Isolation of Lactic Acid Bacteria (LAB) from Salmonids for Potential Use as Probiotics: In Vitro Assays and Toxicity Assessment of Salmo trutta Embryonated Eggs.

This research investigates the potential of lactic acid bacteria (LAB) from freshwater salmonids as prospective probiotics for application in aquaculture. LAB and pathogenic bacteria were obtained from mucus and tissues of Oncorhynchus mykiss and Salmo trutta from fish farms in northeast Spain that had not used antibiotics for the six months preceding the study. Isolates were identified using Gram staining and sequencing of 16S rRNA and ITS-1. To assess the safety of the LAB, antibiotic susceptibility tests (ASTs) against 23 antimicrobials were performed. In vitro antagonism assays were conducted to evaluate the inhibitory effects of living LAB using the agar diffusion test method and their metabolites using the agar well diffusion method. The assays targeted six specific pathogens: Aeromonas salmonicida subsp. salmonicida, Carnobacterium maltaromaticum, Vagococcus salmoninarum, Yersinia ruckeri, Lactococcus garvieae, and the marine pathogen Vibrio jasicida. Additionally, a toxicity assay was conducted on embryonic eggs of S. trutta. The ASTs on probiotic LAB candidates revealed varied responses to antimicrobials, but no resistance to oxytetracycline or florfenicol, which are two antibiotics commonly used in aquaculture, was detected. The in vitro assays indicate that LAB exhibit antagonistic effects against pathogens, primarily when directly stimulated by their presence. In applications involving embryonic eggs or larvae, certain live strains of LAB were found to have adverse effects, with some isolates resulting in higher mortality rates compared to the control group or other isolates. Furthermore, the potential pathogenicity of certain LAB strains, typically considered safe in salmonids, warrants deeper investigation.

Development and verification of double antibody sandwich ELISA for determination of ovalbumin / 中国生物制品学杂志

@#Objective To develop and verify a double antibody sandwich ELISA detection method for the determination of ovalbumin(OVA),in order to determine the OVA content in influenza vaccines.Methods The rabbit anti-OVA polyclonal antibody was used as coating antibody and HRP labeled rabbit anti-OVA polyclonal antibody as detection antibody to develop a double antibody sandwich ELISA for OVA.The antibody concentration(2,1,0.5 and 0.25 pg/mL) for coating,enzyme-labeled antibody concentration(0.5,0.25 and 0.125 μg/mL),and kinds of blocking reagent(blocking with1% BSA,blocking with 2% BSA,blocking with 1% BSA and 1% sucrose,and blocking with 2% BSA and 2% sucrose,using nonblocking as control) were optimized,and the Cut-off value was determined as the judgment standard.The developed method was verified for the linear range and detection limit,specificity,repeatability and accuracy.Results The optimum detection conditions were as follows:the concentration of coating antibody was 1 μg/mL,the concentration of enzyme-labeled antibody was 0.25 μg/mL;The blocking reagent was 2% BSA and 2% sucrose;The Cut-off value was 0.051 66.The linear range of the method was 5～0.313 ng/mL,and the detection limit was 0.078 ng/mL;It did not react with influenza virus,bovine serum albumin(BSA) and Vero cell supernatant;The intraplate and interplate coefficient of variation(CV) were between 2.562%～13.887% and 4.000%～16.497% respectively;The coincidence rate between the results of this method and the Germany SERAMUN OVA quantitative test kit ranged from 93.79% to 107.05%.Conclusion The developed OVA double antibody sandwich ELISA has good specificity,repeatability,linear range and accuracy with convenience and lower cost,which might be used for the quantitative detection of OVA.

Estudo do rendimento viral na produção da vacina influenza, com base no uso de ovos de galinha embrionados com diferentes idades

