Made available in DSpace on 2016-04-28T20:17:26Z (GMT). No. of bitstreams: 1 2006- Glenda Ribeiro de Oliveira.pdf: 475801 bytes, checksum: 8ca6884856073fd041daad94f50fca0e (MD5) Previous issue date: 2006-11-24 / The intake of mycotoxin-contamined feeds may lead to nutrient losses and can have adverse effects on animal health and on productivity. The aims of this study were: 1) to determine the mycobiota present in poultry feed samples, obtained from three factories in the region of S?o Jos? do Vale do Rio Preto, RJ, Brazil; 2) to evaluate the natural occurrence of aflatoxin B1 (AFB1), fumonisin B1 (FB1) and zearalenone using the method enzyme linked immunosorbent assay (ELISA) competitive, commercial kits of Beacon Analytical Systems Inc, USA and 3) to verify the data results by the ELISA to AFB1 and FB1 comparing them with the high precision liquid chromatography methodology (HPLC) Fungal counts were similar between all culture media tested (103 CFU.g-1). Penicillium spp. (41,26%) was the most prevalent genus followed by Aspergillus spp. (33,33%) and Fusarium spp. (20,63%). Aflatoxin B1 was found in 66,27% samples examined by ELISA kits and values ranged between 2 and 21&#956;g.kg-1. Fumonisin B1 was found in 97,87% and levels ranged between 0,3 and 9,1&#956;g.g-1, cooccurring with aflatoxin B1 in 66,7% of the samples. Low concentrations of zearalenone were found (<1 &#956;g.g-1) in 77,1% poultry feed samples. Correlation between the ELISA kits (AFB1 and FB1) and the HPLC analysis was obtained. Values were r2= 0,9922 and r2=0,9823, respectively. In general, the highest mycotoxin levels were found during the hottest month of sampling. The present study shows the simultaneous occurrence of two carcinogenic mycotoxins, aflatoxin B1 and fumonisin B1 together with another Fusarium mycotoxin (zearalenone) in feed intended for poultry consumption. Many samples contained AFB1 levels near the maximum permissible and it could affect young animals. A synergistic toxic response is possible in animals under simultaneous exposure. / A ingest?o de ra??es contaminadas por micotoxinas pode acarretar perdas no seu valor nutritivo e causar efeitos adversos ? sa?de animal e ? produtividade. Os objetivos desse estudo foram: 1)estabelecer a freq??ncia e a preval?ncia da micobiota presente em amostras de ra??es de frangos de corte provenientes de tr?s f?bricas em S?o Jos? do Vale do Rio Preto-RJ, 2)verificar a ocorr?ncia natural de aflatoxina B1 (AFB1), fumonisina B1 (FB1) e zearalenona (ZEA), utilizando a t?cnica biol?gica de imunoensaios-ELISA competitivo, com kits comerciais (Beacon Analytical Systems Inc, USA) e 3)verificar os resultados obtidos pelo m?todo de ELISA, comparando-os com a t?cnica de Cromatografia L?quida de Alta Efici?ncia (CLAE) para AFB1 e FB1. A contagem dos prop?gulos f?ngicos foi similar entre todos os meios de cultura testados (103 UFC g-1). Penicillium spp (41,26%) foi o g?nero de maior preval?ncia seguido por Aspergillus spp (33,33%) e Fusarium spp. (20,63%). Aflatoxina B1 foi encontrada em 66,27% das amostras analisadas pelos kits de ELISA,com valores variando entre 2 e 21&#956;g.kg-1. Fumonisina B1 foi encontrada em 97,87% e os valores variaram entre 0,3 e 9,1&#956;g.g-1, co-ocorrendo com aflatoxinaB1 em 66,7% das amostras. Baixas concentra??es de zearalenona foram encontradas (< 1&#956;g.g-1)em 77,1% das amostras de ra??o. Foi obtida correla??o entre os kits de ELISA (AFB1 e FB1) e as an?lises em CLAE, com valores de r2= 0,9922 e 0,9823, respectivamente. Em geral, os maiores n?veis de micotoxinas foram encontrados durante os meses mais quentes do ano. O presente estudo mostra a ocorr?ncia simult?nea de duas micotoxinas carcinog?nicas, AFB1 e FB1, juntamente com outra Fusarium micotoxina (zearalenona) em ra??es destinadas ao consumo de frangos. Algumas amostras apresentaram n?veis de AFB1 pr?ximos ao m?ximo permitido, o que poderia afetar aves jovens. Uma resposta t?xica sin?rgica ? poss?vel em animais sob exposi??o simult?nea.