Stable gastric pentadecapeptide BPC 157 in honeybee (Apis mellifera) therapy,to control Nosema ceranae invasions in apiary conditions

Nosema ceranae can cause major problems, such as immune suppression, gut epithelial cell degeneration, reduced honeybee lifespan, or suddenly colony collapse. As a novel approach in therapy, we hypothesize the stable gastric pentadecapeptide BPC 157 in honeybee therapy, to control N. ceranae invasions in apiary conditions: BPC 157 treated sugar syrup (0.25 L sugar syrup supplemented with 0.1 μg/ml BPC 157), as well as the pure sugar syrup (0.25 L sugar syrup; control), was administered to honeybee colonies in feeders situated under the roof of the hives, during 21 consecutive days, at the end of beekeeping season. The strength of honeybee colonies was increased 20 and 30 days after initial feeding with BPC 157 supplement (Day 1, 36.100 ± 698; Day 20, 64.860 ± 468; Day 30, 53.214 ± 312 estimated number of honeybees), in field conditions. The similar successful outcome occurs with the N. ceranae spore loads counted in the homogenates of sampled adult honeybees (Day 1, 6.286 ± 2.336; Day 20, 3.753 ± 1.835; Day 30, 2.005 ± 1.534 million spores/bee). Accordingly, with the noted increased strength of the colonies fed with sugar syrup supplemented with BPC 157, the number of N. ceranae spores per honeybee gradually decreased as well. Besides, honeybees infected with N. ceranae fed with sugar syrup exhibited severe damage of midgut wall layers and epithelial cells. By contrast, in honeybees infected with N. ceranae fed with sugar syrup supplemented with BPC 157, all damages were markedly attenuated, damages of the outer muscular coat, in particular. In conclusion, the results of the first field trial on diseased honeybee colonies with BPC 157 indicate significant therapeutic effects with the used oral therapy with BPC 157 supplementation.     

Nosema spp. infection and its negative effects on honey bees (Apis mellifera iberiensis) at the colony level

Nosemosis caused by the microsporidia Nosema apis and Nosema ceranae are among the most common pathologies affecting adult honey bees. N. apis infection has been associated with a reduced lifespan of infected bees and increased winter mortality, and its negative impact on colony strength and productivity has been described in several studies. By contrast, when the effects of nosemosis type C, caused by N. ceranae infection, have been analysed at the colony level, these studies have largely focused on collapse as a response to infection without addressing the potential sub-clinical effects on colony strength and productivity. Given the spread and prevalence of N. ceranae worldwide, we set out here to characterize the sub-clinical and clinical signs of N. ceranae infection on colony strength and productivity. We evaluated the evolution of 50 honey bee colonies naturally infected by Nosema (mainly N. ceranae) over a one year period. Under our experimental conditions, N. ceranae infection was highly pathogenic for honey bee colonies, producing significant reductions in colony size, brood rearing and honey production. These deleterious effects at the colony level may affect beekeeping profitability and have serious consequences on pollination. Further research is necessary to identify possible treatments or beekeeping techniques that will limit the rapid spread of this dangerous emerging disease.     

The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade

Microsporidiosis caused by infection with Nosema apis or Nosema ceranae has become one of the most widespread diseases of honey bees and can cause important economic losses for beekeepers. Honey can be contaminated by spores of both species and it has been reported as a suitable matrix to study the field prevalence of other honey bee sporulated pathogens. Historical honey sample collections from the CAR laboratory (Centro Apícola Regional) were analyzed by PCR to identify the earliest instance of emergence, and to determine whether the presence of Nosema spp. in honey was linked to the spread of these microsporidia in honey bee apiaries. A total of 240 frozen honey samples were analyzed by PCR and the results compared with rates of Nosema spp. infection in worker bee samples from different years and geographical areas. The presence of Nosema spp. in hive-stored honey from naturally infected honey bee colonies (from an experimental apiary) was also monitored, and although collected honey bees resulted in a more suitable sample to study the presence of microsporidian parasites in the colonies, a high probability of finding Nosema spp. in their hive-stored honey was observed. The first honey sample in which N. ceranae was detected dates back to the year 2000. In subsequent years, the number of samples containing N. ceranae tended to increase, as did the detection of Nosema spp. in adult worker bees. The presence of N. ceranae as early as 2000, long before generalized bee depopulation and colony losses in 2004 may be consistent with a long incubation period for nosemosis type C or related with other unknown factors. The current prevalence of nosemosis, primarily due to N. ceranae, has reached epidemic levels in Spain as confirmed by the analysis of worker honey bees and commercial honey.     

