Aschalew Z. Bekele

Survival of Airborne MS2 Bacteriophage Generated from Human Saliva, Artificial Saliva, and Cell Culture Medium

ABSTRACT Laboratory studies of virus aerosols have been criticized for generating airborne viruses from artificial nebulizer suspensions (e.g., cell culture media), which do not mimic the natural release of viruses (e.g., from human saliva). The objectives of this study were to determine the effect of human saliva on the infectivity and survival of airborne virus and to compare it with those of artificial saliva and cell culture medium. A stock of MS2 bacteriophage was diluted in one of three nebulizer suspensions, aerosolized, size selected (100 to 450 nm) using a differential mobility analyzer, and collected onto gelatin filters. Uranine was used as a particle tracer. The resulting particle size distribution was measured using a scanning mobility particle sizer. The amounts of infectious virus, total virus, and fluorescence in the collected samples were determined by infectivity assays, quantitative reverse transcription-PCR (RT-PCR), and spectrofluorometry, respectively. For all nebulizer suspensions, the virus content generally followed a particle volume distribution rather than a number distribution. The survival of airborne MS2 was independent of particle size but was strongly affected by the type of nebulizer suspension. Human saliva was found to be much less protective than cell culture medium (i.e., 3% tryptic soy broth) and artificial saliva. These results indicate the need for caution when extrapolating laboratory results, which often use artificial nebulizer suspensions. To better assess the risk of airborne transmission of viral diseases in real-life situations, the use of natural suspensions such as saliva or respiratory mucus is recommended.

Molecular Epidemiology of Novel Pathogen “Brachyspira hampsonii” Reveals Relationships between Diverse Genetic Groups, Regions, Host Species, and Other Pathogenic and Commensal Brachyspira Species

ABSTRACT Outbreaks of bloody diarrhea in swine herds in the late 2000s signaled the reemergence of an economically significant disease, swine dysentery, in the United States. Investigations confirmed the emergence of a novel spirochete in swine, provisionally designated “Brachyspira hampsonii,” with two genetically distinct clades. Although it has since been detected in swine and migratory birds in Europe and North America, little is known about its genetic diversity or its relationships with other Brachyspira species. This study characterizes B. hampsonii using a newly developed multilocus sequence typing (MLST) approach and elucidates the diversity, distribution, population structure, and genetic relationships of this pathogen from diverse epidemiological sources globally. Genetic characterization of 81 B. hampsonii isolates, originating from six countries, with our newly established MLST scheme identified a total of 20 sequence types (STs) belonging to three clonal complexes (CCs). B. hampsonii showed a heterogeneous population structure with evidence of microevolution locally in swine production systems, while its clustering patterns showed associations with its epidemiological origins (country, swine production system, and host species). The close genetic relatedness of B. hampsonii isolates from different countries and host species highlights the importance of strict biosecurity control measures. A comparative analysis of 430 isolates representing seven Brachyspira species (pathogens and commensals) from 19 countries and 10 host species depicted clustering by microbial species. It revealed the close genetic relatedness of B. hampsonii with commensal Brachyspira species and also provided support for the two clades of B. hampsonii to be considered a single species.

A case report of Nosema ceranae infection in honey bees in Minnesota, USA

In May June of 2012, two newly established European honey bee colonies in Minnesota experienced swarming, ceased to produce eggs and the colony population progressively decreased. Evidence of diarrhea was observed in one of the colonies in Fall of 2012 and the colonies collapsed in late Fall 2012. Samples of whole bodies of adult and larval bees (n D 30) and a pool of fecal samples from affected colonies were submitted to the Minnesota Veterinary Diagnostic Laboratory for disease diagnosis. Whole bodies of bees were ground and diluted in a 1:1 proportion with distillated water. In addition, a 10% suspension of fecal sample was prepared in distilled water. After centrifugation, the supernatants were examined under light microscope. For histopathology, whole bodies of adult bees were fixed in 10% buffered formalin followed by processing and embedding in paraffin. Five micron thick sections were stained with hematoxylin and eosin (H&E) and examined microscopically (Figure 1 (a)). Histological examination of paraffin embedded tissue sections stained with H&E revealed loss of reserve body fat tissue. The intestine had a dilated lumen and intestinal epithelial cells had a distended cytoplasm with multitudes of spore-forming microorganisms (Figure 1 (b)). The intestinal mucosa was diffusely and severely affected. The rectum was distended and contained spore-forming microorganisms intermixed with intestinal contents. These preparations demonstrated the presence of myriads of spore-like microorganisms consistent with microsporidian species. Upon electron microscopic examination of homogenized tissues using iTEM software (Olympus SIS, Munster, Germany), the microsporidian spores appeared oval in shape approximately measuring 3085.00 £ 1713.70 nm (4269.1

