Vrednovanje metode RT-PCR za određivanje sudanskih serotipova virusa epizootske hemoragijske bolesti.

Epizootic hemorrhagic disease (EHD) is an infectious non-contagious disease of deer and cattle. At least 2 serotypes of EHD virus, designated EHDV-4 and EHDV-318, are enzootic in the Sudan. To facilitate clinical disease investigation and control of the disease a rapid diagnostic assay is urgently needed. A reverse transcriptase (RT) polymerase chain reaction (RT-PCR) protocol, previously reported for detection of the United States EHDV serotypes 1 and 2 ribonucleic acid (RNA) in cell culture and clinical specimens, was evaluated for detection of the Sudanese EHDV serotypes. RNA from Sudanese isolates of EHDV-4 and EHDV-318, propagated in cell cultures, were detected by the described RT-PCR-based assay. The specific 387 bp PCR products were visualized on ethidium bromide stained agarose gel. Amplification product was not detected when the EHDV RT-PCR-based assay was applied to RNA from Sudanese bluetongue virus (BTV) serotype 4 (BTV-4) or total nucleic acid extracts from uninfected BHK-21 cells. The scientific observations reported in this paper indicated that the previously described EHDV RT-PCR assay could be applied for detection of EHDV infection among the Sudanese susceptible animal populations.Epizootska hemoragijska bolest je zarazna nekontagiozna bolest jelena i goveda. U Sudanu se enzootski javljaju najmanje 2 serotipa virusa te bolesti i to EHDV-4 i EHDV-318. Za njezinu pouzdanu dijagnostiku i kontrolu potrebne su brze suvremene metode. Za dokaz serotipova izdvojenih u Sudanu vrednovana je lančana reakcija polimerazom uz predhodnu reverznu transkripciju (RT-PCR), opisana u SAD-u za dokaz RNA serotipova 1 i 2 virusa epizootske hemoragijske bolesti u staničnoj kulturi i kliničkom materijalu. RNA sudanskih izolata EHDV-4 i EHDV-318 umnoženih u staničnoj kulturi dokazana je tom metodom. Specifični proizvodi PCR-a od 387 parova baza dokazani su u agaroznom gelu pomoću bojanja etidijevim bromidom. Umnažanje specifičnog slijeda nije dokazano kad je kao izvor nukleinske kiseline rabljen serotip 4 virusa bolesti plavog jezika ili ekstrakti nukleinske kiseline iz neinficirane stanične kulture BHK-21. Na temelju dobivenih rezultata zaključuje se da je prije opisani test u potpunosti prikladan i za određivanje sudanskih serotipova virusa epizootske hemoragijske bolesti

Development of real-time PCR assay for simultaneous detection and genotyping of cystic echinococcosis in humans and livestock

Objective: To develop and evaluate a single-tube one-step real-time quantitative PCR (qPCR) assay for simultaneous diagnosis and genotyping of cystic echinococcosis (CE) in humans and livestock in the Sudan, and to compare it with conventional PCR assay. Methods: Hydatid cysts were obtained from slaughtered animals and from humans after surgical interventions. DNA from the hydatid cysts and associated germ layers was extracted using a commercially available kit. The mitochondrial NADH dehydrogenase subunit 1 (NAD1) was used as a target for PCR amplifications. qPCR and conventional nested PCR assays were compared in this study. Results: The qPCR assay amplified the NAD 1 gene of hydatid cysts on melting temperature generated at 80 °C. Ten-folds serial dilutions of DNA with known dilution of 1 × 106 to 1 × 101 (1 ng–1 fg) resulted in detection of as little as 1 fg of DNA with an R2 value equivalent to 0.997. Similar sensitivities were encountered from both qPCR and the conventional nested PCR. The two assays did not amplify DNAs from Fasciola gigantica, Taenia saginata, Schistosoma bovis and DNA-free samples (negative controls). The PCR amplified products were purified for subsequent sequencing. The sequence data were analysed to insure the specificity of the amplified PCR products and to identify the genotype(s) of hydatid cysts. All cysts were identified as E. canadensis genotype 6 (G6). Conclusions: The developed qPCR should be used as a rapid and reliable assay for diagnosis and genotyping of CE. The assay is highly recommended for the epidemiological surveillance in humans and livestock in endemic countries

Vrednovanje metode RT-PCR za određivanje sudanskih serotipova virusa epizootske hemoragijske bolesti.

