Gel Diffusion Tests Research Articles

Structural, optical and antimicrobial properties of pure and Ag-doped ZnO nanostructures

In the present work, zinc oxide (ZnO) and silver (Ag) doped ZnO nanostructures are synthesized using a hydrothermal method. Structural quality of the products is attested using X-ray diffraction, which confirms the hexagonal wurtzite structure of pure ZnO and Ag-doped ZnO nanostructures. XRD further confirms the crystallite orientation along the c-axis, (101) plane. The field emission scanning electron microscope study reveals the change in shape of the synthesized ZnO particles from hexagonal nanoparticles to needle-shaped nanostructures for 3 wt% Ag-doped ZnO. The optical band gaps and lattice strain of nanostructures is increased significantly with the increase of doping concentration of Ag in ZnO nanostructure. The antimicrobial activity of synthesized nanostructures has been evaluated against the gram-positive human pathogenic bacteria, Staphylococcus aureus via an agarose gel diffusion test. The maximum value of zone of inhibition (22 mm) is achieved for 3 wt% Ag-doped ZnO nanostructure and it clearly demonstrates the remarkable antibacterial activity.

Prevalence of selected viral infections in various age groups of village chickens in Borno State, Nigeria

A preliminary survey for antibodies against some selected viruses in village chickens of various age groups was carried out in Borno State, Nigeria. Three hundred and twenty samples consisting of 160 sera from adult chickens, 60 sera from chicks and 100 yolk samples from embryonated en eggs were tested against Newcastle disease (ND), influenza (FLU) A and B viruses, and egg drop syndrome (EDS) - 76 viruses using haemagglutination inhibition (HI) test; and infectious bursal disease (IBD) virus using the agar gel diffusion test (AGDT). Eighty-five percent of the samples tested positive to one or more viruses, with IBD having the highest percentage prevalence (45.7%) followed by FLU A (26.5%), ND (22.4%), EDS-76 (19.3%) and FLU B (0%). The percentage of mixed infectious among different age groups was highest among chicks. The importance of village chickens in the spread and maintenance of viral diseases of poultry are discussed from an epidemiological point of view.

Molecular and serological characterization of Riemerella isolates associated with poultry in Australia

ABSTRACT A total of 62 isolates of Riemerella-like organisms, originally isolated from Australian poultry (10 from chickens, 46 from ducks, five from unknown hosts and one vaccine strain), were included in this study. On the basis of two published polymerase chain reaction (PCR) assays that are reported to be specific for Riemerella anatipestifer, 51 of the isolates were identified as R. anatipestifer. Forty-six of these isolates had a detailed history and were sourced from ducks, while five were of unknown origin. The 11 remaining isolates failed to yield a positive reaction in either PCR with 10 originating from chickens and one from a duck. Amplification and sequencing of the 16S rRNA gene of these isolates identified the duck isolate as Moraxella lacunta. Phylogenetic analysis of the 10 chicken isolates identified one as R. columbina and the remaining nine isolates as Riemerella-like taxon 2. The 51 Australian R. anatipestifer isolates were assigned by gel diffusion test to serovars 1 (26 isolates), 6 (seven isolates), 8 (five isolates), 9 (two isolates), 13 (one isolate) and 14 (one isolate) while nine isolates gave no reaction to any antiserum. A commercial system was used to perform DNA fingerprinting using rep-PCR analysis, which revealed different clusters with a lack of a clear relationship between the clusters and the serovars.

MICROSCOPICAL AND SEROLOGICAL STUDIES WITH ULTRASTRUCTURE DESCRIPTION OF SARCOCYSTIS SPECIES IN SHEEP IN ASSIUT

This study was conducted to determine theinfection rate of Sarcocystis species in sheep in Assiut. A total of 100 slaughtered sheep examined for Sarcocystis, no macroscopic cysts were observed, 93% (93/100) were positive by microscopical examination and 28% (28/100) were positive by using Agar gel diffusion test.Theinfection rate of Sarcocystis had very high significant differences between microscopic examination and Agar gel diffusion test. High statistical significant effect was found on the infection rate of Sarcocystis in different examined muscles of sheep. The highest infection rate was recorded in oesophagus (71%), followed by diaphragm (65%), tongue (58%), skeletal muscles (53%) and heart muscles (43%). The infection rate of Sarcocystis in males was 95.2% (40/42) while in females were 91.4% (53/58) by microscopical examination. Higher infection rate of Sarcocystis were detected in sheep 6 months - 2 years age 95% (38/40) than that of sheep between 2-4 years old 92.7% (38/41) and those equal or older than 4 years 89.5% (17/19). No significant difference between infection and age groups or sex of animals was observed. The use of transmission electron microscope (TEM) allowed the identification of S. tenella and S. arieticanis in sheep in Assiut. Examination of S. tenella cyst wall with TEM showed palisade-like villar protrusions while, examination of S. arieticanis cyst wall showed hair-like villar protrusions parallel to cyst wall.

