Gel Diffusion Precipitation Research Articles

ŞEROLOGICAL EVIDENCE OF INFECTIOUS BURSAL DISEASE VIRUS INFECTION IN GREY BREASTED HELMET GUINEA FOWLS (Numida meleagris galeata Pallas) IN AN ARID ZONE OF NIGERIA

A survey of guineafowl sera from Maiduguri area of Borno State for antibody to infectious bursal disease (IBD) virus was carried out between the months of January to April, 1991. Agar gel diffusion precipitation test (AGDT) was the serological method employed for the investigation. Out of the total of 196 sera tested, only 16 were found to contain precipitating antibodies to IBD. This represents 9.1% infection rate.

Antiviral Activity of Allokin-Alpha against Herpesvirus in Birds

The article reports the efficacy assessment for the Allokin-alpha (Allokin) preparation by modeling the experimental viral infection in vivo in poultry (day-old chickens). The experimental infection included the following steps: vaccination using the vaccine HVT strain, the intramuscular injection of Allokin, and the preparation of feather follicle extracts. The specific antigen was detected in the feather follicle epithelium using the method of gel diffusion precipitation reaction (DPR). A decrease in the number of positive DPR test results for HVT antigen was observed in the case of Allokin-alpha treatment. It has been demonstrated that the optimal time for the Allokin-alpha administration was on the tenth day after the vaccination with HVT as soon as the number of positive DPR results was 33%, while the number of positive results was 50% when the tested preparation was administered 5 and 15 days after the vaccination (samples were collected on day 28 after the vaccination). Since Allokin-alpha has been successfully used to treat human papillomavirus for 15 years, it may also work well as an antiviral agent in the experimental viral infection simulation by the inoculation of vaccine HVT strain; the preparation is not species specific.

Tissue tropism and molecular characterization of a Japanese encephalitis virus strain isolated from pigs in southwest China

Since September 2012, an epidemic has been spreading among swine in a pig farm located in Sichuan province, southwest China, which has resulted in abortion, stillbirth, and fetal mummification. The brains of stillborn pigs were collected and a previously unknown Japanese encephalitis virus (JEV), namely SCYA201201, was isolated. According to the results of agarose gel diffusion precipitation, indirect immunofluorescence analysis, neutralization testing, reverse transcription PCR (RT-PCR) amplification, and physical and chemical testing, the virus was conformed to have the characteristics of JEV. The virus titer in BHK-21 cells was 108.47PFU/ml and the median lethal dose (LD50) to 3-week-old and 7-day-old mice was 1.99 log10 and 1.02 log10 PFU/LD50, respectively. The results of tissue tropism for mice showed that the viral load in the brain was significantly higher than other organs, indicating that the isolate was strongly neurotropic. Additionally, the complete genome sequence of the isolate was determined and compared with other JEV strains. Phylogenetic analysis showed that the isolate belongs to genotype I and may be an imported virus. The isolate had 88.4% nucleotide identity with the Chinese vaccine strain SA14-14-2. However, there were 69 amino acid substitutions compared with the strain SA14-14-2. Some substitutions indicated that SCYA201201 was highly neurovirulent and infective, in accordance with the results of animal testing.

Outbreak of Marek's disease in a vaccinated broiler breeding flock during its peak egg-laying period in China.

BackgroundOutbreaks of Marek’s disease (MD), caused by Marek’s disease virus (MDV), primarily occur in 10–12-week-old hens.Case presentation We report a case of MD in a breeding flock of 24–30-week-old vaccinated broilers in China. The clinical signs in the affected chickens appeared at 24 weeks, and the incidence of tumours peaked at 30 weeks. The morbidity and mortality of the hens were 5 % and 80 %, respectively. Hematoxylin–eosin staining of the tissues showed the typical characteristics of MD. MDV infection was confirmed in the hens with an agar gel diffusion precipitation assay for the MD antigen in the feather follicle epithelium. An MDV strain, designated AH1410, was isolated from the blood lymphocytes. Sequence analyses of the pp38, meq, and gB genes revealed that strain AH1410 had molecular features consistent with a virulent, previously identified MDV.ConclusionOur data provide evidence that not only is MDV becoming more virulent, but that the period of its onset in chickens is expanding. These findings provide the basis the molecular surveillance and further study of virulent MDV mutants and control strategies for MD in China.

