Role of the lactoferrin-binding protein in pathogenesis of streptococcus uberis

The pathogenesis of the bovine environmental pathogen Streptococcus uberis has been extensively studied, but it remains incompletely understood. The organism produces a surface protein capable of binding lactoferrin (Lbp), originally identified as an M-like streptococcal protein. We hypothesized that Lbp may play a role as a virulence factor. Structural similarity and amino acid sequence homology of Lbp to Mrelated proteins of Group A streptococci suggested a possible functional similarity between Lbp and M-like proteins, which are involved in evasion of host antibacterial defenses, adhesion to host epithelial cells and intracellular invasion by the bacteria. Alternatively, high-affinity binding of the abundant iron-chelating component of the host milk (lactoferrin) suggests that Lbp of S. uberis might play role in iron acquisition by the bacterium. Finally, Lbp might serve as a receptor for signal transduction in the bacterial cell or alter host cell signalling during infection, when the bacteria with surface-bound lactoferrin adhere to or invade the host epithelial cells. In order to test the hypothesis that Lbp is a virulence factor of S. uberis, a mutant strain of S. uberis unable to express the lactoferrin-binding protein was generated and the role of the protein was studied in comparative analyses of the mutant and the parent strains. The results of our study indicated that unlike many streptococcal M-like proteins, the lactoferrin-binding protein of S. uberis did not appear to play a role in overcoming host innate and acquired immune antibacterial responses. Both S. uberis and its lbp mutant were ingested by bovine blood neutrophils and were similar in their ability to survive in fresh bovine blood regardless of the presence either of lactoferrin or of anti-Lbp antibodies. Lbp did not promote bacterial adhesion to host epithelial cells and it was not essential for the internalization of the bacteria by host epithelial cells, since both S. uberis and lbp mutant were found capable of adhering, invading, intracellular survival and intracellular growth when the bacteria were co-cultured with bovine mammary epithelial cells. No significant differences in numbers of adherent or internalized bacteria per host cell were found between wild type and lbp mutant cells. S. uberis requirements for iron were determined to be low and Lbp was not essential for iron acquisition by the organism from iron-saturated lactoferrin. To study the role of Lbp in bacterial virulence during infection of bovine mammary glands, dairy cows in the second half of their lactation periods were challenged with the wild type S. uberis and with the lbp mutant. The results of in vivo infection suggested that expression of Lbp by the bacteria was not essential for colonization of the host mammary gland and that expression of Lbp was not associated with differences in severity of mastitis or with different levels of shedding of the bacteria by infected animals. To study the role of Lbp in signal transduction, differential bacterial cellular protein phosphorylation in the presence of bovine lactoferrin was analyzed. Since no differences in protein phosphorylation profiles were detected between S. uberis and the lbp mutant, it was concluded that Lbp is probably not a part of a classical bacterial twocomponent signalling pathway. However, we demonstrated that the expression of host genes potentially involved in cell morphogenesis, motility and signal transduction was regulated depending on the expression of Lbp by S. uberis. Down-regulation of the expression of selected host genes was verified by quantitative reverse transcription PCR. Putative iron responsive elements were identified in mRNA of several of these genes. Down-regulation of these genes in the cells overloaded with ferric iron was demonstrated by RT PCR. These results indicate that Lbp of S. uberis may interfere with host cellular signalling pathways by inducing perturbations in the cell iron status. This suggests that Lbp of S. uberis may be a virulence factor, playing a role in signal transduction or in the regulation of gene expression in host cells

SARS coronavirus protein 7a interacts with human Ap4A-hydrolase

The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase. In this study we present data demonstrating that the SARS-CoV 7a protein interacts with human Ap4A-hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17). Ap4A-hydrolase is responsible for metabolizing the "allarmone" nucleotide Ap4A and therefore likely involved in regulation of cell proliferation, DNA replication, RNA processing, apoptosis and DNA repair. The interaction between 7a and Ap4A-hydrolase was identified using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation from cultured human cells transiently expressing V5-His tagged 7a and HA tagged Ap4A-hydrolase. Human tissue culture cells transiently expressing 7a and Ap4A-hydrolase tagged with EGFP and Ds-Red2 respectively show these proteins co-localize in the cytoplasm

Does hive strength predispose honey bees to European foulbrood disease?