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-08-24T19:19:55Z No. of bitstreams: 1 2016 - Marli Sidoni.pdf: 1735106 bytes, checksum: 03907fc3437a44bf96b94d6040184407 (MD5) / Made available in DSpace on 2018-08-24T19:19:55Z (GMT). No. of bitstreams: 1 2016 - Marli Sidoni.pdf: 1735106 bytes, checksum: 03907fc3437a44bf96b94d6040184407 (MD5) Previous issue date: 2016-12-01 / This work focused on deploying IgY technology into developing ELISA immunoenzymatic test to FIV diagnose, contributing to the establishment of a production model for a kit in the field of veterinary medicine. The first stage of this work consisted in analyzing the literature of the veterinary documents aimed to diagnose animal diseases, as stated in our current legislation. The access to both national and international optimized the enlightenment of developing parameters and method validation criteria for the development of this new product, named ELISA r-p24 IgY. The second stage consisted in establishing purification production and physicochemical characterization of the recombinant protein p24 of FIV. The biological activity maintenance was proved by way of Western Blot test with the banda presence of approximately 25 kDa, referring to the p24 protein. The third stage was to obtain IgY cat anti-IgG, derived from hens inoculation. The kinetics were monitored by ELISA and the outcome demonstrated that as of the second week, there was a gradual increase in antibody in the yolk, and remained high throughout the period of five months. Reference to the chicken 1, the average concentration was 40,1 mg/mL e for the chicken 2 was 32,2 mg/mL, throughout the period of 5 months. The fourth stage was the use of IgY technology to develop, standardize e validate the r-p24 IgY ELISA related to its use to diagnose the infection caused by FIV. The results were: 99% accuracy, 97.7% sensitivity, 99.5% specificity and 99.1% kappa index. In the fifth stage was carried out a comparative study between ELISA r-p24 IgY and ELISA r-p24 IgG, and it was demonstrated superior performance of the ELISA r-p24 IgY. The ELISA r-p24 IgY to have favorable characteristics from a commercial perspective, such as high precision and maintenance of reactivity for a minimum period of 12 months. Therefore, the above described procedure was efficient and enabled the development of a FIV test. The predominance of IgY technology may contribute to research and development of new tests, following both international regulation related to animal welfare and validation, thus boosting national development of diagnostic kits, for the benefit of human health or animal. / O objetivo deste trabalho foi aplicar a tecnologia IgY no desenvolvimento de um teste imunoenzim?tico, ELISA, para o diagn?stico do FIV, contribuindo no estabelecimento de um modelo de produ??o para um kit nacional na ?rea de medicina veterin?ria. A primeira etapa do desenvolvimento deste trabalho consistiu na revis?o da literatura dos documentos pr?prios para produtos de uso veterin?rio, destinados a diagnosticar doen?as dos animais, disponibilizados na legisla??o vigente. A consulta aos documentos nacionais e internacionais potencializou o esclarecimento de par?metros de desempenho ou crit?rios de valida??o de m?todos, para o desenvolvimento deste novo produto, denominado ELISA r-p24 IgY. A segunda etapa consistiu no estabelecimento da produ??o, purifica??o e caracteriza??o f?sico-qu?mica da prote?na recombinante p24 do FIV. A preserva??o da atividade biol?gica foi demonstrada por Western Blot com a presen?a de uma banda pept?dica de aproximadamente 25 kDa, referente ? prote?na r-p24. A terceira etapa deste trabalho, consistiu na obten??o de anticorpos IgY anti-IgG de gato, a partir da inocula??o em galinhas poedeiras. A cin?tica foi acompanhada por ELISA demonstrando um aumento gradativo do t?tulo de anticorpos na gema a partir da segunda semana, com um aumento significativo no 2? m?s, e mantendo-se elevado durante todo o per?odo de cinco meses. As concentra??es m?dias de prote?nas na galinha 1 foi de 40,1 mg/mL a partir de uma gema e na galinha 2 foi de 32,2 mg/mL por gema, no per?odo de 5 meses. A quarta etapa deste trabalho consistiu no emprego da tecnologia IgY para o desenvolvimento, padroniza??o e a valida??o do teste de Elisa r-p24 IgY para o diagn?stico da infec??o causada pelo FIV. Os resultados obtidos foram: a acur?cia de 99%, a sensibilidade de 97,7%, a especificidade