive.Therefore,initial screening of serum samples should be carried out by I-ELISA followed by confirmation with AGID.

关键词： Brucella canis SDS-PAGE Western blotting Agar gel immunodiffusion test Dot-ELISA I-ELISA

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Retrospective Study of clinical and hematological aspects associated with dogs naturally infected by Hepatozoon canis in Ludhiana,Punjab,India

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Asian Pacific Journal of Tropical Biomedicine 2013年第6期3卷 483-486页

作者： Sushma Chhabra Sanjeev Kumar Uppal Lachhman Das Singla Department of Veterinary Medicine College of Veterinary ScienceGuru Angad Dev Veterinary and Animal Sciences University Department of Veterinary Parasitology College of Veterinary ScienceGuru Angad Dev Veterinary and AnimalSciences University

Objective:To evaluate clinical and hematological aspects of dogs naturally infected with Hepatozoon canis(H.canis)presented at the Small Animal Clinics of Guru Angad Dev Veterinary and Animal Sciences University,Ludhiana.Methods:Blood films of 34 naturally infected dogs were examined for haematological alterations and parasitaemia.Signalment and clinical signs were recorded from the animals.Clinical histories were filled oul during the consultation.Results:Of the 34 positive dogs by Giemsa stained peripheral blood films.88.23%presented parasilaemia by H.canis only,while 11.77%had the combination of H.canis,Babesia sp.and Ehrlichia sp.Young male dogs less than oue-year-old,of nondescript breed,were the most commonly affected.And 26.47%were presented with anorexia/inappetence as the only clinical symptom.Other clinical symptoms were mild to moderate fever,pale mucosae and lethargy;a few were also showing the signs of vomiting and diarrhoea.Haematological alterations showed mainly normochromic-normocytic anaemia,leukocytosis and neutrophilia.Conclusions:The findings of this study substantiate that H.canis caused clinical and haematological alterations of the varied intensity in dogs,even with low parasilaemia.should he taken into consideration.

关键词： Clinical signs Dogs Haematology Hepatozoon canis

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Factors Determining Coyote (canis latrans) Diets

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Open Journal of Ecology 2017年第13期7卷 650-666页

作者： Lauren N. Watine William M. Giuliano Department of Wildlife Ecology and Conservation University of Florida Gainesville FL USA

Although studies have documented the potential for coyote (canis latrans) food use to negatively affect wildlife populations and domesticated animals, they are often equivocal, possibly because most are of small spatial extent, and little is known of factors determining coyote diets. Our objectives were to quantify the diet and identify factors determining coyote food use, particularly game species and livestock, over a large spatial and temporal extent. Contents of gastrointestinal tracts were identified from 263 coyotes opportunistically obtained from hunters, trappers, and as road-kills throughout Florida, 2011-2015. We employed logistic regression in an information-theoretic framework to understand determinants of coyote food use. Coyotes were opportunistic and omnivorous foragers with a diverse diet of vegetation, insects, birds, reptiles, amphibians, and more than 25 species of mammals (including important game species and livestock). They commonly consumed 11 food items (Virginia opossum [Didelphis virginiana], non-mast vegetation, feral hog [Sus scrofa], northern raccoon [Procyon lotor], insects, rabbits (Sylvilagus spp.), skunks [Mephitis mephitis and Spilogale putorius], white-tailed deer (Odocoileus virginianus), mast, birds, and cows [Bos taurus]). Food use was determined by coyote age, sex, and body mass, season of the year, deer hunting and fawning seasons, livestock calving season, and coyote collection method and location/region. As coyotes expand their range and numbers, conservationists may find it useful to understand how this opportunistic and adaptable predator uses available food sources to reduce conflict across the landscape.