Partial biological and molecular characterization of a Cucumber mosaic virus isolate naturally infecting Cucumis melo in Iran.

Melon seedlings showing systemic chlorotic spots and mosaic symptoms were collected in central part of Iran, and a virus was isolated from diseased plants by mechanical inoculation. The virus systemically infected the most inoculated test plants by inducing mosaic symptoms, while, in the members of Fabaceae family and Chenopodium quinoa induced local lesions. Agar gel diffusion test using a polyclonal antiserum against a squash Cucumber mosaic virus (CMV) isolate showed the presence of CMV in the mechanically inoculated plants (designated CMV-Me). The virus was purified by polyethylene glycol precipitation and differential centrifugation. A polyclonal antiserum was produced against the virus that reacted specifically with virus antigen in ELISA and agar gel diffusion tests. The virus was molecularly characterized by PCR amplification of the full length of the coat protein gene using cucumovirus genus specific primer pair CPTALL-3/CPTALL-5 and sequence analysis of the resulting product. No RNA satellite was detected using the primer pair CMVsat3H/sat5T7P. Phylogenetic analysis based on the coat protein amino acid sequences showed that CMV-Me belongs to Subgroup IB. These results may be helpful in melon breeding programs, focusing on plant resistance to plant viruses including CMV.

Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test.

We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2.

Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin.

Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals.

Longevity and Infectivity of Trypanosoma evansi Isolated from the Gut of the Stable Fly Stomoxys calcitrans (Diptera: Muscidae)

Stable fly Stomoxys calcitrans is a blood-sucking fly prevalent in tropical and subtropical countries. The objective of this study was to determine the longevity and infectivity of Trypanosoma evansi that isolated at different intervals from the gut of experimentally infected stable fly with T .evansi . Two generations of flies were used aged 24 and 48 hours (hr). The flies were fed on blood of mice infected with T. evansi through the chicken’s skin placed on glass container in a water bath at 37 °C. Ten fed flies were dissected under the stereo-microscope of intervals; immediately, 1, 12, 24, 26, 27, 28, 29 and 30 hr post-feeding (pf). Smears were prepared from the gut’s isolated trypanosomes and simultaneously inoculated intraperitoneally into four mice for each interval. Three serological tests were employed to detect the antigenic variations of the T. evansi ; agglutination, indirect fluorescent antibody (IFA) and gel diffusion. All mice were infected with T. evansi for all intervals except 27, 28 and 29 hr pf. Infectivity was decreased with time of feeding. Binary fission was observed at 12 hr pf and the parasites disintegrated at 30 hr pf. IFA test showed cross-reaction of various isolates of T. evansi in comparison with agglutination and gel diffusion tests.

A Study of Sexual Assaults in Northern Range of Himachal Pradesh

Background: Rape is among the highest forms of underreported crime experienced by women in all sections of the society and obtaining an accurate measurement of sexual assault is a challenge. An increasing rate of sexual assaults constitutes a large number of cases registered under different sections of the Indian law, their medico-legal examination and forensic investigations.Methods: The study was conducted on 35 sexual assaults cases received for examination at RFSL, Northern Range, Dharamshala, Himachal Pradesh, India. A self-innovated proforma including the details pertaining to socio-demographic profile, medical examination, and results of specimens of victims collected during forensic analysis was designed. Benzidine test, gel-diffusion test, acid phosphatase test and microscopic examinations were conducted in the lab.Results: The common location of offence was house of the accused (31.42%) followed by house of the victim (20.0%). 48.57% of the alleged rape victims were students. Highly affected age group was 11-20 years. Fifteen girls (42.85%) were below eighteen years of age, which was the age of invalid consensual rape. Forcible rape and invalid consensual rape was reported in 68.56% cases. 77.14% victims were unmarried and 22.85% were married. 85.71% victims were Hindus followed by Muslims (11.42%) and Christian (2.85%). 80% victims knew the assailant. Rupture of hymen was found in 88.57% cases and hymen was intact in 11.42% cases. Delay of medico-legal examination of victim was noted. 31.42% victims had combined genital and extra genital injuries. Acid phosphatase test was found positive in 34.28% cases and positivity for spermatozoa in 22.85% cases. Blood was detected on clothes, undergarments and vaginal swabs in 42.85% cases. In 8.55% cases rape was committed by family members. Only one victim became pregnant following the act of sexual intercourse.Conclusion: Cases were reported from Hindu, Muslim and Christian communities and adolescents were more at risk. Majority of the victims have acquaintance with the assailant and the residence of the accused was the most common location of sexual assault. Delay in medico-legal examination caused problems in detection of semen and spermatozoa. Most of the victims had bath and genital wash after the sexual act, before medical examination and destroyed the evidence.