de 99,5%, e o ?ndice kappa de 99,1%. Na quinta etapa deste trabalho realizou-se o estudo comparativo do ELISA r-p24 IgY frente ao ELISA r-p24 IgG, e foi demonstrado o desempenho superior no ELISA r-p24 IgY. A valida??o do ELISA r-p24 IgY mostrou caracter?sticas desej?veis para o uso comercial, tais como alta precis?o e manuten??o da reatividade por um per?odo m?nimo de 12 meses. Conclui-se que o procedimento elaborado foi eficiente e possibilitou o desenvolvimento de um teste para o diagn?stico do FIV. O dom?nio da Tecnologia IgY poder? contribuir com a pesquisa e o desenvolvimento de novos ensaios atendendo ?s normas e diretrizes nacionais e internacionais, tanto de bem-estar animal como de valida??o, impulsionando o desenvolvimento nacional de kits diagn?sticos de interesse em sa?de humana ou animal.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-09-12T17:52:35Z No. of bitstreams: 1 2017 - Elisa Domingues Padua.pdf: 1950908 bytes, checksum: d9644f85d3b5935e0ad3f149d04d4f4b (MD5) / Made available in DSpace on 2018-09-12T17:52:35Z (GMT). No. of bitstreams: 1 2017 - Elisa Domingues Padua.pdf: 1950908 bytes, checksum: d9644f85d3b5935e0ad3f149d04d4f4b (MD5) Previous issue date: 2017-07-25 / Despite some reports of the occurrence of Leishmaniasis in felines, the literature is scarce with regard to its research on populations of cats from areas endemic to the disease. In this way, the present study aimed to investigate the possibility of infection in cats by means of a rapid qualitative test of the antibody test in Serop?dica and Itagua?, metropolitan region of Rio de Janeiro, an endemic area for tegumentary and visceral canine leishmaniasis Anti-Leishmania chagasi by indirect immunoenzymatic assay (ELISA) and indirect immunofluorescence (RIFI). For this purpose, blood samples were collected and the serum of 255 cats was sent to the Escola Nacional de Sa?de P?blica (FIOCRUZ). In this population of cats five animals (1.9%, 5/255) were reactive to the rapid test (TR DPP?), 44 (17.3%) had anti-Leishmania spp. Identified by the RIFI. The main clinical signs observed in the seropositive animals were ocular secretion (16/44 - 36%), nasal secretion (16/44 - 36%), weight loss (7/44 - 15.9%), alopecia (6/44 - 13 , 6%) and ulcerated skin lesions (5/44 - 11.3%), in addition to hepatosplenomegaly (2/44 - 4.5%), lymphadenomegaly (5/44 - 11.3%), corneal opacity 2/44 - 4.5%) and gingivitis (2/44 - 4.5%). It was not possible to establish a standardization for the ELISA. Detection of anti-Leishmania spp. in felines, calls attention to this species that must be further investigated in relation to its power as a source of infection in relation to the parasite. / Apesar de alguns relatos da ocorr?ncia de Leishmaniose em felinos, a literatura ? escassa no que diz respeito ? sua pesquisa em popula??es de gatos de ?reas end?micas para a doen?a. Desta forma, o presente estudo teve por objetivo pesquisar, em Serop?dica e Itagua?, regi?o metropolitana do Rio de Janeiro, ?rea end?mica para Leishmaniose tegumentar e visceral canina, a possibilidade de infec??o em gatos, por meio de teste r?pido qualitativo, da pesquisa de anticorpos anti-Leishmania chagasi pelas t?cnicas de ensaio imunoenzim?tico indireto (ELISA) e rea??o de imunofluoresc?ncia indireta (RIFI). Para tanto, foram colhidas amostras de sangue e soro de 255 gatos, os quais foram encaminhados ? Escola Nacional de Sa?de P?blica (FIOCRUZ). Nessa popula??o, cinco gatos (1,9%, 5/255) foram reagentes ao teste r?pido (TR DPP?), 44 (17,3%) apresentaram anticorpos anti-Leishmania spp. identificados pela RIFI. Os principais sinais cl?nicos observados nos animais soropositivos foram secre??o ocular (16/44 ? 36%), secre??o nasal (16/44 ? 36%), emagrecimento (7/44 ? 15,9%), alopecia (6/44 ? 13,6%) e les?o ulcerada na pele (5/44 ? 11,3%), al?m de hepatoesplenomegalia (2/44 ? 4,5%), linfadenomegalia (5/44 ? 11,3%), opacidade de c?rnea (2/44 ? 4,5%) e gengivite (2/44 ? 4,5%). N?o foi poss?vel estabelecer uma padroniza??o para o ELISA. A detec??o de anticorpos anti-Leishmania spp. em felinos chama a aten??o para essa esp?cie que deve ser mais investigada em rela??o ao seu poder como fonte de infec??o em rela??o ao parasito.

