Comparison of individual and pooled sampling methods for estimation of Vairimorpha (Nosema) spp. levels in experimentally infected honey bee colonies.

The microsporidian pathogens Vairimorpha apis and V. ceranae are known to cause intestinal infection in honey bees and are associated with decreased colony productivity and colony loss. The widely accepted method for determining Vairimorpha colony infection level for risk assessment and antibiotic treatment is based on spore counts of 60 pooled worker bees using light microscopy. Given that honey bee colonies consist of as many as 1,000 times more individuals, the number of bees collected for Vairimorpha detection may significantly impact the estimated colony infection level, especially in the case of uneven distribution of high- and low-infected individuals within a hive. Hence, we compared the frequency and severity of Vairimorpha infection in individual bees to pooled samples of 60, 120, and 180 bees, as well as compared the Vairimorpha spp. prevalence in pooled samples of 60 and 180 bees. Overall, we did not find significant differences in spore counts in pooled samples containing incremental numbers of bees, although we observed that, in less-infected colonies, a low frequency of highly infected individuals influenced the estimated colony infection level. Moreover, Vairimorpha spp. prevalence did not differ significantly among the pooled bee samples tested. Increasing the number of pooled bees from the recommended 60 bees to 180 bees did not yield a more accurate representation of colony infection level for highly infected colonies, but the clinical importance of a low frequency of highly infected individuals in less-infected colonies needs to be addressed in future studies.

Investigation of Melissococcus plutonius isolates from 3 outbreaks of European foulbrood disease in commercial beekeeping operations in western Canada.

European foulbrood (EFB) disease is an economically important bacterial disease of honey bee larvae caused by enteric infection with Melissococcus plutonius. In this study, we investigated 3 clinical outbreaks of EFB disease in commercial beekeeping operations in western Canada in the summer of 2020 and characterized the Melissococcus plutonius isolates cultured from these outbreaks according to genetic multi-locus sequence type and i n vitro larval pathogenicity. We isolated M. plutonius sequence type 19 from EFB outbreaks in British Columbia and Alberta, and a novel M. plutonius sequence type 36 from an EFB outbreak in Saskatchewan. In vitro larval infection with each M. plutonius isolate was associated with decreased larval survival in vitro by 58.3 to 70.8% (P < 0.001) compared to non-infected controls. Further elucidation of mechanisms of virulence of M. plutonius, paired with epidemiologic investigation, is imperative to improve EFB management strategies and mitigate risks of EFB outbreaks in western Canada.

Enquête sur des isolats de Melissococcus plutonius provenant de trois éclosions de loque e uropéenne dans des exploitations apicoles commerciales de l'Ouest canadien. La loque européenne (EFB) est une maladie bactérienne économiquement importante des larves d'abeilles mellifères causée par une infection entérique par Melissococcus plutonius. Dans cette étude, nous avons enquêté sur trois éclosions cliniques de la maladie EFB dans des exploitations apicoles commerciales dans l'ouest du Canada à l'été 2020 et caractérisé les isolats de Melissococcus plutonius cultivés à partir de ces éclosions selon le typage génomique multilocus et la pathogénicité larvaire in vitro. Nous avons isolé le type de séquence 19 de M. plutonius des éclosions d'EFB en Colombie-Britannique et en Alberta, et une nouvelle séquence de type 36 de M. plutonius d'une éclosion d'EFB en Saskatchewan. L'infection larvaire in vitro avec chaque isolat de M. plutonius était associée à une diminution de la survie larvaire in vitro de 58,3 à 70,8 % (P < 0,001) par rapport aux témoins non infectés. Une élucidation plus poussée des mécanismes de virulence de M. plutonius, associée à une enquête épidémiologique, est impérative pour améliorer les stratégies de gestion de l'EFB et atténuer les risques d'épidémies d'EFB dans l'Ouest canadien.(Traduit par Dr Serge Messier).

Establishment of apiary-level risk of American foulbrood through the detection of Paenibacillus larvae spores in pooled, extracted honey in Saskatchewan.

