Gel Diffusion Precipitin Test Research Articles

Exploring Antigenic Properties and Immune Responses in Bufo himalayanus: Implications for Conservation and Biodiversity Protection

Background: The objective of the study is to investigate antigenic property of soluble antigens of Nematodes Cosmocercoides dukae isolated from infected Bufo himalayanus (Class Amphibia, order: Anura) and to investigate the nature of protective immune response in laboratory rats. Bufo himalayanus is a species found in the districts of Darjeeling, Sikkim and adjoining hills of the sub-Himalayan belt, at an altitude of 6000 ft. above the sea level and known to be heavily infested by natural gut dwelling nematode infections. The present study is a small step towards protection and conservation of this amphibian species.
 Results: study of morphology of the nematode parasite confirmed it to be Cosmocercoides dukae. A crude whole body homogenate (antigenic protein) of the parasite was injected in healthy non-infected rats, spleen histo-architecture of treated rats differed from control rats. Humoral immune response assay by Gel Diffusion Precipitin (GDP) Test revealed that the parasite antigen invoked very little to no response. Study of the crude nematode antigenic protein extract concentration was 6.09 mg/ml and protein profile assay by SDS-PAGE showed seven major and several minor bands.
 Discussion: Though humoral immune response assay of parasite in experimental rats evoked very little or no immune response, this would not mean absence of antibodies, rather the antibody titers were presumably very low. It is to be noted that spleen of treated rats showed a distinctive histological feature. GDP test with infected toad serum also showed negative result. But protein profile assay of antigenic protein by SDS-PAGE showed many bands of proteins, which could be isolated and tested for further characterization.
 Conclusion: In order to maintain the Himalayan biodiversity, this amphibian species needs protection from such natural infective nematode parasites. If we are able to focus any light to this area, this knowledge might help the future research workers to plan for the protection of this species.

EPIDEMIOLOGY AND CONTROL OF GENITAL PASTEURELLOSIS IN BREEDING RABBITS

This study aimed to investigate the epidemiology and the possible ways of control of Pasteurella multocida (P. multocida) microorganism as a genital and a respiratory pathogen in rabbit bucks during the period from 2012-2016. Two hundred semen and nasal samples randomly collected from 23-35-week-old apparently healthy breeding bucks representing 30 rabbit farms located in different districts at Giza Governorate. The examined rabbit bucks suffered from low reproductive activity. Serological identification was performed by using slide agglutination test (SAT), gel-diffusion precipitin test (GDPT) and enzyme linked immunosorbant assay (ELISA). Molecular identification of the isolated strains was performed using polymerase chain reaction (PCR). The correlation between the presences of P. multocida and reproductive disorders in rabbit bucks was investigated by measuring semen parameters (advanced sperm motility %, live spermatozoa %, Sperm concentration x 106 / ml and sperm abnormalities %) in alternative husbandry systems. The results revealed that isolation of 7 isolates of P. multocida with an incidence of 3.5 %. The in vitro antibiotic sensitivity test revealed that the isolated strains were highly sensitive to thiamphenicole, gentamycin, ciprofloxacin, amoxycillin, amoxycillin + clavulanic acid, clindamycin, colistin sulphate, tetracycline, enrofloxacin, lincomycin and vancomycin and moderately sensitive to neomycin and oxytetracycline. On the other hand isolates were resistant to ampicillin, erythromycin, penicillin G, sulphamethozole-trimethoprim and streptomycin. Experimental infection of 6-8-month-old rabbit bucks with isolated strain (A: 12) of P. multocida from semen followed by treatment trials using thiamphenicole and gentamycin with ciprofloxacin were carried out. Clinical signs, postmortem gross lesions and semen evaluation with re-isolation of P. multocida strains in pure form were discussed in details. Histopathological examination of tissue sections from different organs of experimentally infected bucks revealed less severe lesions after treatment with thiamphenicole and gentamycin with ciprofloxacin, when compared with infected non treated rabbit groups.

