Kinetika tvorbe protutijela u teladi imunizirane glutation-S transferazom metilja Schistosoma mansoni.

Six calves were immunized with Schistosoma mansoni glutathione-S transferase (GST) antigen vaccine, expressed in E. coli (GST-coli) or yeast (GST-yeast). Six calves were injected with extracts of E. coli or yeast and kept as controls. Two experimentally infected calves were kept as source of infection and to provide positive antiserum to Schistosoma bovis. Kinetics of antibody response of the immunized calves was monitored by Western blots and enzyme-linked immunosorbent assay (ELISA). Using Western blots, the immunized calves produced specific IgG antibodies, which recognized S. mansoni GST (S.mGST) antigen, whereas the control calves did not. Using ELISA, antibodies to S.mGST antigen were detected in sera from S.mGSTcoli and GST-yeast vaccinated calves, but not in sera from control calves or S. bovis experimentally infected calves. In addition, the ELISA failed to detect S.mGST specific antibodies in sera from immunized calves or their controls when S. bovis adult worm extract was used as an antigen. The results of this study indicated that S.mGST antigen has no diagnostic potential for detection of bovine schistosomosis, caused by S. bovis infection, in susceptible ruminants.Šestero teladi bilo je imunizirano antigenom pripravljenim od glutation-S-transferaze (GST) metilja Schistosoma mansoni, koji je bio proizveden u bakteriji E. coli (GST-coli) ili kvascu (GST-kvasac). Kontrolnih šestero teladi dobilo je ekstrakt bakterije E. coli ili kvasca. Dva pokusno invadirana teleta držana su kao izvor invazije te za dobivanje antiseruma za metilj Schistosoma bovis. Kinetika tvorbe protutijela u cijepljene teladi bila je praćena postupkom Western blot i imunoenzimnim testom. Pomoću postupka Western blot u imunizirane teladi ustanovljena su protutijela IgG specifična za antigen S. mansoni (S.mGST), koja nisu dokazana u kontrolne teladi. Imunoenzimnim testom bila su dokazana protutijela za antigen S.mGST u uzorcima seruma teladi imunizirane antigenom S.mGST coli i GST-kvasac, ali ne i u serumima kontrolne teladi i teladi pokusno invadirane metiljom S. bovis. Osim toga, imunoenzimnim testom nisu dokazana protutijela specifična za S.mGST u serumima imunizirane i kontrolne teladi kad je kao antigen bio rabljen ekstrakt adulta metilja S. bovis. Rezultati istraživanja su pokazali da S.mGST kao antigen nije prikladan za dijagnosticiranje shistosomoze goveda uzrokovane metiljom S. bovis u prijemljivih preživača

Dokaz američke serološke skupine orbivirusa upotrebom višestruke RT-PCR.

A multiplex RT-PCR assay, for simultaneous detection and differentiation of United States serogroup of Orbiviruses, including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in cell culture, was developed. Sets of primers were designed to hybridize to genome segment six of EHDV-2 and to genome segment 10 of BTV-10. The RT-PCR assay utilized a single-tube PCR amplification in which EHDV and BTV primers were used simultaneously in a multiplex format. The EHDV primers produced a 387 base pair (bp) specific PCR product from RNA samples of cell culture-adapted EHDV serotypes 1 and 2, but not from BTV serotypes 2, 10, 11, 13, 17; or from total nucleic acid extract of baby hamster kidney (BHK) cells controls. Likewise, the BTV primers generated a 251-bp amplicon from RNA samples of BTV serotypes 2, 10, 11, 13, and 17, whereas EHDV-1 and EHDV-2; and BHK- 21 cells total nucleic acid extract failed to demonstrate the 251-bp specific BTV PCR product. EHDV and BTV PCR amplification products were easily identified on the basis of size differences on ethidium bromide stained agarose gels. This multiplex RT-PCR assay provides supportive diagnostic method for rapid detection of BTV and/or EHDV infections among susceptible ruminants.Rabljen je višestruki RT-PCR (lančana reakcija polimerazom uz prethodnu reverznu transkripciju) za dokazivanje i razlikovanje serološke skupine virusa bolesti plavog jezika (BPJ) i virus epizootske hemoragijske bolesti jelena (EHB) izdvojenih u SAD-u te uzgojenih na staničnoj kulturi. Određeni su parovi početnica za hibridizaciju na odsječak 6 genoma virusa EHB-2 te na odsječak 10 genoma virusa BPJ-10. Za RT-PCR korištena je PCR amplifikacija u jednoj epruveti u kojoj su početnice za virus EHB i virus BPJ istodobno upotrijebljene u višestrukom obliku. Pomoću početnica virusa EHB proizvedeno je 387 parova baza (pb) specifičnog proizvoda PCR iz uzoraka RNA serotipova 1 i 2 toga virusa prilagođenog na staničnu kulturu, ali ne i iz serotipova 2, 10, 11, 13 i 17 virusa BPJ ili iz ekstrakta ukupne nukleinske kiseline kontrolne stanične kulture bubrega hrčka (BHK). Početnice za virus BPJ dovele su do proizvoda od 251 para baza iz uzoraka RNA serotipova 2, 10, 11, 13 i 17 toga virusa, dok serotipovi 1 i 2 virusa EHB i ukupni ekstrakt nukleinske kiseline stanične kulture BHK-21 nisu doveli do specifičnog proizvoda od 251 para baza. Umnoženi proizvodi PCR za viruse EBH i BPJ mogli su se jednostavno identificirati na osnovi razlika u veličini na agaroznom gelu obojenom etidij-bromidom. Postupak višestruke RT-PCR pruža pomoćnu dijagnostičku metodu za brzo dokazivanje infekcija uzrokovanih virusima BPJ i EHB među prijemljivim preživačima