A Real-Time PCR for Detection and Quantification of Mycoplasma ovipneumoniae

A real-time PCR for detection and quantification of M. ovipneumoniae was developed using 9 recently sequenced M. ovipneumoniae genomes and primers targeting a putative adhesin gene p113. The assay proved to be specific and sensitive (with a detection limit of 22 genomic DNA) and could quantify M. ovipneumoniae DNA over a wide linear range, from 2.2 × 10² to 2.2 × 10⁷ genomes.     

Analytical sensitivity of air samplers based on uniform point-source exposure to airborne Porcine reproductive and respiratory syndrome virus and swine influenza virus

Research and surveillance activities involving airborne pathogens rely on the capture and enumeration of pathogens suspended in aerosols. The objective of this study was to estimate the analytical sensitivity (detection threshold) of each of 4 air samplers for Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV). In a 5-min sampling period under controlled conditions, the analytical sensitivity of the AGI-30 (Ace Glass, Vineland, New Jersey, USA), AGI-4 (Ace Glass), SKC BioSampler (SKC, Eighty Four, Pennsylvania, USA), and Midwest Micro-Tek sampler (Midwest Micro-Tek, Brookings, South Dakota, USA) was calculated at 1 × 10^1.1, 1 × 10^1.3, 1 × 10^1.1, and 1 × 10^1.2 median tissue culture infectious dose (TCID₅₀) equivalents for PRRSV and 1 × 10^1.4, 1 × 10^1.1, 1 × 10^1.6, and 1 × 10^1.2 TCID₅₀ equivalents for SIV [per 60 L (5-min sampling period)]. Despite marked differences in sampler design, no statistically significant difference in analytical sensitivity was detected between the samplers for collection of artificially produced aerosols containing cell-culture-propagated PRRSV or SIV.     

Intraoperative fluid therapy for video-assisted ovariohysterectomy in dogs

BackgroundIntraoperative fluids are still poorly studied in veterinary medicine. In humans the dosage is associated with significant differences in postoperative outcomes.ObjectivesThe aim of this study is to verify the influence of three different fluid therapy rates in dogs undergoing video-assisted ovariohysterectomy.MethodsTwenty-four female dogs were distributed into three groups: G5, G10, and G20. Each group was given 5, 10, and 20 mL·kg⁻¹·h⁻¹ of Lactate Ringer, respectively. This study evaluated the following parameters: central venous pressure, arterial blood pressure, heart rate, respiratory rate, temperature, acid-base balance, and serum lactate levels. Additionally, this study evaluated the following urinary variables: urea, creatinine, protein to creatinine ratio, urine output, and urine specific gravity. The dogs were evaluated up to 26 h after the procedure.ResultsAll animals presented respiratory acidosis during the intraoperative period. The G5 group evidenced intraoperative oliguria (0.80 ± 0.38 mL·kg⁻¹·h⁻¹), differing from the G20 group (2.17 ± 0.52 mL·kg⁻¹·h⁻¹) (p = 0.001). Serum lactate was different between groups during extubation (p = 0.036), with higher values being recorded in the G5 group (2.19 ± 1.65 mmol/L). Animals from the G20 group presented more severe hypothermia at the end of the procedure (35.93 ± 0.61°C) (p = 0.032). Only the members of the G20 group presented mean potassium values below the reference for the species. Anion gap values were lower in the G20 group when compared to the G5 and G10 groups (p = 0.017).ConclusionsThe use of lactated Ringer''s solution at the rate of 10 mL·kg⁻¹·h⁻¹ seems to be beneficial in the elective laparoscopic procedures over the 5 or 20 mL·kg⁻¹·h⁻¹ rates of infusion.     

