No description available.

We describe a simple, sensitive, nonradioactive, relatively rapid and relatively inexpensive protocol to measure DNA synthesis in cultured cells by a chemiluminescent bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA). We show that it exhibits similar sensitivity and activity as traditional 3H-thymidine incorporation assays and a commercial chemiluminescent BrdU ELISA kit when tested in commonly used cell lines, such as NIH 3T3 cells, mink lung epithelial cells (Mv1LU), and baby hamster kidney (BHK-21) fibroblasts. This assay also exhibits a wider dynamic range than colorimetric BrdU ELISA methods. Besides being a viable, nonradioactive alternative to 3H-thymidine incorporation assays, our BrdU ELISA is less expensive than a commercial chemiluminescent BrdU ELISA kit.

No description available.

Ornithobacterium rhinotracheale (ORT) is a Gram-negative bacterium that causes respiratory disease in poultry characterized by rhinitis, tracheitis, and pneumonia with mortality averaging 2-3%. In the Shenandoah Valley of Virginia, the seroprevalence for ORT among turkey flocks as determined by enzyme-linked immunosorbent assay (ELISA) was found to be 70.9% (n=175). Additionally, the seroprevalence for hemorrhagic enteritis virus (vaccine induced), Bordetella avium, and paramyxovirus-1 was 100%, 74.8%, and 6.3% respectively. No significant interactions were detected. The type strain of ORT is characteristically non-hemolytic at least for 96 hours at 37°C on Columbia Blood Agar. In recent years, atypical isolates that rapidly produce hemolysis have been isolated with increasing frequency. A variety of in vitro tests were used to determine differences between representative isolates of the hemolytic (H) and non-hemolytic (NH) phenotypes. Findings suggest that the H isolate contains a 4 kb plasmid similar to that found in Reimerella anatipestifer. No plasmid was found in the NH isolate. Differences in growth characteristics and resistance to tetracyclines were also noted. No differences in proteins, biochemical characteristics or 16S rRNA sequences were found, the latter serving as confirmation that the isolates were both ORT. Embryo inoculation was used to assess virulence. No significant differences were observed and most embryos survived through to the day of hatch (pip) despite the fact that ORT could be re-isolated. In turkey poults however, the H phenotype did appear less virulent. A significant depression in weight gain was noted for birds inoculated intratracheally with the NH isolate at 7 days post-inoculation (dpi). NH inoculates also had significantly higher antibody levels on ELISA at 14 and 21 dpi and histopathological lesion scores for lung at 7, 14, and 21 dpi. The NH isolate could be re-isolated from NH-inoculated poults through 21 dpi; whereas the H isolate could only be re-isolated through 14 dpi. In conclusion, there are numerous differences between the NH and H isolates found in the field with the H isolate appearing less virulent and as such, making it a potential vaccine candidate. The phenotypic difference appears to correlate with this, but may not suffice to explain it. / Ph. D.

Background: Hemophilia A is X-linked recessive congenital bleeding disorder. Exogenous infusion of FVIII is the treatment of choice in hemophilia A patients. However, inhibitor development remains the major problem in management of Hemophilia A. It has been showed that IVIG has immunomodulatory effects and it has been being used in the treatment of several autoimmune and inflammatory disorders. Here, we investigated the effect of co-administration of FVIII with IVIG on the development of inhibitor in naive and previously immunized hemophilia A mice. Methods: Initially, hemophiliac mice were immunized by weekly intraperitoneal injection of human recombinant FVIII (rFVIII). The mice then were treated, either by rFVIII/IVIG co-injection or rFVIII alone. In the other experimental group, naive hemophiliac mice were treated with rFVIII/IVIG co-injection for four weeks followed by injection of either rFVIII or rFVIII/IVIG. Plasma's anti-FVIII Ab titer was measured using ELISA. Results: Weekly injection of rFVIII led to the development of anti-FVIII Ab in all previously untreated mice. Treatment of those immunized mice with rFVIII/IVIG co-injection did not reduce the level of pre-existing Ab. On the other hand, naive mice treated with rFVIII/IVIG co-injection showed significantly less Ab titer compared to the mice received rFVIII alone after 4 weeks (mean Ab titre of 1 compared to 39, in rFVIII/IVIG co-injection and rFVIII groups respectively). Although the rFVIII/IVIG-treated mice developed immune response following the injection of rFVIII alone, Ab titer in those that kept receiving rFVIII/IVIG co-injection remained lower compared to other groups during the whole twelve weeks of the experiment. Conclusions: Co-injection of rFVIII with IVIG decreased the anti-FVIII immune response in previously untreated hemophilia A mice. These findings suggest that IVIG co-administration can be effective in management of hemophilia A patients at risk of inhibitor development. / Thesis / Master of Applied Science (MASc)