BC Blueberries, Project Apis m., Boehringer Ingelheim, Mitacs, Costco Wholesale, Saskatchewan Agriculture Development Fund, Agriculture Funding Consortium, Saskatchewan Beekeepers Development CommissionEuropean Foulbrood (EFB) is a bacterial disease of young honey bee larvae, caused by Melissococcus plutonius infection of the larval midgut. It occurs in times of nutritional stress when insufficient food is supplied to the larvae by the nursing bee population. EFB increases larval mortality, thereby limiting the colony’s growth, which can have consequences on the hive’s pollination services, honey production, and ability to reproduce. Recently, increased incidence of EFB has been observed across North America; however, the underlaying factors predisposing colonies to EFB remain largely unknown

Are honey bees a suitable model for fetal alcohol spectrum disorders?

Fetal alcohol spectrum disorders (FASDs) are a continuum of disorders caused by prenatal exposure to ethanol. They affect an estimated 4% of Canadians. FASDs are associated with a host of complications including, but not limited to, cognitive difficulties, developmental delay, increased mortality, smaller birth weight, smaller brain size, as well as gross and fine motor issues. It has been previously established that fruit flies (Drosophila melanogaster) are a suitable invertebrate model for FASDs. Honey bees (Apis mellifera) share many similarities to Drosophila as a research model, but with the distinct advantage of highly social behaviour, similar to that of humans. In this project we exposed honey bees to incremental, sublethal concentrations of ethanol during larval development and monitored their survival, developmental rate, and weight at adult emergence. We found that larval honey bees exposed to ≥6% ethanol experienced significantly higher mortality, developmental delay, and lower body weight at emergence. Accordingly, these results, in combination with ongoing neurobehavioural analyses of adult bees exposed to ethanol as larvae, suggest that honey bees may be an ideal model for human FASDs

НАУКОВІ ПІДХОДИ ТА ПРАКТИЧНІ АСПЕКТИ ЗАСТОСУВАННЯ МЕТОДУ ДОСЛІДЖЕННЯ ВПЛИВУ ПРОБІОТИКІВ НА ОРГАНІЗМ МЕДОНОСНИХ БДЖІЛ (Apis Mellifera) IN VITRO

Одним із сучасних і перспективних напрямків вирішення проблем оздоровлення пасіки, зокрема, безмедикаментозним методом, є застосування пробіотиків. Майже усі існуючі  пробіотичні штами, мають статус GRAS (Generally regarded as safe) і визнані як безпечні для використання людиною. Проте питання взаємодії макроорганізму і пробіотиків вимагає більш детального вивчення. Особливої уваги заслуговує метод дослідження впливу пробіотиків на ріст і розвиток личинок робочих особин медоносної бджоли in vitro, як модель з мінімальною варіативною складовою (D.R. Schmehl, 2016). Один із найважливіших аспектів цієї методики - це створення умов, за яких експозиція досліджуваного фактору проявляє максимальний і безпосередній вплив на ріст і розвиток личинок аж до появи імаго. Аналізуючи результати досліджень впливу пробіотичної добавки «Апіпротект-плюс» на ріст і розвиток личинок та лялечок робочих особин бджоли медоносної було виявлено, що концентрація пробіотичної добавки «Апіпротект-плюс» у дозі 1×106 КУО в 1 мл дієти В і С не викликала відхилення у рості і розвитку личинок, хоча спостерігалися спорадичні випадки затримки їх розвитку, в межах допустимого методикою до 5%. Застосування пробіотичної добавки «Апіпротект-плюс» у концентрації 1×109 КУО в 1 мл дієти В і С призводило до порушень перебігу процесів метаморфозу в личинок робочих особин бджоли медоносної, які характеризувалися вираженим відставанням у рості і розвитку (Р<0,001), що на завершальному етапі призводило до їх загибелі. Очевидно подібний ефект викликаний як безпосереднім впливом самих мікроорганізмів на личинок, так і продуктів їх метаболізму на склад компонентів дієти. Таким чином, можна припустити, що застосування методу вирощування личинок робочих медоносних бджіл in vitro дозволяє отримати додаткові відомості про вплив на їх ріст і розвиток пробіотичних груп мікроорганізмів та встановити їх допустимі концентрації, які можуть бути використані як in vitro, так in vivo.Одним із сучасних і перспективних напрямків вирішення проблем оздоровлення пасіки, зокрема, безмедикаментозним методом, є застосування пробіотиків. Майже усі існуючі  пробіотичні штами, мають статус GRAS (Generally regarded as safe) і визнані як безпечні для використання людиною. Проте питання взаємодії макроорганізму і пробіотиків вимагає більш детального вивчення. Особливої уваги заслуговує метод дослідження впливу пробіотиків на ріст і розвиток личинок робочих особин медоносної бджоли in vitro, як модель з мінімальною варіативною складовою (D.R. Schmehl, 2016). Один із найважливіших аспектів цієї методики - це створення умов, за яких експозиція досліджуваного фактору проявляє максимальний і безпосередній вплив на ріст і розвиток личинок аж до появи імаго. Аналізуючи результати досліджень впливу пробіотичної добавки «Апіпротект-плюс» на ріст і розвиток личинок та лялечок робочих особин бджоли медоносної було виявлено, що концентрація пробіотичної добавки «Апіпротект-плюс» у дозі 1×106 КУО в 1 мл дієти В і С не викликала відхилення у рості і розвитку личинок, хоча спостерігалися спорадичні випадки затримки їх розвитку, в межах допустимого методикою до 5%. Застосування пробіотичної добавки «Апіпротект-плюс» у концентрації 1×109 КУО в 1 мл дієти В і С призводило до порушень перебігу процесів метаморфозу в личинок робочих особин бджоли медоносної, які характеризувалися вираженим відставанням у рості і розвитку (Р<0,001), що на завершальному етапі призводило до їх загибелі. Очевидно подібний ефект викликаний як безпосереднім впливом самих мікроорганізмів на личинок, так і продуктів їх метаболізму на склад компонентів дієти. Таким чином, можна припустити, що застосування методу вирощування личинок робочих медоносних бджіл in vitro дозволяє отримати додаткові відомості про вплив на їх ріст і розвиток пробіотичних груп мікроорганізмів та встановити їх допустимі концентрації, які можуть бути використані як in vitro, так in vivo

