1.
Evaluation Of Insecticide Resistance And Biochemical Mechanisms In Anopheles Subpictus In District Kasur, Pakistan
by Huma Naeem | Prof. Dr. Kamran Ashraf | Dr. Imran Rashid | Prof. Dr.
Material type: ; Format:
print
; Literary form:
not fiction
Publisher: 2013Dissertation note: Mosquito are major vectors, considering the havoc they play by transmitting many diseases which have greatly affected human beings worldwide. The ecological, socioeconomic conditions of different regions and the emergence of insecticide resistance in main vectors are strongly responsible for determining the geographical distributions and incidence of vector-borne diseases. Among mosquitoes, Anopheles species are responsible for transmission of filariasis, Japanese encephalitis virus and malaria in subtropical region. An. subpictus is a confirmed vector in many countries of South and Southeast Asia. The present study was designed due to limited earlier records regarding insecticide susceptibility status of An. subpictus from district Kasur, Punjab. During the first half of the study, three insecticides i.e. DDT 4%, deltamethrin 0.05% and permethrin 0.75% were testing by using WHO susceptibility bioassay. A total approximately (n=1000) different types of mosquitoes were caught from district Kasur. Mosquitoes belonging to three genra Anopheles, Culex and Aedes were found in our collection. After species identification, An. subpictus was separated for further processing. It was highly abundant species among Anophelines species captured. Field collected adult blood fed females of An. subpictus was reared in the insectary for F1 generation. Two to three days old non engorged adults of An. subpictus were evaluated by using WHO susceptibility bioassays. Both male and female mosquitoes have shown resistance against all three insecticides tested. Probit analysis was used to check the time-response values. Percentage mortalities were recorded against DDT, deltamethrin and permethrin (mortality range 29.47 %-76.28 %.) with higher KT50 and KT95 values.Second half of the study was undertaken to quantify detoxifying enzymes in An. subpictus following WHO biochmical assays. Biochemical analysis of detoxifying enzymes in An. subpictus has revealed that there is significant alteration/elevation of metabolic enzymes when compared with the lab strain might be a contributing factor in conferring insecticides resistance. Elevated levels of GST (0.469 ± 0.115), MFOs (11.665 ± 4.165), ? esterases (1.5808 ± 0.7657) and ? esterase (3.9682 ± 2.311) were detected in An. subpictus. There was a significant high alteration of AChE enzyme activity detected in this species with 57.52% (± 9.234) mean percentage propoxur inhibition. These enzymes are implicated in the metabolism of DDT, pyrethroids and carbamate insecticides.
Availability: Items available for loan: UVAS Library [Call number: 1823,T] (1).
3.
Development Of DNA Based Diagnosis Of Ancylostoma Caninumin Dogs And Its Specificity With Traditional Fecal Microscopy
by Abida Rehman (2012-VA-648) | Dr. WasimShehzad | Mr. Akhtar Ali | Dr. Imran Rashid.
Material type: ; Literary form:
not fiction
Publisher: 2014Dissertation note: The blood feeding, canine hookworms have gained importance due to their potential to cause a variety of diseases in animals and human communities (Traversa 2012). Almost all types of canine hookworms are involved in causing zoonotic diseases(Traub et al. 2008), infecting more than half a billion people worldwide (Fenwick 2012), which result in ~65000 deaths annually (Plotkin et al. 2008). Ancylostoma caninum is the most prevalent and pathogenic intracellular obligate hookworm parasite of dogs (Bojar and Klapec 2012). In Pakistan, parasitic infection byA. caninum is widely prevalent with variable distributions in different parts of the country. A microscopic based study in the Lahore areashowed 59.1% ofA.caninuminfestation in dogs (Ashraf et al. 2008).
Clinically,A. caninum has been responsible for often neglected disease ancylostomiasis in its host.In this condition, it principally attacks on the mucosal layer of small intestine through its buccal capsule for sucking blood (Marquardt et al. 2000). Secretory anticoagulant proteins of A. caninum help in this process by blocking a wide variety of blood clotting factors including Xa. The inhibition of blood clotting factors causes greater blood loss which ranges from 1-2ml/worm/day (Cappello et al. 1995; Georgi et al. 1969; Stassens et al. 1996). The disease is indicated by the symptomsof weight loss, lethargy, roughness of the hair coat,infected pale mucous membranes, tarry feces and excretion of eggs (Marquardt et al. 2000).In chronic situation,A. caninum producesa devastating condition of iron deficiency anemia with intestinal bleeding (Loukas and Prociv 2001).A. caninum infection in the dog isaccompanied by other life threatening pathological conditions, these includegastrointestinal infections, hypoproteinemia, mental retardation,pneumonitis andacute fatalities (Schwenkenbecher and Kaplan 2007).Especially, puppies are more suscepted to aforementioned diseases because of transmammary transmission,low levels of body immunity and higher egg count(Anderson 2000; Olsen 1986).