Universidade Federal do Pará, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários. / The brazilian Amazon region maintains the largest variety of arboviruses and the state of Pará is responsible for 26% of this territory, so the major goal of this work was to determine the prevalence and distribution of HI antibodies to 19 domestic arbovirus in domestic herbivores in the state of Pará and to standardize an indirect sandwich IgG ELISA test to serum samples of equines, cattle, buffaloes and sheep. In all species studied and throughout the State of Pará a large prevalence of HI antibodies to all arbovirus analyzed was observed and SLEV, ILHV, EEEV, MAGV and WEEV, showed higher prevalence, where the SLEV was the most prevalent. Regarding the virus families HI antibodies to MAGV was the most prevalent Bunyaviridae in all species, the most prevalent Flaviviridae was SLEV in all species and in the family Togaviridae the EEEV was more prevalent in horses. In order to analyze the prevalence of HI antibodies by animal species was observed that horses did not show significant differences compared to buffaloes, however, showed significant difference compared to cattle and sheep; there was not observed significant difference between the ruminant species. Using sandwich indirect IgG ELISA a large number of crossed reactions were found. This enzymatic test can be used to detect IgG antibodies among families of arboviruses studied. / A região Amazônica brasileira mantém a maior variedade de arbovírus e o estado do Pará corresponde a 26% desse território, o presente trabalho teve como objetivo determinar a prevalência e a distribuição de anticorpos detectados por inibição de hemaglutinação (IH) para 19 arbovírus em herbívoros domésticos no estado do Pará e padronizar testes de ELISA sanduíche indireto em equinos, bovinos, bubalinos e ovinos. Em todas as espécies de animais estudadas e em todo estado do Pará ocorreu detecção de anticorpos para todos os arbovírus analisados dentre os quais os SLEV, ILHV, EEEV, MAGV e WEEV apresentaram maior prevalência de anticorpos IH, sendo o SLEV o mais prevalente. Na detecção de anticorpos para diferentes famílias de arbovírus o MAGV foi o mais prevalente da família Bunyaviridae em todas as espécies, o SLEV foi o mais prevalente da família Flaviviridae em todas as espécies, na família Togaviridae o EEEV foi mais prevalente em equinos. Ao analisar a prevalência de anticorpos IH por espécie animal foi observado que os equinos não apresentaram diferença significativa em relação aos bubalinos, porém, apresentaram diferença significativa maior em comparação aos bovinos e ovinos, não havendo diferença significativa entre as espécies de ruminantes. O uso de ELISA IgG sanduíche indireto apresentou grande frequência de reações sorológicas cruzadas entre as famílias de arbovírus estudadas.

