Serodiagnosis of nasal myasis in camels (Camelus dromedaries) in Egypt using third larval instar affinity-purified glycoprotein.

The larvae of Cephalopina titillator cause nasopharyngeal myiasis in camels, which parasitize the living tissues of the nasal and paranasal sinuses, pharynx, and larynx. C. titillator infestation adversely affects camel health, meat, and milk production, and can even cause death. In our study, to improve the immunodiagnosis of camel nasal myiasis, a sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed and evaluated using the Concanavalin-A (Con-A) affinity purification for the C. titillator-N-acetylglucosamine (Ct-GlucNAc) glycoprotein fraction from third larval instars as an antigen for detecting C. titillator antibodies. Crude antigens were prepared from larval instars of C. titillator and evaluated by indirect ELISA. The third C. titillator larval antigen (L3Ct) had the highest protein content (P < 0.001) and the best diagnostic value; chi-square = 235 (P < 0.001). Four glycoprotein fractions were purified separately from the L3Ct antigen by Con-A purification and evaluated. The Ct-GlucNAc glycoprotein fraction was the fraction of choice with the highest diagnostic accuracy (P < 0.05). Using Ct-GlucNAc as a coating antigen, indirect ELISA showed a 99.3% sensitivity for positive results in camel myiasis samples and 100% specificity for negative results in healthy camel samples. The diagnostic accuracy was 99.7%, and no cross reactivity was detected for other parasitic diseases. The indirect ELISA results were confirmed by the western immunoblotting which was characterized by comparing sera from naturally infested dromedary camels with C. titillator, sera from healthy camels and sera from camels with other parasitic infections (Echinococcus granulosus, Fasciola gigantica, Hard ticks; Hyalomma dromedarii, Trichostronglid sp., Eimeria spp., and Cryptosporidium sp.). Immunoreactive antigenic bands of 63, 50, 30 and 18 kDa were predominantly detected in sera from camels with nasopharyngeal myiasis and didn't react with healthy and camel's sera from other parasitic infections. However, seven immunoreactive bands appeared at 120, 70, 63, 48, 35, 29, and 19 kDa in the crude L3Ct antigen. In addition, a positive rate of C. titillator immunodiagnosis was detected by indirect ELISA (48.6%, chi-square = 483, P < 0.001), which was significantly greater than that of postmortem diagnosis (31%). In conclusion, the current study introduces a new diagnostic immunoaffinity glycoprotein fraction of C. titillator 3rd larval instar-based ELISA as a highly accurate, simple and fast method to detect specific antibodies of nasal myiasis in camels.

A Novel Designed Sandwich ELISA for the Detection of Echinococcus granulosus Antigen in Camels for Diagnosis of Cystic Echinococcosis.

