Direct detection of chicken genomic DNA for gender determination by thymine-DNA glycosylase.

1. Birds, especially nestlings, are generally difficult to sex by morphology and early detection of chick gender in ovo in the hatchery would facilitate removal of unwanted chicks and diminish welfare objections regarding culling after hatch. 2. We describe a method to determine chicken gender without the need for PCR via use of Thymine-DNA Glycosylase (TDG). TDG restores thymine (T)/guanine (G) mismatches to cytosine (C)/G. We show here, that like DNA Polymerase, TDG can recognise, bind and function on a primer hybridised to chicken genomic DNA. 3. The primer contained a T to mismatch a G in a chicken genomic template and the T/G was cleaved with high fidelity by TDG. Thus, the chicken genomic DNA can be identified without PCR amplification via direct and linear detection. Sensitivity was increased using gender specific sequences from the chicken genome. 4. Currently, these are laboratory results, but we anticipate that further development will allow this method to be used in non-laboratory settings, where PCR cannot be employed.

High-performance thin-layer chromatographic determination of rabeprazole sodium and domperidone in combined dosage form.

A new, simple, high-performance thin-layer chromatographic method for determination of rabeprazole sodium (RAB) and domperidone (DOM) in combined tablet dosage form has been developed and validated. The mobile phase was toluene-acetone-methanol (4.5 + 4.5 + 0.5, v/v/v) with UV detection at 285 nm. The retention factors for RAB and DOM were found to be 0.53 +/- 0.12 and 0.32 +/- 0.20. The method was validated with respect to linearity, accuracy, precision, and robustness. Beer's law was obeyed in the concentration range of 50-800 ng/band for both RAB and DOM. The method has been successfully applied for the analysis of drugs in a pharmaceutical formulation.

Use of live and inactivated vaccines in the control of West Nile fever in domestic geese.

The recent epizootic of West Nile fever in Israel affected predominantly young domestic geese between three and eight weeks old. Clinically, the birds presented paralytic signs while morbidity and mortality were severe in affected flocks. The condition was encountered from early September through late November on goose farms located throughout the country. Losses incurred by goose flocks were sufficiently great as to warrant investigation of ways to protect young geese against the neurological form of the disease. We have conducted a series of vaccination trials in which three-week old geese were immunized with an attenuated, commercial flavivirus vaccine derived from Israel turkey meningoencephalitis virus (TME). Birds were challenged two weeks later with a low Vero cell passage of West Nile virus by the intracerebral route. In a second group of experiments, inactivated and live TME vaccines were given in tandem at an interval of two weeks and challenged two weeks later. The third vaccination trial was based on West Nile virus (WNV) harvested from infant mouse brain, inactivated with formalin and oil adjuvanted. A single injection given either subcutaneously or intramuscularly resulted in 75% protection of the vaccinated groups, while two injections spaced two weeks apart resulted in 94% protection. Groups of geese, vaccinated at the farms and challenged under controlled conditions in the laboratory, showed levels of protection ranging from 39% to 72% for TME vaccine and 52% and 80% for WNV vaccine. The lower levels of protection are attributable to flocks being affected with intercurrent infections at the time of vaccination.

Day-old vaccination with live-in-oil vaccines: Newcastle disease (ND) and infectious bursal disease (IBD) in chicks and ND in turkey poults.

One-day-old broiler chicks were vaccinated with live Newcastle disease (ND) vaccine incorporated in oil alone or in killed-in-oil ND vaccine. Incorporation of live vaccine in oil emulsions was carried out just prior to vaccination. Live-in-oil ND vaccine containing 106.0 median embryo lethal doses (ELD50/dose induced the same protection following challenge and the same level of antibody at 42 days post-vaccination as did commercial killed-in-oil ND vaccine containing about 250 times as much antigen (108.4 ELD50/dose). Incorporation of live ND vaccine in killed-in-oil vaccine contributed markedly to protection rates and antibody levels, as compared to those obtained following vaccination with killed-in-oil vaccine only. One-day-old turkey poults also showed the advantage of incorporation of live ND vaccine in killed-in-oil vaccine when challenged 3 months post-vaccination. One-day-old broiler chicks, vaccinated with live ND and infectious bursal disease vaccine (IBD) incorporated in killed-in-oil combined ND + IBD vaccine, showed better protection against challenge with IBDV and higher antibody levels to NDV as compared to vaccination with killed-in-oil vaccine alone.

Vaccination of turkeys in the wattles (dewlap) with turkey meningo-encephalitis live vaccine and Pasteurella multocida killed-in-oil vaccine.