La paratuberculose est une maladie enzootique contagieuse des ruminants due à Mycobacterium avium ssp. paratuberculosis (Map). La longue période d'incubation et les faibles performances diagnostiques des tests limitent l'efficacité des plans de maîtrise. Les grands effectifs des troupeaux ovins limitent l'approche par dépistage individuel exhaustif, et les plans de maîtrise s'orientent vers la vaccination, dont les effets n'ont pas été évalués dans les troupeaux français. Á partir d'échantillons de sérum et de fèces de près de 4000 brebis issues de 30 élevages du Lot, les performances diagnostiques de deux trousses sérologiques ELISA et d'une qPCR sur fèces ont été estimées par des modèles à classes latentes bayésiens et fréquentistes. Nos résultats confirment la faible sensibilité et le défaut de spécificité des sérologies ELISA pour la détection des ovins infectés ; la qPCR présentant de meilleures performances diagnostiques. Par ailleurs nous avons évalué les performances diagnostiques relatives des ELISA et de la qPCR appliquées à des mélanges d'échantillons. Dans les deux cas les animaux forts répondeurs étaient détectés de façon systématique, les animaux faiblement positifs étant détectés de façon moins constante. Sur la base de simulations, nous avons évalué les performances des stratégies de dépistage et de suivi basées sur les analyses de mélanges d'échantillons à l'échelle des troupeaux. Alors que la sérologie ELISA est apparue insuffisamment spécifique, l'analyse de mélange de fèces par qPCR semble être une approche prometteuse, permettant de détecter des faibles prévalences d'infection. Enfin les travaux menés dans les troupeaux vaccinés ont précisé dans quelles mesures leur situation épidémiologique pouvait être approchée par l'emploi d'analyses en mélanges. / Paratuberculosis is a contagious enzootic disease in ruminants caused byMycobacteriumavium ssp. paratuberculosis (Map). The long incubation period and the low informative values of antemortem diagnostic tests limit the effectiveness of control plans. In large sheep flocks, exhaustive individual testing is unfeasible and control plansmainly focus on vaccination, the effects of which have not yet been evaluated in French flocks. Using blood and feces samples from nearly 4000 ewes in 30 sheep flocks from the French department of Lot, the diagnostic performances of two serum ELISA and one fecal qPCR kits were estimated using bayesian and frequentist latent class modeling. Our results confirm the low sensitivity and non-perfect specificity of serum ELISA for the detection of subclinically infected animals, while the diagnostic performances of fecal qPCR were better. We also evaluated the relative diagnostic performances of pooled-sample analysis for both tests. Highly qPCR/ELISA positive samples were invariously detected while low positive ones were associated with lower detection rates. The flock-level epidemiological performances of screening strategies based on pooled-sample analysis were evaluated by simulation studies. Pooled serum ELISA appeared lowly specific. Conversely, pooled fecal qPCR appeared promising, allowing the detection of low infection prevalence. Finally, the work carried out in the vaccinated flocks made it possible to better know their epidemiological status and to specify to what extent it could be approached using pooled-sample analysis.

Florida's mild climate supports year round enzootic transmission of arthropod-borne (arbo) viruses, such as St. Louis Encephalitis virus (SLEV), West Nile virus (WNV), and Eastern Equine Encephalomyelitis virus (EEEV). First isolated in 1960 from two Florida blue jays, Highlands J virus (HJV) is endemic to the state and vectored by the same mosquitoes as EEEV (Henderson et al, 1962). EEEV and HJV are both alphaviruses, but HJV is not pathogenic to humans, occasionally causes encephalitis in horses, and is a recognized pathogen in some bird species (turkeys, emus, etc) (Cilnis et al, 1996). The Florida Sentinel Chicken Arboviral Surveillance Program, established in 1978, utilizes sentinel chickens to detect arboviral activity throughout the state. Current serologic antibody detection methods include the hemagglutination inhibition (HAI), IgM antibody capture enzyme-linked immunosorbent (MAC-ELISA), and serum neutralization plaque reduction (PRNT) assays (Blackmore et al, 2003). In 2003, sentinel chickens detected significantly greater alphavirus activity than seen in the previous 15 years (Stark & Kazanis, 2003). This increase raised concerns that bridging into the human population had become a serious threat as well as an important issue for veterinary health. The objective of this study was to determine if cross-reactions with Highlands J virus were impacting the serologic diagnostic tests routinely performed for identification of EEEV. For 2003, the HAI test detected 476 alphavirus positive sentinels. We tested 316 of these chickens in the PRNT, which identified 176 EEEV positive sentinels and 75 HJV positive sentinels. Our results indicate that Highlands J virus is extensively cross-reactive in the HAI test and that the MAC-ELISA is more specific for the detection of antibodies solely to EEEV. We demonstrated that EEEV antibody titers in the HAI test were positively correlated to antibody titers in the PRNT assay. Analysis of alphaviral activity by county indicates widespread transmission of HJV across the northern and panhandle regions of the state; however EEEV activity was greater than HJV activity in all but four counties. Consequently, distinguishing between the two agents can reduce the expenditure of resources on unnecessary vector control and medical alerts to protect the public health from Highlands J virus.

