Sperm Use During Egg Fertilization in the Honeybee (Apis Mellifera)

A technique to quantify sperm use in honeybee queens (Apis mellifera) was developed and used to analyze the number of sperm used in different groups of honeybee queens. To do this a queen was placed on a frame with worker cells containing no eggs, and an excluder box was placed around her. The frame was put back into the colony and removed after two and a half hours. This method reduced stress on the queen so that she felt comfortable enough to lay eggs and did not require the queen to be killed so that she could be sampled multiple times to look at effects of age and time after introduction into a colony. The eggs were guaranteed to be fresh and sperm number present on the egg could be counted with a fluorescence microscope by using DAPI to dye the DNA in the sperm heads. The queens were found to be very economic in their sperm use and an overall median of three sperm per egg was found. Individual queens were found to vary significantly in sperm used per egg, however no queen had a median of over ten sperm per egg. Days after introduction into the colony also had a significant effect on sperm use when looking at the queens individually and it was found that newly introduced queens used more sperm than established queens. Overall, the study shows that freshly laid eggs can be collected and sperm on these eggs can be counted directly. This is an important finding for further research into the factors affecting different patterns of sperm use and possibly for beekeepers to breed queens who use their sperm most efficiently, and who will therefore be able to maintain a successful colony for the longest time

Estudo de marcadores imunológicos para o diagnóstico e avaliação terapêutica da toxocaríase

In human toxocariasis, there are few approaches using immunological markers for diagnosis and therapeutic assessment. An immunoblot (IB) assay using excretory-secretory Toxocara canis antigen was standardized for monitoring IgG, IgE and IgA antibodies in 27 children with toxocariasis (23 visceral, three mixed visceral and ocular, and one ocular form) for 22-116 months after chemotherapy. IB sensitivity was 100% for IgG antibodies to bands of molecular weight 29-38, 48-54, 95-116, 121-162, >;205 kDa, 80.8% for IgE to 29-38, 48-54, 95-121, >; 205 kDa, and 65.4% for IgA to 29-38, 48-54, 81-93 kDa. Candidates for diagnostic markers should be IgG antibodies to bands of low molecular weight (29-38 and 48-54 kDa). One group of patients presented the same antibody reactivity to all bands throughout the follow-up study; in the other group, antibodies decayed partially or completely to some or all bands, but these changes were not correlated with time after chemotherapy. Candidates for monitoring patients after chemotherapy may be IgG antibodies to >; 205 kDa fractions, IgA to 29-38, 48-54, 81-93 kDa and IgE to 95-121 kDa. Further identification of antigen epitopes related to these markers will allow the development of sensitive and specific immunoassays for the diagnosis and therapeutic assessment of toxocariasis.Métodos imunológicos desempenham papel importante no diagnóstico da toxocaríase, entretanto há poucos estudos sobre marcadores diagnósticos e de acompanhamento terapêutico. Foi padronizado ensaio de immunoblot (IB) empregando antígeno de excreção-secreção de Toxocara canis para pesquisa de anticorpos IgG, IgE e IgA em 27 crianças com toxocaríase nas formas visceral (23), mista visceral e ocular (3) e ocular (1), por 22-116 meses após quimioterapia. Foram observados dois perfis de reatividade dos anticorpos: permanência contra todas as frações no decorrer do estudo; diminuição ou negativação contra algumas ou todas as frações, porém, essas mudanças não se correlacionaram com tempo de tratamento. A sensibilidade do IB foi 100,0% para anticorpos IgG específicos para frações de massa molecular de 29-38, 48-54, 95-116, 121-162, >; 205 kDa, 80,8% para IgE específicos para 29-38, 48-54, 95-121, >; 205 kDa e 65,4% para IgA específicos para 29-38, 48-54, 81-93 kDa. Anticorpos IgG específicos para frações de baixa MM (29-38 e 48-54 kDa) podem ser sugeridos como candidatos a marcadores diagnósticos. Por sua vez, anticorpos IgG para fração >; 205 kDa, IgA para 29-38, 48-54, 81-93 kDa e IgE para 95-121 kDa podem ser candidatos a marcadores terapêuticos. A identificação de epítopos antigênicos relacionados a estes marcadores poderá ser importante para o desenvolvimento de ensaios altamente sensíveis e específicos no diagnóstico e avaliação terapêutica da toxocaríase

Detecção de larvas de Toxocara canis em leite: um estudo experimental em coelhas.

