Alternative BSE risk assessment methodology for beef and beef offal imported into Japan

The Food Safety Commission (FSC) of Japan, established in July 2003, has its own initiative to conduct risk assessments on food stuffs known as "self-tasking assessment". Within this framework, the FSC decided to conduct a risk assessment of beef and beef offal imported into Japan from countries with no previous BSE reports; thus, a methodology was formed to suit to this purpose. This methodology was partly based on the previous assessments of Japanese domestic beef and beef imported from U.S.A./Canada, but some modifications were made. Other organizations' assessment methods, such as those used for BSE status assessment in live cattle by the OIE and EFSA's GBR, were also consulted. In this review, the authors introduce this alternative methodology, which reflects (1) the risk of live cattle in the assessed country including temporal risks of BSE invasion and domestic propagation, with the assessment results verified by surveillance data, and (2) the risk of beef and beef offal consisting of cumulative BSE risk by types of slaughtering and meat production processes implemented and the status of mechanically recovered meat production. Other possible influencing factors such as atypical BSE cases were also reviewed. The key characteristic of the current assessment is a combination of the time-sequential risk level of live cattle and qualitative risk level of meat production at present in an assessed country.     

Detection of carrot red leaf virus-RNA in carrot seeds by multiplex RT-nested PCR

A practical method to detect carrot red leaf virus (CtRLV)-RNA in carrot seeds using a multiplex RT-nested PCR was developed. Total RNA was extracted from carrot seeds by the guanidine thiocyanate method. RT-PCR was performed with a one step RT-PCR kit with CtRLV primers and carrot ubiquitin primers for the internal control, and the second PCR was performed with CtRLV nested primers. Ubiquitin were detected by the first PCR in all samples, while distinct CtRLV-RNA was detected by the 2nd PCR. CtRLV-RNA was detected from carrot seeds from three cultivars. The multiplex RT-nested PCR method was able to detect CtRLV-RNA from a single carrot seed.     

FC-20 Efficacy of griseofulvin for juvenile cellulitis in dogs

Medical records of 97 dogs with pemphigus foliaceus were evaluated. The average age of onset was 6.3 years (range 0.5–16 years). Crusts were the most common lesions in 79 dogs; pustules were observed in 36 dogs. No gender predisposition was identified. The trunk was the most commonly involved area (51 dogs), followed by the inner pinnae (46), dorsal muzzle (37), footpads (32), periocular area (26), outer pinnae (23) and planum nasale (23). Facial involvement only was noted in 15 dogs. Of the 48 dogs in which cytology was recorded, concurrent infections were identified in 32 dogs, acantholytic cells were seen in 37 dogs, numerous neutrophils in 35 dogs, and numerous eosinophils in eight cases. Final control of the disease was achieved with: glucocorticoids (24 dogs); azathioprine (9); chlorambucil (1); aurothioglucose (1); a combination of glucocorticoids and azathioprine (31); glucocorticoids and aurothioglucose (2); tetracycline/doxycycline and niacinamide (8); prednisolone, tetracycline and niacinamide (1); fatty acid supplementation (2); and tacrolimus (1). One dog was completely tapered off drugs and stayed in remission. Average time to improvement was 6 weeks, and average time to remission was 9.3 months. Forty-three dogs were followed for <12 months, and 12 of these were euthanized: eight for other diseases and four due to a lack of response or adverse effects of treatment. In 54 dogs, the follow up was >12 months; four of these dogs were euthanized (one due to an unrelated cause, one due to neoplastic disease and two related to pemphigus foliaceus).
Funding: Self-funded.     

P‐5 Importance of psychogenic factors in canine recurrent pyoderma

Recurrent pyoderma is a relapsing bacterial infection of the skin. Evaluation of the primary predisposing factors is essential, but they are not always easy to determine. We reported that psychogenic factors should be considered as one cause of canine dermatoses featuring pruritic behavior such as grooming and scratching, and predicted that it could be added as a cause of recurrent pyoderma. The purpose of this study was to evaluate the roles of psychogenic factors in canine recurrent pyoderma. This study examined 21 dogs with recurrent pyoderma, which showed relapsing but antibiotic‐responsive skin lesions. Predisposing factors, especially allergy, infection, endocrine disorders, congenital factors and improper treatments, were carefully ruled out with standard diagnostic procedures. Diagnosis of psychogenic factors was based on fulfillment of the following features: (1) distinctive area of broken hairs or asymmetric erythema, (2) incidental pruritic behavior related to emotionally unstable situation or physiological intervention, (3) existence of environmental antecedent triggers or concurrent psychiatric symptoms, and (4) requirement for psychogenic treatment, consisting of behavior modification and psychopharmacological therapy in some. The incidence of psychogenic factors and the efficacy of the treatment in recurrent pyoderma were evaluated. In these 21 dogs, 10 cases (47.6%) were compatible with the presence of psychogenic factors, and seven cases (33.3%) were improved with behavioral treatment. Psychogenic factors should be considered in the aetiology of so‐called idiopathic canine recurrent pyoderma. Further investigation is needed to understand the relationship between skin barrier dysfunction and psychogenic factors. Funding: Self‐funded.     