SURVEY OF ARIEMERELLA ANATIPESTIFEROUTBREAK IN SOUTHERN TAIWAN DUCK FARMS

A septicemic outbreak in southern Taiwan duck farms in 2014 resulted in high mortality of ducklings. Samples from oral or cloacal sites of affected Muscovy and Pekin ducks were collected and the identity of the field isolates was confirmed using Riemerella anatipestifer (RA) 16S rRNA and outer membrane protein A (OmpA)-specific primers in polymerase chain reactions (PCR), with 15 isolates found positive for both 16S rRNA and OmpA. Detection of both the 16S rRNA and OmpA genes could be a rapid PCR test for RA. Serotyping of the isolates using gel-diffusion precipitin test identified serotypes 1, 4, 6, 17, and 19 while a number of isolates were unidentifiable. Sequence analysis of the OmpA gene found high identity (99.0–99.7%) among isolates in Taiwan. These results indicate that RA remains as a significant cause of duck septicemic disease in southern Taiwan.

An outbreak of chronic fowl cholera in broiler breeder chickens in Korea

Fowl cholera is a contagious acute and chronic disease caused by Pasteurella multocida in both domesticated and wild birds. Acute fowl cholera in both chickens and wild birds has recently been documented in Korea, but the chronic form has not been reported in Korea until now. This study describes the first outbreak of chronic fowl cholera in 13-week-old Arbor Acre broiler breeder chickens submitted to the College of Veterinary Medicine, Kyungpook National University in April 2006. The clinical signs of the affected flock of 9,621 chickens were lameness caused by swollen hock joints, diarrhea, ruffled feathers, and an average weekly mortality of 1.0%. At necropsy, purulent or caseous exudates were found in the hock and wing joints, humerus, and eyes, and severe pneumonia and pericarditis were discovered. Eleven bacterial isolates obtained from the liver, joint, infraorbital sinus and sternal bursa of the submitted chickens were all identified as Pasteurella multocida based on their physiological and biochemical characteristics. Five isolates were examined for antimicrobial susceptibility against 21 different antimicrobial agents including ampicillin. All were resistant to kanamycin, neomycin, and streptomycin, and some were resistant to gentamicin. The tested isolates were all susceptible to the other 17 antimicrobial agents. All 11 isolates were capsular serogroup A based on multiplex polymerase chain reaction. In addition, two of five isolates used in the antimicrobial susceptibility test were identified as somatic serotype 1 by an agar gel diffusion precipitin test, while the others were non-typable.

Evaluation of Pasteurella multocida isolated from rabbits by capsular typing, somatic serotyping, and restriction endonuclease analysis.

The goal of this study was to characterize Pasteurella multocida isolated from rabbits. Five hundred and fifty-three apparently healthy rabbits were sampled for this study. Nasal swabs were collected from each rabbit for P. multocida isolation and identification. Isolates were further characterized by capsular and somatic antigens and genomic DNA fingerprinting. Thirty-nine P. multocida isolates were recovered from 553 rabbits (7%). Capsular typing was done by depolymerization of P. multocida capsule by Staphylococcus aureus hyaluronidase and by disc diffusion with mucopolysaccharidase enzymes (heparinase III, chondroitinase AC, and hyaluronidase). Thirty-one (79%) of the isolates were capsular type A, and 8 isolates (21%) had untypable (UT) capsules. The gel-diffusion precipitin test was used to determine the somatic type of P. multocida isolates. Nineteen isolates were somatic serotype 3 (49%), 12 were serotype 1 (31%), 1 was serotype 2, 2 were serotype 5, 2 were serotype 12 with a weak reaction to antiserum raised against serotype 7 (5%), and 1 was serotype 4. Two of the isolates (5%) were UT. Restriction endonuclease analysis of the DNA of the isolates revealed 7 distinct profiles by digestion with HindIII, and 12 profiles were obtained with HpaII, whereas digestion with EcoRI did not differentiate between any of the P. multocida DNA isolates studied. The DNA restriction endonuclease enzyme HpaII was found more useful for differentiating between DNA fingerprints of P. multocida rabbit isolates. However, no correlation between capsular type, somatic serotypes, and DNA fingerprints was seen in this study.