Chronic High-Dose Neonicotinoid Exposure Decreases Overwinter Survival of Apis mellifera L.

Overwinter colony mortality is an ongoing challenge for North American beekeepers. During winter, honey bee colonies rely on stored honey and beebread, which is frequently contaminated with the neonicotinoid insecticides clothianidin and thiamethoxam. To determine whether neonicotinoid exposure affects overwinter survival of Apis mellifera L., we chronically exposed overwintering field colonies and winter workers in the laboratory to thiamethoxam or clothianidin at different concentrations and monitored survival and feed consumption. We also investigated the sublethal effects of chronic thiamethoxam exposure on colony pathogen load, queen quality, and colony temperature regulation. Under field conditions, high doses of thiamethoxam significantly increased overwinter mortality compared to controls, with field-realistic doses of thiamethoxam showing no significant effect on colony overwinter survival. Under laboratory conditions, chronic neonicotinoid exposure significantly decreased survival of winter workers relative to negative control at all doses tested. Chronic high-dose thiamethoxam exposure was not shown to impact pathogen load or queen quality, and field-realistic concentrations of thiamethoxam did not affect colony temperature homeostasis. Taken together, these results demonstrate that chronic environmental neonicotinoid exposure significantly decreases survival of winter workers in the laboratory, but only chronic high-dose thiamethoxam significantly decreases overwinter survival of colonies in the field

Table_1_Are fungicides a driver of European foulbrood disease in honey bee colonies pollinating blueberries?.XLSX

IntroductionBlueberry producers in Canada depend heavily on pollination services provided by honey bees (Apis mellifera L.). Anecdotal reports indicate an increased incidence of European foulbrood (EFB), a bacterial disease caused by Melissococcus plutonius, is compromising pollination services and colony health. Fungicidal products are commonly used in blueberry production to prevent fungal diseases such as anthracnose and botrytis fruit rot. Pesticide exposure has been implicated in honey bee immunosuppression; however, the effects of commercial fungicidal products, commonly used during blueberry pollination, on honey bee larval susceptibility to EFB have not been investigated.MethodsUsing an in vitro infection model of EFB, we infected first instar honey bee larvae with M. plutonius 2019 BC1, a strain isolated from an EFB outbreak in British Columbia, Canada, and chronically exposed larvae to environmentally relevant concentrations of fungicide products over 6 days. Survival was monitored until pupation or eclosion.ResultsWe found that larvae chronically exposed to one, two, or three fungicidal products [Supra® Captan 80WDG (Captan), low concentration of Kenja™ 400SC (Kenja), Luna® Tranquility (Luna), and/or Switch® 62.5 WG (Switch)], did not significantly reduce survival from EFB relative to infected controls. When larvae were exposed to four fungicide products concurrently, we observed a significant 24.2% decrease in survival from M. plutonius infection (p = 0.0038). Similarly, higher concentrations of Kenja significantly reduced larval survival by 24.7–33.0% from EFB (p DiscussionThese in vitro results suggest that fungicides may contribute to larval susceptibility and response to M. plutonius infections. Further testing of other pesticide combinations is warranted as well as continued surveillance of pesticide residues in blueberry-pollinating colonies.</p