This experimental study proposes to carry out a data collection regarding the viral yield of the production of the monovalent influenza virus, using embryonated chicken eggs with different ages. Considering that it is an experimental work, the main justification for its realization is the constant search for the improvement of industrial production processes, specifically, the possibility of increasing the production capacity of the factory of monovalent influenza viruses. For this, the determination of the best embryonic age will enhance a greater replication of the viral population existing in the microenvironment of the egg surrounding the embryo. All stages of the study procedures, which included the use of various modern equipment and laboratory materials, as well as specific reagents inherent to the process, were in accordance with the parameters established by the Quality Assurance department of the Butantan Institute, being in-process controls essential for the different stages of the artificial incubation stage of embryonated chicken eggs. Embryonated chicken eggs aged between 37 and 44 weeks, strains B/Phuket/3073/2013, B/Austria/1359417/2021, A/Darwin/9/2021, respectively, were used, totaling 9,000 (nine thousand) eggs with 9 (nine), 10 (ten), 11 (eleven) and 12 (twelve) day-old embryos for interpretation of the respective statistical data. The study was carried out in the Laboratory for the Production of Influenza Banks (PBI) building, since it was in compliance with regard to the nature of the laboratory activities monitored by the Institution's support disciplines, as well as being in line with the Good Laboratory Practices, Good Manufacturing Practices and Good Biosafety Practices. The eggs were incubated in specific artificial incubation machines and subjected to a temperature range between 33°C and 35°C, as well as a humidity of approximately 55%, which were the in-process controls chosen for effective viral replication and subjected to following unit operations: receipt of eggs, primary candling, primary incubation, preparation of viral inoculum, test of hemagglutinating units of viral inoculum, viral inoculation, secondary incubation, secondary candling, primary cooling, collection of Allantoic Liquid, secondary cooling, sample purification , determination of sucrose content, UHA and Optical Density of 14 purified samples. The results of this experimental study were submitted to statistical analysis to enhance discussions about the implementation of the use of embryonated chicken eggs, with 11 days and/or 12 days of life, better viral yields found, as strategies to establish new specifications of embryonated eggs, as raw material for the production process.

Este estudo experimental propõe a realização de um levantamento de dados no tocante ao rendimento viral da produção do monovalente do vírus influenza, utilizando ovos de galinha embrionados e com diferentes idades. Considerando que é um trabalho experimental, a principal justificativa para sua realização é pela constante busca do aperfeiçoamento dos processos produtivos industriais, especificamente, na possibilidade de aumentar a capacidade produtiva da fábrica de monovalentes de vírus influenza. Para isto, a determinação da melhor idade embrionária potencializará uma maior replicação da população viral existente no microambiente do ovo circundante ao embrião. Todas as etapas dos procedimentos do estudo, que contou com a utilização de diversos equipamentos modernos e materiais laboratoriais, bem como reagentes específicos inerentes ao processo, estavam em conformidade com os parâmetros estabelecidos pelo departamento de Garantia da Qualidade do Instituto Butantan, sendo controles em processo essenciais para os diferentes estágios da etapa de incubação artificial dos ovos de galinha embrionados. Foram utilizados ovos embrionados de galinha com idades entre 37 e 44 semanas, das cepas B/Phuket/3073/2013, B/Austria/1359417/2021, A/Darwin/9/2021, respectivamente, totalizando 9.000 (nove mil) ovos com embriões de 9 (nove), 10 (dez), 11 (onze) e 12 (doze) dias de idade para interpretação dos respectivos dados estatísticos. O estudo foi realizado no prédio do Laboratório da Produção de Bancos Influenza (PBI), uma vez que estava dentro da conformidade no tocante a natureza das atividades de cunho laboratorial acompanhadas pelas disciplinas de apoio da Instituição, assim como mantinham-se em consonância com as Boas Práticas de Laboratório, Boas Práticas de Fabricação e Boas Práticas de Biossegurança. Os ovos foram incubados em máquinas específicas de incubação artificial e submetidos ao range de temperatura compreendido entre 33°C e 35°C, bem como a umidade de aproximadamente 55%, que foram os controles em processo escolhidos para a efetiva replicação viral e submetidos às seguintes operações unitárias: recebimento dos ovos, ovoscopia primária, incubação primária, preparo do inóculo viral, teste de unidades 12 hemaglutinantes do inóculo viral, inoculação viral, incubação secundária, ovoscopia secundária, resfriamento primário, colheita do Líquido Alantóico, resfriamento secundário, purificação das amostras, determinação do teor de sacarose, UHA e Densidade Óptica das amostras purificadas. Os resultados deste estudo experimental foram submetidos a análises estatísticas para potencializar as discussões sobre a implementação do uso de ovos de galinha embrionados, com 11 dias e/ou 12 dias de vida, melhores rendimentos virais encontrados, como estratégias para estabelecer novas especificações dos ovos embrionados, na condição de matéria prima para o processo produtivo.

Propagation of avian influenza virus in embryonated ostrich eggs.