Temperature has been known to be an important factor for in vitro studies where human cell cultures are infected with HRV (human Rhinovirus). The mechanisms behind the temperature effect on the struggle between virulence and cellular defense, are still largely unknown and may be a crucial part in finding a treatment to the common cold. In this study we focused on a few cellular key elements in this struggle and observed behavior changes in regards to the pre-infection growth temperature and the temperature during the viral infection. Past studies have focused mainly on the temperature post inoculation, but here we also wanted to correlate virulence to the growth temperatures preceding the viral infection. We found that the growth temperature of the cell did indeed affect its response to the HRV. If the cells had been growing in an optimal body temperature of 37°C before getting virally infected at 33°C, the viability of the cells did decrease in comparison to cells that had been growing in 33°C from before the viral infection. We could also observe a significant temperature dependence regarding IL-8 release upon HRV inoculation. HRV strive to block induction of inflammatory cytokines such as interferons and IL-1. It may be that impaired IL-8 release at lower temperatures will prevent important danger signals alerting the immune system when cytokine signaling is otherwise hampered by viral intervention.

Memoria para optar al Título Profesional de Médico Veterinario / Con el objetivo de realizar una evaluación serológica de la respuesta inmune de pollos de engorde en condiciones de campo frente un programa no convencional de vacunación contra Bronquitis Infecciosa con dos vacunas de serotipos diferentes, se realizaron muestreos para obtener sueros de aves vacunadas con la cepa Ma5 del virus de la Bronquitis infecciosa al día de edad y una vacuna de la cepa 4/91 a los 14 días de edad para detectar anticuerpos a través de la técnica de ELISA. Para esto se muestrearon seis grupos (de 20 pollos cada uno) de aves comerciales en condiciones de campo a los días 14, 28 y 35 de edad en cada día, además de utilizar dos grupos en condiciones experimentales, uno bajo el mismo protocolo no convencional de vacunación y el segundo grupo como control negativo (sin vacunas) muestreados en los mismos días y la misma cantidad de individuos. Además, se muestrearon 20 pollos al día de edad para determinar el nivel de anticuerpos maternos. El resultado de los títulos de anticuerpos fue una baja considerable en el día 14 de edad (dos semanas post vacunación) en comparación a los títulos de anticuerpos maternos, con un leve aumento en el día 28 de edad (4 semanas post primera vacunación y dos semanas post segunda vacunación), y con un mayor aumento en el día 35 presentándose todas las medias geométricas positivas, excepto por el grupo Control negativo experimental. Se determinó la existencia de diferencias significativas entre los grupos en el día 35 de muestreo a través de la prueba de ANOVA para luego identificar a través de la prueba de Tukey entre cuáles grupos había diferencias, observándose que el grupo Control negativo experimental se diferencia del resto y el grupo Experimental vacunado no presenta diferencias significativas con la mayoría de los grupos de campo / In order to perform a serological evaluation of the immune response of broilers in field conditions using an unconventional program of vaccination against Infectious Bronchitis with two vaccines of different serotypes, samples were taken to obtain sera from birds vaccinated with the Ma5 strain of infectious bronchitis virus at day of age and a vaccine of the strain 4/91 at 14 days old to detect antibodies through the ELISA technique. For this, six groups (of 20 chickens each) of commercial birds were sampled under field conditions on days 14, 28 and 35 old, for each day, in addition two groups were used under experimental conditions, one under the same protocol of non conventional vaccination and the second group as negative control (without vaccines) sampled in the same days and the same number of individuals per group were used. In addition, 20 chickens were sampled at one day old to determine the level of maternal antibodies. The result of the antibody titres showed a considerable decrease on 14 day old (two weeks post vaccination) compared to the maternal antibody titers, with a slight increase on 28 day old (4 weeks post first vaccination and two weeks post second vaccination), and with a greater increase on 35 day old showing all groups positive averages, except for the experimental negative control group. We determined the existence of significant differences between the groups on day 35 of sampling through the ANOVA test and then identify through the Tukey test between which groups there were differences, observing that the experimental negative control group is different from the rest and the Experimental vaccinated group does not present significant differences with most of the field groups