A field evaluation of two vaccines against Mycoplasma hyopneumoniae infection in pigs

Background

A field trial was carried out with two Mycoplasma hyopneumoniae vaccines in order to investigate the benefit of vaccination under field conditions in modern Danish pig production facilities with pigs being positive for M. hyopneumoniae. The M. hyopneumoniae infection of the herd was confirmed through blood samples that were positive for antibodies against M. hyopneumoniae combined with gross lesions of the lungs related to M. hyopneumoniae at slaughter and detection of M. hyopneumoniae by polymerace chain reaction in these lesions.

Results

A total of 2,256 pigs from two herds were randomly divided into three groups. Group 1 received 2 mL ThoroVAX^®VET, Group 2 received 1 mL Ingelvac^®MycoFLEX, and Group 3 was a non-vaccinated control group. The vaccination was performed by a person who was not involved in the rest of the trial and vaccination status thereby blinded to the evaluators.The prevalence of lung lesions related to M. hyopneumoniae were significantly lower for pigs vaccinated with ThoroVAX^®VET but not for pigs vaccinated with Ingelvac^®MycoFLEX^®, when compared to non-vaccinated pigs. There was no significant effect of vaccination on growth rate, antibiotic consumption or mortality.

Conclusion

This trial demonstrated that vaccination with Thoro^®VAX VET was effective in reducing the prevalence of lung lesion in pig units infected with M. hyopneumoniae.     

Formulation of a rational dosage regimen of ceftiofur hydrochloride oily suspension by pharmacokinetic-pharmacodynamic (PK-PD) model for treatment of swine Streptococcus suis infection

BackgroundOur previously prepared ceftiofur (CEF) hydrochloride oily suspension shows potential wide applications for controlling swine Streptococcus suis infections, while the irrational dose has not been formulated.ObjectivesThe rational dose regimens of CEF oily suspension against S. suis were systematically studied using a pharmacokinetic-pharmacodynamic model method.MethodsThe healthy and infected pigs were intramuscularly administered CEF hydrochloride oily suspension at a single dose of 5 mg/kg, and then the plasma and pulmonary epithelial lining fluid (PELF) were collected at different times. The minimum inhibitory concentration (MIC), minimal bactericidal concentration, mutant prevention concentration (MPC), post-antibiotic effect (PAE), and time-killing curves were determined. Subsequently, the area under the curve by the MIC (AUC_0–24h/MIC) values of desfuroylceftiofur (DFC) in the PELF was obtained by integrating in vivo pharmacokinetic data of the infected pigs and ex vivo pharmacodynamic data using the sigmoid E_max (Hill) equation. The dose was calculated based on the AUC_0–24h/MIC values for bacteriostatic action, bactericidal action, and bacterial elimination.ResultsThe peak concentration, the area under the concentration-time curve, and the time to peak for PELF''s DFC were 24.76 ± 0.92 µg/mL, 811.99 ± 54.70 μg·h/mL, and 8.00 h in healthy pigs, and 33.04 ± 0.99 µg/mL, 735.85 ± 26.20 μg·h/mL, and 8.00 h in infected pigs, respectively. The MIC of PELF''s DFC against S. suis strain was 0.25 µg/mL. There was strong concentration-dependent activity as determined by MPC, PAE, and the time-killing curves. The AUC_0–24h/MIC values of PELF''s DFC for bacteriostatic activity, bactericidal activity, and virtual eradication of bacteria were 6.54 h, 9.69 h, and 11.49 h, respectively. Thus, a dosage regimen of 1.94 mg/kg every 72 h could be sufficient to reach bactericidal activity.ConclusionsA rational dosage regimen was recommended, and it could assist in increasing the treatment effectiveness of CEF hydrochloride oily suspension against S. Suis infections.     