Polymerase chain reaction–restriction fragment length polymorphism analysis of the genes involved in the biosynthesis of the lipopolysaccharide of Pasteurella multocida

The lipopolysaccharide, also known as the somatic antigen or O-antigen, is an important virulence factor of Pasteurella multocida. In the current study, the genes involved in the biosynthesis of the outer core region of the lipopolysaccharide, which were obtained from somatic type reference strains and field strains of P. multocida, were subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The PCR-RFLP analysis classified 11 out of the 16 serotypes into 5 PCR-RFLP types (I-V). Types I and V contain strains belong to serotypes 1 and 13, respectively. The rest of the PCR-RFLP types contain strains belong to certain groups of serotypes. Typing of 38 field strains from poultry using PCR-RFLP analysis and the gel diffusion precipitation test showed consistent results. These results indicate that the PCR-RFLP analysis can be a useful tool for rapid somatic typing of some strains of P. multocida.

Biomimetic modulation of crystal morphology using gel: From nano to micron-scale architectures

Various in vitro studies have been used to elucidate the roles of matrix molecules in the processes of biomineralization. Among these, gel-diffusion–precipitation methodology is unique. This effective method is capable of characterizing the effects of matrix molecules on mineralization while only using very small quantities of material. Here we discuss the versatility of agar–agar and agarose gel in morphosynthesis of different calcium and barium minerals. Crystalline nano to micron-scale superstructures of these minerals were characterized by means of scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), FT-IR, thermo gravimetric analysis (TGA) and differential scanning calorimetric (DSC) analysis.

Some Unusual Presentations of Hepatic Amoebiasis in Enugu, Nigeria

Eighty-two cases of hepatic amoebiasis were diagnosed and treated at the University of Nigeria Teaching Hospital during a 4-year period. Typical or classical features were observed in 75 (91.4%) of the cases while the remaining seven presented atypically or unusually. These were obstructive jaundice (three cases), coma (two cases) and pyrexia of unknown origin (two cases). All the cases had positive gel diffusion precipitation test for amoebic antibodies, and in all but one there was demonstration of an abscess cavity. In the three cases of obstructive jaundice, the abscess cavities were located close to the hilum and compressed the main bile duct. There was no mortality recorded especially in the jaundiced cases despite the grave prognosis associated with jaundice in hepatic amoebiasis. This study demonstrates the need to consider hepatic amoebiasis in an area when patients present with either obstructive jaundice, coma or pyrexia of unknown origin and to do ultrasonography as an initial investigation in such patients.

DEMONSTRATION OF ACID PHOSPHATASE ACTIVITY IN ANTIGENIC GLYCOPROTEIN FRACTIONS OBTAINED FROM THE CULTURE FILTRATE OF PSEUDOMONAS PSEUDOMALLEI

Pseudomonas pseudomallei, the causative microorganism of melioidosis, was grown in Mueller-Hinton liquid medium, and glycoprotein fractions were separated from the culture filtrate by ammonium sulfate precipitation, gel-filtration with Sephadex G-75, and column chromatography with DEAE-cellulose. The fractions revealed acid phosphatase activity, and reacted to the sera from melioidosis patient in gel-diffusion precipitation assay.

Parasitological and serological studies on amoebiasis and other intestinal parasitic infections in the rural sector around Recife, Northeast Brazil

Parasitological examinations were carried out during July to December, 1989, on 485 inhabitants of four villages in São Lourenço da Mata, 25 km northwest of Recife, Pernambuco, Brazil. Approximately 99.6% of the inhabitants were infected with at least one species of intestinal parasites. A high prevalence of Schistosoma mansoni (82.1%), hookworm (80.2%) Trichuris trichiura (69.9%), Ascaris lumbricoides (61.9%) and Entamoeba coli (36.7%) infections were demonstrated. Test tube cultivation revealed that the most common species of hookworm in this region was Necator americanus (88.4%), and also that the prevalence of Strongyloides stercoralis was 5.8%. Three hundred and thirty-four sera were serologically examined for amoebiasis by the gel diffusion precipitation test (GDP) and enzyme-linked immunosorbent assay (ELISA). No positive reaction was observed in all sera as examined by GDP, while 24 sera were positive by ELISA.

Pathogenicity of an isolate of infectious bursal disease virus in local Nigerian ducks

Three to six-week old local Nigerian ducks were inoculated orally and intraocularly with an isolate of infectious bursal disease virus (IBDV) serotype 1 which was pathogenic for chickens. Neither clinical signs nor gross or microscopic lesions were observed. IBDV antigen was not detected in the bursa by the agar gel diffusion precipitation test. Precipitins and neutralising antibodies to IBDV were also not detected in the serum samples and IBDV was not isolated from the bursa, spleen and liver of the ducks. The above observations suggest that the ducks might be resistant to IBDV infection.