The Australian paralysis tick, Ixodes holocyclus causes a potentially fatal paralysis in domestic animals, livestock and humans with companion animals (mainly dogs) most commonly affected. Current treatment regimes include administration of a commercial tick anti-serum (TAS), prepared as hyperimmune serum in dogs, to neutralise the effects of the toxin. However, each new batch must be standardised using an expensive and highly subjective bioassay performed in neonatal mice. There is currently an urgent need for a more cost effective and rapid in vitro assay that can be more objectively and accurately quantified. Further understanding of the composition of the toxin molecule is also required to develop toxin-specific reagents necessary for these assays. One of the main objectives of this study was to develop a suitable immunoassay to replace the existing mouse bioassay for assessing batches of tick anti-sera for use in tick paralysis therapy in dogs. Initially an enzyme-linked immunosorbent assay (ELISA) was established to detect and quantify antibody specific for I. holocyclus toxin in dog sera. Using a partially purified antigen extracted from I. holocyclus salivary glands, good discrimination was achieved between reactive (hyperimmune) and non-reactive (naïve) sera. The hyperimmune dog sera reacted very strongly with the antigen compared to negligible reactions of serum from dogs not exposed to I. holocyclus. The reactions of hyperimmune sera were also significantly weaker to a non-toxin antigen control extracted from the salivary glands of the non-toxic tick Rhipicephalus microplus, indicating the assay was detecting toxin-specific responses. Furthermore, each of the hyperimmune sera that reacted strongly and specifically with the I. holocyclus antigen in the ELISA also strongly neutralised toxin in the mouse bioassay. Together these findings support the suitability of this ELISA for assessing the potency of batches of commercial dog hyperimmune sera for use as therapy for tick paralysis in dogs. Sera from dogs that were experimentally infested with ticks and sera from patient dogs, presenting at veterinary clinics with signs of tick paralysis, were also screened for antibodies to I. holocyclus antigen using the ELISA. Twenty-eight out of 29 sera from animals with single or multiple exposures to ticks failed to recognise the I. holocyclus antigen indicating the ELISA is not suitable as a diagnostic test to detect toxin-specific antibodies in animals with limited exposure to I. holocyclus infestation. A panel of toxin-specific monoclonal antibodies (mAbs) was produced as research tools to analyse and purify tick toxin components. Rats were successfully immunised against tick toxin using a combination of inoculation of partially purified salivary gland antigen and exposure to tick infestation. The latter approach preserved the native confirmation of the toxin using a natural route of immunisation and rats were chosen due to their high tolerance of multiple tick infestations over several days. While fusion of rat spleen cells with mouse myeloma cells has been reported several times in the literature, the resulting hybridomas are unstable with fastidious culture requirements. Optimisation of the culture conditions revealed that most rat-mouse hybridoma lines grew best in serum-free medium supplemented with 5% foetal bovine serum. Of 600 hybridomas produced, only 12 were shown to be specific for the Ixodes antigen, as determined by ELISA. A selection of these hybridomas representing various patterns of affinity and/or antigen specificity were further analysed for toxin-neutralising ability in a mouse bioassay. Notably, the most potent toxin-neutralising mAb in mice, showed a specific but relatively moderate reaction to Ixodes antigen in the ELISA. The most potent toxin-neutralising mAbs inactivated toxin as strongly as the commercial TAS used for immunotherapy in dogs with tick paralysis. This suggests that mAbs may present an alternative source of immunotherapy, providing a potentially endless supply of a highly consistent reagent and negating the need to use live animals for both the production of tick antiserum and the continual testing of reagent batches. The toxin-neutralising mAbs were also used to analyse I. holocyclus toxin in polyacrylamide gel electrophoresis (PAGE) and Western blot to identify specific toxin proteins. The most potent neutralising mAbs consistently recognised high MW proteins (100-200 kDa) in a smeared pattern. Although this was contrary to previous reports of low molecular weight components (3-5 kDa) in holocyclotoxin, this study was the first to use mAbs prepared to native toxin. The large molecular weight structures likely represent presucursors to, or complexes of the smaller peptides, previously identified. When the Toxin-neutralising mAbs were assessed as ligands to affinity purify toxin components from crude Ixodes SG extracts, toxin components of 110 and 32 kDa were consistently identified. These purified proteins represent good candidates for N-terminal sequencing to further identify the toxin components in I.holocyclus salivary glands.