Paenibacillus larvae, the causative agent of American foulbrood (AFB), produces spores that may be detectable within honey. We analyzed the spore content of pooled, extracted honey from 52 large-scale (L) and 64 small-scale (S) Saskatchewan beekeepers over a two-year period (2019-2020). Our objectives were: (i) establish reliable prognostic reference ranges for spore concentrations in extracted honey to determine future AFB risk at the apiary level; (ii) identify management practices as targets for mitigation of risk. P. larvae spores were detected in 753 of 1476 samples (51%). Beekeepers were stratified into low (< 2 spores/gram), moderate (2- < 100 spores/gram), and high (≥ 100 spores/gram) risk categories. Of forty-nine L beekeepers sampled in 2019, those that reported AFB in 2020 included 0/26 low, 3/18 moderate, and 3/5 high risk. Of twenty-seven L beekeepers sampled in 2020, those that reported AFB in 2021 included 0/11 low, 2/14 moderate, and 1/2 high risk. Predictive modelling included indoor overwintering of hives, purchase of used equipment, movement of honey-producing colonies between apiaries, beekeeper demographic, and antimicrobial use as risk category predictors. Saskatchewan beekeepers with fewer than 2 spores/gram in extracted honey that avoid high risk activities may be considered at low risk of AFB the following year.

Comparison of individual hive and apiary-level sample types for spores of Paenibacillus larvae in Saskatchewan honey bee operations.

Three commercial honey bee operations in Saskatchewan, Canada, with outbreaks of American foulbrood (AFB) and recent or ongoing metaphylactic antibiotic use were intensively sampled to detect spores of Paenibacillus larvae during the summer of 2019. Here, we compared spore concentrations in different sample types within individual hives, assessed the surrogacy potential of honey collected from honey supers in place of brood chamber honey or adult bees within hives, and evaluated the ability of pooled, extracted honey to predict the degree of spore contamination identified through individual hive testing. Samples of honey and bees from hives within apiaries with a recent, confirmed case of AFB in a single hive (index apiaries) and apiaries without clinical evidence of AFB (unaffected apiaries), as well as pooled, apiary-level honey samples from end-of-season extraction, were collected and cultured to detect and enumerate spores. Only a few hives were heavily contaminated by spores in any given apiary. All operations were different from one another with regard to both the overall degree of spore contamination across apiaries and the distribution of spores between index apiaries and unaffected apiaries. Within operations, individual hive spore concentrations in unaffected apiaries were significantly different from index apiaries in the brood chamber (BC) honey, honey super (HS) honey, and BC bees of one of three operations. Across all operations, BC honey was best for discriminating index apiaries from unaffected apiaries (p = 0.001), followed by HS honey (p = 0.06), and BC bees (p = 0.398). HS honey positively correlated with both BC honey (rs = 0.76, p < 0.0001) and bees (rs = 0.50, p < 0.0001) and may be useful as a surrogate for either. Spore concentrations in pooled, extracted honey seem to have predictive potential for overall spore contamination within each operation and may have prognostic value in assessing the risk of future AFB outbreaks at the apiary (or operation) level.

Testicular Changes of Honey Bee Drones, Apis mellifera (Hymenoptera: Apidae), During Sexual Maturation.

The normal developmental anatomy and histology of the reproductive tract of the honey bee drone, Apis mellifera (Linnaeus, 1758), has been well documented. The post-emergence maturation changes of the accessory glands are likewise well understood, but the normal histological changes of the testicle undergoing physiologic atrophy are not well characterized. To address this knowledge gap, herein we describe the anatomy and sequential histological stages of normal testicular atrophy of drones sampled daily from emergence to sexual maturity in the spring (June) and early summer (July). Testicular histological changes during maturation are characterized by the following stages: I) conclusion of spermiogenesis; II) evacuation of spermatodesms from tubular lumens; III) progressive follicular cell atrophy, and IV) complete atrophy and collapse of testicular parenchyma. Tubular changes occur in a basilar to apical direction where segments closer to the vas deferens are histologically more mature than corresponding apical segments. In addition, the rate of testicular maturation was found to change with seasonal progression. This description of physiologic testicular atrophy should be useful for future studies investigating potential pathological effects of stressors on drone testes during sexual maturation.