Vaccination of turkeys via the wattle has been introduced as a novel route of vaccination using attenuated live viral turkey meningo-encephalitis (TME) and killed-in-oil bacterial (Pasteurella multocida) vaccines. The efficacy of the immunization was evaluated by the haemagglutination-inhibition test for TME and by challenge for TME and P. multocida. Immunization via the wattle route was comparable or better as compared with the conventional routes, intramuscular and subcutaneous, for P. multocida and TME, respectively. These results were obtained by wattle vaccination administered either by injection, punching with a needle as used for fowl pox vaccination or by topical application. The advantages of wattle vaccination are: no local untoward reactions (P. multocida), which might frequently occur in the muscles following improper subcutaneous mass vaccination, less time and labour consuming, and less stress for the turkeys. It is suggested to test the wattle route of vaccination with other viral and bacterial vaccines in turkeys and other avian species.

Host factors affecting the homologous and heterologous immune response of cattle to FMDV: genetic background, age, virus strains and route of administration.

Sixty bulls were tested for antibodies to the heterologous serotype C1 of FMDV following repeatable vaccinations with a commercial trivalent vaccine (O1, A22, Asia1). Six (10%) bulls were found to possess rather high levels of heterologous neutralizing antibodies which showed accumulative trend with age. Two high positive and two negative bulls for the heterologous serotype C1 were selected for progeny test involving ten daughters of each bull. The four bulls, either positive or negative for the heterologous serotype C1, showed significant phenotypic correlation between their heterologous and homologous titers (O1, A22, Asia1). This correlation between heterologous and homologous antibody titers was not found in the daughters of these bulls. However, two of ten daughters of one positive bull, to C1 showed individual high titers (> or = 1.5). The intradermal (ID) as compared to subcutaneous (SC) route of administration resulted in higher rate of responders to both heterologous serotypes C1 and SAT1. The heterologous immune response to FMDV in Israeli-Friesian cattle was found to be related to the age of the host, multiplicity of vaccinations, route of vaccination, kind and numbers of the antigens used in the vaccine. The homologous immune response is also controlled by genetic factors.

Homologous and heterologous antibody response of cattle and sheep after vaccination with foot and mouth disease and influenza viruses.

Homologous and heterologous antibody response to FMD and influenza vaccines was studied in 37 calves and 45 lambs at the age of 2 months. The FMD and influenza monovalent killed vaccines were administered simultaneously twice. Another group of 18 calves was vaccinated twice, first at the age of 2 months and second at the age of 6 months, with trivalent FMD vaccine. The antibody titers were measured by ELISA and HI after second vaccination, for FMDV and influenza, respectively. The conclusions of this study are summarized as follows. Individuals, lambs and calves, that cross-respond to one heterologous serotype are liable to respond to another heterologous serotype of the same virus. Individuals, lambs and calves, showing double cross-reactivity to one virus (FMDV), are highly liable to show double cross-reactivity to entirely another virus (Influenza). Multivalent vaccines of FMDV are expected to elevate the antibody titers for at least one heterologous serotype (not included in the vaccine) and to detect antibodies for an additional heterologous serotype, not detected otherwise following monovalent vaccination. These results indicate the important role of the host in the spectrum of the specific immune response.

Molecular characterization of an unassigned Israeli Newcastle disease virus isolate.

Detection of Newcastle disease virus (NDV) and avian pathotyping of NDV isolates are extremely important because the appearance of virulent virus has significant economic consequences in terms of vaccination, eradication, and the ability to export poultry products. By using nucleotide and amino acid (aa) homology analysis, we could demonstrate that a NDV broiler isolate is a velogenic virus. This analysis was done after mean death time and intracerebral pathogenicity index tests gave inconsistent results. By establishing a nucleotide sequence dendrogram, we found that the disputed Ber-Tuvia was clearly in the same group as the known Herev-Laet, a velogenic isolate. The difference between Ber-Tuvia 92 and the Herev-Laet velogenic isolate was 6% as opposed to > 16% of the meso- and lentogenic isolates. The Ber-Tuvia isolate contains the Arg/Arg and Lys/Arg aa at positions 112, 113 and 115, 116, respectively, in the fusion protein cleavage aa sequence, which is typical for virulent NDV isolates.

Enhanced antibody response in cattle against Leptospira hardjo by intradermal vaccination.

A commercial killed Leptospira hardjo vaccine (with adjuvant) and non-adjuvanted preparation of the same vaccine were used in comparison of the effectiveness of the intradermal (i.d.) and subcutaneous (s.c.) routes for these vaccines. The tests were conducted in 50 females aged 6-14 months. After the first vaccination, both types of vaccine elicited a very poor antibody response by both routes of vaccination. However, after booster vaccination, the commercial vaccine (with adjuvant) elicited a remarkable immune response which was twice as high by i.d. compared with s.c. vaccination. No local or general adverse reactions were observed after i.d. vaccination with the adjuvanted commercial vaccine (potassium aluminium sulphate).

Comparison of dimethyl dioctadecyl ammonium bromide, Freund's complete adjuvant and mineral oil for induction of humoral antibodies, cellular immunity and resistance to Newcastle disease virus in chickens.