Influenza A viruses (IAVs) are typically isolated and cultured by successive passages using 9- to 11-day-old embryonated chicken eggs (ECEs) and in 14-day old ECEs for virus mutational studies. Real-time reverse transcription-polymerase chain reaction tests (RT-PCRs) are commonly used for IAV diagnosis, but virus isolation remains invaluable in terms of its high sensitivity, providing viable isolates for further studies and the ability to distinguish between viable and nonviable virus. Efforts at isolating ostrich-origin IAVs from RT-PCR positive specimens using ECEs have often been unsuccessful, raising the possibility of a species bottleneck, whereby ostrich-adapted IAVs may not readily infect and replicate in ECEs, yet the capacity of an ostrich embryo to support the replication of influenza viruses has not been previously demonstrated. This study describes an optimised method for H5 and H7 subtype IAV isolation and propagation in 28-day old embryonated ostrich eggs (EOEs), the biological equivalent of 14-day old ECEs. The viability of EOEs transported from breeding sites could be maximised by pre-incubating the eggs for 12 to 14 days prior to long-distance transportation. This method applied to studies for ostrich-adapted virus isolation and in ovo studies will enable better understanding of the virus-host interaction in ostriches and the emergence of potentially zoonotic diseases.

Expanding Mouse-Adapted Yamagata-like Influenza B Viruses in Eggs Enhances In Vivo Lethality in BALB/c Mice.

Despite the yearly global impact of influenza B viruses (IBVs), limited host range has been a hurdle to developing a readily accessible small animal disease model for vaccine studies. Mouse-adapting IBV can produce highly pathogenic viruses through serial lung passaging in mice. Previous studies have highlighted amino acid changes throughout the viral genome correlating with increased pathogenicity, but no consensus mutations have been determined. We aimed to show that growth system can play a role in mouse-adapted IBV lethality. Two Yamagata-lineage IBVs were serially passaged 10 times in mouse lungs before expansion in embryonated eggs or Madin-Darby canine kidney cells (London line) for use in challenge studies. We observed that virus grown in embryonated eggs was significantly more lethal in mice than the same virus grown in cell culture. Ten additional serial lung passages of one strain again showed virus grown in eggs was more lethal than virus grown in cells. Additionally, no mutations in the surface glycoprotein amino acid sequences correlated to differences in lethality. Our results suggest growth system can influence lethality of mouse-adapted IBVs after serial lung passaging. Further research can highlight improved mechanisms for developing animal disease models for IBV vaccine research.

In vivo effects of aflatoxin B1 and benzo[a]pyrene on the heart muscle of chicken embryos.

The purpose of the present study was to evaluate the effects of aflatoxin B1 (AFB1) and benzo[a]pyrene (BaP) on the heart muscle of chicken embryos of the broiler line Ross 308. The benzo[a]pyrene in the organic oil solution was injected in ovo on the 6th day of the incubation in doses of: 0.1, 0.5, and 1 mg/kg weight of eggs; the aflatoxin B1 in the organic oil solution was injected in ovo on the 6th day of the incubation into the yolk in doses of 80, 120 and 240 ng/kg weight of eggs. Multiple biochemical and hepatic parameters have been observed, including sodium, potassium, chloride, cholesterol, uric acid, total proteins, aminotransferase aspartate, and aminotransferase alanine. A low dose of AFB1 and BaP administered in ovo during early embryonic development had a significant impact on chicken embryonic development, as demonstrated by alterations in biochemical, mineral, and hepatic parameters.

Potency Testing of Venoms and Antivenoms in Embryonated Eggs: An Ethical Alternative to Animal Testing.

Venoms are complex mixtures of biologically active molecules that impact multiple physiological systems. Manufacture of antivenoms (AVs) therefore requires potency testing using in vivo models to ensure AV efficacy. As part of ongoing research to replace small animals as the standard model for AV potency testing, we developed an alternate in vivo method using the embryonated egg model (EEM). In this model, the survival of chicken embryos envenomated in ovo is determined prior to 50% gestation, when they are recognized as animals by animal welfare legislation. Embryos were found to be susceptible to a range of snake, spider, and marine venoms. This included funnel-web spider venom for which the only other vertebrate, non-primate animal model is newborn mice. Neutralization of venom with standard AV allowed correlation of AV potency results from the EEM to results from animal assays. Our findings indicate that the EEM provides an alternative, insensate in vivo model for the assessment of AV potency. The EEM may enable reduction or replacement of the use of small animals, as longer-term research that enables the elimination of animal use in potency testing continues.