Echinococcus spp. are important cosmopolitan zoonotic parasitic tapeworms that cause a disease called hydatidosis or cystic echinococcosis (CE), which has remarkable economic losses. The objective of our study was to develop a specific IgG polyclonal antigen-based ELISA (Sandwich ELISA; capture ELISA) method for the detection of circulating Echinococcus granulosus (E. granulosus) antigens in camels infected with hydatid cysts before slaughtering and its application in serodiagnosis of CE in animals to assess the positive rate of hydatidosis in camels slaughtered in Giza governorate abattoirs in Egypt. In this study, molecular identification of Echinococcus sp. isolate was performed based on the NADH dehydrogenase subunit 1 (NAD1) gene, revealing the isolate (GenBank: OQ443068.1), which is identical to the G6 E. granulosus sensu lato genotype. The positive rate of hydatid cysts was determined in slaughtered camels' organs (n = 587). The results revealed that hydatid cysts were found in 46.5% (273/587) of the examined camels. Pulmonary echinococcosis was significantly more prevalent in the slaughtered camels (60%, 164/273) than hepatic echinococcosis (39.9%, 109/273), (p = 0.001, Chi Square = 11.081). Cyst fertility rates were higher in hepatic (90.8%, 99/109) than in pulmonary cysts (83.5%, 137/164) and the most viable protoscoleces were recorded from fertile the hepatic cysts (67.85 ± 12.78). In this study, hydatid cyst germinal layer antigen (GlAg) was isolated and used for the immunization of rabbits to raise IgG polyclonal antibodies (anti-Echinococcus GlAb IgG). These IgG polyclonal antibodies were purified by affinity chromatography using a protein A column, then labeled with horseradish peroxidase. Electrophoretic analysis of IgG polyclonal antibodies and crude GlAg was performed in 10% polyacrylamide gels. The SDS-PAGE revealed four bands at molecular weights of 77 kDa, 65 kDa, 55 kDa, and 25 kDa. The Sandwich ELISA was performed to evaluate the sensitivity and specificity and cross-reactivity of the prepared IgG polyclonal antibodies. The circulating hydatid antigen was found in 270 out of the 273 samples with hydatidosis, with a sensitivity of 98.9% (270/273), a specificity of 94.9% (296/312) and a diagnostic efficacy of 96.8%. Regarding the cross reactivity, anti-Echinococcus GlAb IgG showed a low cross-reactivity with Fasciola gigantica infected camel sera (3/8), and Myiasis (Cephalopina titillator larvae; 3/20). No cross-reactivity was recorded with uninfected camel sera (negative sera for E. granulosus), and no cross-reactivity was found with antigens of Eimeria spp., Toxoplasma gondii, Cryptosporidium sp., and Hyalomma dromedarii (ticks' infestation). Then, Sandwich ELISA was conducted again to detect E. granulosus antigen in all the collected camel sera, which resulted in a 48.7% (286/587) positive rate of CE compared to 46.5% (273/587) using a postmortem inspection (PM diagnosis) (p = 0.5, Chi Square = 0.302). In conclusion, the Sandwich ELISA technique introduced in this study appears to be a sufficiently sensitive diagnostic assay for the detection of camels' echinococcosis using anti-Echinococcus GlAb IgG. In addition, it might offer a significant medical and veterinary importance in helping the early detection of hydatidosis, as well as its early treatment.

Molecular characterization of some equine vector-borne diseases and associated arthropods in Egypt.

Equine vector-borne diseases (EVBDs) are emerging and re-emerging diseases, and most of them are zoonotic. This study aimed to investigate EVBDs in equines and associated arthropods (ticks and flies) from Egypt using molecular analyses, in addition to a preliminary characterization of associated ticks and flies by the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and molecular techniques. In this study, 335 blood samples were obtained from equines that appeared to be in good health (320 horses and 15 donkeys) in Cairo and Beni Suef provinces, Egypt. From the same animals, 166 arthropods (105 sucking flies and 61 ticks) were collected. Ticks and flies were preliminary characterized by the MALDI-TOF and molecular tools. Quantitative PCR (qPCR) and standard PCR coupled with sequencing were performed on the DNA of equines, ticks, and flies to screen multiple pathogens. The MALDI-TOF and molecular characterization of arthropods revealed that louse fly (Hippobosca equina) and cattle tick (Rhipicephalus annulatus) infesting equines. Anaplasma platys-like (1.6%), Anaplasma marginale (1.6%), Candidatus Ehrlichia rustica (6.6%), a new Ehrlichia sp. (4.9%), and Borrelia theileri (3.3%) were identified in R. annulatus. Anaplasma sp. and Borrelia sp. DNAs were only detected in H. equina by qPCR. A. marginale, Anaplasma ovis, and Theileria ovis recorded the same low infection rate (0.6%) in donkeys, while horses were found to be infected with Theileria equi and a new Theileria sp. Africa with recorded prevalence rates of 1.2% and 2.7%, respectively. In conclusion, different pathogens were first detected such as A. platys-like, Candidatus E. rustica, and a new Ehrlichia sp. in R. annulatus; A. marginale, A. ovis, and T. ovis in donkeys; and a new Theileria sp. "Africa" in horses.

Copro-microscopical and immunological diagnosis of cryptosporidiosis in Egyptian buffalo-calves with special reference to their cytokine profiles.