Epizootic hemorrhagic disease (EHD) is an infectious non-contagious disease of deer and cattle. At least 2 serotypes of EHD virus, designated EHDV-4 and EHDV-318, are enzootic in the Sudan. To facilitate clinical disease investigation and control of the disease a rapid diagnostic assay is urgently needed. A reverse transcriptase (RT) polymerase chain reaction (RT-PCR) protocol, previously reported for detection of the United States EHDV serotypes 1 and 2 ribonucleic acid (RNA) in cell culture and clinical specimens, was evaluated for detection of the Sudanese EHDV serotypes. RNA from Sudanese isolates of EHDV-4 and EHDV-318, propagated in cell cultures, were detected by the described RT-PCR-based assay. The specific 387 bp PCR products were visualized on ethidium bromide stained agarose gel. Amplification product was not detected when the EHDV RT-PCR-based assay was applied to RNA from Sudanese bluetongue virus (BTV) serotype 4 (BTV-4) or total nucleic acid extracts from uninfected BHK-21 cells. The scientific observations reported in this paper indicated that the previously described EHDV RT-PCR assay could be applied for detection of EHDV infection among the Sudanese susceptible animal populations.Epizootska hemoragijska bolest je zarazna nekontagiozna bolest jelena i goveda. U Sudanu se enzootski javljaju najmanje 2 serotipa virusa te bolesti i to EHDV-4 i EHDV-318. Za njezinu pouzdanu dijagnostiku i kontrolu potrebne su brze suvremene metode. Za dokaz serotipova izdvojenih u Sudanu vrednovana je lančana reakcija polimerazom uz predhodnu reverznu transkripciju (RT-PCR), opisana u SAD-u za dokaz RNA serotipova 1 i 2 virusa epizootske hemoragijske bolesti u staničnoj kulturi i kliničkom materijalu. RNA sudanskih izolata EHDV-4 i EHDV-318 umnoženih u staničnoj kulturi dokazana je tom metodom. Specifični proizvodi PCR-a od 387 parova baza dokazani su u agaroznom gelu pomoću bojanja etidijevim bromidom. Umnažanje specifičnog slijeda nije dokazano kad je kao izvor nukleinske kiseline rabljen serotip 4 virusa bolesti plavog jezika ili ekstrakti nukleinske kiseline iz neinficirane stanične kulture BHK-21. Na temelju dobivenih rezultata zaključuje se da je prije opisani test u potpunosti prikladan i za određivanje sudanskih serotipova virusa epizootske hemoragijske bolesti

Experimental bovine schistosomiasis in zebu calves Esquistossomose bovina experimental em bezerros zebuínos

Five calves were each experimentally infected with 30,000 cercariae of Schistosoma bovis and three calves were kept as controls. S. bovis eggs first appeared in feces of the infected animals by week 5 post infection and all animals were shedding by week 6 post infection. Between week 7 and 9 post infection, where fecal egg counts were highest, the infected animals developed mucoid and then hemorrhagic diarrhoea and they became dull and depressed. Packed cell volumes and hemoglobin concentrations of the infected animals showed progressive reductions compared to the uninfected control calves. The animals were necropsied and perfused at week 12 post infection and tissue egg densities and worm burden were determined.Cinco bezerros foram infectados experimentalmente com 30.000 cercarias de Schistosoma bovis e três bezerros foram usados como controle. Ovos de S. bovis apareceram inicialmente nas fezes dos animais infectados pela 5ª semana após infecção e todos os animais estavam eliminando ovos na 6ª semana após infecção. Entre as 7ª e 9ª semanas após infecção, nas quais a quantidade fecal de ovos era mais alta, os animais infectados manifestaram diarréia com muco seguida por disenteria e os animais apresentando-se morimbundos e depressivos. PCV e concentração da hemoglobina dos animais infectados demonstrou redução progressiva em comparação com os bezerros não infectados. Os animais foram necropsiados na 12ª semana para determinação da densidade de ovos nos tecidos e quantidade de parasitas

ELISA FOR BOVINE SCHISTOSOMIASIS VACCINE EVALUATION: A PRELIMINARY REPORT

Six calves were immunized with schistosomula of Schistosoma bovis irradiated at 3 or 20 Kilorad (Krad) and three calves were kept as controls. Twenty four weeks post immunization, three calves (one from the 20 Krad and two from the 3 Krad group) were challenged with normal cercaria of S. bovis. The immune response was monitored by agar gel immunodiffusion (AGID) and the Enzyme Linked Immunosorbent Assay (ELISA) using adult worm antigen. Using AGID, precipitin lines were observed only with sera from challenged animals. Using ELISA, the immune response of the vaccinated calves was first detected by 2-3 weeks, peaking by 6-8 weeks post vaccination. The immune response of the three challenged calves was elevated by 2 weeks post challenge, peaking at 8-10 weeks post challenge and remained high throughout the experimental period. This study suggests that ELISA could be used for diagnosis of bovine schistosomiasis

Development and evaluation of real-time loop-mediated isothermal amplification assay for rapid detection of cystic echinococcosis

Cystic echinococcosis (CE) or hydatidosis, caused by the larval stage of Echinococcus granulosus (EG)-complex, is a neglected parasitic disease of public health importance. The disease is endemic in many African and Mediterranean countries including the Sudan. The objective of the present study was to develop and evaluate a real-time loop-mediated isothermal amplification (LAMP) assay for simple and rapid detection of CE in humans and domestic live stock in Sudan. A set of six LAMP primers, designed from the mitochondrial NADH-1 gene of EG cattle strain of genotype 5 (G5), was used as a target for LAMP assay. The assay was performed at a constant temperature (63 °C), with a real-time follow-up using a LightCycler and fluorochrome dye. Following amplification cycles in a simple water bath, LAMP products were observed for color change by naked eye and were visualized under UV light source using agarose gel electrophoresis. The real-time LAMP assay identified a variety of hydatid cysts strains recovered in the Sudan, including Echinococcus canadenses (G6) and Echinococcus ortleppi (G5). Real-time LAMP positive results were detected by the presence of an amplification curve, whereas negative results were indicated by absence of fluorescence detection. Positive LAMP results appeared as a bluish-colored reaction as observed by naked eye, whereas negative LAMP results were observed as purple-colored reaction. The sensitivity studies indicated that the LAMP assay detected as little as a 10 fg of parasite DNA. There was 100 % agreement between results of the LAMP assay and our previously described nested PCR when testing 10-fold serial dilution of DNA extracted from EG-complex hydatid cyst. However, there was no cross-reactivity with other parasites including cysticercus bovis, Fasciola gigantica, and Schistosoma bovis and nucleic acid free samples. The developed LAMP assay would be expected to prove highly significant in epidemiological surveys of CE in developing countries or areas of resource-poor settings for both ease of use and cos