Experimentally induced lameness in turkeys inoculated with a newly emergent turkey reovirus.

Newly emergent turkey arthritis reoviruses (TARVs) have been isolated from cases of lameness in male turkeys over 10 weeks of age. In a previous study, experimental inoculation of TARV in one-week-old turkey poults produced lymphocytic tenosynovitis at four weeks post inoculation but without causing clinical lameness. This study was undertaken to determine if TARV infection at an early age can lead to clinical lameness in birds as they age. One-week-old male turkeys were inoculated orally with a TARV (strain TARV-O’Neil) and monitored for the development of gait defects until 16 weeks of age. At 4, 8, 12 and 16 weeks of age, a subset of birds was euthanized followed by the collection of gastrocnemius tendon, digital flexor tendon, and intestines for virus detection by rRT-PCR and for histologic inflammation scoring. Clinical lameness was first displayed in TARV-infected turkeys at 8 weeks of age and ruptured gastrocnemius tendons with progressive lameness were also seen at 12–16 weeks of age. The virus was detected in gastrocnemius tendon of 4- 8- and 12-week-old turkeys but not in 16-week-old turkeys. Histologic inflammation scores of tendons at each of the four time points were significantly higher in the virus-inoculated group than in the control group (p < 0.01). Lesions began as lymphocytic tenosynovitis with mild synoviocyte hyperplasia at four weeks of age and progressed to fibrosis as the birds aged. These results demonstrate the potential of TARV to infect young turkeys and to produce subclinical tenosynovitis that becomes clinically demonstrable as the turkeys age.

Genotypes, Antibiotic Resistance, and ST-8 Genetic Clone in Campylobacter Isolates from Sheep and Goats in Grenada

Rectal swabs from 155 sheep and 252 goats from Grenada were evaluated to determine the prevalence of Campylobacter spp., antibiotic resistance, and multilocus sequence types. Fifteen Campylobacter isolates were obtained (14 C. jejuni and 1 C. coli). The prevalence (3.7%) did not differ significantly between sheep (4.5%) and goats (3.2%). Among the seven antimicrobials tested, resistance was only detected for tetracycline (30.8%) and metronidazole (38.5%). Campylobacter isolates showed no significant difference between sheep and goats for type of antimicrobial resistance or percent of resistant isolates. Twelve of the isolates were successfully genotyped consisting of four recognized clonal complexes and three novel sequence types. Importantly, one isolate from one goat was identified as the C. jejuni sequence type-8, a zoonotic and tetracycline-resistant clone reported to be a highly virulent clone associated with ovine abortion in the USA. Although most samples were from comingled sheep and goat production units, there were no shared sequence types between these two host species. None of the sequence types identified in this study have previously been reported in poultry in Grenada, suggesting sheep- and goat-specific Campylobacter clones in Grenada. This is the first report of genotyping of Campylobacter isolates from sheep and goats in the Eastern Caribbean.