Cryptosporidiosis is considered to be one of the most devasting gastrointestinal diseases in calves. The aim of this study was to investigate Cryptosporidium parvum infection (C. parvum) in buffalo-calves with both copro-microscopic examination and enzyme linked immunosorbent assay (ELISA) using two C. parvum prepared antigens with regards to their cytokines profile; interferon- γ (IFN-γ), interleukin (IL)-12 and IL-14 to achieve a proper diagnosis. All collected buffalo- calves' fecal samples were examined by modified Ziehl-Neelsen staining technique. ELISA was performed to evaluate the diagnostic accuracy of the two C. parvum prepared antigens; crude whole oocyst (CWO) and crude sonicated oocyst (CSO) in detection of anti-C. parvum IgG in buffalo-calves' sera. As well, concentrations of INF-γ, IL-12 and IL-14 in the buffalo-calves' serum samples were estimated. The results revealed that the overall parasitological incidence of cryptosporidiosis was 40%. However, the serological diagnosis by ELISA assay showed 53.75% and 27.5% when using CWO and CSO antigen, respectively. Also, the diagnostic efficacy parameters of both antigens; CWO and CSO showed a significant high specificity (83.3%) achieved by CSO antigen and a high sensitivity (71.8%) by CWO antigen. The levels of INF-γ, IL-12 and IL-14 were significantly increased in positive Cryptosporidium infected group by both coprological and serological assays followed by the group which was positive for cryptosporidiosis by copro-microscopic examination only. The present study concluded that a combination of coprological and serological examination with reference to the cytokines profile is needed for proper diagnosis of cryptosporidiosis in buffalo-calves.

Cellular immune response and scanning electron microscopy in the evaluation of Moringa leaves aqueous extract effect on Cryptosporidium parvum in buffalo intestinal tissue explants.

Cryptosporidium is an apicomplexan parasite of human and animals and is considered as an important co-factor in neonatal diarrhea. In this study, an explant culture was used as an in vitro model of buffalo intestine to evaluate the effect of Moringa leaves extract on Cryptosporidium parvum (C. parvum) oocysts using light and scanning electron microscopy and measuring IFN-γ, IL-12 and IL-14 in the culture supernatants. C. parvum oocysts were collected from naturally-infected calf feces, isolated, excysted and then co-inoculated with ileal tissue explants culture medium. The prepared Moringa leaves extract was then introduced to the infected tissues in the concentrations of 100 mg/ml and 300 mg/ml. After 24 h, tissues were collected and processed for light and scanning electron microscopy. Also, culture supernatants were collected for cytokines measurement. C. parvum parasitophorous vacuoles were found attached to the surface of tissue in Cryptosporidium-infected ileal tissue explants. High magnification imaging of ileal tissue explants using scanning electron microscopy showed that Moringa leaves extracts had a great effect on Cryptosporidium-infected ileal tissue explants. There was a high significant (P < 0.001) increase in IFN-γ, IL-12 and IL-14 (375, 275 and 90 pg/ml, respectively) in the supernatants of infected non-treated ileal tissue explant culture plate wells compared to the control non-infected ones (74.66, 75 and 50 pg/ml, respectively). A concentration of 100 mg/ml Moringa extract exhibited the highest anticryptosporidial effect causing a significant decrease in IFN-γ, IL-12 and IL-14 levels (225, 150 and 65 pg/ml, respectively) compared with supernatants of infected non-treated ileal explant culture plate wells. In this study, explant culturing of buffalo ileal tissues allowed investigating the pathogenesis of cryptosporidiosis using light and scanning electron microscopy and studying changes in cytokine levels in tissues with and without Moringa leaves extract treatment. This model could help to understand the regulation of intestinal secretory and inflammatory responses, and could be useful for the screening of potential anticryptosporidial candidate compounds.

Protective effect of Eimeria stiedae coproantigen against hepatic coccidiosis in rabbits.

Immunization of rabbits against hepatic coccidiosis was tried. The animals were immunized twice with Eimeria stiedae coproantigen in freund's adjuvant with two week intervals. The rabbits were challenged orally with sporulated E. stiedae oocysts one week post last injection. The protection was assessed by number of oocysts output, number of focal liver lesions, clinical sings and antibody response. The immunization resulted in 70% protection from infection and decline in oocysts count. High level of IgG response in immunized rabbits than control infected ones was occurred and being responsible for the recorded protection. The electrophoretic make up of the coproantigen and oocyst antigen showed different patterns of separation. Common as well as specific bands to each antigen were identified. Using the rabbit sera after 3 weeks post vaccination in immunoblot assay, immunogenic components were detected of molecular weight 155, 103, 74, 66, 44, 22KD with coproantigen and 155, 115, 57, 26 KD with oocyst antigen. While, the rabbit sera after 2 and 4 weeks post challenge reacted with oocyts antigen, in immunoblot assay, revealing immunogenic bands of molecular weight 155, 115, 57, 26, 22KD and 155, 115, 57, 25 KD respectively. Bands of 22KD and 155KD are partially responsible for eliciting host protective immune response where they were recognized by immunized sera in coproantigen and by immunized infected sera in oocyst antigen.