Dimethyl dioctadecyl ammonium bromide (DDA), a lipophilic quaternary amine, was evaluated in adult chickens for potentiation of immunological responses to subcutaneously administered inactivated Newcastle disease virus (NDV) vaccines. DDA enhanced humoral and cell-mediated immune (CMI) responses to levels which were significantly higher than those induced by the vaccine alone The haemagglutination inhibition antibody titers induced by DDA were slightly lower than those induced by mineral oil although neutralizing antibody titers seemed to be higher. DDA induced strong CMI (DTH and lymphocyte proliferation) responses, more than those induced by Freund's complete adjuvant and mineral oil. Both DDA and mineral oil induced comparable high levels of protection to challenge doses of 200,000 LD50 per chicken. No toxic effects or local tissue damage were observed in any of the inoculated chickens.

Vaccination of chickens with live fowl pox (FP) vaccine in oil.

Live fowl pox (FP) vaccine was adjuvanted in oil just prior to the subcutaneous (SC) vaccination of one day old chicks and adult chickens. The birds were challenged by the wing web (WW) method and absence of "takes" were considered as protection. On 21 day post challenge, 90%-100% of the chicks or chickens were protected while on day 9 post challenge 60% were protected. Full protection of the live-in-oil adjuvanted vaccine is probably somewhat delayed as compared to protection endowed by the liquid vaccine. Incorporation of live FP vaccine in two different kinds of commercial Newcastle disease (ND) killed vaccine in oil, was shown to endow full protection following SC administration.

Immunization of chickens with live Newcastle disease vaccine adjuvanted with oil.

Chickens of various ages and breeds were vaccinated subcutaneously with Newcastle disease live-in-oil vaccines. These vaccines were prepared immediately prior to the vaccination by mixing the lyophilized live vaccine with the oil adjuvant, which was kept at room temperature. The live-in-oil vaccines were shown to be 30-50 times more effective in efficacy tests than either the same vaccines reconstituted in water or killed-in-oil vaccines. In addition to its biological advantage, the method of preparation of live-in-oil vaccine saves the expensive space of cold storage and shipment necessary for conventional killed-in-oil vaccines.

Differences in protection between heavy and light breeds of chickens following vaccination with Newcastle disease vaccines-a survey of data, 1971 to 1990.

Data obtained over 20 years of Newcastle disease vaccine testing were statistically analyzed. The protection afforded heavy and light breeds of chickens was compared following challenge (efficacy) after vaccination with live and inactivated vaccines produced from different virus strains. Standard challenge virus was used throughout the period. The data show that the heavy breeds were significantly inferior in their protectability when compared with the light breeds. This inferiority was shown after vaccination with all types of vaccines. It is suggested that heavy and light breeds of chickens differ genetically in their acquired resistance to Newcastle disease virus, although difference in susceptibility to the virus as a pathogen cannot be ruled out entirely.

Biological and antigenic characterization of Netivot virus, an unusual new Orbivirus recovered from mosquitoes in Israel.

The antigenic and biological characteristics of a new Orbivirus, designated Netivot virus, are described. This agent was originally recovered in cultures of the C6/36 clone of Aedes albopictus cells from a pool of Culex pipiens captured in Israel. Netivot virus is not pathogenic for newborn mice, nor did it initially produce detectable cytopathic effect (CPE) in Vero cells. It is closely related antigenically to Umatilla and Llano Seco viruses; these 3 agents appear to constitute a new serogroup within the genus Orbivirus. Netivot virus is also more distantly related to a number of other orbiviruses in the blue-tongue, epizootic hemorrhagic disease of deer, and Eubenangee serogroups. Netivot virus replicated to high titer and produced CPE in a variety of mosquito cell cultures, but it did not grow in 2 sand fly cell lines. Inoculation of Ae. aegypti and Ae. albopictus with Netivot virus resulted in almost 100% mortality in both species within 15 days after infection. The recovery of this and a number of other yet unidentified viral agents from field-collected mosquitoes in cultures of C6/36 cells, but not in the conventional vertebrate assay systems, suggests the existence in nature of many yet unrecognized mosquito-associated viruses. It also demonstrates the value of using new isolation methods in arbovirus studies.

Isolation of viruses from mosquitoes of the Negev, Israel.

In a survey of the mosquito population of the Negev carried out between July 1982 and September 1984, over 85,000 insects belonging to 10 species were tested for the presence of viruses. They yielded 91 virus isolates in C-6/36 mosquito cell cultures; 20 of the isolates were recovered also in Vero cell cultures and in suckling mice inoculated intracerebrally. Of the 20 isolates recovered in the vertebrate systems 17 were identified as Sindbis, and three as West Nile viruses. 71 viruses which have been isolated only in mosquito cell cultures remain unidentified. Sindbis and West Nile arboviruses were isolated only from Culex pipiens and from Cx perexiguus, while the unidentified viruses were isolated from these and from five other mosquito species.