Predicting Distribution of Aedes Aegypti and Culex Pipiens Complex, Potential Vectors of Rift Valley Fever Virus in Relation to Disease Epidemics in East Africa.

The East African region has experienced several Rift Valley fever (RVF) outbreaks since the 1930s. The objective of this study was to identify distributions of potential disease vectors in relation to disease epidemics. Understanding disease vector potential distributions is a major concern for disease transmission dynamics. DIVERSE ECOLOGICAL NICHE MODELLING TECHNIQUES HAVE BEEN DEVELOPED FOR THIS PURPOSE: we present a maximum entropy (Maxent) approach for estimating distributions of potential RVF vectors in un-sampled areas in East Africa. We modelled the distribution of two species of mosquitoes (Aedes aegypti and Culex pipiens complex) responsible for potential maintenance and amplification of the virus, respectively. Predicted distributions of environmentally suitable areas in East Africa were based on the presence-only occurrence data derived from our entomological study in Ngorongoro District in northern Tanzania. Our model predicted potential suitable areas with high success rates of 90.9% for A. aegypti and 91.6% for C. pipiens complex. Model performance was statistically significantly better than random for both species. Most suitable sites for the two vectors were predicted in central and northwestern Tanzania with previous disease epidemics. Other important risk areas include western Lake Victoria, northern parts of Lake Malawi, and the Rift Valley region of Kenya. Findings from this study show distributions of vectors had biological and epidemiological significance in relation to disease outbreak hotspots, and hence provide guidance for the selection of sampling areas for RVF vectors during inter-epidemic periods

A Spatial Analysis of Rift Valley Fever Virus Seropositivity in Domestic Ruminants in Tanzania

Rift Valley fever (RVF) is an acute arthropod-borne viral zoonotic disease primarily occurring in Africa. Since RVF-like disease was reported in Tanzania in 1930, outbreaks of the disease have been reported mainly from the eastern ecosystem of the Great Rift Valley. This cross-sectional study was carried out to describe the variation in RVF virus (RVFV) seropositivity in domestic ruminants between selected villages in the eastern and western Rift Valley ecosystems in Tanzania, and identify potential risk factors. Three study villages were purposively selected from each of the two Rift Valley ecosystems. Serum samples from randomly selected domestic ruminants (n = 1,435) were tested for the presence of specific immunoglobulin G (IgG) and M (IgM), using RVF enzyme-linked immunosorbent assay methods. Mixed effects logistic regression modelling was used to investigate the association between potential risk factors and RVFV seropositivity. The overall RVFV seroprevalence (n = 1,435) in domestic ruminants was 25.8% and species specific seroprevalence was 29.7%, 27.7% and 22.0% in sheep (n = 148), cattle (n = 756) and goats (n = 531), respectively. The odds of seropositivity were significantly higher in animals sampled from the villages in the eastern than those in the western Rift Valley ecosystem (OR = 1.88, CI: 1.41, 2.51; p<0.001), in animals sampled from villages with soils of good than those with soils of poor water holding capacity (OR = 1.97; 95% CI: 1.58, 3.02; p< 0.001), and in animals which had been introduced than in animals born within the herd (OR = 5.08, CI: 2.74, 9.44; p< 0.001). Compared with animals aged 1-2 years, those aged 3 and 4-5 years had 3.40 (CI: 2.49, 4.64; p< 0.001) and 3.31 (CI: 2.27, 4.82, p< 0.001) times the odds of seropositivity. The findings confirm exposure to RVFV in all the study villages, but with a higher prevalence in the study villages from the eastern Rift Valley ecosystem