Vaccine Brucella abortus strain RB51, unlike the wild strain 2308 and another vaccine strain (strain 19) does not induce anti-O-chain antibodies. An efficacious vaccine strain that fails to produce an O-chain and thus a lack of an anti-O-chain humoral response greatly simplifies identification of vaccinated versus field strain infected animals. The three primary objectives of this research were the following: 1) to develop a serological assay to detect anti-RB51 antibodies in vaccinated elk (Cervus elaphus nelsoni), 2) to identify potential antigenic alterations in RB51 after vaccination of elk and BALB/c mice, and 3) to confirm the general stability of RB51. Elk were divided into four groups based upon gender and the route of inoculation (subcutaneous or ballistic) of RB51 bacteria. This study developed a highly reliable ELISA (using a monoclonal anti-bovine IgG 1 antibody and acetone killed whole RB51 bacteria) which can identify RB51-vaccinated elk. Also, isolates recovered from RB51-vaccinated elk were inoculated into female BALB/c mice whose spleens were then cultured. All elk and mice isolates were bacteriologically, biochemically, and serologically evaluated. This study showed that RB51 is a highly stable strain, which does not revert to smooth morphology or initiate synthesis of LPS-O-chain, maintains it biochemical characteristics, does not undergo detectable antigenic variations, and remains attenuated even after successive passages in elk and mice. Overall, this research indicates that RB51 is a vaccine candidate for the prevention of brucellosis in elk. Further studies are needed to determine the protective capabilities of RB51 in elk. / Master of Science

Background: The monitoring of infectious diseases like the porcine reproductive and respiratory syndrome (PRRS) using pen-wise oral fluid samples becomes more and more established. The collection of individual oral fluid, which would be useful in the monitoring of PRRSV negative boar studs, is rather difficult. The aim of the study was to test two methods for individual oral fluid collection from pigs and to evaluate the specificity of a commercial ELISA for detection of PRRSV antibodies in these sample matrices. For this reason, 334 serum samples from PRRSV negative pigs (group 1) and 71 serum samples from PRRSV positive pigs (group 2) were tested for PRRSV antibodies with a commercial ELISA. Individual oral fluid was collected with a cotton gauze swab from 311 pigs from group 1 and 39 pigs from group 2. Furthermore, 312 oral fluid samples from group 1 and 67 oral fluid samples from group 2 were taken with a self-drying foam swab (GenoTube). The recollected oral fluid was then analysed twice with a commercial ELISA for detection of PRRSV antibodies in oral fluid.

