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

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

Dokazivanje bakterije Mycobacterium avium subspecies paratuberculosis pomoću ugniježđene lančane reakcije polimerazom (nPCR).

Mycobacterium avium subspecies paratuberculosis, the cause of paratuberculosis (Johne’s disease) is a chronic debilitating infection in ruminants. The disease is one of the most widespread bacterial infections of ruminants throughout the world. Recently, the organism was reported to be associated with enteric infection in humans and hence the disease is of public health importance. In the present study, a nested Polymerase Chain Reaction (nPCR) was developed and evaluated for detection of Mycobacterium avium subspecies paratuberculosis. The insertion sequence (IS) 1311 was used as a target DNA for the nested PCR amplification. First, a pair of primers (P1 and P2) was designed from IS1311 nucleotide sequences to produce 549 bp PCR products. Next, a second pair of internal (nested) primers (P3 and P4) was used to produce a 260 bp PCR product. The nested amplification was necessary to increase the sensitivity of the PCR assay and to confirm the identity (specificity) of the first amplified PCR product. Because it is a highly sensitive technique the nPCR could be used for detection of Johne’s disease at an early stage of the infection. In addition, the described nPCR assay could be used as a supplement, or as an alternative, to conventional bacteriological procedures currently used for diagnosis of the disease in susceptible humans or animal populations.Mycobacterium avium subspecies paratuberculosis uzrokuje paratuberkulozu (Johneovu bolest) koja se očituje kroničnim tijekom u preživača. To je jedna od najraširenijih bakterijskih zaraza preživača diljem svijeta. Nedavno je objavljeno da je uzročnik povezan s crijevnom infekcijom u ljudi te je bolest postala važna za javno zdravstvo. U radu je rabljena ugniježđena lančana reakcija polimerazom (nPCR) za dokazivanje Mycobacterium avium subspecies paratuberculosis. Sekvencija 1311 je korištena kao ciljna DNK za unutarnju amplifikaciju pomoću lančane rekacije polimerazom. Najprije je određen par početnica (P1 i P2) od sekvencije s 1311 nukleotida za proizvodnju 549 pb PCR proizvoda. Zatim je upotrijebljen drugi par unutarnjih početnica (P3 i P4) za proizvodnju 260 pb PCR proizvoda. Ugniježđena reakcija je bila potrebna da poveća osjetljivost PCR-a i potvrdi identitet (specifičnost) prvotno umnoženog PCR proizvoda. Budući da je nPCR vrlo osjetljiva metoda može se koristiti za otkrivanje Johneove bolesti u ranom stadiju infekcije. Opisana nPCR metoda može se rabiti kao dodatna ili alternativna metoda u odnosu na uobičajene bakteriološke postupke koji se svakodnevno rabe za dijagnosticiranje bolesti u ljudi i/ili životinja

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

Prevalence of bluetongue virus infection and associated risk factors among cattle in North Kordufan State, Western Sudan

Abstract Background Bluetongue virus causes febrile disease in sheep and a fatal hemorrhagic infection in North American White-tailed deer. However, in cattle the disease is typically asymptomatic and no clinical overt disease is associated with bluetongue infection. Bluetongue virus activity has been detected in Khartoum, Sennar and South Darfur states of the Sudan. Currently, no information is available in regard to previous exposure of livestock to Bluetongue virus in North Kordufan State, the largest livestock producing region in the country. The present study was conducted to determine the prevalence of bluetongue antibodies and to identify the potential risk factors associated with the presence of bluetongue antibodies among cattle in North Kordufan State, Sudan. A total of 299 bovine blood samples were collected randomly from six localities in North Kordufan State and were tested by enzyme-linked immunosorbent assay (ELISA) for detection of BTV-specific immunoglobulin G (IgG) antibodies. Results The serological evidence of Bluetongue virus infection was observed in 58 out of 299 cows, accounting for a 19.4% prevalence rate among cattle in North Kordufan State. Older cattle (&gt;2 years of age) had four times the odds to be infected with BTV compared to young cattle (OR = 4.309, CI = 1.941-9.567, p-value = 0.01). Application of preventive measures, such as spraying or dipping with insecticide protects cattle against Bluetongue infection. Application of vector control measures decreased the odds for bluetongue seropositivity by 7 times (OR = 7.408, CI = 3.111-17.637, p-value = 0.01). Conclusions The results of this study indicated that age and application of routine insecticides are influential risk factors for seroprevalence of Bluetongue in cattle. Surveillance of Bluetongue virus should be extended to include other susceptible animals and to study the distribution of the insect vectors in the region to better predict and respond to BTV outbreak in the State of North Kordufan, Sudan. </jats:sec

A nosocomial transmission of crimean-congo hemorrhagic fever to an attending physician in north kordufan, Sudan

<p>Abstract</p> <p>Background</p> <p>Crimean-Congo hemorrhagic fever (CCHF), a tick-borne disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is a member of the genus Nairovirus in the family Bunyaviridae. Recently, CCHFV has been reported as an important emerging infectious viral pathogen in Sudan. Sporadic cases and multiple CCHF outbreaks, associated with nosocomial chain of transmission, have been reported in the Kordufan region of Sudan.</p> <p>Aims</p> <p>To confirm CCHF in an index patient and attending physician in North Kordufan region, Sudan, and to provide some information on virus genetic lineages.</p> <p>Methods</p> <p>Antibody captured ELISA, reverse transcription PCR, partial S segment sequences of the virus and subsequent phylogenetic analysis were used to confirm the CCHFV infection and to determine the virus genetic lineages.</p> <p>Results</p> <p>CCHF was confirmed by monitoring specific IgM antibody and by detection of the viral genome using RT-PCR. Treatment with oral ribavirin, replacement with fluid therapy, blood transfusion and administration of platelets concentrate resulted in rapid improvement of the health condition of the female physician. Phylogenetic analysis of the partial S segment sequences of the 2 CCHFV indicates that both strains are identical and belong to Group III virus lineage, which includes viruses from Africa including, Sudan, Mauritania, South Africa and Nigeria.</p> <p>Conclusion</p> <p>Further epidemiologic studies including, CCHFV complete genome analysis and implementation of improved surveillance are urgently needed to better predict and respond to CCHF outbreaks in the Kordufan region, Sudan.</p