Investigation of clinical outbreaks of American foulbrood in honey-bee operations in Saskatchewan.

Four outbreaks of American foulbrood were investigated in honey-bee operations in Saskatchewan during the summer of 2019. Clinical signs were confirmed by the Saskatchewan Provincial Specialist in Apiculture and the causative agent was cultured and identified through matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Evaluation of management practices revealed off-label metaphylactic use of oxytetracycline in 3 of 4 operations and a discontinuation of antibiotic use in the fourth. Recent regulatory changes regarding access to medically important antimicrobials has provided an opportunity for veterinarians to promote evidence-based use of antimicrobials in apiculture while safe-guarding the health of commercial honeybee populations and the economic viability of their producers.

Enquête sur des poussées de cas cliniques de loque américaine dans des opérations d'abeilles mellifères en Saskatchewan. Quatre poussées de cas de loque américaine furent investiguées dans des opérations d'abeilles mellifères en Saskatchewan durant l'été 2019. Les signes cliniques furent confirmés par le Spécialiste provincial en apiculture de la Saskatchewan et l'agent causal fut cultivé et identifié par spectroscopie de masse par ionisation laser assistée par une matrice et analyse à temps de vol (MALDI-TOF MS). Une évaluation des pratiques de gestion a révélé l'utilisation métaphylactique en dérogation d'oxytétracycline dans trois des quatre opérations et un arrêt de l'utilisation d'antibiotique dans la quatrième. Des changements réglementaires récents concernant l'accès à des antimicrobiens importants médicalement ont fourni une opportunité aux vétérinaires de faire la promotion de l'utilisation factuelle des antimicrobiens en apiculture tout en conservant la santé des populations d'abeilles mellifères et la viabilité économique des apiculteurs.(Traduit par Dr Serge Messier).

Use of serum amyloid A in serum and synovial fluid to detect eradication of infection in experimental septic arthritis in horses.

While serum amyloid A (SAA) has been investigated as a potential marker for septic arthritis in horses, no study has reported on whether SAA can be used to detect eradication of joint infection. Therefore, the objective of this study was to investigate whether the eradication of joint infection in experimentally induced septic arthritis in horses can be detected using serum and synovial fluid SAA. A total of 17 horses were randomly assigned to 3 groups. A middle carpal joint of each horse was injected with saline (control group, n = 3), lipopolysaccharide (LPS) (nonseptic synovitis group, n = 6), or Escherichia coli (septic arthritis group, n = 8) on day 0. Starting on day 1, horses underwent treatment for septic arthritis. Sequential samples of serum and synovial fluid were collected, and quantification of SAA was carried out. Concentrations of serum and synovial fluid SAA were compared among groups and time points. A concurrent study was conducted and determined that infection was eradicated on day 4 in this experimental model of septic arthritis. Concentrations of serum and synovial fluid SAA rapidly increased after inoculation of E. coli and were highest on day 3 and day 4, respectively. Thereafter, both serum and synovial fluid SAA decreased with eradication of joint infection, although they remained significantly increased from baseline until day 9 and day 10, respectively. Serum and synovial fluid SAA did not increase in the control or nonseptic synovitis group. These findings suggest that serial measurements rather than a single measurement of SAA are required to determine eradication of infection from septic arthritis in horses.