Invasive aspergillosis in cancer.

Ten confirmed cases of invasive aspergillosis (IA) in cancer patients were analysed retrospectively. Eight were pulmonary, one was sinonasal and one was cutaneous. The majority of patients had haematological malignancies (7), the remaining three were cases of solid tumours. Fever was present in all 10 cases. Cough and lung signs were present in all eight cases of invasive pulmonary aspergillosis. Haemoptysis was encountered in three of nine cases of pulmonary and sinonasal aspergillosis. Mortality was low (2%). While corticosteroids, antibiotics and anticancer chemotherapy/radiotherapy were factors predisposing the patients to IA, neutropenia was perhaps responsible for their mortality. Seven of the patients had other associated pathogens isolated in culture in addition to Aspergillus spp. Aspergillus fumigatus was the predominant species, followed by A. flavus, A. glaucus, A. nidulans and A. niger. Direct microscopic examination (in six of seven cases) and culture (six of seven cases) correlated well with radiographic and clinical findings in cases with lung involvement. Serology for anti-Aspergillus antibodies performed by gel diffusion precipitin test was positive in one case of sinonasal aspergillosis, wherein only one precipitin band was observed. Correlation of clinical symptoms, consistent radiographic findings and microbiological work-up (the latter including a triad of direct microscopy, culture and serology) are required to arrive at a diagnosis of IA, especially where histology cannot form the mainstay of diagnosis.

A potential new serotype of Riemerella anatipestifer isolated from ducks in Thailand

Eighty isolates of Riemerella anatipestifer representing 71 outbreaks of riemerellosis in Thailand between 1994 and 1999 were serotyped using the gel diffusion precipitin test. Based on the precipitation patterns, 25 serological profiles containing one to three antigenic determinants were recognized. Heat-stable antigens of the organism reacted with antisera raised against 16 known serotypes and an untypable strain 698/95. The most prevalent serotype appeared to be serotype 7, followed by serotypes 5, 10, 21 and 1. Further study demonstrated that the untypable strain probably represents a new serotype. Analysis of the polymerase chain reaction-amplified rrs genes for restriction fragment length polymorphisms verified the inclusion of strain 698/95 within the species R. anatipestifer and supported earlier work excluding strain 670/89, which had originally been designated the reference strain of serotype 20. Therefore, it is suggested that the strain 698/95 could be adopted as a replacement for the reference strain of serotype 20. Attention should be paid to strains with multiple antigenic factors as they may be useful for the preparation of vaccines.

Detection of antibodies against virus infection associated (VIA) antigen of foot and mouth disease virus using counterimmuno electrophoresis technique

The antibodies to virus associated (VIA) antigen of the foot and mouth disease (FMD) virus were detected in sera samples of experimental infected calves using counterimmunoelectrophoresis (CIEP) in comparison with Agar Gel Diffusion Precipitin (AGDP) test. The precipitation bands appeared in 60 minutes with CIEP while the results of AGDP read after 7 days. Moreover, the use of CIEP technique proved simple to perform and very economic in the use of reagents.

Physiological characteristics, antimicrobial susceptibility and serotypes of Pasteurella anatipestifer isolated from ducks in Thailand

Twenty isolates of Pasteurella (Moraxella) anatipestifer from ducks with serositis and septicemia in Thailand between 1988 and 1989 were characterized by various tests. Eighteen isolates fermented glucose and maltose, 3 fructose and 1 each mannose, arabinose, trehalose or sorbitol. All isolates produced gelatinase but not urease, while 2, 3, 5 and 6 produced indole, were CAMP positive, and were proteolytic for milk and coagulated serum respectively. Seven enzymes, phosphatase alkaline, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, phosphatase acid and phosphoamidase were detected from all the isolates. The isolates were highly susceptible to ampicillin, erythromycin, penicillin G and tylosin. Gel-diffusion precipitin tests demonstrated that serotype 1 was most prevalent (60%) and serotype 6 followed (5%). Seven isolates (35%) were untypable. These results indicated that P. anatipestifer of serotype 1 played an important role in recent outbreaks of the disease in Thailand.

