Detection of Tobamoviruses from Soils by Non-precoated Indirect ELISA

A method for detecting tobamoviruses from field soils was developed using non-precoated indirect enzyme-linked immunosorbent assay (Id-ELISA). Absorbance values in Id-ELISA were relatively low after directly applying Pepper mild mottle virus (PMMoV)-infested soil extract. However, heat treating the soil extract before application greatly enhanced the absorbance values. The heat treatment was essential for the Id-ELISA detection of tobamoviruses from infested soil, although the efficiency of virus recovery varied depending on the properties of soil. The number of local lesions in the infectivity assay was consistent with the absorbance values in Id-ELISA. Moreover, the absorbance values in Id-ELISA were correlated with the incidence of soil transmission of PMMoV. Thus, Id-ELISA combined with heat treatment is a practical technique for the diagnosis of infestation with Tobamovirus in field soils, Gray Lowland soil and Sand-dune Regosol. Received 4 October 1999/ Accepted in revised form 9 December 1999     

Immunoreactivity and morphological changes of bursal follicles in chickens infected with vaccine or wild-type strains of the infectious bursal disease virus

Infectious bursal disease (IBD) is characterized by immunosuppression due to the depletion of lymphocytes in the atrophied bursa of Fabricius (BF). We have sometimes encountered contradictory findings: chickens infected with the vaccine IBD virus (IBDV) strain have sometimes exhibited a highly atrophied BF, but not immunosuppression. In this study, chickens administered vaccine or wild-type strains of IBDV were later vaccinated with the B1 strain of the Newcastle disease virus (NDV). Bursal changes were examined histologically with a focus on the bursal follicle. The immunoreactivity to NDV was also evaluated with the hemagglutination inhibition test. In gross examination, we observed a few chickens with a severely atrophied BF in vaccine strain-administered groups (vaccine groups), and the level of severity was the same as that in the wild-type strain-administered group (wild-type group). However, these chickens retained humoral antibody responses to NDV and were revealed to possess a higher number of bursal follicles than those of the wild-type group. These results indicated that macroscopic evaluation dose not accurately reflect the immunoreactivity and degree of bursal damage in IBDV-administered chickens. We also found non-immunosuppressed chickens in the wild-type group. These non-immunosuppressed chickens retained a significantly higher number of normal follicles and total follicles according to our statistical analysis. Furthermore, a high correlation coefficient between the NDV-HI titer and the number of normal follicles was found in the wild-type group. These results implied that the retained number of normal follicles is important for the immunoreactivity of chickens infected with IBDV.     

Preparation of Anti-Rabies Virus N Protein IgYs by DNA Immunization of Hens Using Different Types of Adjuvants

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 µg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund''s complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16–19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.     

Development of Increased Oxolinic Acid-resistance in Burkholderia glumae

Genomic DNA, partially digested with Sau3AI, of Burkholderia glumae Pg-13, resistant to oxolinic acid (OA), was ligated into pUC118, and Escherichia coli DH5α resistant to 2.5 μg/ml OA was transformed with the plasmid. After incubation on a medium supplemented with 20 μg/ml OA, a clone harboring pB′46 was selected. The nucleotide sequence of the 724-bp insert of pB′46 had no homology to any characterized gene. B. glumae Pg-5, resistant to 10 μg/ ml OA, was transformed with plasmid pUCD3101B′46 containing the insert. An obtained transformant, B25, was resistant to 50 μg/ml OA and had greater resistance to other quinolones than did Pg-5. Transformants of OA-sensitive B. glumae with pUCD3101B′46 had OA sensitivity similar to that of the parental isolates. Received 18 September 2000/ Accepted in revised form 18 October 2000     

Congenital malformations of the external and middle ear accompanied by temporal bone anomaly in a calf

A 7-month-old female Holstein calf presented with bilateral microtia and absent external acoustic meatus. The real-time polymerase chain reaction test was negative for bovine viral diarrhea virus and bovine leukemia virus. The calf’s dam had a normal reproductive history. Computed tomography confirmed bilateral atresia of external auditory canals, aplasia of tympanic cavities and the ossicular chain, and temporomandibular joint abnormality. Necropsy revealed a severe malformation of the temporal bone. In the tympanic region, the external acoustic pore, tympanic bulla, and muscular process were absent bilaterally. The bilateral inner ear structure was normal. Based on these findings, we diagnosed the present case as congenital malformations of the external and middle ear accompanied by temporal bone anomaly.     

A Comparative Study on Egg Yolk IgY Production with Different Adjuvants and their Inhibitory Effects on Staphylococcus aureus

Objectives: Atopic dermatitis (AD) is one of the most common skin disorders in infants and children and is often aggravated by increased Staphylococcus aureus (S. aureus) colonization. An inhibitory effect of a specific egg yolk antibody (IgY) on S. aureus growth was demonstrated in this study. Furthermore, the effects of water- or oil-based adjuvants on the preparation of anti-S. aureus IgY and hen immunization were compared.Methods: Hens were immunized intramuscularly with formalin-killed S. aureus mixed with either a water-soluble polysaccharide λ-carrageenan, oil-based Freund''s complete adjuvant (FCA), or Freund''s incomplete adjuvant (FIA). Anti-S. aureus IgYs (FIA-IgY, FCA/FIA-IgY, and λCarra-IgY) were purified from the egg yolk of immunized hen eggs, and the activity of the IgY against S. aureus antigen was measured by ELISA. The proportion of each IgY that was absorbed by S. aureus was also determined. Then, the effect of purified anti-S. aureus IgY on S. aureus growth inhibition was investigated in vitro.Results: The yolk of eggs and purified FIA-IgY from the FIA group showed the highest antibody activity, followed by FCA/FIA-IgY and λCarra-IgY. The proportion of each IgY that was absorbed by S. aureus antigen was as follows: FIA-IgY (18.1%), FCA/FIA-IgY (12.9%), and λCarra-IgY (7.0%). Only FIA-IgY significantly inhibited S. aureus growth in liquid medium.Conclusion: A specific IgY that was produced using the FIA adjutant inhibited S. aureus growth. Although water-soluble λ-carrageenan showed an adjuvant effect on anti-S. aureus IgY induction in egg yolk, but did not inhibit S. aureus growth. The use of the oil adjuvant FIA was necessary in the preparation of anti-S. aureus IgY as a treatment for AD symptoms.     

Characterization of vpr vector constructed from chimeric simian and human immunodeficiency virus

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful tool for investigating AIDS pathogenesis and for development of vaccine. We constructed a SHIV-vpr vector (designated as SHIV-3sj) by replacing vpr region with restriction enzyme sites. SHIV-3sj was designed to express inserted gene along with its viral replication. Five cytokine genes were inserted into SHIV-3sj, and ability of viral replication and expression of the inserted genes were examined. The short insert including RANTES and IL-5 resulted in the successful expression from SHIV-3sj, while the construct having longer genes including IL-2, IL-6 and IL-12p35 failed to become replication competent. These results suggest that the length of the insert is an important factor for the replication ability of SHIV-3sj vector.