Bien que l'amyloïde sérique (SAA) fut étudiée comme marqueur potentiel pour l'arthrite septique chez les chevaux, aucune étude n'a rapporté si SAA peut être utilisée pour détecter l'élimination d'une infection articulaire. Ainsi, l'objectif de la présente étude était d'examiner si l'élimination d'une infection articulaire lors d'arthrite septique induite expérimentalement chez les chevaux peut être détectée en utilisant la SAA du sérum et du liquide synovial. Un total de 17 chevaux fut réparti de manière aléatoire en trois groupes. Une articulation carpienne médiale de chaque cheval fut injectée avec de la saline (groupe témoin, n = 3), du lipopolysaccharide (LPS) (groupe synovite non-septique, n = 6) ou Escherichia coli (groupe arthrite septique, n = 8) au jour 0. En débutant au jour 1, les chevaux furent soumis à un traitement pour arthrite septique. Des échantillons séquentiels de sérum et de liquide synovial furent prélevés et la quantification de SAA effectuée. Les concentrations de SAA dans le sérum et le liquide synovial furent comparées parmi les groupes et à différents temps. Une étude concomitante était menée et a déterminé que l'infection était éliminée au jour 4 dans ce modèle expérimental d'arthrite septique. Les concentrations de SAA dans le sérum et le liquide synovial ont rapidement augmenté après l'inoculation d'E. coli et étaient maximales au jour 3 et au jour 4, respectivement. Par la suite, les concentrations de SAA du sérum et du liquide synovial ont diminué avec l'élimination de l'infection articulaire, bien qu'elles soient demeurées augmentées significativement par rapport au seuil de base jusqu'au jour 9 et jour 10, respectivement. Les concentrations de SAA du sérum et du liquide synovial n'ont pas augmenté dans les groupes témoin et synovite non-septique. Ces résultats suggèrent que des mesures en série plutôt qu'une mesure unique de SAA sont requises pour déterminer l'élimination de l'infection lors d'arthrite septique chez les chevaux.(Traduit par Docteur Serge Messier).

In Vitro Effects of Pesticides on European Foulbrood in Honeybee Larvae.

Neonicotinoid and fungicide exposure has been linked to immunosuppression and increased susceptibility to disease in honeybees (Apis mellifera). European foulbrood, caused by the bacterium Melissococcus plutonius, is a disease of honeybee larvae which causes economic hardship for commercial beekeepers, in particular those whose colonies pollinate blueberries. We report for the first time in Canada, an atypical variant of M. plutonius isolated from a blueberry-pollinating colony. With this isolate, we used an in vitro larval infection system to study the effects of pesticide exposure on the development of European foulbrood disease. Pesticide doses tested were excessive (thiamethoxam and pyrimethanil) or maximal field-relevant (propiconazole and boscalid). We found that chronic exposure to the combination of thiamethoxam and propiconazole significantly decreased the survival of larvae infected with M. plutonius, while larvae chronically exposed to thiamethoxam and/or boscalid or pyrimethanil did not experience significant increases in mortality from M. plutonius infection in vitro. Based on these results, individual, calculated field-realistic residues of thiamethoxam and/or boscalid or pyrimethanil are unlikely to increase mortality from European foulbrood disease in honeybee worker brood, while the effects of field-relevant exposure to thiamethoxam and propiconazole on larval mortality from European foulbrood warrant further study.

Effects of chronic dietary thiamethoxam and prothioconazole exposure on Apis mellifera worker adults and brood.

BACKGROUND: Chronic exposure of honey bees (Apis mellifera Linnaeus) to the neonicotinoid thiamethoxam and the fungicide prothioconazole is common during foraging in agricultural landscapes. We evaluated the survival and hypopharyngeal gland development of adult worker honey bees, and the survival of the worker brood when chronically exposed to thiamethoxam or thiamethoxam and prothioconazole in combination. RESULTS: We found that 30 days of exposure to 40 µg kg-1 of thiamethoxam significantly (P < 0.001) increased the frequency of death in worker adults by four times relative to solvent control. The worker brood required 23 times higher doses of thiamethoxam (1 mg L-1 or 909 µg kg-1 ) before a significant (P = 0.04), 3.9 times increase in frequency of death was observed relative to solvent control. No additive effects of simultaneous exposure of worker adults or brood to thiamethoxam and prothioconazole were observed. At day 8 and day 12, the hypopharyngeal gland acinar diameter was not significantly different (P > 0.05) between controls and adult workers exposed to thiamethoxam and/or prothioconazole. CONCLUSION: These results indicate that chronic exposure to field-realistic doses of thiamethoxam and/or prothioconazole are unlikely to affect the survival of adult workers and brood. © 2019 Society of Chemical Industry.