Trichinella spiralis: affinity purified antigen based diagnosis and immunoprophylaxis.

The current research introduces a trial to develop vaccine candidate against trichenosis. A method of affinity chromatography was adopted to purify a Trichinella spiralis larval extract. The isolated fraction resolved into six bands of 148 KDa, 133 KDa, 118.5 KDa, 101 KDa 98.5 KDa and 79.5 KDa as observed by SDS-PAGE. The diagnostic value of this fraction was checked against antibodies regularly collected from rats experimentally infected with trichinosis compared with that of crude larval extract by ELISA. The crude extract detected the antibodies as early as one week post infection and the maximum level was recorded four weeks post infection. The advantage of the isolated fraction over the crude extract in trichinosis diagnosis was clearly observed at high serum dilution reached to 1:4000. The protective value of the isolated fraction was also investigated. Rats immunized subcutaneously with affinity purified larval extract with Freund's adjuvant showed reduction in worm burden reached to 82%. IgG antibody response in immunized rats was higher than that of control infected animals as measured by ELISA. This response might be partially responsible for the observed protection.

Identification and diagnostic evaluation of cross-reactive antigen between hydatid cyst fluid of Echinococcus granulosus and Trichinella spiralis larval extract.

A cross reactive fraction was isolated from hydatid cyst fluid antigen of E. granulosus using CNBr Sepharose 4B affinity chromatography in which anti-T. spiralis antibodies were coupled with the column. Biochemical characterization of the isolated fraction included the use of SDS-PAGE, isoelectric focusing and amino acid analysis. The fraction showed 5 polypeptides of 165, 95.5, 63.5, 30.6 and 24 KDa. The isoelectric points of these polypeptides were 7.8, 7.2, 6.7, 6.2 and 5.7. The fraction exhibited 17 amino acids and was rich in tyrosine (20.81) and glutamic (15.28) ug/100 mg. The fraction proved higher potency in the diagnosis of experimental trichinellosis in rats than echinococcosis in dogs by ELISA. All experimentally infected animals reacted positively, recording 100% diagnostic sensitivity. Collectively, the present study proved that the hydatid cyst fluid cross-reactive fraction could be used in the diagnosis of trichinellosis at different intervals of infection and as early as 1 week post infection.

Cross-protection induced by cross-reactive antigen against Fasciola gigantica and Trichinella spiralis infections.

Trichinella spiralis /Fasciola gigantica cross-reactive fraction was purified from T. spiralis larval extract by affinity column chromatography in which CNBr-Sepharose 4B was coupled with F. gigantica antibodies. The fraction consisted of six polypeptides of 191KDa, 178KDa, 149KDa, 106KDa, 101KDa and 32 KDa as revealed by SDS-PAGE. Analysis of the free amino acids of the fraction revealed 17 amino acids with high proportions of tyrosine and glutamic. Immunization of rabbits subcutaneously with the cross-reactive fraction in Freund's adjuvant followed by challenge with F. gigantica metacercariae resulted in reduction in worm burdens reached to 47.8%. While immunization of rats with the same fraction in Freund's adjuvant followed by infection with T. spiralis larvae resulted in reduction in worm count reached to 74%. IgG antibody response in rabbits increased due to immunization to reach its maximum value at the time of infection and then decreased gradually up to the end of the experiments. But. remained higher than the level in non vaccinated control animals. In rat sera, IgG level increased due to vaccination but the level recorded its optimum value one week post infection and then decreased. Thus the cross-reactive antigen proved cross-protection with the protection inducing capability against both diseases.

Fasciola gigantica: immunization of rabbits with proteins isolated from coproantigen.