Equine Influenza Culture Methods.

Equine influenza viruses are cultured in embryonated chicken eggs or in mammalian cells, generally Madin-Darby canine kidney (MDCK) cells, using methods much the same as for other influenza A viruses. Mutations associated with host adaptation occur in both eggs and MDCK cells, but the latter show greater heterogeneity and eggs are the generally preferred host. Both equine-1 H7N7 and equine-2 H3N8 viruses replicate efficiently in 11-day-old eggs, but we find that equine-1 viruses kill the embryos whereas equine-2 viruses do not.

Uptake, Deposition, and Metabolism of Triphenyl Phosphate in Embryonated Eggs and Chicks of Japanese Quail (Coturnix japonica).

The toxicokinetics of triphenyl phosphate (TPHP) in vivo including the uptake, deposition, and biotransformation into the metabolite diphenyl phosphate (DPHP) is presently reported in embryonated eggs and chicks of Japanese quail. Quail were dosed with TPHP at 3 concentrations by air cell egg injection on embryonic day 0, followed by daily oral dosing after chicks hatched (5 d). Vehicle-only exposed controls were also used. In dosed eggs, only 33% of the TPHP remained 2 d after injection (no hepatic development); after 10 d (post-hepatogenesis), only 2% remained. The estimated TPHP half-lives in the eggs ranged from 1.1 to 1.8 d for the 3 dosed groups. In all exposed eggs and chicks, DPHP significantly increased with dose (0.001 < p < 0.044). It appears that DPHP is an important metabolite in quail, making up 41 to 74% of all metabolites formed in embryonated eggs. In chicks, at medium and high doses, DPHP concentrations significantly exceeded those of TPHP (p ≤ 0.007), making up 67 and 76% of the total burden, respectively. Our findings suggest that rapid TPHP metabolism occurred in chicks and embryonated quail eggs but that this may vary with the age of the embryonated egg and the stage of embryo development, which should be considered when evaluating concentrations of TPHP and DPHP measured in eggs of wild birds. Environ Toxicol Chem 2020;39:565-573. © 2019 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry © 2019 SETAC.

The Role of the IACUC in the Absence of Regulatory Guidance.

Institutional Animal Care and Use Committees (IACUCs) occasionally face regulatory requirements for which clear guidance may not be available. Either the regulating body has chosen not to provide such guidance or the guidance may be minimal or even ambiguous. Such guidance may be desirable when institutions have research needs, in which case IACUCs are left to their own interpretation to develop internal policies, procedures, and documents. Typically, this is approached by an IACUC working with partners in the laboratory animal community and may involve input from regulators who can provide context as well as parameters to consider. Over time, shared institutional experiences and documentation coalesce to create a general framework that provides a baseline for others to consider as templates for further policy elaboration or development. The strength of this approach relies on the ability to share freely, including having unobstructed access to such documents.

An In Vitro Assay of Disinfectants on the Viability of Heterakis gallinarum Eggs.

Nematodes are widespread and common in poultry. Disinfectants are used to reduce infection rates in poultry houses, but there is little documentation of their effectiveness. An in vitro assay was developed to test the efficacy of products to damage Heterakis gallinarum eggs, and nine disinfectants and chemicals commonly used in the poultry industry were tested. Embryonated eggs of H. gallinarum were pipetted into wells of plastic cell culture plates (250-300 eggs/well in water). Measured amounts of test articles were added to the suspensions for 2, 4, 6, or 24 hr. After exposure, eggs were washed with water and treated with trypan blue (1 ml of 0.4% solution, added to each well) at room temperature for 2 min. Eggshell integrity was determined microscopically by counting the number of eggs that were clear (intact) or that contained blue dye (compromised). As a test of embryo viability, five eggs per well from treatments containing compromised eggs were transferred to a Petri dish and hatched manually, using forceps to open the eggshell. Released larvae were then observed for signs of controlled movement. In a test of Clorox bleach (NaOCl), Green Klean, Decon7, Kem San, PLT, Virkon S, NaCl, dry limestone (CaCO3), and diesel fuel, only NaOCl (bleach) and Green Klean damaged the eggshell, and only 20,625 ppm of NaOCl rendered the larvae nonviable.