Deformed Wing Virus Infection in Honey Bees (Apis mellifera L.).

Deformed wing virus (DWV) is a single-stranded RNA virus of honey bees (Apis mellifera L.) transmitted by the parasitic mite Varroa destructor. Although DWV represents a major threat to honey bee health worldwide, the pathological basis of DWV infection is not well documented. The objective of this study was to investigate clinicopathological and histological aspects of natural DWV infection in honey bee workers. Emergence of worker honey bees was observed in 5 colonies that were clinically affected with DWV and the newly emerged bees were collected for histopathology. DWV-affected bees were 2 times slower to emerge and had 30% higher mortality compared to clinically normal bees. Hypopharyngeal glands in bees with DWV were hypoplastic, with fewer intracytoplasmic secretory vesicles; cells affected by apoptosis were observed more frequently. Mandibular glands were hypoplastic and were lined by cuboidal epithelium in severely affected bees compared to tall columnar epithelium in nonaffected bees. The DWV load was on average 1.7 × 106 times higher (P < .001) in the severely affected workers compared to aged-matched sister honey bee workers that were not affected by deformed wing disease based on gross examination. Thus, DWV infection is associated with prolonged emergence, increased mortality during emergence, and hypoplasia of hypopharyngeal and mandibular glands in newly emerged worker honey bees in addition to previously reported deformed wing abnormalities.

Use of standard diagnostic techniques to determine eradication of infection in experimental equine septic arthritis.

Septic arthritis is an important disease in horses, necessitating aggressive and prolonged therapy. In order to guide therapy, reliable methods of detecting the eradication of infection are needed. Therefore, the objective of this study was to investigate detection of eradication of infection in an experimental model of equine septic arthritis using standard diagnostic techniques. For this purpose, 17 adult horses were assigned to 3 experimental groups. The middle carpal joint of each horse was injected with Escherichia coli (Septic group, n = 8), lipopolysaccharide (LPS) (LPS group, n = 6), or sterile saline (Control group, n = 3) at day 0. Contralateral joints were not injected. Standard therapy was applied to all joints except non-injected joints in the Control group at day 1. Sequential samples of synovial fluid (SF) were collected for bacterial culture using 3 culture media [Columbia blood agar (CBA), brain heart infusion broth (BHI), and Signal blood culture medium] and for cytological evaluation [percentage neutrophils (PN), total nucleated cell count (TNCC), and total protein (TP)]. Escherichia coli-specific polymerase chain reaction (PCR) was carried out to detect E. coli DNA in synovial fluid. Culture and PCR were positive for E. coli in all joints injected with E. coli at day 1 and 1 joint was positive on BHI at day 4. Based on the results of bacterial culture, PCR, and TNCC, the elimination of infection in our experimental model occurred by day 4 post-infection in 6 out of 7 cases. Total protein (TP) and PN remained elevated at clinical threshold used for diagnosis of septic arthritis until day 14. In our experimental model of E. coli-induced arthritis, we conclude that TP and PN may not be good indicators for detecting the eradication of bacterial infection caused by E. coli from infected and subsequently treated joints.