Two fractions were isolated from coproantigen by ion-exchange chromatography in which DEAE cellulose was utilized. Both fractions and crude antigen were characterized by SDS-polyacrylamide gel electrophoresis which revealed 13 bands of molecular weight ranged from 205-31 in crude coproantigen. While fraction I resolved into six bands of molecular weight 198, 178, 148, 111, 101 & 45. Fraction II showed seven bands of 191 KDa, 178KDa, 166KDa, 118KDa, 98.5KDa, 72KDa & 32KDa. Fraction 1I was higher immunoreactivity than fraction by ELISA. Three immunoreactive bands of 191KDa, 118KDa & 98.5KDa were identified in fraction II using immunoblot assay. Five bands of 178KDa, 148KDa, 111KDa, 101KDa & 45KDa were detected in fraction I. Immunization of rabbits twice with fraction II in Freund's adjuvant with two weeks interval followed by challenge with F. gigantica metacercariae resulted in 66.6% protection from infection. The protection was assessed by detect-ion of hepatic damage, worm recoveries and antibody response. High level of IgG response in vaccinated rabbits than control infected ones occurred and being responsible for the recorded protection.

Induction of protective antibody response in rabbits against fascioliasis with Toxocara/Fasciola cross- reactive defined antigen.

A method of affinity chromatography purification of Toxocara vitulorum antigen cross- reacts with Fasciola gigantica antiserum is described. Characterization of the isolated cross- reactive fraction by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and amino acid analysis resulted in a fraction consists of five polypeptides of 137.7KDa, 81KDa, 75KDa, 48KDa and 21.6KDa with isoelectric points of 8, 7.5, 7.2, 6.7 and 6.6. Seventeen amino acids were identified in the fraction with high proportions of only two of them (tyrosine and glutamic). Rabbits immunization with this identified T. vitulorum cross- reactive antigen in Freund's adjuvant followed by challenge with F. gigantica metacercariae resulted in 60% reduction in worm burden over control infected rabbits. Higher IgG level was detected in vaccinated rabbits four weeks post first immunization than control infected ones and remained high up to the end of the trial.

Cross-reaction: a common trait among helminthes.

Cross-reaction between three important zoonotic helminths, Fasciola gigantica, Trichinella spiralis and Echinococcus granulosus, was proved by ELISA. Cross-binding activities in the prepared antisera were strongly directed towards protoscolices and hydatid fluid antigens of E. granulosus rather than to F. gigantica and T. spiralis antigens. Two sets of polypeptides were identified in each antigen by immunoblot; species-specific and cross-reactive. Cross-reactive components in F. gigantica antigen were 205 KD, 178 KD, 166 KD, 106 KD, 100 KD, 65 KD, 45 KD and 32 KD. While, cross-reactive molecules in T. spiralis antigen were 205 KD, 191 KD, 166 KD, 148 KD, 132 KD, and 32 KD. In protoscolices antigen six cross-reactive components were identified, 205 KD, 191 KD, 149 KD, 106 KD, 45 and 32 KD. Moreover, 205 KD, 190 KD, 177 KD, 149 KD, 103 KD and 33 KD were detected in hydatid fluid antigen by heterologous antisera. Interestingly, three polypeptides of 205 KD, 149 KD and 32 KD showed broad immunogenicity with the developed antisera raising the prospect of being putative common immunoprophylactic components.

Comparative immunodiagnostic approach of toxocariasis in buffalo calves.

Five Toxocara vitulorum antigens were utilized to diagnose natural toxocariasis in buffalo calves by ELISA. Adult antigen was proved to be the most potent one. The second potent antigen was egg antigen followed by excretory secretory antigen of male worms then female worms. The coproantigen was the least potent one. The electrophoretic make-up of the antigens, examined by SDS-PAGE, revealed different patterns of separation. Common as well as specific component(s) to each antigen were identified. Employing naturally infected buffalo calf sera in immunoblot assay, five immunogenic components were detected in adult antigen of molecular weight 191 KD, 166 KD, 102 KD, 65 KD and 54 KD. The reactive polypeptides in egg antigen were 191 KD, 105 KD, 99 KD and 79 KD. In coproantigen, six bands were identified. These components were of molecular weight 191 KD, 178 KD, 166 KD, 124 KD, 96 KD and 65 KD. Five components of molecular weight 191 KD, 166 KD, 102 KD, 96 KD and 65 KD were immunogenic in excretory/secretory antigen of male worms. Only four polypeptide of 191 KD, 166 KD, 102 KD and 66 KD were identified in excretory/secretory female antigen. Of interest is the immunogenic component of 54 KD expressed only by adult extract in immunoblot assay. This component could be responsible for the immunodiagnostic advantage of adult worm antigen and it deserves further studies to evaluate its diagnostic value.