The life cycle of A. caninumis almost same both in human and dog. Itis most complex and critical for them as compared to other members of its genus. The cycle starts with the production of eggs by adult worms within intestine of an infectedhost which are passed out with feces. These eggs survive in soil without damages by variable environmental conditions,can become a source of reinfection for host species. Theinfective filariform larvae (hatch from eggs) get their route ina host bodyby penetrating through hair follicles on skin contact. In puppies these are mostly transmitted through transmammary or prenatal routes. After penetration, these larvae take way to lungs through the blood or lymphatic circulation. From here, these can be swallowed towards intestine where they attached to feed on blood and mucous (Marquardt et al. 2000). They also migrate to skeletal muscles via somatic circulation where they depositedas hypobiotic larvae. Warm and moist conditions cause their reactivation and migration towards gut (Prociv and Luke 1995; Traub et al. 2014).
It is seenthat a healthy pet doggetsA. caninuminfection mainly due to lackof proper veterinary care, large population of infected stray dogs, poor sanitation, and by contamination of public parks and streets (Klimpel et al. 2010; Zewduet al. 2010). Infective dogs are responsible for transmission of this parasite to humans either playing role aspets, stray or rescue animals (Jafri and Rabbani 1999; Szabova et al. 2007) by shedding millions of eggs(Epe 2009). Children areparticularly and frequently attacked byA. caninumbecause they used to play in open contaminated areas (Farooqi et al. 2014).
Transmission of this parasite to humans is mostly by penetration through the skin, when it comes in contact with its filariform larval stages present in contaminated soil.Humans also get infection through larval ingestion, and larvae canbe transmitted from the fur coat ofinfected companion animals (Caumes 2000; Hochedez and Caumes 2007; Provic 1998). In human, penetration of A. caninumfilariform larvae causessevere cutaneous larva migrans (CLM). It is the most devastating hypersensitivity reaction, characterized by long follicular, pustular, ephemeral lesions at infection sites with intense itching and pain (Kirby-Smith et al. 1926; Little et al. 1983). These sites can be attacked by bacterial species leading tothe damages ofsoft tissues (Chaudhry and Longworth 1989). CLM caused by A. caninum has achievedmore attentionmainly due to associated pathological conditions. These include eosinophillic enteritis, pneumonitis, and growth defects, etc. (Bowmanet al. 2003; Garcia et al. 2008; Tu et al. 2008). In childrenheart problems and mental retardation also havebeen reported (Albonico et al. 1999; Crompton 2000).
As, A.caninumiscausing serious healthhazards, so improved and proper control of its pathogenesis is mandatory.The accurate diagnosisof infection helps to prevent its transmission by selection of appropriate precautions and vaccines. Specific identificationis alsohelpful to minimizedthe chances of anthelminticdrugresistance by A.caninumas reported in different studies (Bethony et al. 2006; Kopp et al. 2007; Peeling et al. 2008; Roeber et al. 2013).
Different diagnostic methods are used to identify parasitic hookworms. Currently, fecal microscopy is the widely applied diagnosticmethod for the identification of hookworm infection,which depends on morphological based analysis of eggs (Katz et al. 1972; Ngui et al. 2012b). The main advantage of this traditional approach is its tendency to analyze sampleboth qualitatively (floatation, sedimentation) and quantitatively (McMaster, FLOTAC, Katokatz)(Cringoli et al. 2011; Eberl et al. 2002). However, the identifications based on morphological characteristics may prone to inaccurate diagnosis, because eggs of many hookworms e.g. Ancylostoma, Trichostrongylus, Unicinariastenocephala, Oesophagostomum, Necator americanus and Terniden species are morphologically indistinguishable,thiscan variate specificity and sensitivity of microscopy (Bajwa et al. 2014; Monis et al. 2002;Tan et al. 2014).
Improvement in microscopic detection can be made by usingcopro-culture and immunodiagnostic techniques especially when results are confounding. In the copro-culture method, eggs are raisedto larval stages in appropriate growth conditions in the laboratory andthen classified up to genus level (Reiss et al. 2007). But in this technique as microscopic examination, morphological similarities between larval stages of related species hampers specific identification. Moreover, it is time consuming (takes about 7-14 days) and requires experienced technicians to handle larvae. Similarly, it is very difficult to evaluate exact burden of worms as there is significant variations in the number of excreted eggs in chronic cases, which is another reasonable disadvantage ofthis diagnostic technique (Booth et al. 2003; Schar et al. 2013). Immunological assays, including ELISA,based on the detection of coproantigens in the feces or serum of the infected dog using captured IgG have been used to increase the sensitivity of analysis (Kwon et al. 2003;Loukas et al. 1992). These methodsallowed effective and quick detection of parasistes as compared to fecal microscopy and coproculture technique.But issue of cross reactivity with other antigens like of Strongyloides stercoralis in mixed infections isan associated problem (Lindo et al. 1994). Moreover, immunological assays failed to provide information about past or current infection and also do not differentiate between species in mixed infection (Basuni et al. 2011).
All the problems of traditional methods can effectively addressed by the adoption of DNA based molecular approaches. These methods have beenproved as worthwhile alternatives to identify parasitic species in any developmental stage (Muldrew 2009; Ndao 2009; Vasoo and Pritt 2013). Specifically, these techniques are very feasible for specific identification of genusAncylostomabecause ithasambiguous features.Advanced highly sensitive DNA based diagnostic procedures have potential to identify the causative agentfromminute quantity of DNA (from 0.2g of egg), as in low worm burden with no symptom of disease(de Carvalho et al. 2012; Wong et al. 2014).Rapid detection of parasites can be made in only one day even in case of mixed infections(Gasser et al. 2009).