Rapid Small‐Scale Preparation Method of Cell Surface Polysaccharides

A rapid small-scale method of extraction of lipopolysaccharide (LPS) and capsular polysaccharides was developed for the purpose of identification of chemotypes of LPS and serotypes of capsular antigens. Cell surface polysaccharides were prepared within less than 2 hr from 1.5 ml of broth or suspension of colonies cultured overnight. The preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for LPS, and by double diffusion gel precipitation (Ouchterlony) test and blotting to nitrocellulose membrane for capsular polysaccharide. The analyses with the preparations obtained by the method could provide adequate results capable of identifying chemotypes of LPS and serotypes of capsular antigens.

Unusual Precipitins in Pathological Human Sera

In conducting double diffusion gel precipitation tests, unexpected, strong precipitation reactions were noticed between a serum originating from a renal graft recipient and many pathological human sera. The highest frequency of positive reactions were produced by sera of patients with infectious mononucleosis (83%), chronic Epstein-Barr virus infections (72%), rheumatoid arthritis (57%), lepromatous leprosy (57%), and AIDS (44%). The precipitin in these pathological sera was identified as an antibody of IgM variety and the precipitinogen in the transplantation serum was shown to be a thermostable component with beta-globulin mobility. No explanation of the nature of reactions observed can be given at present.

Parasitological and serological studies on amoebiasis and other intestinal parasitic infections in Recife and its suburban area, northeast Brazil.

Parasitological examinations were carried out during April to August, 1987, with 187 out-patients of the IMIP hospital, located in the center of Recife City, and 464 inhabitants of several villages around Cabo City, 50 Km southeast of Recife, Pernambuco, Brazil. Approximately 71% of the IMIP patients and 92% of the Cabo inhabitants were infected with at least one species of intestinal parasite. There was minimum difference in the prevalence rate of Trichuris trichiura between two areas, whereas the prevalence rates of Ascaris lumbricoides, hookworms, Strongyloides stercoralis, Schistosoma mansoni and Entamoeba histolytica were higher in the inhabitants of the Cabo City area. Only Giardia lamblia was more prevalent in the out-patients of IMIP hospital. Test tube cultivation revealed that the prevalence rate of Necator americanus in both areas was much higher than that of Ancylostoma duodenale , and also that the prevalence rate of S. stercoralis of the IMIP patients and Cabo inhabitants were 4.5% and 9.6%, respectively. Six hundred and fifteen sera were serologically examined for amoebiasis by the gel diffusion precipitation test (GDP) and enzyme linked immunosorbent assay (ELISA) using the antigen prepared from axenically cultured trophozoite of E. histolytica (strain HM-ITMSS). No positive reaction was observed in all of the sera as examined by GDP, while 32 out of 615 sera were positive on ELISA.

Antibodies to Saline Extractable Tissue Antigen in Sera of Patients with Renal Diseases

Multiple serum samples originating from 110 renal allograft recipients were examined against saline extract of normal human kidney by means of double diffusion gel precipitation. Eleven recipients were found to be positive; 99 of 106 sera from these patients were positive. Pretransplantation sera were available from 7 of these recipients and 6 patients were found "positive." The precipitation reaction was composed of one line. Identity reactions were formed between the lines produced by sera from all patients except 1. Sera of patients from end-stage renal disease produced similar reaction; however, only 3 of 234 sera from patients with nonrenal diseases precipitated the kidney extract. None of 154 normal sera were positive. Several positive sera also were positive in complement fixation tests with human kidney extract. Evidence was presented that the antibodies under study combined with a nonorgan-specific but species-restricted tissue antigen. The hypothesis was advanced that these antibodies are autoantibodies formed in response to a sequestered antigen released as a result of tissue damage. Apparently, the antigen is released frequently in immunogenic form from injury to kidney but infrequently from injury to other organs.