Studies on the Detection of Antibody to Duck Hepatitis Virus by Enzyme-Linked Immunosorbent Assay

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to duck hepatitis virus (DHV) is described. The results of ELISA were compared with those of an agar gel diffusion precipitin (AGDP) test and a serum-neutralization (SN) test. The specificity of the ELISA was in accordance with the specificity of the AGDP and SN tests, but there was a difference in sensitivity. The positive detection rates of ELISA, SN test, and AGDP test for 93 clinical samples were 68.8%, 68.8%, and 18.8%, respectively. A positive/negative (P/N) value larger than or equal to 2.1 plus an absorbance value larger than or equal to 0.4 was used as a comprehensive positive standard for the ELISA. This eliminated false-positive reactions. The results showed that the ELISA was a rapid, sensitive, and accurate method for detecting antibody to DHV.

Proteins and antigens of Pasteurella multocida serotype 1 from fowl cholera

The protein profiles of Pasteurella multocida serotype 1 isolates and the response of chickens to serotype 1 antigens were investigated using SDS-PAGE. Patterns obtained with Coomassie blue staining of soluble protein extracts were similar. The major difference between isolates was the position of one of the major proteins in the 34–38 kDa region. When chickens were experimentally infected with a clinical isolate of P. multocida serotype 1 various proteins were recognised by immunoblotting, including one with a relative molecular weight of 34 kDa; however, no reactions were observed in the region where LPS is known to migrate. When these infection sera were used in an EIA with purified LPS obtained from Heddleston serotype 1 type strain (X-73) they reacted strongly. Serum used for serotyping isolates in the gel diffusion precipitin test recognised many antigens in common with sera from infected birds, but some antigens were specific to typing sera.

Prevalence and characterization of Pasteurella multocida in rabbits and their environment in Japan.

Prevalence and some properties of Pasteurella multocida in rabbits kept at laboratory animal facilities and commercial rabbitries, and in their environment were investigated. A total of 1,147 nasal swab samples from 1,147 rabbits and 126 samples from their environment were subjected to the isolation of P. multocida. The bacteria were isolated from 199 (29.8%) of 668 rabbits in laboratory animal facilities and from 1 (0.2%) of 479 rabbits in the rabbitries. Isolation rate of P. multocida was low (0.9%) or high (44.9%) in the facilities with or without the monitoring for the presence of the bacteria, respectively. The highest rate of the isolation from rabbits was recorded at 10 to 12 months of their housing time. Thirty-nine cultures (31.0%) of air and the surfaces of floors, tips of water bottles, and cages were positive for P. multocida and isolation rate of the bacteria was high (78.6%) in the air. Biological and biochemical properties of the isolates were identical except for indole production and raffinose fermentation. The isolates were susceptible to antibiotics tested except for clindamycin, serologically similar in the gel-diffusion precipitin test and weakly virulent for mice. The present results suggested that these P. multocida isolates were the causal agent of rabbits rhinitis (snuffles) in Japan.

Serological reactions in sheep and cattle experimentally infected with three Australian isolates of bluetongue virus

Serums from 103 sheep and 24 cattle experimentally infected with one of 3 serotypes of bluetongue virus isolated in Australia were tested for antibody to bluetongue virus in the serum neutralisation test and the agar gel diffusion precipitin test. Antibody to bluetongue virus was first detected by these tests 8 to 10 days after intravenous infection in 4 sheep that were bled daily for serum analysis. The agar gel diffusion test failed to detect antibody in 28% (29/103) of sheep which had seroconverted in the serum neutralisation test. A further 7% (7/103) of sheep serums were negative in both tests 14 to 22 d after infection. Both tests detected antibody to bluetongue virus in all cattle serums by 10 days after detection of viraemia. In comparison with the intravenous route of infection, extended prepatent periods for the commencement of viraemia resulting from intradermal, subcutaneous and intrauterine routes of infection in the cattle caused corresponding delays in the detection of antibody. For example, one cow that was infected by intrauterine inoculation did not become viraemic until 22 d after inoculation and antibody was not detected until 32 d after inoculation.