L'arthrite septique est une pathologie importante chez les chevaux, nécessitant une thérapie agressive et prolongée. Afin de guider la thérapie, des méthodes fiables pour détecter l'éradication de l'infection sont requises. Ainsi, l'objectif de la présente étude était d'examiner la détection de l'éradication de l'infection dans un modèle expérimental d'arthrite septique équine en utilisant des techniques diagnostiques standards. À cet effet, 17 chevaux adultes ont été assignés à trois groupes expérimentaux. L'articulation carpienne moyenne de chaque cheval a été injectée avec Escherichia coli (groupe septique, n = 8), du lipopolysaccharide (LPS) (groupe LPS, n = 6), ou de la saline stérile (groupe témoin, n = 3) au jour 0. Les articulations contra-latérales n'ont pas été injectées. Au jour 1, une thérapie standard fut appliquée à toutes les articulations sauf les articulations non-injectées dans le groupe témoin. De manière séquentielle des échantillons de liquide synovial (LS) furent prélevés pour culture bactérienne en utilisant trois milieux de culture [gélose au sang Columbia (CBA), bouillon coeur-cerveau (BHI), et hémoculture Signal] et pour évaluation cytologique [pourcentage de neutrophiles (PN), dénombrement total de cellules nucléées (DTCN), et la quantité de protéines totales (PT)]. Une réaction d'amplification en chaîne par la polymérase (ACP) spécifique à E. coli a été réalisée afin de détecter l'ADN d'E. coli dans le LS. La culture et l'ACP étaient positives pour E. coli dans toutes les articulations injectées avec E. coli au jour 1 et une articulation était positive avec le BHI au jour 4. Sur la base des résultats des cultures bactériennes, de l'ACP, et du DTCN, l'élimination de l'infection dans notre modèle expérimental est survenue au jour 4 post-infection dans 6 des 7 cas. Les valeurs de PT et de PN sont demeurées élevées au seuil clinique utilisé pour diagnostiquer une arthrite septique jusqu'au jour 14. Dans notre modèle expérimental d'arthrite induite par E. coli, nous concluons que les valeurs de PT et de PN ne seraient pas de bons indicateurs pour détecter l'éradication de l'infection bactérienne causée par E. coli dans des articulations infectées et subséquemment traitées.(Traduit par Docteur Serge Messier).

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.

Reduced activities of thiamine-dependent and cytochrome c oxidase enzymes in cerebral cortex of cattle affected by sulfur-induced polioencephalomalacia.

Sulfur-induced polioencephalomalacia (PEM) is an important disease affecting cattle in certain geographical regions. However, the pathogenesis of brain damage is not completely understood. We previously observed that excess dietary sulfur may influence thiamine status and altered thiamine metabolism may be involved in the pathogenesis of sulfur-induced PEM in cattle. In this study, we evaluated the activities of thiamine-dependent enzymes [α-ketogluterate dehydrogenase (α-KGDH) and pyruvate dehydrogenase (PDH)] and cytochrome c oxidase (COX) in the cerebral cortex of sulfur-induced PEM-affected cattle (n = 9) and clinically normal cattle (n = 8, each group) exposed to low or high dietary sulfur [LS = 0.30% versus HS = 0.67% sulfur on a dry matter (DM) basis]. Enzyme activities in PEM brains were measured from the brain tissue regions and examined using ultraviolent (UV) light illumination to show fluorescence or non-fluorescence regions. No gross changes under regular or UV light, or histopathological changes indicative of PEM were detected in the brains of cattle exposed to LS or HS diets. The PDH, α-KGDH, and COX activities did not differ between LS and HS brains, but all enzymes showed significantly lower (P < 0.05) activities in UV-positive region of PEM brains compared with LS and HS brains. The UV-negative regions of PEM brain had similar PDH activities to LS and HS brains, but the activities of α-KGDH and COX were significantly lower than in LS and HS brains. The results of this study suggest that reduced enzyme activities of brain PHD, α-KGDH, and COX are associated with the pathogenesis of sulfur-induced PEM.