Susceptibility of Laboratory Rats and Guinea-pigs to Infectious Bursal Disease Virus Infection

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Comparison of virus neutralizing and precipitating antibodies to infectious bursal disease virus and their effect on susceptibility to challenge

A comparison was made between the tissue culture virus neutralization (TCVN) test and the quantitative gel diffusion (QGD) test for infectious bursal disease (IBD). The TCVN test was demonstrated to be considerably more sensitive than the QGD test. When TCVN titres of 2 6 or less were regarded as negative, the QGD test gave false positives (11 · 0%) and negatives (52 · 9%) as measured by susceptibility to IBD challenge. Qualitative agar gel diffusion precipitation gave more false negatives (79 · 4%) but no false positives.

Antigenic relationship between Candida parapsilosis and Candida albicans serotype B.

We examined the antigenic relationship between Candida parapsilosis and C. albicans serotype B with respect to antigenic factors 13 and 13b, specific for the former species and common to both species, respectively. Acetolysis of C. albicans serotype B cell-wall mannan gave six oligosaccharides. Their chemical structure was determined by 1H-nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and partial acid hydrolysis. The structure of the hexasaccharide derived from C. albicans serotype B mannan was alpha-D-Manp-(1-2)-alpha-D-Manp-(1-3)-alpha-D-Manp-(1- 2)-alpha-D-Manp-(1-2)- alpha-D-Manp-(1-2)-D-Man (M6) which is identical to that from C. parapsilosis mannan. Inhibition of two precipitin reaction systems (anti-C. albicans serotype B serum and anti-C. parapsilosis serum to the respective homologous mannan), by oligosaccharides from homologous and heterologous mannans indicated that M6 from either C. albicans serotype B or C. parapsilosis was the most effective inhibitor. Moreover inhibition of the agglutination reaction between factor serum containing anti-factors 13 and 13b and C. albicans serotype B or C. parapsilosis cells by oligosaccharides from both mannans also indicated that the M6s were the most effective inhibitors. These results suggest that the M6s derived from the two species are identical in their chemical structure, although the structures of the whole mannans of the two species are not identical as demonstrated by gel diffusion precipitation patterns, and that M6s may be involved in the specificities of antigenic factors 13 and 13b. The amount of M6 is larger in C. parapsilosis cell-wall mannan, suggesting that high repeating frequency of M6 fragment may induce the antibody specific for C. parapsilosis.

Distribution of P2Antigen in Neural and Non-Neural Bovine Tissues

Rabbit antisera to bovine nerve preparations were used to study the tissue distribution in the ox of P2, an antigen specific for the peripheral nervous system. Double diffusion gel precipitation tests were able to demonstrate P2 in spinal nerves, trigeminal nerve, spinal cord, medulla oblongata, and pons, but not in higher centers of the CNS, optic nerve, or non-neural tissues. A highly sensitive inhibition of enzyme immunoassay was developed to detect and quantitate low levels of P2. By this assay, P2 was found to be most concentrated in peripheral nerves, with decreasing amounts found in the spinal cord, medulla oblongata, pons, cerebellum, and cerebral peduncle. No P2 was found in the thalamus, cerebrum, or optic nerve; however, low levels of P2 were detected in the adrenal medulla, a non-neural tissue composed largely of cells derived from the embryonic neural crest region.

Serological types of Pasteurella multocida isolated from turkeys and chickens in Canada.

Outbreaks of fowl cholera continue to plague the Canadian poultry industry despite widespread immunization against the causative agent, Pasteurella multocida. Fowl cholera bacterins currently employed by domestic poultry growers contain three serological types, namely, serotypes 1, 3, and 4. In this study a total of 84 strains of P. multocida were isolated in Canada from outbreaks of fowl cholera in turkeys and chickens. Serotyping was accomplished using the gel diffusion precipitin test. Based on the gel diffusion precipitation patterns, 27 serotypes containing one to six antigenic determinants were recognized. The most prevalent serotype both in turkeys and chickens appeared to be type 3. Significantly, greater than 20% of P. multocida isolates failed to react with antisera raised against serotypes 1, 3, and 4.

Affinity diffusion. I. Method for measuring dissociation constants of precipitating antibodies.

Dissociation constant (Kd) of antigen-antibody reactions can be obtained from the rates (measured by the progression of the precipitate vs. time) with which antigens diffuse into antibody-containing gels, as a function of antibody-concentration. A bovine serum albumin vs. rabbit anti-bovine serum albumin system was studied with whole antiserum and with its purified IgG fraction. A value was found for Kd of approximately 1.0 x 10-5 moles per liter. It is note- worthy that in monodimensional single diffusion gel precipitation systems of this type, the rate of progression of the precipitate front is significantly faster than the molecular diffusion coefficient of the antigen.