关键词： canis latrans Coyote Determinants Diet Factors

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Inhibition of Ehrlichia canisand Babesia canistransmission among ticks fed together on dogs vaccinated with Bm86 antigen

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Open Journal of Animal Sciences 2013年第3期3卷 24-32页

作者： Alina Rodríguez-Mallon Gervasio H. Bechara Rosangela M. Zacarias Efrain Benavides-Ortiz Jose Luis Soto-Rivas Arlen Patricia Gómez-Ramírez Javier Andrés Jaimes-Olaya Mario Pablo Estrada-García Centro de Ingeniería Genética y Biotecnología Habana Cuba Facultad de Ciencias Agrarias Universidad de Ciencias Comerciales Altamira Nicaragua Facultad de Ciencias Agropecuarias Universidad de La Salle Bogotá DC Colombia Universidade Estadual Paulista-UNESP Jaboticabal Brazil

GAVAC (Heber Biotec S.A, Havana, Cuba) is a commercially available vaccine developed with the Rhipicephalus (Boophilus) microplus Bm86 recombinant antigen. Bm86 is a “concealed” antigen that is present in the plasmatic membrane of tick gut epithelial cells with unknown function so far. It is well known that after vaccination in the last fifteen years in Cuba, there was a significant decrease of babesiosis (Babesia bovis and Babesia bigemina) and anaplasmosis (Anaplasma marginale) in cattle. A reduced transmission capacity of ticks fed on tick-immune animals and humans has been reported for several tick-borne pathogens. Recent experiments have demonstrated that an anti-tick vaccine may contribute to the control of tick-borne pathogens not only by decreasing the exposure of susceptible hosts to ticks, but also by reducing the vector capacity of ticks. In this study, the potential of Bm86 vaccination to interfere with pathogen transmission among ticks was evaluated by using as experimental model the brown dog tick Rhipicephalus sanguineus and the tickborne Babesia canis and Ehrlichia canis pathogens. Dogs, vaccinated and not vaccinated, were infested with pathogen-infected ticks and noninfected nymphs of R. sanguineus. After feeding, the pathogen transmission to newly molted adults from co-feeding uninfected nymphs was studied by conventional PCR and qPCR. Results suggest that the anti-Bm86 antibodies could be able to block the transmission of B. canis and/or E. canis from infected to non-infected ticks.

关键词： Rhipicephalus sanguineus Ehrlichia canis Babesia canis Tick-Borne Diseases Dogs Bm86

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Multifocal granulomata in presumed Toxocara canis infection in adult

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World Journal of Ophthalmology 2013年第4期3卷 38-40页

作者： Lakshmi Kuniyal Jyotirmay Biswas Department of Vitreoretina Sankara Nethralaya Chennai TN 600006 Andhra Pradesh India Department of Uvea Sankara Nethralaya Chennai TN 600006 Andhra Pradesh India

Human infection of Toxocara canis in eye is usually an outcome of accidental ingestion of the embryonated eggs. The average age at diagnosis of ocular Toxocariasis is 7.5 years, ranging from 2 to 31 years. It constitutes 1%-2% of uveitis in children. Diagnosis is based upon the clinical features observed in a young patient and confirmed by the presence of specific Ig G in the serum or aqueous humor by Enzyme-linked immunosorbent assay test. We report a case of Presumed Toxocara infection in 45-year-old male which is unique in presentation with multifocal granulomata in retina. Our Pub Med search could not produce case with similar presentation. Probably this is the first reported case of multifocal granulomata in presumed ocular Toxocara in any age

关键词： Presumed Toxocara canis Ocular Multifocal Granulomata

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Antidermatophytic Activity of Essential Oils against Locally Isolated Microsporum canis—Gaza Strip

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Natural Science 2014年第9期6卷 676-684页

作者： Emad Khalil Abou Elkhair Biology Department Faculty of Science Al Azhar University-Gaza Gaza Strip Palestine

There is a need for new antimicrobial drugs due to the continuous development of resistance. Natural antimicrobials are of utmost importance due to safety issue and availability. The antifungal activity of four essential oils (Mentha piperta, Thymus vulgaris, Cymbopogon citratus, and Oreganum majoranum oils) against locally isolated Microsporum canis was determined by disc diffusion method and minimum inhibitory concentration was determined using broth dilution method. Mentha piperta oil showed the highest antifungal activity among tested oils in this study. The total inhibition attached when 0.046% was used with M. piperta, the total inhibition obtained with the 0.133% of C. citratus oil, which is accounted as the second essential oils, also T. vulgaris and O. majoranum oils achieved reductions at 0.133%. The present work has revealed that all oils have been used in low concentrations and produced promising results in comparison to the positive control (Clotrimazole).