Comparison of inoculation regimes for the experimental production of swine erysipelas arthritis: II Serological findings in a gel diffusion precipitin test and enzyme‐linked immunosorbent assay

The serological response of pigs to Erysipelothrix rhusiopathiae inoculation was monitored by a gel diffusion precipitin test (GDPT) using a crude, serotype-specific, autoclaved antigen and an enzyme-linked immunosorbent assay (ELISA) using a heat-extracted, alcohol precipitated and molecular seived antigen previously shown to react with serum from pigs infected with serotypes 1 or 2. All pigs receiving 3 or 5 weekly intravenous inoculations of either a highly virulent (VRS 229) or a lowly virulent isolate (VRS 252) produced GDPT-reactive antibody within 3 weeks, but only 44% were still reactive at 8 to 9.5 weeks. The ELISA response was significantly higher in pigs inoculated with the highly virulent strain, and was similar in pigs receiving 3 or 5 doses of either strain. In a dose-response trial, after 3 doses of VRS 229, GDPT reactivity occurred earlier and was stronger in pigs given higher doses of E. rhusiopathiae, but the response peaked 3 to 5 weeks after the start of challenge and was short lived. GDPT reactivity correlated with dose, but not with the severity of arthritis. The ELISA demonstrated specific IgG antibody was present by 2 weeks, and persisted to at least 11 weeks. The ELISA reactivity was significantly higher in pigs with arthritis than in pigs that received low doses and were not arthritic. Within groups of pigs with arthritis a significant, dose dependent, linear ELISA response developed but did not correlate with the presence or degree of arthritis at slaughter. Non-arthritic pigs had similar low ELISA responses to uninoculated controls.

Bluetongue and related viruses in New South Wales: isolations from, and serological tests on samples from sentinel cattle.

Sentinel cattle at a number of localities in northern and central coastal New South Wales were sampled over the summer and autumn seasons of the years 1979, 1980 and 1981. A total of 118 orbiviruses were isolated; 99 were of the Palyam group, 15 were of the epizootic haemorrhagic disease (EHD) of deer group, and 4 of the bluetongue group. The Palyam group viruses were identified by serotype as 68 Bunyip Creek, 23 CSIRO Village, 7 D'Aguilar and one was not typed. The EHD viruses were identified as 13 type 5 and 2 type 6. All 4 bluetongue viruses were type 21. There was also convincing serological evidence that bluetongue type 1 infection occurred in 1980. Antibody to the bluetongue group, as demonstrated in a gel diffusion precipitin test, was often transient. It appeared to be mostly cross-reactive with, and induced by, other orbivirus infections, particularly those of the EHD group. Viruses of the Palyam group also seemed to be implicated in some circumstances. Where infections by viruses of the bluetongue group were demonstrated, the precipitating antibody responses to a bluetongue group antigen were not noticeably stronger than many which followed EHD virus infection. The results generally confirm previous conclusions, deduced from serological surveys, regarding the frequency of orbivirus infections, the presence of bluetongue viruses, and the transient nature of many bluetongue group antibody reactions.

Correlation of positive syphilis serology with invasive amebiasis in Japan.