A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels. Amplification products were not detected when the PCR-based assay was applied to DNA from other blood parasites including Thieleria annulata, Babesia bigemina or nucleic acid free samples. Application of this nPCR-based assay to clinical samples resulted in direct detection of T. evansi from a variety of tissue samples collected from experimentally infected mice and blood from naturally infected camels. The described nPCR-based assay provides a valuable tool to study the epidemiology of T. evansi infection in camels and other susceptible animal populations

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

Dokaz sudanskih izolata serološke skupine virusa bolesti plavog jezika pomoću lančane reakcije polimerazom (PCR).

The potential use of the recently reported reverse transcriptase (RT) polymerase chain reaction (RT-PCR)-based assay for detection of North American isolates of bluetongue virus (BTV) ribonucleic acid in cell culture was evaluated for detection of Sudanese isolates of BTV. Two pairs of oligoribonucleotide primers, selected from non-structural protein 1 (NS1) gene of BTV-17, were used for RT-PCR amplification. The BTV RT-PCR produced an 826 base pair (bp) amplification product. RNA from Sudanese BTV serotypes 4 and 16, propagated in cell cultures, were detected by this RT-PCR-based assay. Amplification product was not detected when the nested BTV RT-PCR based assay was applied to RNA from closely related Sudanese Orbivirus, epizootic hemorrhagic disease virus (EHDV) serotype 4 and total nucleic acid extracts from uninfected Vero cells. The results of this study indicated that our previously described BTV RT-PCR assay could be used for detection of the Sudanese BTV isolates and possibly other serotypes of BTV serogroup from different continents.Lančana reakcija polimerazom uz reverznu transkripciju (RT-PCR) prethodno je razvijena za dokazivanje ribonukleinske kiseline sjevernoameričkih izolata virusa bolesti plavog jezika (BPJ) umnoženih u staničnoj kulturi te je ujedno rabljena i za dokazivanje sudanskih izolata virusa BPJ. Za RT-PCR amplifikaciju odabrana su dva para oligonukleotidnih početnica na genu koji kodira za nestrukturni protein 1 (NS1) virusa BPJ-17. Pomoću početnica dobiven je amplifikacijski proizvod od 826 parova baza (pb). Ovim RT-PCR postupkom dokazana je RNA sudanskih serotipova 4 i 16 umnoženih u staničnim kulturama. Amplifikacijski proizvod nije bio dokazan kad je metoda bila primijenjena za dokazivanje RNA usko srodnog serotipa 4 sudanskog orbivirusa epizootske hemoragijske bolesti i nukleinske kiseline izdvojene iz neinficiranih stanica Vero. Rezultati ovog istraživanja pokazuju da se ranije opisani RT-PCR za virus BPJ može koristiti za dokazivanje sudanskih izolata virusa BPJ i moguće drugih serotipova serološke skupine virusa BPJ s različitih kontinenata

Syndromic approach to arboviral diagnostics for global travelers as a basis for infectious disease surveillance

Background Arboviruses have overlapping geographical distributions and can cause symptoms that coincide with more common infections. Therefore, arbovirus infections are often neglected by travel diagnostics. Here, we assessed the potential of syndrome-based approaches for diagnosis and surveillance of neglected arboviral diseases in returning travelers. Method To map the patients high at risk of missed clinical arboviral infections we compared the quantity of all arboviral diagnostic requests by physicians in the Netherlands, from 2009 through 2013, with a literature-based assessment of the travelers’ likely exposure to an arbovirus. Results 2153 patients, with travel and clinical history were evaluated. The diagnostic assay for dengue virus (DENV) was the most commonly requested (86%). Of travelers returning from Southeast Asia with symptoms compatible with chikungunya virus (CHIKV), only 55% were tested. For travelers in Europe, arbovirus diagnostics were rarely requested. Over all, diagnostics for most arboviruses were requested only on severe clinical presentation. Conclusion Travel destination and syndrome were used inconsistently for triage of diagnostics, likely resulting in vast under-diagnosis of arboviral infections of public health significance. This study shows the need for more awareness among physicians and standardization of syndromic diagnostic algorithm