Image_1_Are fungicides a driver of European foulbrood disease in honey bee colonies pollinating blueberries?.JPEG

IntroductionBlueberry producers in Canada depend heavily on pollination services provided by honey bees (Apis mellifera L.). Anecdotal reports indicate an increased incidence of European foulbrood (EFB), a bacterial disease caused by Melissococcus plutonius, is compromising pollination services and colony health. Fungicidal products are commonly used in blueberry production to prevent fungal diseases such as anthracnose and botrytis fruit rot. Pesticide exposure has been implicated in honey bee immunosuppression; however, the effects of commercial fungicidal products, commonly used during blueberry pollination, on honey bee larval susceptibility to EFB have not been investigated.MethodsUsing an in vitro infection model of EFB, we infected first instar honey bee larvae with M. plutonius 2019 BC1, a strain isolated from an EFB outbreak in British Columbia, Canada, and chronically exposed larvae to environmentally relevant concentrations of fungicide products over 6 days. Survival was monitored until pupation or eclosion.ResultsWe found that larvae chronically exposed to one, two, or three fungicidal products [Supra® Captan 80WDG (Captan), low concentration of Kenja™ 400SC (Kenja), Luna® Tranquility (Luna), and/or Switch® 62.5 WG (Switch)], did not significantly reduce survival from EFB relative to infected controls. When larvae were exposed to four fungicide products concurrently, we observed a significant 24.2% decrease in survival from M. plutonius infection (p = 0.0038). Similarly, higher concentrations of Kenja significantly reduced larval survival by 24.7–33.0% from EFB (p DiscussionThese in vitro results suggest that fungicides may contribute to larval susceptibility and response to M. plutonius infections. Further testing of other pesticide combinations is warranted as well as continued surveillance of pesticide residues in blueberry-pollinating colonies.</p

Image_2_Are fungicides a driver of European foulbrood disease in honey bee colonies pollinating blueberries?.TIF

IntroductionBlueberry producers in Canada depend heavily on pollination services provided by honey bees (Apis mellifera L.). Anecdotal reports indicate an increased incidence of European foulbrood (EFB), a bacterial disease caused by Melissococcus plutonius, is compromising pollination services and colony health. Fungicidal products are commonly used in blueberry production to prevent fungal diseases such as anthracnose and botrytis fruit rot. Pesticide exposure has been implicated in honey bee immunosuppression; however, the effects of commercial fungicidal products, commonly used during blueberry pollination, on honey bee larval susceptibility to EFB have not been investigated.MethodsUsing an in vitro infection model of EFB, we infected first instar honey bee larvae with M. plutonius 2019 BC1, a strain isolated from an EFB outbreak in British Columbia, Canada, and chronically exposed larvae to environmentally relevant concentrations of fungicide products over 6 days. Survival was monitored until pupation or eclosion.ResultsWe found that larvae chronically exposed to one, two, or three fungicidal products [Supra® Captan 80WDG (Captan), low concentration of Kenja™ 400SC (Kenja), Luna® Tranquility (Luna), and/or Switch® 62.5 WG (Switch)], did not significantly reduce survival from EFB relative to infected controls. When larvae were exposed to four fungicide products concurrently, we observed a significant 24.2% decrease in survival from M. plutonius infection (p = 0.0038). Similarly, higher concentrations of Kenja significantly reduced larval survival by 24.7–33.0% from EFB (p DiscussionThese in vitro results suggest that fungicides may contribute to larval susceptibility and response to M. plutonius infections. Further testing of other pesticide combinations is warranted as well as continued surveillance of pesticide residues in blueberry-pollinating colonies.</p