La polio-encéphalomalacie (PEM) induite par le souffre est une maladie importante affectant les bovins dans certaines régions géographiques. Toutefois, la pathogenèse des dommages cérébraux n'est pas complètement comprise. Nous avons observé antérieurement qu'un excès de souffre alimentaire peu influencer le statut de la thiamine et le métabolisme altéré de la thiamine pourrait être impliqué dans la pathogenèse de la PEM induite par le souffre chez les bovins. Dans la présente étude nous avons évalué les activités d'enzymes dépendant de la thiamine [α-kétoglutarate déshydrogénase (α-KGDH) et pyruvate déshydrogénase (PDH)] et du cytochrome c oxydase (COX) dans le cortex cérébral de bovins affectés de PEM induite par le souffre (n = 9) et de bovins cliniquement normaux (n = 8, chaque groupe) exposés à des quantités faibles (LS) ou élevés (HS) de souffre alimentaire (LS = 0,30 % vs HS = 0,67 % souffre sur une base de matière sèche). L'activité enzymatique dans les cerveaux PEM était mesurée à partir de régions du tissu cérébral et examinée à l'aide d'une lampe à rayons ultraviolets (UV) pour montrer les régions fluorescentes et non-fluorescentes. Aucun changement macroscopique n'était apparent à l'examen sous éclairage régulier ou lumière UV, et aucun changement histopathologique indicateur de PEM ne fut détecté dans les cerveaux des bovins exposés à des diètes LS ou HS. L'activité de PDH, de α-KGDH, et de COX ne différait pas entre les cerveaux LS et HS, mais tous les enzymes montraient une activité significativement plus faible (P < 0,05) dans les régions positive aux UV dans les cerveaux PEM comparativement aux cerveaux LS et HS. Les régions UV négative des cerveaux PEM avaient des activités PDH similaires à celles des cerveaux LS et HS, mais les activités de α-KGDH et de COX étaient significativement plus faibles que dans les cerveaux LS et HS. Les résultats de cette étude suggèrent que la réduction d'activités de PDH, α-KGDH et de COX du cerveau est associée avec la pathogenèse de PEM induite par le souffre.(Traduit par Docteur Serge Messier).

Evaluation of inhibition of F4ac positive Escherichia coli attachment with xanthine dehydrogenase, butyrophilin, lactadherin and fatty acid binding protein.

BACKGROUND: Neonatal and post-weaning colibacillosis caused by enterotoxigenic E. coli is responsible for substantial economic losses encountered by the pork industry. Intestinal colonization of young piglets by E. coli depends on the efficiency of bacterial attachment to host gastrointestinal epithelium that is mediated by fimbriae. We tested the effect of porcine individual milk fat globule membrane (MFGM) proteins on F4ac positive E. coli attachment to porcine enterocytes in vitro. RESULTS: Butyrophilin, lactadherin and fatty acid binding protein inhibited fimbriae-dependent adherence of E. coli to enterocytes in vitro, while xanthine dehydrogenase did not. The inhibiting activity was dose-dependent for all three proteins, but the inhibiting efficiency was different. CONCLUSIONS: The results indicate that MFGM proteins may interfere with attachment of E. coli to porcine neonatal intestinal mucosa.

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.

Optimization of a DNA vaccine against SARS.

Severe acute respiratory syndrome coronavirus (SARS-CoV) first appeared in Southern China in November 2002, and then quickly spread to 33 countries on five continents along international air travel routes. Although the SARS epidemic has been contained, there is a clear need for a safe and effective vaccine should an outbreak of a SARS-CoV infection reappear in human population. In this study, we tested four DNA-vaccine constructs: (1) pLL70, containing cDNA for the SARS-CoV spike (S) gene; (2) pcDNA-SS, containing codon-optimized S gene for SARS-CoV S protein (residues 12-1255) fused with a leader sequence derived from the human CD5 gene; (3) pcDNA-St, containing the gene encoding the N-portion of the codon-optimized S gene (residues 12-532) with the CD5 leader sequence; (4) pcDNA-St-VP22C, containing the gene encoding the N-portion of the codon-optimized S protein with the CD5 leader sequence fused with the C-terminal 138 amino acids of the bovine herpesvirus-1 (BHV-1) major tegument protein VP22. Each of these plasmids was intradermally administered to C57BL/6 mice in three separate immunizations. Analysis of humoral and cellular immune responses in immunized mice demonstrated that pcDNA-SS and pcDNA-St-VP22C are the most immunogenic SARS vaccine candidates.