Nota de Investigación- Un ensayo in vitro de desinfectantes sobre la viabilidad de los huevos de Heterakis gallinarum. Los nematodos son comunes y están generalizados en la avicultura. Los desinfectantes se usan para reducir las tasas de infección en las granjas avícolas, pero existe poca documentación de su efectividad. Se desarrolló un ensayo in vitro para probar la eficacia de los productos para afectar los huevos de Heterakis gallinarum, y se probaron nueve desinfectantes y productos químicos comúnmente utilizados en la industria avícola. Los huevos embrionados de H. gallinarum se pipetearon en pozos de placas plásticas de cultivo celular (250­300 huevos/pozo en agua). Se agregaron cantidades medidas de los compuestos a probar a las suspensiones durante dos, cuatro, seis o 24 horas. Después de la exposición, los huevos se lavaron con agua y se trataron con azul tripán (1 ml de solución al 0.4%, se agregaron a cada pozo) a temperatura ambiente durante dos minutos. La integridad de la cáscara del huevo se determinó microscópicamente al contar el número de huevos que estaban claros (intactos) o los que contenían colorante azul (afectado). Como prueba de la viabilidad de los embriones, se transfirieron cinco huevos por pozo de los tratamientos que contenían huevos afectados a una placa de Petri y se eclosionaron manualmente utilizando fórceps para abrir la cáscara del huevo. Las larvas liberadas fueron observadas para detectar signos de movimiento controlado. En una prueba con blanqueador Clorox (NaOCl), Green Klean, Decon7, Kem San, PLT, Virkon S, NaCl, caliza seca (CaCO3) y combustible diesel, solo NaOCl (lejía) y Green Klean dañaron la cáscara del huevo, y solo 625 ppm de NaOCl provocaron que las larvas no fueran viables.

Safety of viable embryonated eggs of the whipworm Trichuris suis as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on viable embryonated eggs of the whipworm Trichuris suis as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The applicant proposes to use the NF as a food supplement in the format of a 15-mL bottle containing 250 viable embryonated eggs of T. suis. The target population for the NF is the general population. Considering the compositional data and proposed conditions of use, the consumption of the NF is considered of no nutritional relevance. Available data suggest that most larvae of T. suis after hatching in the intestinal tract of humans remain immature and live for several weeks in the gastrointestinal tract of the human host. Nevertheless, under certain circumstances, T. suis can be invasive in human, being able to mature into adult size and reproduce in humans. Human studies have also shown that administration of T. suis ova may increase the incidence of adverse gastrointestinal reactions. The Panel considers that there are no studies available that demonstrate the safety of this NF intended for the general population at a proposed intake of 250 viable embryonated eggs of T. suis ova per day. Based on the available information, the Panel cannot establish a safe dose at which no safety concerns would be expected. The Panel concludes that the safety of the NF has not been established.

Propagation and Titration of Influenza Viruses.

Influenza viruses are constantly circulating among humans, in which they cause seasonal epidemics of severe respiratory disease. Additionally, these zoonotic viruses infect different mammals and birds, from which new antigenic variants are occasionally transmitted to humans leading to devastating global pandemics. Surveillance programs, in which viruses from the main reservoir (waterfowl), intermediate hosts (like pigs and other farm animals), and other affected species are isolated and characterized, are crucial for the global influenza prevention strategy. This chapter gives an overview of the most commonly used methods for the propagation and titration of influenza viruses, which are key steps in surveillance procedures, as well as in vaccine development and basic research. Depending on the host and the viral strain, primary isolates are obtained from biological samples of different origin and subsequently amplified in embryonated chicken eggs or cell cultures. These propagation procedures are the focus of the first part of this chapter. Once the initial isolates have been amplified, virus titration methods based on particular characteristics of influenza viruses, such as their ability to agglutinate red blood cells (RBCs) or to induce cytopathic effects (CPE) in cell monolayers, are used to estimate the amount of viral particles. Such approaches, like the hemagglutination assay (HA assay), 50% tissue culture infectious dose (TCID50), or plaque assay, are included in the second part of this chapter. Although they are simple and cost-effective, some of these techniques have been partially replaced by faster and more sensitive methods based on the quantification of viral genomes, such as the quantitative real-time reverse transcription PCR (RT-qPCR), which is presented at the end of this section. The different protocols are explained in detail in order to facilitate the preparation and quantification of infectious virus stocks.

Phytochemicals reduce aflatoxin-induced toxicity in chicken embryos.