Phenotypic characterisation of the cellular immune infiltrate in placentas of cattle following experimental inoculation with Neospora caninum in late gestation

Despite Neospora caninum being a major cause of bovine abortion worldwide, its pathogenesis is not completely understood. Neospora infection stimulates host cell-mediated immune responses, which may be responsible for the placental damage leading to abortion. The aim of the current study was to characterize the placental immune response following an experimental inoculation of pregnant cattle with N. caninum tachyzoites at day 210 of gestation. Cows were culled at 14, 28, 42 and 56 days post inoculation (dpi). Placentomes were examined by immunohistochemistry using antibodies against macrophages, T-cell subsets (CD4, CD8 and γδ), NK cells and B cells. Macrophages were detected mainly at 14 days post inoculation. Inflammation was generally mild and mainly characterized by CD3⁺, CD4⁺ and γδ T-cells; whereas CD8⁺ and NK cells were less numerous. The immune cell repertoire observed in this study was similar to those seen in pregnant cattle challenged with N. caninum at early gestation. However, cellular infiltrates were less severe than those seen during first trimester Neospora infections. This may explain the milder clinical outcome observed when animals are infected late in gestation.     

Bacillus amyloliquefaciens CECT 5940 improves performance and gut function in broilers fed different levels of protein and/or under necrotic enteritis challenge

Two studies were conducted to investigate the effect of Bacillus amyloliquefaciens CECT 5940 (BA) as a probiotic on growth performance, amino acid digestibility and bacteria population in broiler chickens under a subclinical necrotic enteritis (NE) challenge and/or fed diets with different levels of crude protein (CP). Both studies consisted of a 2 × 2 factorial arrangement of treatments with 480 Ross 308 mix-sexed broiler chickens. In study 1, treatments included 1) NE challenge (+/−), and 2) BA (1.0 × 10⁶ CFU/g of feed) supplementation (+/−). In study 2, all birds were under NE challenge, and treatments were 1) CP level (Standard/Reduced [2% less than standard]) and 2) BA (1.0 × 10⁶ CFU/g of feed) supplementation (+/−). After inducing NE infection, blood samples were taken on d 16 for uric acid evaluation, and cecal samples were collected for bacterial enumeration. In both studies, ileal digesta was collected on d 35 for nutrient digestibility evaluation. In study 1, the NE challenge reduced body weight gain (BWG), supressed feed conversion ratio (FCR) and serum uric acid levels (P < 0.001). Supplementation of BA increased BWG (P < 0.001) and reduced FCR (P = 0.043) across dietary treatments, regardless of challenge. Bacillus (P = 0.030) and Ruminococcus (P = 0.029) genomic DNA copy numbers and concentration of butyrate (P = 0.017) were higher in birds fed the diets supplemented with BA. In study 2, reduced protein (RCP) diets decreased BWG (P = 0.010) and uric acid levels in serum (P < 0.001). Supplementation of BA improved BWG (P = 0.001) and FCR (P = 0.005) and increased Ruminococcus numbers (P = 0.018) and butyrate concentration (P = 0.033) in the ceca, regardless of dietary CP level. Further, addition of BA reduced Clostridium perfringens numbers only in birds fed with RCP diets (P = 0.039). At d 35, BA supplemented diets showed higher apparent ileal digestibility of cystine (P = 0.013), valine (P = 0.020), and lysine (P = 0.014). In conclusion, this study suggests positive effects of BA supplementation in broiler diets via modulating gut microflora and improving nutrient uptake.     