关键词： Antifungal Microsporum canis Mentha piperta Essential Oils Gaza Strip

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Molecular detection of Ehrlichia canisin ticks population collected on dogs in Meshkin-Shahr, Ardebil Province, Iran

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Journal of Biomedical Science and Engineering 2013年第7期6卷 1-5页

作者： Atefeh Khazeni Zakieh Telmadarraiy Mohammad Ali Oshaghi Mehdi Mohebali Zabiholah Zarei Seyed Mohammad Abtahi Department of Medical Entomology and Vector Control School of Public Health Tehran University of Medical Sciences (TUMS) Tehran Iran Department of Medical Parasitology School of Public Health Tehran University of Medical Sciences (TUMS) Tehran Iran Department of Parasitology School of Medicine Isfahan University of Medical Sciences and Health Services Isfahan Iran

Canine monocytic ehrlichiosis is a disease which can cause death in dogs and cats by the Ehrlichia canis and transmitted by hard ticks. The main vector is Rhipicephalus sanguineus which is a common tick in Iran and common ectoparasite on dogs. Few researches has been done to show Ehrlichiosis situation in dog’s ticks. Animal husbandry in Meshkin-Shahr County from Ardebil province (North-West Iran) is the main job of people. Specimens were collected on dogs’ ears, neck, shoulder and toes and they were transferred to the Entomology Laboratory, School of Public Health, Tehran University of Medical Sciences. After DNA extraction, Ehrlichia agent was detected by nested-PCR, 16 s rRNA amplification. Determination of sequence homologies have been done in Gen-Bank. 146 ticks were identified which included 29.44% female and 47.94% male. Rhipicephalus sanguineus were the most prevalent ticks. Ehrlichia spp and Anaplasma spp were found in 43.84% of all the specimens containing Anaplasma ovis, and Ehrlichia sp. and Herlichia canis. Rhipicephalus sanguineus is widely spread and the most common ticks on dogs. As far as we know, this is the first report of E. canis in vector from Iran. Nested PCR showed that hard ticks can contain Anaplasma ovis, Ehrlichia spp and E. canis. These results warrant studying on vector competence of ticks for the Ehrlichiosis agents.

关键词： Ehrlichiosis Tick Dog Anaplasma ovis Ehrlichia canis Ardebil Iran

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Characterization of insect-fauna of the free-ranging urban dog, canis domesticus (L.) carcass in tropical region of Pakistan: A tool for forensic entomology

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Advances in Entomology 2013年第2期1卷 29-37页

作者： Farzana Perveen Anzela Khan Department of Zoology Shaheed Benazir Bhutto University (SBBU) Main Campus Sheringal Pakistan

As the authors’ request, the following paper published in Vol.1, No.2, 29-37, 2013 has been withdrawn from the website.

关键词： canis domesticus Carcass Decomposition Ecological Succession Pattern Forensic Entomology Insect Fauna Takht Bhai

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Disseminated toxocariasis in an immunocompetent host

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Asian Pacific Journal of Tropical Biomedicine 2014年第10期4卷 838-840页

作者： Madan Raj Aryal Paras Karmacharya Amrit Pokharel Smith Giri Ranjan Pathak Richard Alweis Department of Medicine Reading Health SystemPA 19611USA Department of Medicine Tribhuvan University Teaching HospitalKathmanduNepal Department of Medicine University of Tennessee Health Science CenterTN 38163USA

Toxocariasis is a zoonotic infection caused by Toxocara canis,or less commonly,Toxocara cati,which is one of the most common zoonotic infections worldwide.It commonly affects the pediatric and immunocompromised population;however,it has rarely been reported in the immunocompetent adults.Two of the well-recognized syndromes in children are visceral larva migrans and ocular larva migrans.Infection in adults usually ranges from asymptomatic to nonspecific symptoms which makes the diagnosis challenging.A case of 36 year-old male was presented with disseminated toxocariasis with pulmonary and hepatic involvement and striking peripheral eosinophilia.

关键词： Toxocariasis Toxocara canis Mebendazole Albendazole

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