Microplate Agglutination Test for Canine Brucellosis Using Recombinant Antigen-Coated Beads

Brucella canis, a facultative intracellular pathogen, is the causative agent of canine brucellosis. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods, including agglutination and gel diffusion tests. In this study, four recombinant antigens, heat shock protein 60, rhizopine-binding protein, Cu-Zn superoxide dismutase, and hypothetical protein (Ag 4), were constructed. These antigens were coated on latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. All recombinant antigens showed specific reaction with sera from B. canis-infected dogs in Western blotting. In a microplate agglutination test, mixing sera from B. canis-infected dogs, but not sera from B. canis-free dogs, with single or multiple antigens-coated latex beads produced clear agglutination. Moreover, the antigen-coated latex beads did not show nonspecific agglutination in hemolyzed serum samples. A survey of canine serum samples conducted by the microplate agglutination test using single antigen-coated latex beads showed that this method would be useful in the serological diagnosis of canine brucellosis. Further investigations using more serum samples are required to confirm the usefulness of our method.

PARASITOLOGICAL AND SEROLOGICAL STUDY ON FASCIOLA DIAGNOSIS IN CATTLE AND BUFFALOES IN ASSIUT GOVERNORATE

Received at: 30/3/2014 Accepted: 16/4/2014 The present study was carried out to investigate the prevalence of fascioliasis among 100 cattle and buffaloes of different age and sex in Assiut Governorate. Samples were examined by microscopic examination, Agar Gel Diffusion Test AGDT and ELISA techniques to detect Fasciola species eggs and antibodies, respectively. Prevalence of Fasciola was 8% by microscopic examination, 32% by AGDT and 60.86% by ELISA test. Cattle showed higher prevalence of infection 61.4% than buffaloes 29.4%. Female cattle and buffaloes were more susceptible to the infection 64.55% than male 23.8%. Fasciola was more frequently recorded in 2-4 years old cattle and buffaloes 76% followed by more than 4 years 46.7% then less than 2 years of age 20%. All positive results with fecal egg examination and AGDT were also positive with ELISA. Serological examination by AGDT and ELISA confirms the microscopical examination. The results of this study revealed that the agar gel diffusion test and ELISA could become a useful tools to diagnose fascioliasis in cattle and buffaloes.

Seroconversion and semen shedding in rams experimentally infected with Brucella ovis

AIM: To determine the time taken for rams to develop antibodies to Brucella ovis in serum, shed B. ovis in semen and develop lesions of epididymitis following infection with B. ovis.METHODS: Fifteen 19-month-old rams were artificially infected with B. ovis by inoculation of infected semen onto the nasal and rectal mucus membranes (Day 0). Serum was collected from each ram at 2 to 8-day intervals and tested at commercial laboratories using a complement fixation test (CFT) and an ELISA. Cut-off values for the CFT were 0–4/4 negative; 1/8–3/8 suspicious and 4/8–4/128 positive, and for the ELISA were <10% negative; ≥10 to <50% suspicious and ≥50% positive. Selected serum samples were also tested using a gel diffusion test (GDT). At 7 to 8-day intervals semen was collected for bacterial culture and the scrotal contents were palpated to identify lesions of epididymitis. The study was terminated after 56 days.RESULTS: On Day 28 B. ovis was isolated from the semen of one ram and by Day 49 it was isolated from the semen of 10 rams. All 10 rams had suspicious or positive ELISA or CFT titres by Day 36 and 56, respectively. The GDT results were all negative on Day 36 and in general did not become positive in individual rams until 7–28 days after semen shedding commenced. Epididymitis was detected in one ram on Day 36; by Day 56 eight rams had epididymitis detectable by scrotal palpation.CONCLUSIONS: The B. ovis ELISA test identified infected rams at an earlier stage than the CFT; this was at 19–36 days after exposure. Rams can begin shedding B. ovis in semen as early as 28 days after exposure and lesions of epididymitis develop as early as 36 days after exposure.CLINICAL RELEVANCE: During a test and slaughter campaign for the control of B. ovis, the most appropriate serological re-testing interval is likely to be around 28 days (4 weeks) using the ELISA with or without the CFT, although caution is required in interpretation of “suspicious” ELISA results. Following a B. ovis breakdown, two negative CFT or ELISA tests 60 days apart are recommended to confirm freedom from infection, supporting current guidelines.