Influence of Neospora caninum intra-specific variability in the outcome of infection in a pregnant BALB/c mouse model

Previous assays in pregnant animals have demonstrated the effect of different host factors and timing of infection on the outcome of neosporosis during pregnancy. However, the influence of Neospora caninum isolate itself has been poorly investigated. Here, we compared the effects on clinical outcome and vertical transmission observed in a pregnant mouse model following infection with 10 different N. caninum isolates. The isolates in our study included the Nc-Liv isolate and nine N. caninum isolates obtained from calves. Female BALB/c mice were inoculated with 2 × 10⁶ tachyzoites at day 7 of pregnancy. Morbidity and mortality, in both dams and offspring during the course of infection, and transmission to progeny at day 30 postpartum were evaluated. The serum IgG1 and IgG2a production in dams were also examined. All dams showed elevated IgG1 and IgG2a responses, confirming N. caninum infection, although signs of disease were only exhibited in dams infected with 4 of the 10 isolates (Nc-Spain 4H, Nc-Spain 5H, Nc-Spain 7 and Nc-Liv). In neonates, clinical signs were observed in all N. caninum-infected groups, and neonatal mortality rates varied from greater than 95% with the isolates mentioned above to less than 32.5% with the other isolates. Vertical transmission rates, as assessed by parasite PCR-detection in neonate brains, also varied from 50% to 100% according to the isolate implicated. These results confirm the wide pathogenic and transmission variability of N. caninum. The intra-specific variability observed herein could help us explain the differences in the outcome of the infection in the natural host.     

Quantification of Mycobacterium avium subspecies in pig tissues by real-time quantitative PCR

Background

Mycobacterioses in animals cause economical losses and certain Mycobacterium avium subspecies are regarded as potential zoonotic agents. The evaluation of the zoonotic risk caused by M. avium subspecies requires information about the quantities of Mycobacterium strains in infected animals. Because M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing, we tested the suitability of a culture-independent real-time quantitative PCR (qPCR) assay for this purpose.

Methods

Mycobacterial DNA was extracted from porcine tissues by a novel method and quantified by Mycobacterium genus specific qPCR assay targeting the 16S rRNA gene.

Results

The response of the qPCR assay to the amount of M. avium subspecies avium mixed with porcine liver was linear in the range of approximately log10⁵ to log10⁷Mycobacterium cells per 1 g of liver. The assay was validated with three other M. avium subspecies strains. When the assay was applied to porcine lymph nodes with or without visible lesions related to Mycobacterium avium subspecies infections, around 10⁴–10⁷ mycobacterial genomes per gram of lymph nodes were detected.

Conclusions

The qPCR assay was found to be suitable for the quantification of Mycobacterium avium subspecies in porcine lymph nodes and liver.     

Pathological changes,distribution and detection of Brucella melitensis in foetuses of experimentally-infected does

BackgroundBrucellosis of goats is caused by Brucella melitensis. It is a re-emerging zoonotic disease in many countries due to transmission from domestic animals and wildlife such as ibex, deer and wild buffaloes.ObjectiveTo describe the pathological changes, identification and distribution of B. melitensis in foetuses of experimentally infected does.MethodsTwelve female goats of approximately 90 days pregnant were divided into 4 groups. Group 1 was exposed intra-conjunctival to 100 µL of sterile PBS while goats of Groups 2, 3 and 4 were similarly exposed to 100 µL of an inoculum containing 10⁹ CFU/mL of live B. melitensis. Goats of these groups were killed at 15, 30 and 60 days post-inoculation, respectively. Foetal fluid and tissues were collected for bacterial identification (using direct bacterial culture, PCR and immuno-peroxidase staining) and histopathological examination.ResultsBilateral intra-conjunctival exposure of pregnant does resulted in in-utero infection of the foetuses. All full-term foetuses of group 4 were either aborted or stillborn, showing petechiations of the skin or absence of hair coat with subcutaneous oedema. The internal organs showed most severe lesions. Immune-peroxidase staining revealed antigen distribution in all organs that became most extensive in group 4. Brucella melitensis was successfully isolated from the stomach content, foetal fluid and various other organs.ConclusionVertical transmission of caprine brucellosis was evident causing mild to moderate lesions in different organs. The samples of choice for isolation and identification of B. melitensis are stomach content as well as liver and spleen tissue.     