<p>Dosada&scaron;nji objavljeni literaturni podaci ukazuju da aflatoksini, usled njihovog dokazanog toksičnog, mutagenog, teratogenog i kancerogenog efekta na organizam ljudi i životinja, predstavljaju najpoznatiju i najrizičniju grupu mikotoksina. Nasuprot navedenom, dostupna naučna i stručna literatura iz Republike Srbije ukazuje na nedostatak sistematski sprovedenih monitoringa učestalosti pojave aflatoksina. Kao sirovine za ispitivanje pojave aflatoksina, u ovoj doktorskoj disertaciji, odabrani su kukuruz i mleko. Kukuruz je odabran jer predstavlja jednu od najzastupljenijih žitarica u Srbiji. Navedena činjenica dodatno povećava potrebu za istraživanjem, jer pojava aflatoksina u velikoj meri utiče i na kvalitet kukuruza, kao i smanjenje prinosa i ekonomskog profita zemlje. Cilj da se ispita mleko proistekao je iz činjenice da ono usled velike frekventnosti u ishrani, predstavlja namirnicu sa najvećim potencijalnim rizikom za unos aflatoksina u organizam ljudi.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Analiza pojave aflatoksina u kukuruzu obuhvatila je 1000 uzoraka, sakupljenih tokom perioda od četiri godine (2009-2012.). Dobijeni rezultati ukazali su da aflatoksini nisu detektovani u uzorcima kukuruza iz 2009., 2010. i 2011. godine. Međutim, od 700 analiziranih uzoraka kukuruza iz 2012., čak 553 (79%) bilo je kontaminirano aflatoksinima. Značajne razlike u pojavi aflatoksina u kukuruzu, iz različitih proizvodnih godina, mogu se objasniti izuzetno toplim i suvim vremenskim uslovima zabeleženim u 2012. godini. Su&scaron;ni vremenski uslovi bili su povoljni za razvoj plesni i sintezu aflatoksina u kukuruzu, a takođe su u velikoj meri doprineli i visokom nivou pojave aflatoksina M1 (AFM1) u mleku. Analiza 200 uzoraka različitih kategorija kravljeg mleka pokazala je da je AFM1 sa koncentracijama od 0,01 do 1,20 ug/kg detektovan u 198 (98,7%) uzoraka. Na osnovu određenih koncentracija AFM1 u uzorcima mleka, prikupljenih informacija o prosečnom unosu mleka, telesnoj težini različitih starosnih kategorija i polova 1500 ispitanika, procenjen je rizik od unosa AFM1. Dobijeni rezultati ukazali su da su sve ispitane starosne kategorije, a posebno deca, izložena visokom riziku od unosa AFM1 u organizam preko kontaminiranog mleka.<br />Usled svega navedenog, proizilazi potreba za kontinuiranim praćenjem pojave aflatoksina u kukuruzu i AFM1 u mleku, sa ciljem za&scaron;tite stanovni&scaron;tva od rizika povezanih sa njihovim dokazanim toksičnim i kancerogenim efektima. Tokom poslednjih godina, do&scaron;lo je do povećane tražnje za razvojem osetljive, tačne, jednostavne i brze metode, čijom primenom će se obezbediti pouzdano određivanje različitih aflatoksina pri niskim koncentracijama. Usled toga, ELISA metode, kao i metode tečne hromatografije sa različitim detektorima (HPLC-FLD, HPLC-UV light-FLD, LC-MS/MS) su razvijene, optimizovane i validovane. Validacija je izvr&scaron;ena prema smernicama odgovarajućih evropskih Regulativa, analizom sertifikovanih referentnih materijala i spajkovanih uzoraka. Dobijeni parametri validacije potvrdili su da se sve primenjene metode sa visokom pouzdano&scaron;ću mogu koristiti za određivanje aflatoksina u kukuruzu, odnosno AFM1 u mleku. Validovane metode su zatim primenjene za analizu aflatoksina i AFM1 u prirodno kontaminiranim uzorcima kukuruza i mleka. Analizom prirodno kontaminiranih uzoraka kukuruza uočeno je da određena koncentracija aflatoksina u velikoj meri zavisi od neravnomerne raspodele aflatoksina unutar čvrstih uzoraka. Pored toga, analiza uzoraka kukuruza iz skladi&scaron;ta, takođe je ukazala na neravnomernu raspodelu ukupnih plesni, vrste Aspergillus flavus, kao i aflatoksina. S druge strane, dobijeni koeficijenti korelacije (&gt; 0,9) između primenjenih metoda za određivanje AFM1 u mleku, ukazali su da među dobijenim rezultatima postoji snažna korelacija, koja može poticati i od relativno homogenije raspodele AFM1 unutar tečnih uzoraka.</p> / <p>Recent studies have demonstrated that аflatoxins (AFs) represent the best known and most intensively researched mycotoxins in the world, with proven toxic, mutagenic, teratogenic and carcinogenic effects on human and animal health. However, up to the author&#39;s knowledge, there are no investigations concerning the occurrence of AFs in food or feed from Republic of Serbia. For that purpose, maize and cow&acute;s milk samples were chosen as matrixes for investigation. Maize is selected because it represents one of the major crops grown in Serbia and presence of AFs leads to reduction of maize quality as well as significant economical losses. On the other hand, milk has the greatest demonstrated potential for AFs introducing into the human diet since it represents one of the main foodstuffs in human nutrition.<br />An investigation regarding occurrence of AFs in maize included 1000 samples collected during four years period (2009-2012). Obtained results indicate that AFs were not detected in maize samples collected during 2009-2011 period. However, among 700 analyzed maize samples from 2012 even 553 (79%) samples contained AFs. Significant differences in occurrence of AFs in maize could be contributed to extremely hot and dry weather conditions noted during maize growing season 2012. Described weather conditions were favorable for mould growth and AFs synthesis in maize and also could be the possible reason for high contamination frequency of aflatoxin M1 (AFM1) in milk. Analysis of 200 cow&acute;s milk samples showed that AFM1 were detected in 198 (98.7%) samples in concentrations ranged from 0.01 to 1.20 &mu;g/kg. On the basis of the obtained AFM1 concentrations in cow&rsquo;s milk samples, collected information about average milk intake and mean body weight (bw) for different age&rsquo;s categories, mean ingestion of AFM1 in ng/kg per bw per day were estimated. Obtained results showed that all age&rsquo;s categories, especially children, are exposed with high risk related to presence of AFM1 in milk.<br />Based on everything stated above, presence of AFs in maize and AFM1 in milk should be continuously controlled in order to protect the population from risks associated with its proven toxicity and carcinogenicity. During recent years, there has been an increase in demand for development of sensitive, accurate, simple and fast method which is reliable to detect AFs and AFM1 at low concentrations. For that purpose, enzyme linked immunosorbent asssay (ELISA), high performance liquid chromatography with fluorescence detector (HPLC-FLD; HPLC-UV light-FLD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were optimized and validated. The method validation was performed according European Union Regulative using certificated reference material as well as spiked blank maize and milk samples. The obtained validation parameters indicate that all applied methods are suitable for determination of AFs in maize as well as AFM1 in milk samples. Afterwards, validated methods were applied for analysis of natural contaminated maize and milk samples. Analysis of natural contaminated maize samples indicate that determined AFs concentration mostly depends of uneven distribution of AFs in solid samples. Furthermore, analysis of maize samples from different warehouses indicates an uneven distribution of moulds, Aspergillus flavus and AFs. On the other hand, obtained correlation coefficients (&gt;0.9) indicate that strong correlation exists between applied methods for AFM1 determination. Relatively homogeneous distribution of AFM1 in liquid samples could be the possible reason for obtained strong correlation between applied methods.</p>