Purification and Structural Characterization of a Type II Arabinogalactan-Protein from Champagne Wine

Some arabinogalactan-proteins (AGPs) isolated from red wine have interesting physicochemical and sensory properties. An AGP was isolated from a Champagne wine by anion-exchange, affinity, and size exclusion chromatography. Glycosyl residue analysis showed that it included arabinose (12.8%), galactose (68.5%), glucuronic acid (5.9%), and trace rhamnose (0.7%). Champagne AGP reacted with Yariv reagent in gel diffusion tests. The protein part of the AGP primarily contained hydroxyproline (22% of total amino acids), serine (17.6%), alanine (10.8%), glutamic acid (10.3%), and threonine (8.6%). Methylation analysis revealed that the carbohydrate moiety of t he glycoprotein had a h ighly branched structure. T he core consisted of 3-linked �A -d-galactopyranose units carrying side chains of 6-linked �A-d-galactopyranose, partly substituted in position 3 and 4 by l-arabinofuranosyl residues. Galactose and arabinose as well as rhamnose and glucuronic acid occurred in terminal positions. Size exclusion chromatography with multiangle laser light scattering of the Champagne AGP revealed apparent average molecular weight mass of 1.1 �~ 105 g/mol, characterized by a narrow polydispersity index (Mw/Mn 1.09) and a very low intrinsic viscosity ([�A] = 0.1 dL/g).

Haemophilus parasuisSerovars Isolated from Pathological Samples in Northern Italy

From January 2007 to December 2011, a total of 106 Haemophilus parasuis strains isolated from pigs were serotyped by agar gel diffusion test (DG). Serovar 4 was the most prevalent (24.5%), followed by serovar 13 (19.8%) and serovar 5 (11.3%). Twenty-nine strains were non-typeable (27.3%). The strains were divided into two groups, depending on whether they were isolated from specific pathological lesions of systemic disease such as polyserositis, arthritis or meningitis (73 cases of 106) or from the lower respiratory tract of pigs suffering from bronchopneumonia (33 cases of 106). Serovars 4 and 13 had a higher prevalence in systemic infection (polyserositis) than in respiratory disease only. Pasteurella multocida (14/106), Streptococcus suis (7/106), Actinobacillus pleuropneumoniae (4/106), Bordetella bronchiseptica (3/106) and Arcanobacterium pyogenes (3/106) were isolated in association with H. parasuis.

Replication and attenuation of foot and mouth disease virus in chick embryo

This study includes replication and attenuation of foot and mouth disease virus type O which isolated from infected calves. Many passages for the virus in chick-Embryo were established as a substitute method to the tissue culture which is highly caustic in contrast to the chick embryo. The virus passed ten consequent passages which lead to the reduce of the titer of the virus from 106.53 TCID50/ 0.1 ml in cattle testis tissue culture to 103 TCID50/ 0.1 ml. the pathogenecity of attenuated FMD virus were also studied in both chick-embryo and guinea pigs. Using agar gel diffusion test precipitation antibodies was detected in guinea pig serum after 14 and 21 days post exposure to the attenuated virus. The inoculated guinea pig group with the chick-embryo attenuated virus appear resistance to the challenge virus. The result suggested the efficacy of attenuation of foot and mouth disease virus by using chick-embryo system for immunization against this disease on the level of laboratory Animal.

Replication and attenuation of foot and mouth disease virus in chick embryo

This study includes replication and attenuation of foot and mouth disease virus type O which isolated from infected calves. Many passages for the virus in chick-Embryo were established as a substitute method to the tissue culture which is highly caustic in contrast to the chick embryo. The virus passed ten consequent passages which lead to the reduce of the titer of the virus from 106.53 TCID50/ 0.1 ml in cattle testis tissue culture to 103 TCID50/ 0.1 ml. the pathogenecity of attenuated FMD virus were also studied in both chick-embryo and guinea pigs. Using agar gel diffusion test precipitation antibodies was detected in guinea pig serum after 14 and 21 days post exposure to the attenuated virus. The inoculated guinea pig group with the chick-embryo attenuated virus appear resistance to the challenge virus. The result suggested the efficacy of attenuation of foot and mouth disease virus by using chick-embryo system for immunization against this disease on the level of laboratory Animal.