Intracellular localization of the SARS coronavirus protein 9b: evidence of active export from the nucleus.

Open reading frame 9b (ORF 9b) encodes a 98 amino acid group-specific protein of severe acute respiratory syndrome (SARS) coronavirus (CoV). It has no homology with known proteins and its function in SARS CoV replication has not been determined. The N-terminal part of the 9b protein was used to raise polyclonal antibodies in rabbits, and these antibodies could detect 9b protein in infected cells. We analyzed the sub-cellular localization of recombinant 9b protein using fluorescence microscopy of live transfected cells and indirect immunofluorescence of transfected fixed cells. Our findings indicate that the 9b protein is exported outside of a cell nucleus and localizes to the endoplasmic reticulum. Our data also suggest that the 46-LRLGSQLSL-54 amino acid sequence of 9b functions as a nuclear export signal (NES).

Short nucleotide polymorphic insertions in the MCL-1 promoter affect gene expression.

We have recently reported novel short nucleotide (six and eighteen) polymorphic insertions, in the MCL-1 promoter and their association with higher mRNA and protein levels. The aim of the present study was to test the hypothesis that these insertions directly affect MCL-1 gene expression. Haematopoietic and epithelial human cell lines were transfected with +0, +6, or +18 MCL-1 promoter fragments positioned upstream of the Firefly luciferase reporter gene. The cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and granulocyte macrophage colony-stimulating factor (GM-CSF). Compared to +0, both polymorphic insertions (+6 and +18) were associated with increased promoter activity. Although chromatin immunoprecipitation assay showed that there are Sp1/Sp3 binding sites in the MCL-1 promoter, electrophoretic mobility shift assay showed that it is unlikely that these sites are in the region harboring these insertions. These results provide further evidence for the biological effect of MCL-1 promoter polymorphisms on gene expression.

G125A single-nucleotide polymorphism in the human BAX promoter affects gene expression.

Earlier we had reported a guanine to adenosine substitution at position 125 (G125A) in the BAX promoter, and its association with higher stage of the disease and failure to achieve complete response to treatment in chronic lymphocytic leukemia (CLL) patients. The aim of this study was to test the hypothesis that G125A leads to a reduction in the transcription of the BAX gene and that this is a direct cause of altered BAX mRNA and protein expression. In lymphocytes of CLL patients, BAX mRNA expression was determined by RNase protection assay and Bax protein was detected by immunoblotting. The presence of G125A in the BAX promoter was associated with lower BAX mRNA (P=0.004) and protein (P=0.024) levels. In transient expression assays using wild-type and mutant BAX promoter sequences linked to Luciferase as a reporter, the G125A polymorphism reduced expression of the BAX promoter by 2.6-fold. These studies suggest a mechanism for the biological effect of the G125A.

Characterization of a bovine lactoferrin binding protein of Streptococcus uberis.

The interaction between Streptococcus uberis and bovine lactoferrin (bLf) has been characterized. The binding of 125I-bLf to S. uberis was time-dependent and displaceable by unlabeled bLf. The Scatchard plot was linear and approximately 7,800 binding sites were expressed by each bacterial cell, with an affinity of 1.0 x 10(-7) M. Both heat and protease treatment of bacterial cells reduced bLf-binding significantly, indicating the presence of a cell surface localized protein receptor for the glycoprotein. One protein was identified from the cell wall of S. uberis as the functionally active bLf-binding protein and it existed in both monomeric and dimeric forms. The recombinant protein expressed in E. coli cells was able to bind bLf and had molecular weights similar to that of native S. uberis. Deletion analysis located the bLf-binding domain to a 200 amino acid region at the amino terminus of Lbp. Analysis of the primary and secondary structure suggested that Lbp is an M-like protein. An isogenic mutant of S. uberis lacking the internal sequence of the lbp gene was constructed by allele replacement. Adherence experiments with wild type S. uberis and the lbp mutant revealed that Lbp is not responsible for attachment of S. uberis to host epithelial cells.