Acid-insoluble ash is a better indigestible marker than chromic oxide to measure apparent total tract digestibility in pigs

The aim of this study was to determine the apparent total tract digestibility (ATTD) of nutrients in cottonseed meal (CSM) and soybean meal (SBM) in simple carbohydrate and more complex wheat-based diets using 2 indigestible markers and total faecal collection. Twenty-five Large White × Landrace boars (57.8 kg) were randomly allocated to either a pure wheat diet, 40% CSM or SBM in either a sugar-starch- (1:1) or wheat-based diet for 18 d. Acid-insoluble ash (AIA) and chromic oxide (Cr₂O₃) were included in all diets as indigestible markers. Diets were offered (1,800 g/d per pig) in 3 meals/d from d 1 to 11 and 8 meals/d from d 12 to 17. On d 9, the pigs were moved to individual metabolism cages to allow total faecal collection. On d 18, the pigs were fed hourly for 8 h. After the 8th meal, pigs were anaesthetized and digesta sampled from the terminal ileum and rectum before lethal injection. There were no differences between ATTD of nitrogen (N) determined using AIA as a marker and measured by total faecal collection. On the other hand, the ATTD of N of diets containing CSM in sugar-starch- or wheat-based diets and the pure wheat diet determined using Cr₂O₃ as a marker was less (−3.11%, −4.46% and −6.59%; P < 0.001) than that measured by total faecal collection. The ATTD of N determined using AIA as a marker was highly correlated with that measured using total faecal collection (P < 0.001; R² = 0.95). Similarly, the ATTD of N determined using Cr₂O₃ as a marker was correlated with that measured using total faecal collection, although the correlation was not quite as strong as using AIA (P < 0.001; R² = 0.87). Also, the slope of the regression line and the intercept were closer to unity and zero for the relationship when the ATTD of N was determined using AIA compared to Cr₂O₃ as an indigestible marker. The ATTD of organic and dry matter behaved similarly. These data demonstrate that the basal diet and choice of indigestible marker can substantially influence the ATTD and that the use of AIA as an indigestible marker is more suitable than Cr₂O₃ in digestibility studies in pigs.     

Analytical performance and method comparison of a quantitative point-of-care immunoassay for measurement of bile acids in cats and dogs

Point-of-care analyzers (POCAs) for quantitative assessment of bile acids (BAs) are scarce in veterinary medicine. We evaluated the Fuji Dri-Chem Immuno AU10V analyzer and v-BA test kit (Fujifilm) for detection of feline and canine total serum BA concentration. Results were compared with a 5th-generation assay as reference method and a 3rd-generation assay, both run on a bench-top analyzer. Analytical performance was assessed at 3 different concentration ranges, and with interferences. For method comparison, samples of 60 healthy and diseased cats and 64 dogs were included. Linearity was demonstrated for a BA concentration up to 130 µmol/L in cats (r = 0.99) and 110 µmol/L in dogs (r = 0.99). The analyzer showed high precision near the lower limit of quantification of 2 µmol/L reported by the manufacturer. Intra- and inter-assay coefficients of variation were < 5% for both species and all concentrations. Interferences were observed for bilirubin (800 mg/L) and lipid (4 g/L). There was excellent correlation with the reference method for feline (r_s = 0.98) and canine samples (r_s = 0.97), with proportional biases of 6.7% and −1.3%, respectively. However, a large bias (44.1%) was noted when the POCA was compared to the 3rd-generation assay. Total observed error was less than total allowable error at the 3 concentrations. The POCA reliably detected feline and canine BA in clinically relevant concentrations.     

东方蜜蜂微孢子虫对蜜蜂健康的影响

自1996年首次报道以来,东方蜜蜂微孢子虫(Nosema ceranae)已被证实可以影响蜜蜂个体的行为、定向、飞行和寿命等,使其成为蜜蜂健康与保护领域的研究热点.本文整理了近10年N.ceranae的研究情况,从N.ceranae的传播途径、对蜜蜂健康的影响、与其他病原的相互作用进行综述,提出目前N.ceranae研...     