Background: In this study, six enzyme-linked immunosorbent assays (ELISA), intended for routine porcine reproductive and respiratory syndrome virus (PRRSV) herd monitoring, are tested for their ability to detect PRRSV specific antibodies in the serum of pigs after vaccination with an inactivated PRRSV type 1 vaccine and subsequent infection with a highly pathogenic (HP) PRRSV field strain. For this reason, ten piglets (group V) from a PRRSV negative herd were vaccinated twice at the age of 2 and 4 weeks with an inactivated PRRSV vaccine. Ten additional piglets (group N) from the sameherd remained unvaccinated. Three weeks after second vaccination, each of the piglets received an intradermal application of an HP PRRSV field strain. Serum samples were taken before first vaccination as well as before and 3, 7, 10 and 14 days after HP PRRSV application. All serum samples were tested for PRRSV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as well as for PRRSV antibodies with all six study ELISAs. Results: At the beginning of the study (before vaccination), all of the piglets were PRRSV antibody negative with all study ELISAs. They also tested negative for PRRSV RNA measured by RT-qPCR. From day 3 after HP PRRSV application until the end of the study, a viremia was detected by RT-qPCR in all of the piglets. On day 0 (day of HP PRRSV application), nine out of ten piglets of the pre-vaccinated group tested PRRSV antibody positive with one of the tested ELISAs, although with lower S/P values than after infection. On day 10 after HP PRRSV application, all study ELISAs except one had significantly higher S/P or OD values, respectively more positive samples, in group V than in group N. Conclusions: Only one of the tested ELISAs was able to detect reliably PRRSV antibodies in pigs vaccinated with an inactivated PRRSV vaccine. With most of the tested ELISAs, higher S/P values respectively more positive samples after PRRSV infection were seen in the pre-vaccinated group than in the non-vaccinated.