Aflatoxins (AF) are toxic metabolites produced by molds, Aspergillus flavus and Aspergillus parasiticus, which frequently contaminate poultry feed ingredients. Ingestion of AF-contaminated feed by chickens leads to deleterious effects, including decreased bird performance and reduced egg production. Moreover, AF residues in fertilized eggs result in huge economic losses by decreasing embryo viability and hatchability. This study investigated the efficacy of 2 generally recognized as safe phytochemicals, namely carvacrol (CR) and trans-cinnamaldehyde (TC), in protecting chicken embryos from AF-induced toxicity. Day-old embryonated eggs were injected with 50 ng or 75 ng AF with or without 0.1% CR or TC, followed by incubation in an incubator for 18 d. Relative embryo weight, yolk sac weight, tibia weight, tibia length, and mortality were recorded on d 18 of incubation. The effect of phytochemicals and methanol (diluent) on embryo viability was also determined. Each experiment had ten treatments with 15 eggs/treatment (n = 150 eggs/experiment) and each experiment was replicated 3 times. Both phytochemicals significantly decreased AF-induced toxicity in chicken embryos. At 75 ng of AF/egg, CR and TC increased the survival of chicken embryo by ∼55%. Moreover, CR and TC increased relative embryo weight by ∼3.3% and 17% when compared to eggs injected with 50 ng or 75 ng AF, respectively. The growth of embryos (tibia length and weight) was improved in phytochemical-treated embryos compared to those injected with AF alone (P < 0.05). Phytochemical and methanol treatments did not adversely affect embryo survival, and other measured parameters as compared to the negative control (P > 0.05). Results from this study demonstrate that CR and TC could reduce AF-induced toxicity in chicken embryos; however, additional studies are warranted to delineate the mechanistic basis behind this effect.

A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination.

BACKGROUND: Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. METHODS: We established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3'-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. RESULTS: Targeted RNA recombination allowed for the modification of the 3'-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52. CONCLUSIONS: Targeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general.

Detection of Specific Antibody Reactivity to Toxocara Larval Excretory-secretory Antigens in Asthmatic Patients (5-15 Years).

BACKGROUND & PURPOSE: Humans act as an intermediate host for Toxocara canis and Toxocara cati. Toxocara may be an important risk factor for asthma in humans. The aim of the present study was to evaluate immunoglobulin G (IgG) anti-Toxocara canis antibody, using enzyme-linked immunosorbent assay (ELISA) in asthmatic patients (aged 5-15 years), referring to a clinic of pulmonary diseases in Arak, Iran. MATERIALS & METHODS: In this bi-group cross sectional study, serum samples were collected from 110 children with confirmed asthma and 70 children without asthma within one year. IgG anti-Toxocara antibody was detected viaELISA method. The collected data were analyzed, using SPSS. RESULTS: The seroprevalence of antibodies against Toxocara species was estimated at 1.8% (two males) in asmathic children viaELISA method; however, no antibodies against Toxocara canis were detected in the control group. There was no significant correlation between the frequency of antibodies against Toxocara and variables such as age, gender, or place of residence (P>0.05). Moreover, the frequency of antibodies against Toxocara was not significantly correlated with contact with dogs, consumption of unwashed fruits and vegetables, or use of raw/undercooked sheep liver (P>0.05). CONCLUSION: The present study showed anti-Toxocara antibody in 1.8% of asthmatic children and determined the seroprevalence of toxocariasis in asthmatic children and adolescents in Arak, Iran. Based on the findings, the low rate of infection with Toxocara among asthmatic children may be attributed to acceptable personal hygiene and religious considerations.

Bacteria-induced hatching of Trichuris muris eggs occurs without direct contact between eggs and bacteria.

Using three isolates of the murine parasitic nematode Trichuris muris, E, E/J (the E isolate maintained in Japan), and S, I have previously demonstrated that when the embryonated eggs of the E/J and E isolates are incubated with the intestinal bacteria Escherichia coli and Staphylococcus aureus, they are induced to hatch in vitro. However, the eggs of the S isolate are unresponsive to these bacteria. In the present study, I investigated whether direct contact between the embryonated eggs of the E/J and E isolates and bacteria is required to induce their hatching. To do so, a new co-culture system for eggs and bacteria (E. coli or S. aureus) was developed to block any direct contact between the eggs and the bacteria. In the hatching experiment using the new system, when direct contact between the eggs and bacteria was completely prevented, the eggs still hatched. However, the peak levels of hatching without direct contact were about 20 % lower than those with direct contact, and peak hatching occurred later without direct contact. This evidence suggests that hatching occurs without direct contact between the eggs and bacteria, and that unidentified material derived from active bacteria induces the hatching of embryonated eggs of the E/J and E isolates of T. muris in vitro.