One-Step Real-Time Reverse Transcription–PCR for the Detection of Turkey Reoviruses

SUMMARY During late 2010 and early 2011, an unusual problem of lameness and swollen hock joints in commercial turkeys was reported in the upper Midwest, which continues to this day. The disease caused substantial economic losses to turkey producers. Reovirus was isolated from tendons and joint fluids of lame turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory. This study was undertaken to develop a TaqMan real-time reverse transcription-PCR (rRT-PCR) assay for the early detection of turkey reoviruses (both enteric and lameness strains). A primer probe set was designed from the conserved region of the S4 segment of the turkey reovirus genome. The newly developed rRT-PCR was specific for the detection of turkey reoviruses. The detection limit of this assay was 10 genome copies per reaction. For the TARV-MN4 strain of turkey arthritis reovirus, one 50% tissue culture infectious dose was equivalent to 11.6 ± 0.2 genome copies. The highest coefficient of variation for intraexperimental and interexperimental variability was 0.08 and 0.06, respectively, indicating the reproducibility of the assay. This new test should be useful for the detection of turkey enteric and arthritis reoviruses. RESUMEN Método de transcripción reversa y PCR en un solo paso para la detección de reovirus de pavos. Durante finales del año 2010 y principios del 2011, se informó de un problema poco común de cojera e inflamación de corvejones en pavos comerciales en el Medio Oeste de los Estados Unidos, que continúa hasta este momento. La enfermedad ha causado substanciales pérdidas económicas a los productores de pavo. Se aisló un reovirus de los tendones y de los fluidos de las articulaciones de los pavos con cojera enviados al Laboratorio de Diagnóstico Veterinario de Minnesota. Este estudio se realizó para desarrollar un método de transcripción reversa y PCR en tiempo real (rRT-PCR) con sondas TaqMan para la detección temprana de los reovirus de pavo (cepas entéricas y cepas causantes de cojera). Se diseñó un conjunto de iniciadores para la región conservada del segmento S4 del genoma del reovirus de pavo. El método de rRT-PCR recientemente desarrollado era específico para la detección de reovirus de pavo. El límite de detección de este ensayo fue de 10 copias de genoma por reacción. Para la cepa TARV-MN4 de reovirus de pavo causante de artritis, una doses infectante 50% en cultivo de tejidos fue equivalente a 11.6 ± 0,2 copias del genoma. Los mayores coeficientes de variación para la variabilidad intraexperimental e interexperimental fueron de 0.08 y 0.06, respectivamente, indicando la reproducibilidad del ensayo. Esta nueva prueba puede ser útil para la detección de reovirus de pavos entéricos y causantes de artritis.

Efficacy of Five Commonly Used Disinfectants Against Turkey Arthritis Reovirus

SUMMARY Since late 2009, an unusual problem of reovirus-related lameness has been seen in market-age tom turkeys in the upper Midwest area of the United States. In this study, we determined the efficacy of five commonly used disinfectants (Virocid, Keno X5, Synergize, One Stroke, and Tek Trol) against turkey arthritis reoviruses (TARVs). For comparison, turkey enteric reovirus (TERV) and chicken arthritis reovirus (CARV) were also included. At their recommended concentrations, all five disinfectants were found to be effective virucidals, inactivating 99.99% of all viruses within 10 min. However, oxidizing agents and quaternary ammonium compounds + aldehyde types of disinfectants were more effective, killing the viruses in a shorter time (2–5 min) than the other types of disinfectants. These results indicate that these disinfectants can be an effective tool in the control of these viruses. RESUMEN Eficacia de cinco desinfectantes comúnmente utilizados contra reovirus que producen artritis en pavos. Desde finales del 2009, se ha observado un problema poco común de cojeras relacionado con reovirus en los pavos machos a la edad de mercado en el área norte del Medio Oeste de los Estados Unidos. En este estudio, se determinó la eficacia de cinco desinfectantes de uso común (Virocid, Keno X5, Synergize, One Stroke y Tek Trol) contra reovirus que producen artritis en pavos (TARVs). Con fines de comparación, también se incluyeron reovirus entéricos de los pavos (TERV) y reovirus que producen artritis en el pollo (CARV). En sus concentraciones recomendadas, se encontró que los cinco desinfectantes fueron viricidas eficaces, inactivando el 99.99% de todos los virus en 10 minutos. Sin embargo, los agentes oxidantes y compuestos de amonio cuaternario + desinfectantes del tipo de los aldehídos fueron más efectivos, matando los virus en un tiempo más corto (2–5 minutos) que los otros tipos de desinfectantes. Estos resultados indican que estos desinfectantes pueden ser una herramienta eficaz en el control de estos virus.