<p>Transition metals such as nickel (Ni), cobalt (Co), palladium (Pd), chromium (Cr) and gold (Au) are widely used as alloys in jewelry and biomaterials such as orthodontic and orthopaedic appliances. These metals also cause cell-mediated allergic contact dermatitis (ACD) reactions in a significant proportion of the population upon prolonged direct exposure. The immune mechanisms underlying the response to these metals are not yet well defined. In the studies described in this thesis we therefore investigated the profile of cytokine responses to various metal ions <i>in vitro</i> and the relationship with the ACD reaction <i>in vivo</i>. In the first study, we investigated the relationship between the profile and magnitude of Ni²⁺-induced cytokine responses <i>in vitro</i> and the degree of <i>in vivo</i> reactivity to Ni²⁺. PBMC from Ni²⁺-reactive (ACD) and non-reactive control subjects were cultured with or without NiCl₂. The numbers of IL-4-, IL-5- and IL-13-producing cells and the concentrations of IFN-γ, IL-10 and IL-13 produced were analysed by ELISpot and ELISA respectively. Ni²⁺ elicited a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects with a positive correlation observed between the levels of the elicited cytokines and the degree of patch test reactivity. Hence, suggesting an involvement of both Th1- and Th2-type cytokines in ACD to Ni²⁺ and a direct association between the magnitude of the Ni²⁺-induced cytokine response overall and the <i>in vivo</i> reactivity to Ni²⁺. The impact of the regulatory cytokine IL-10 on Ni²⁺-induced Th1- and Th2-type cytokine responses in human PBMC was investigated in the next study. PBMC from blood donors with a history of Ni²⁺ reactivity and non-reactive control donors were stimulated with Ni²⁺ <i>ex vivo</i> with or without addition of human recombinant IL-10 (rIL-10) or neutralising mAb to IL-10. Depletion/enrichment experiments were performed to phenotype the Ni²⁺-specific cytokine producing cells. Exogenous rIL-10 significantly down-regulated the production of all cytokines but with a more pronounced effect on IFN-γ. IL-10 neutralisation, on the other hand, enhanced the levels of Ni²⁺-induced IFN-γ only. Ni²⁺-specific cytokine-producing cells in PBMC were found to be predominantly CD4⁺ T cells. Thus, IL-10 may play a regulatory role <i>in vivo</i> by counteracting the ACD reactions mediated by CD4⁺ T cells producing Th1-type cytokines. In the third study, we investigated the relationship between <i>in vivo</i> patch test reactivity to a number of metals (Ni, Co, Pd, Cr and Au) included in the standard and/or dental patch test series and <i>in vitro</i> responses to the metals in question. PBMC from metal patch test positive and negative control subjects were stimulated with a panel of eight metal salts and cytokine responses analysed by ELISpot and/or ELISA. A mixed Th1- (IL-2 and/or IFN-γ) and Th2-type (IL-4 and/or IL-13) cytokine profile was observed in PBMC from most metal allergic subjects upon <i>in vitro</i> stimulation with the metal(s) to which the subject was patch test positive. Our data suggest that other metals included in the standard and dental patch test series, just like Ni2⁺, induce a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects <i>in vitro</i>. We further developed a simplified ELISpot protocol utilising plates precoated with capture monoclonal antibodies (mAb) and subsequent detection in one step using enzyme-labelled mAb, for enumerating the frequency of allergen (Ni²⁺)-specific cytokine producing cells. This was compared with a regular ELISpot protocol, with an overnight incubation for capture mAb adsorbtion and detection with biotinylated mAb followed by enzyme-labelled streptavidin. PBMC from Ni²⁺-reactive and non-reactive subjects were incubated with or without NiCl₂ and the enumeration of cells producing IFN-γ, IL-4 or IL-13 by the two protocols were compared. PBMC from Ni²⁺-reactive subjects showed significantly higher Ni²⁺-induced IL-4 and IL-13 responses and the number of antigen-specific cytokine-producing cells determined by the two ELISpot protocols correlated well. In a nutshell, our data point to the potential use of <i>in vitro</i> cytokine assays as diagnostic tools in distinguishing ACD subjects sensitised to different metals and non-sensitised subjects.</p>