Efficacy of fumigation with Trans-cinnamaldehyde and eugenol in reducing Salmonella enterica serovar Enteritidis on embryonated egg shells.

This study investigated the efficacy of two GRAS (generally regarded as safe)-status, plant-derived antimicrobials (PDAs), namely trans-cinnamaldehyde (TC) and eugenol (EUG) applied as a fumigation treatment in reducing SE on embryonated egg shells. Egg shells of day-old embryonated eggs were spot inoculated with a 4-strain mixture of SE (∼6.5 log CFU/egg) and subjected to fumigation with the aforementioned PDAs (0 or 1% concentration) for 20 minutes in a hatching incubator. SE on the shell and embryo was enumerated on days 1, 3, 6, 9, 13, 16 and 18. On day 13, the eggs were re-inoculated, followed by fumigation treatment for 20 minutes. Since the two PDAs were dissolved in ethanol (final concentration 0.04%), eggs fumigated with ethanol were included as a control.Approximately 6 log CFU/egg of SE were recovered from the shell of untreated, inoculated eggs on days 1 and 13. The fumigation of embryonated egg shells with the two PDAs was more effective in reducing SE on the shell and embryo compared to controls (P < 0.05). On day 18, the eggs fumigated with ethanol were SE positive on the shell, whereas no pathogen was detected on eggs subjected to fumigation with TC and EUG. Similarly, although the embryos of eggs subjected to fumigation with ethanol yielded 1 log CFU/egg of SE on day 18, the embryos of TC and EUG treated eggs were devoid of the pathogen. This study demonstrated that TC and EUG dissolved in 0.04% ethanol could potentially be used as a fumigation treatment for reducing SE on embryonated egg shell, however, quality traits of eggs, including the hatchability need to be ascertained.

Isolation of chicken astrovirus from specific pathogen-free chicken embryonated eggs.

Astroviruses have been associated with enteric disorders in many animal species, including chickens. Here, we describe the isolation, propagation, and pathological characteristics of chicken astrovirus (CAstV) in specific pathogen free (SPF) chicken embryonated eggs (CEE) from chickens with diarrhea and runting-stunting syndrome. The CEE were inoculated via the yolk sac route. Viral confirmation was carried out using PCR techniques and transmission electron microscopy negative staining with ammonium molybdate. The intestinal contents were screened for CAstV, and differential diagnostic testing was performed for avian nephritis virus, avian rotavirus, avian reovirus, chicken parvovirus, infectious bronchitis virus, and fowl adenovirus Group I to detect co-infection with other infectious agents. Seven- or 14-day-old CEEs presented with hemorrhages, edema, a gelatinous aspect, deformities, and dwarfism. The supporting membranes did not show any alterations. Here, we have described the isolation of CAstV and its pathological characteristics in SPF CEE.

Dose-dependent establishment of Trichuris suis larvae in Göttingen minipigs.

Embryonated eggs of the pig whipworm Trichuris suis (TSOee) constitute the active pharmaceutical ingredient (API) in a medicinal product explored in human clinical trials against several immune-mediated diseases. The measurement of TSO biological potency (hatchability and infectivity) is a requirement for the assessment of TSO's pharmacological potency in human clinical trials. The present study aims to validate the dose-dependent establishment of T. suis larvae in Göttingen minipigs and eventual clinical implication of a dose range (1000-10,000 TSO). Four groups of 5 minipigs were inoculated with doses of 1000, 2500, 7500, and 10,000 TSOee, respectively, to evaluate a range of concentrations of TSOee in a minipig infectivity model. Unembryonated eggs (TSOue) were added to keep the total egg number in the inoculum constant at 10,000 eggs. Two groups received 2500 and 7500 TSOee per pig without the addition of TSOue as controls. The intestinal larval establishment at 21 days post inoculation (dpi) demonstrated a clear positive linear dose-response relationship between numbers of inoculated TSOee and recovered larvae. There was a low level of variation in larval counts in all study groups. Thus, the infectivity model in minipigs within the tested dose range offers a reliable, sensitive and accurate assay for testing biological potency of TSO.