High level expression and purification of bioactive bovine interleukin-18 using a baculovirus system

Bioactive recombinant bovine interleukin-18 (rboIL-18) was expressed using a baculovirus system. Normally, IL-18 is translated as a precursor form of a 24kDa polypeptide and processed by IL-1beta converting enzyme (ICE) to a mature bioactive form of 18kDa protein. Hence, to express active form IL-18, we constructed two recombinant baculoviruses containing boIL-18 and human ICE (hICE) genes, respectively, and superinfected these viruses into insect cells. Superinfection of both recombinant viruses into the cells resulted in the expression of a 24kDa precursor form and an 18kDa mature form detectable in the supernatant by immunoblotting using anti-porcine IL-18 antibody. Culture supernatant from the superinfected cells showed a synergistic effect with recombinant boIL-12 for production of interferon-gamma (IFN-gamma) in bovine peripheral mononuclear cells. By addition of histidine hexamer at the C-terminal of boIL-18, the mature IL-18 was purified. Bioactivity remained after purification.     

Production and in vivo testing of a recombinant bovine IL-12 as an adjuvant for Salmonella typhimurium vaccination in calves

A recombinant bovine interleukin-12 (boIL-12) that contains a histidine hexamer, rboIL-12His, was produced, purified and administered to calves. We first tried the purification of heterodimer IL-12 from a mixture of p40 homodimer, p40 monomer, and p40-p35 heterodimer with a p35 subunit tagged with a histidine hexamar at its C-terminal (p35His). A recombinant baculovirus expressing p35His was generated and used for superinfection with a recombinant baculovirus expressing p40 subunit. The expressed subunits, p40 and p35His, were assembled into a 70kDa heterodimer in insect cells, released into culture medium, and then purified using a nickel chelate column. The purified rboIL-12His was bioactive for induction of IFN-gamma in bovine peripheral blood mononuclear cells (PBMCs) in vitro.The purified rboIL-12His was then administered to calves with inactivated Salmonella Typhimurium (ST). When sera were assayed by ELISA, specific anti-ST IgG1 antibodies were detected in all ST immunized calves, but, specific anti-ST IgG2 antibodies were detected only in calves administered ST along with rboIL-12His, indicating a possible switch to a Th1 response. Administration of commercially available Salmonella vaccine did not elicit IgG2 antibodies in calves. These results suggest that co-administration of IL-12 with inactivated ST cells could induce a Th1-type response in calves.     

Effects of Temperature and Host on the Generation of Tomato Spotted Wilt Virus Defective Interfering RNAs

ABSTRACT The generation of defective interfering (DI) RNA molecules of tomato spotted wilt tospovirus (TSWV) was studied by serially passaging in-ocula from plant to plant under different controlled conditions. DI RNAs were generated at higher rates in plants at 16 degrees C than in plants incubated at higher temperatures. Another factor promoting the TSWV DI RNA generation was the use of high virus concentrations in the inocula. The solanaceous species Capsicum annuum, Datura stramonium, Lycopersicon esculentum, Nicotiana benthamiana, and N. rustica supported the generation of DI RNAs, whereas the virus recovered from the inoculated composite species, Emilia sonchifolia, remained free of any DI RNA under all conditions tested. This study resulted in a strategy to maintain DI RNA-free TSWV isolates, as well as in an efficient way to produce a large population of different DI RNA species. A single DI RNA species usually became dominant in an isolate after a few rounds of serial inoculations. The possible mechanisms involved in TSWV DI RNA generation under different inoculation circumstances are discussed.     

Divergence of p33/34 gene of Theileria found in Cervus nippon in Japan

The 18S rRNA gene and the piroplasm major immunodominant protein gene (p33/34) of Theileria from various subspecies of sika deer in 8 different locations of Japan were analyzed. The similarity between 633 bp partial sequences of the 18S rRNA gene among various subspecies of sika deer was found to be between 99.7% and 100%. While the percent identities of the 412 bp partial p33/34 gene sequence and deduced amino acid sequences between Theileria of sika deer from Yamaguchi Prefecture and those found in deer from other Prefectures, were comparatively low, 68.7% to 70.1% and 64.1% to 70.0% respectively. These findings suggest that there are at least two genetically distinct strains of Theileria of sika deer in Japan.     

Revised phylogenetic relationships among herpesviruses isolated from sturgeons

Initial phylogenetic comparisons based on a region of the DNA polymerase of seven herpes-like viruses found in sturgeons in North America and Europe indicated the presence of three distinct clades. A revised phylogenetic analysis of the same viruses, based on corrected DNA polymerase sequences and newly obtained sequence data from the putative ATP subunit of the terminase gene, indicate only two clades. These two clades correspond to the historical designations given to these herpes-like viruses from white sturgeon Acipenser transmontanus: white sturgeon herpesvirus type 1 (WSHV-1) and type 2 (WSHV-2). The identification of putative terminase gene sequences for all seven herpes-like viruses from sturgeons confirms their affinity with the family Herpesviridae (because this gene is unique to herpesviruses) and more distantly with T4-like bacteriophages. The two clades of sturgeon herpesviruses are therefore appropriately designated as Acipenserid herpesviruses 1 and 2, which correspond to the previous common names of white sturgeon herpesvirus types 1 and 2.