Toxocariasis, caused most commonly by Toxocara canis, is an important cosmopolitan zoonosis. Paratenic hosts have been employed to provide knowledge regard to the transmission of toxocariasis. Transmammary transmission in murine experimentally infected was observed based on the recovery of larvae from the tissue. The aim of this study was to evaluate the possibility of transmammary transmission of Toxocara canis in rabbits by detecting larvae directly in milk. Seventeen sexually mature virgin white New Zealand female rabbits were divided into two groups. Twelve animals were orally inoculated with 1,000 T. canis embryonated eggs (infected group), and five animals remained uninfected (control group). One month following the infection, the females were mated. Manual collection of 500 ?L of milk from each rabbit was performed on days +7, +14 and +21 of lactation for three consecutive lactations. The recovery of larvae was determined via a centrifuge-sedimentation technique using ether and formalin solutions. ELISA test was run to confirm the production of anti-T. canis antibodies (IgG) by infected rabbits. The presence of larvae was observed in milk samples from 5 (41.7%) of the 12 infected rabbits. The total number of recovered larvae was 20, ranging from 1 to 4 larvae per lactation/rabbit. Larvae were recovered exclusively on days 7 and 14 of lactation. Recovery was verified in different lactations. No significant difference was observed with respect to the number of larvae either in the same lactation period or in different lactation periods. Anti-T. canis antibodies were detected in all infected rabbits. In conclusion, the presence of larvae in rabbit milk samples suggests the possibility of galactogenic transmission of T. canis in paratenic hosts. Moreover, the technique employed in this study allows for the recovery of larvae directly from milk

DNA Electrophoretic Migration Patterns Change after Exposure of Jurkat Cells to a Single Intense Nanosecond Electric Pulse

Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns

Proceedings of the 13th annual conference of INEBRIA

CITATION: Watson, R., et al. 2016. Proceedings of the 13th annual conference of INEBRIA. Addiction Science & Clinical Practice, 11:13, doi:10.1186/s13722-016-0062-9.The original publication is available at https://ascpjournal.biomedcentral.comENGLISH SUMMARY : Meeting abstracts.https://ascpjournal.biomedcentral.com/articles/10.1186/s13722-016-0062-9Publisher's versio

A serological follow-up of toxocariasis patients after chemotherapy based on the detection of IgG, IgA, and IgE antibodies by enzyme-linked immunosorbent assay.

A serological follow-up study was carried out on 27 children (1–12 years old) with\ud visceral and/or ocular toxocariasis, after treatment with thiabendazole. A total of 159 serum samples were collected in a period ranging from 22–116 months. Enzyme-linked\ud immunosorbent assays (IgG, IgA, and IgE ELISA) were standardized, using excretory–secretory antigens obtained from the second-stage larvae of a Toxocara canis culture. The sensitivity found for the IgG, IgA, and IgE ELISA, as determined in visceral toxocariasis patients, was 100%, 47.8%, and 78.3%, respectively. Approximately 84% of the patients presented single or multiple parasitosis, as diagnosed by stool examination, yet such variables did not appear to affect the anti-Toxocara immune response. Titers of specific IgE\ud antibody showed a significant decrease during the first year after treatment, followed by a decrease in the IgA titers in the second year, and in the IgG titers from the fourth year onwards. Sera from all patients presented high avidity IgG antibodies, indicating that they were in the chronic phase of the disease. Moreover, 1 year after treatment, the level of leukocytes, eosinophils, and anti-A isohemagglutinin in patients decreased significantly. The present data suggest that IgE antibodies plus eosinophil counts are helpful parameters for patient followup after chemotherapy.Laboratório de Investigação Médica em Imunologia- LIM 48- Hospital das Clínicas da Faculdade de Medicina da Universidade de São PauloPublicação referente à tese de doutorado em Ciências – Área: Biologia da Relação Patógeno-Hospedeiro pelo Instituto de Ciências Biomédicas da Universidade de São Paulo, intitulada: "Toxocaríase humana: avaliação dos níveis de anticorpos IgG, IgA e IgE anti-Toxocara canis e correlação clínico-laboratorial em pacientes submetidos à terapêutica.

An alternate technique for isolation of Toxocara canis excretory-secretory antigens

The aim of the present study was to test the effectiveness of a sausage-casing membrane for dialysis of Toxocara excretory-secretory antigens (TES). The protein concentrated by the tested membrane was compared with that obtained using a Sigma commercial membrane, as were the protein fractions found by polyacrylamide gel electrophoresis. Standard positive and negative serum samples were evaluated in an ELISA immunoassay, and equivalent data were obtained in all steps, indicating that the sausage-casing membrane is efficient, besides being less expensive to process.<br>O objetivo do presente estudo foi testar a eficácia de uma membrana utilizada para o preparo de embutidos, na obtenção do antígeno de excreção e secreção de Toxocara (TES). A concentração protéica foi comparada com a obtida com a membrana Sigma tanto quanto as frações protéicas separadas por eletroforese em gel de poliacrilamida. Amostras de soros padrão positivo e negativo foram avaliadas no teste imunoenzimático ELISA. Dados equivalentes foram observados em todas as etapas, sugerindo que a membrana possa ser utilizada para diálise por ser eficiente e de menor custo no preparo do antígeno