Approximately 20% of 52 Japanese males with invasive amebiasis confirmed by gel diffusion precipitin test (GDP) were positive by both Treponema pallidum hemagglutination test (TPHA) and nontreponemal antigen tests, and an additional 20% were positive by TPHA alone. However, none of 109 GDP-negative Japanese males without invasive amebiasis were positive on these serologic tests for syphilis. At least 2 bisexuals and 4 homosexuals were among those with invasive amebiasis. All of these biased males were positive on both TPHA and nontreponemal antigen tests. Four of the biased males had liver abscess and 2 had amebic dysentery or colitis. Bisexual or homosexual males were not found in the negative GDP group. These observations suggest that sexually transmitted amebiasis due to Entamoeba histolytica occurs among sexually biased males in Japan.

Immunoreactivity of fractionated antigens obtained from autoclaved extracts of an arthritogenic isolate of Erysipelothrix rhusiopathiae.

The immunoreactive antigens in heat-extracted (autoclaved) preparations of an arthritogenic strain of Erysipelothrix rhusiopathiae (isolate VRS 229, serotype 1a) have been identified by gel diffusion precipitin (GDP) tests and a novel application of the enzyme linked immunosorbent assay (ELISA) procedure. Antigens precipitated by ethanol treatment of autoclaved extracts of this strain were resolved into 4 major peaks (A,B,C and D) after gel permeation chromatography on Sephacryl S200. Peak A was confirmed as a protein peak (Lowry positive) which was excluded from the gel. This peak was identified to be ELISA-reactive when assayed with serum from pigs infected with other isolates corresponding to serotypes 1a, 1b and 2. However, it did not form precipitin lines in GDP tests. Peak B was Lowry-positive and also contained carbohydrates. It was not as reactive in ELISA tests but rapidly formed precipitin lines with serum from pigs infected with the homologous isolate, but only erratically with serums from pigs infected with other serotype 1a and 1b isolates, and not with serotype 2 isolates. Peaks C and D were high in carbohydrate and phosphate content respectively but were both non-reactive in GDP tests and only slightly so by ELISA. Since serotypes 1 and 2 are the most predominant among isolates from infected pigs it is likely that the commonly recognised A antigen is a useful ELISA reagent for the diagnosis of E. rhusiopathiae infection; B antigen on the other hand, would probably be of limited diagnostic value.

Comparison of two gel diffusion precipitin tests in the serodiagnosis of caprine arthritis encephalitis virus infection in goats

Comparison of two gel diffusion precipitin tests in the serodiagnosis of caprine arthritis encephalitis virus infection in goats

A Serotypic Survey of Pasteurella multocida Isolated from Poultry

One hundred forty-eight Pasteurella multocida isolates from four southeastern states and California were serotyped by a gel diffusion precipitin test. The isolates were predominantly from turkeys and chickens. Sixty-eight percent of the isolates had antigenic characteristics of serotypes 3 and 4 (3 X 4). In turkeys, 76% of the isolates were 3 X 4, and serotype 3 was second (17%) in frequency. In chickens, 54% of the isolates were 3 X 4 and 19% were serotype 1.

Serology and semen culture for the diagnosis ofBrucella ovisinfection in chronically infected rams

The serological response to Brucella ovis and the shedding of the organism in semen was followed for a period of 13-14 months in 42 naturally infected rams. Most rams remained chronically infected and excreted the organism in their semen throughout the investigation. B. ovis was isolated from 87.9% of the semen samples from the infected rams. The most common sites from which B. ovis could be isolated at necropsy were the epididymides and accessory sexual glands. In one ram the organism was isolated from lung, spleen, kidney and iliac lymph nodes. Three rams ceased to shed B. ovis in their semen during the course of the investigation. Seventy-five (11%) of 686 sera from infected rams were negative in the complement fixation test (CFT) although 76% and 77% of CFT-negative sera were positive in the gel diffusion precipitin test (GDT) and enzyme labelled immunosorbent assay (ELISA) respectively. The high incidence of CFT-negative infected rams was due to the selection for the investigation of many rams with histories of negative or vacillating CFT titres. Sera from five rams which never shed B. ovis in their semen reacted erratically in the three serological tests. The five rams were from heavily infected flocks and were kept in contact with infected rams throughout the investigation.