There are various available approaches in DNA based methods which can be applied for the detection of Ancylostomaspecies, theseinclude PCR-RFLP (e Silva et al. 2006; Traub et al. 2004), copro-PCR (Sato et al. 2010), single-strand conformation polymorphism (SSCP) (Gasser and Monti 1997; Monti et al. 1998),specific (conventional) PCR (Yong et al. 2007) and multiplex real time PCR (Jonker et al. 2012). The conventional PCR techniquehas worldwideapplications in specie specific identification of parasiticspecies(Gordon et al. 2011). For specific diagnosis,genetic markers have been identified in mitochondrial (cox1 gene) and nuclear genomes (internal transcribed spacers (ITS-1, ITS-2), 5.8S and 28S in ribosomal DNA (rDNA)) (Chilton 2004; Denver et al. 2000; Gobert et al. 2005; Ngui et al 2012a). Preferably, spacers are more suitable for diagnostic purposes because these regions havesequence variationsamong different species. Moreover, these are shorter in length (250-300) in comparison with mitochondrial DNA (Blouin 2002; van Samson-Himmelstjerna et al. 2002). Therefore, the availability of sequencing technologies,specific genetic markers and large amount of genetic data have increased the chances of implementation, development and effectiveness of DNA based diagnostic method for identification ofA. caninum(Taniuchi et al. 2011).
Availability: Items available for loan: UVAS Library [Call number: 2262-T] (1).
6.
In-Vivo Efficacy Of Aqueous And Alcoholic Extracts Of Hedera Helix Against Fasciolosis In Sheep
by Muhammad Faisal Raza (2008-VA-206) | Dr. Muhammad Avais | Dr. Muhammad Hassan Saleem | Dr. Imran Rashid.
Material type: ; Literary form:
not fiction
Publisher: 2015Dissertation note: The liver fluke, Fasciola hepatica, is a common parasite of ruminants in many countries in the temperate climates and often causes severe economic losses. Infection with both F. hepatica and F. gigantica may also occur in occasionally causing large scale epidemics. These parasites adversely affect the health status of animals and cause enormous economic losses Triclabendazole (TCBZ) is a worldwide, one of the most used drugs for the control of fasciolosis but the efficacy may depend on the susceptibility of the strain involved in the drug. However, resistance of the liver fluke to TCBZ in naturally infected sheep has been reported. Plants are being used as anthelmintic as the allopathic drugs today which offer substitute to the use of synthetic chemicals. This study was carried out to evaluate the efficacy of aqueous and methanolic extracts of H. helix against fasciolosis in sheep. For this purpose, adult sheep was used for In vivo trials. Animals were screened for the presence of liver fluke and 30 sheep positive for liver fluke infestation were used for therapeutic trials. They were divided into 5 groups. Group A and B were given single dose of aqueous extract of Hedera helix at concentrations of 1.13 gm/kg and 2.25 gm/kg body weight respectively. Animals in C and D group were given 1.13 gm/kg and 2.25 gm/kg body weight respectively, while sheep in E group were given Triclabendazole at dose rate of 1 ml/5kg body weight. Fecal samples from each animal was collected in the morning, starting from day 0 pre-treatment and at days 4,7 and 14 post-treatment and were evaluated for the EPG through Modified McMaster method.
The comparative efficacy was analysed by two way analysis of variance (ANOVA) using SPSS 20.0. A probability levels ˂0.05 was considered as statistically significant.
During In vivo experiment all the treatments showed significant reduction in EPG. The maximum reduction in EPG showed by crude aqueous extract (CAE) at day 4 post treatment at 1.13 gm/kg dose rate was 20.0012 %. There were no significant reduction in EPG at day 7 and 14 day for aqueous extract. The maximum reduction by aqueous extract at day 4 at dose
Summary
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rate of 2.25 gm/kg was 40.47 % and at day 7 it was 45.24 %, whereas there was no significant reduction at day 14 post treatment. The maximum reduction showed by crude Methanolic extract at dose rate of 1.13 gm/kg at day 4 was 29.54 %. At day 7 post treatment the reduction of EPG was 40.09 % and at day 14 the reduction was 43.18 %. The maximum reduction showed by crude Methanolic extract at dose rate of 2.25 gm/kg at day 4 was 56.09 %. At day 7 post treatment the reduction of EPG was 64.85 % and at day 14 the reduction was 68.29 %. The positive control treated with Triclabendazole showed the maximum reduction of 97.5 % in eggs per gram (EPG).
So it is concluded from the study that the extracts of Hedera helix have the anthelmintic efficacy against liver fluke. The increase in dose rate of extracts may give better results. So there is need to conduct further studied and more trials on Hedera helix extracts under field conditions to achieve maximum efficiency levels. Availability: Items available for loan: UVAS Library [Call number: 2431-T] (1).