A Leishmaniose visceral é uma doença parasitária crônica em homens e cães, causada por protozoários da espécie L. (Leishmania) chagasi. O diagnóstico parasitológico é confirmado pelo achado do agente em aspirados de medula óssea, linfonodo, baço e fígado, enquanto que a sorologia IgG especifica é usada em geral para estudos epidemiológicos, apesar dos altos níveis séricos de anticorpos IgG anti-Leishmania. Existem relatos anedóticos de resultados sorológicos negativos em doença ativa, atribuído à formação de imunocomplexos. Dado que imunocomplexos podem ser dissociados por tratamento ácido, nós buscamos a padronização de um teste simples de dissociação ácida dos imunocomplexos de amostras de soro, por tratamento ácido e neutralização em poços adsorvidos com antígenos de Leishmania, seguida de ELISA (ELISA dissociativo). A confirmação da presença de antígenos foi realizada pela detecção após adsorção ácida por DOT-ELISA, usando soro de coelho hiperimune anti-Leishmania. Amostras de hamsteres infectados experimentalmente com L. (L.) chagasi mostraram a presença e interferência de imunocomplexos na sorologia principalmente nas fases iniciais da infecção, por ELISA dissociativo e DOT-ELISA. Em amostras maiores de áreas endêmicas, o ELISA dissociativo aumentou a soropositividade em 10% em amostras de cães negativas e 3,5% de amostras humanas negativas, com confirmação por DOT-ELISA. Os resultados mostram que este teste poderia ser usado no diagnóstico da LV, como abordagem alternativa para a identificação sorológica de casos assintomáticos e para indicação de métodos parasitológicos invasivos confirmatórios. / Visceral leishmaniasis, caused by Leishmania (Leishmania) chagasi, is a chronic parasitic disease of humans and dogs. Confirmation of the protozoal agent in bone marrow, lymph node or spleen aspirate is diagnostic, while specific-IgG serology is used mainly for epidemiology despite the general presence of high levels of serum immunoglobulins. Anecdotal reports of false-negative serology in active disease cases are known and are ascribed to the formation of immune complexes. Because dissociation of immune complexes can be accomplished by acid treatment, we devised a simple, routine enzyme immunoassay (ELISA) for the dissociation of immune complexes in serum samples using acid treatment and neutralization in wells adsorbed with Leishmania antigen (dELISA). Confirmatory acid DOT-ELISA was also developed for antigen detection by anti-Leishmania rabbit antiserum. In experimental L. (L.) chagasi hamster models, immune complexes interfered with ELISA mostly in the early stages of infection, according to both dELISA and antigen DOT-ELISA results. In larger samples from endemic areas, dELISA increased seropositivity by 10% in negative dog samples (7/70) and 3.5% in negative human samples (3/85), showing that dELISA could be used in the serodiagnosis of visceral leishmaniasis. Moreover, dELISA could be used as an alternative approach to screening asymptomatic visceral leishmaniasis patients, instead of invasive confirmatory testing.