Invasive slug populations (Arion vulgaris) as potential vectors for Clostridium botulinum

Background

Norwegian meadows, including those for silage production, are recently found heavily invaded by the slug Arion vulgaris in exposed areas. As a consequence, large numbers of slugs might contaminate grass silage and cause a possible threat to animal feed quality and safety. It is well known that silage contaminated by mammalian or avian carcasses can lead to severe outbreaks of botulism among livestock. Invertebrates, especially fly-larvae (Diptera), are considered important in the transfer of Clostridium botulinum type C and its toxins among birds in wetlands. C. botulinum form highly resistant spores that could easily be consumed by the slugs during feeding. This study aimed to determine whether Arion vulgaris could hold viable C. botulinum and enrich them, which is essential knowledge for assessing the risk of botulism from slug-contaminated silage. Slug carcasses, slug feces and live slugs were tested by a quantitative real-time PCR (qPCR) method after being fed ≅ 5.8 × 10⁴ CFU C. botulinum type C spores/slug.

Results

Low amounts of C. botulinum were detected by qPCR in six of 21 slug carcasses with an even spread throughout the 17 day long experiment. Declining amounts of C. botulinum were excreted in slug feces up to day four after the inoculated feed was given. C. botulinum was only quantified the first two days in the sampling of live slugs. The viability of C. botulinum was confirmed for all three sample types (slug carcasses, slug feces and live slugs) by visible growth in enrichment media combined with obtaining a higher quantification cycle (Cq) value than from the non-enriched samples.

Conclusions

Neither dead nor live invasive Arion vulgaris slugs were shown to enrich Clostridium botulinum containing the neurotoxin type C gene in this study. Slugs excreted viable C. botulinum in their feces up to day four, but in rapidly decreasing numbers. Arion vulgaris appear not to support enrichment of C. botulinum type C.     

Establishing conditions for the storage and elution of rabies virus RNA using FTA? cards

The Flinders Technology Associates filter paper cards (FTA^® cards) can be used to store nucleic acid from various samples and are easily portable. However, RNA is physicochemically unstable compared with DNA, and appropriate methods have not been established for storage and extraction of RNA from FTA^® cards. The present study investigated the optimum conditions for storage and elution of viral RNA (vRNA) using rabies virus (RABV) applied to FTA^® cards. When TE buffer was used, the elution rates of vRNA increased with the length of the elution time. When the cards were stored at −80°C or −20°C, vRNA was stable over 3 months. Degradation of vRNAs occurred following storage at 4°C and room temperature, suggesting that RNA should be extracted from cards as soon as possible if no freezer is available. When we tried to amplify vRNA from RABV-infected animal brains applied to FTA^® cards and stored at −80°C for 6 months, we did not detect any amplified products with the primer set for 964 bp of RABV N gene. However, we were able to detect amplified products by increasing the elution time of vRNA from FTA^® cards from 30 min to 24 hr or by changing the primer sets to amplify 290 bp of N gene. Thus, we recommend extending the elution time for damaged or low concentration samples in FTA^® cards.     

Development of a nanoparticle-assisted PCR assay to distinguish canine coronaviruses I and II

Nanoparticle-assisted PCR (nanoPCR) is a novel method for the simple, rapid, and specific detection of viruses. We developed a nanoPCR method to detect and differentiate canine coronavirus I (CCoV I) and II (CCoV II). Primer pairs were designed against the M gene conserved region of CCoV I and CCoV II, producing specific fragments of 239 bp (CCoV I) and 105 bp (CCoV II). We optimized the annealing temperature and primer concentrations for the CCoV nanoPCR assay and assessed its sensitivity and specificity. Under optimized nanoPCR reaction conditions, the detection limits were 6.47 × 10¹ copies/μL for CCoV I and 6.91 × 10² copies/μL for CCoV II. No fragments were amplified using other canine viruses as templates. The sensitivity of the nanoPCR assay was 100-fold higher than that of a conventional RT-PCR assay. Among 60 clinical samples collected from Beijing, China, the assay detected 12% positive for CCoV I and 48% positive for CCoV II. Our nanoPCR method is an effective method to rapidly detect CCoV I and CCoV II alone, or as a mixed infection, in dogs.