An outbreak of peste des petits ruminants (PPR) in camels in the Sudan

In mid-August 2004, an outbreak of a previously unknown fatal disease of camels was reported to Kassala State veterinary authorities. Several areas in the state were visited during August–October 2004 to collect epidemiological data and specimens for diagnosis. Clinically the disease was characterized by sudden death of apparently healthy animals and yellowish and later bloody diarrhea and abortion. The disease outbreaks coincided with the seasonal movement of animals towards autumn green pasture. Death was always sudden and proceeded with colic and difficulty in respiration. Mortality rate ranged between 0% and 50% and vary in accordance with the area with a mean of 7.4%. More than 80% of deaths were in pregnant and recently-delivered she-camels. All age, sex and breed groups were affected but more than 50% of deaths were reported in adult animals in comparison to calves and young camels. The main post-mortem findings include lung congestion and consolidation, paleness and fragility of liver, enlarged lymph nodes and congestion and hemorrhage of small intestine and stomach. Agar gel diffusion test (AGDT), RT-PCR and virus isolation in cell culture gave positive results for peste des petits ruminants virus (PPRV), a virus belonging to the Morbillivirus, Genus, member of the family Paramyxoviridae. The effect of this new devastating disease on camel production in the affected area was discussed as well as proposals for future research.

Purification of anatid herpesvirus 1 particles by tangential-flow ultrafiltration and sucrose gradient ultracentrifugation

Anatid herpesvirus 1 (AHV-1) infection causes substantial economic losses to the world-wide waterfowl production. However, little is known about the efficient method used to study the purification of AHV-1 and the negative staining morphology of the purified virus particles. This lack of knowledge is one of the important factors that have affected the progress of research studies on AHV-1 molecular virology to such an extent that they are lagging far behind those on other members of the same family Herpesviridae. Therefore, an efficient method for purifying AHV-1 from cell-culture medium has been developed. Abundant AHV-1 particles, whose morphological features match those of herpesvirus, were obtained by using the following procedures: (1) conventional differential centrifugation for removal of debris after cell disruption, (2) tangential-flow ultrafiltration coupled with sucrose density gradient ultracentrifugation for isolation of the virus, and (3) conventional differential ultracentrifugation for virus concentration. The purified AHV-1 particles were subjected to transmission electron microscopy (TEM), infectivity and recovery tests, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting assay, and agar gel diffusion test (AGDT). The results of examinations revealed that purified AHV-1 particles were free of visible contamination or degradation. The purified AHV-1 particles were biologically active and were successful in initiating infection upon inoculation into susceptible duck embryo fibroblast. The procedures are reliable technically and feasible for purification of large volumes of viruses.

Expression analysis of the molluscan p53 protein family mRNA in mussels ( Mytilus spp.) exposed to organic contaminants

In this study, we report the tissue expression analysis of the p53 protein family mRNA in mussels ( Mytilus spp.) by means of quantitative RT-PCR. The tissue specific response was evaluated after 24 h exposure to a sublethal benzo[a]pyrene (B[a]P) concentration (75 nM), showing a 2.6 fold induction in digestive gland cells and a dramatic gene down regulation in circulating hemocytes. The comet assay and DNA gel diffusion tests showed significant effects in hemocytes and negligible differences in the digestive gland nuclei, implicating p53 in DNA damage of molluscan hemocytes. Finally, the kinetics of p53 protein family mRNA expression in the digestive gland of animals exposed to B[a]P and crude oil (0.5 ppm) showed partially overlapping trends, characterised by a common down regulation after 1 week exposure. These data should be carefully considered in view of the biological effects of organic pollutants and particularly following spills.

토끼풀에서 분리한 Peanut stunt virus의 성질

An isolate of Peanut stunt virus (PSV), named as Tr-PSV, was isolated from white clover (Trifolium repens L) showing mosaic symptom. Tr-PSV systemically infected all plants tested in the Nicotiana spp. and induced local lesions on inoculated leaves of Chenopodium amaranticolor. However, Tr-PSV induced typical mosaic symptoms as ER-PSV on Vigna unguiculata 5 to 6 days after inoculation, while Fny-CMV used as a control virus of Cucumovirus produced local lesions on inoculated leaves. In dsRNA analysis, Tr-PSV consisted of four dsRNAs, but satellite RNA was not detected. The cDNA of coat protein gene of Tr-PSV was amplified by RT-PCR using a Cucumovirus-specific single pair primers that designed to amplify a DNA fragment of approximately 950 bp. By restriction mapping analysis using RFLP of the RT-PCR products and by serological properties of gel diffusion test, Tr-PSV belongs to a typical member of PSV subgroup I. This is the first report on the occurrence of PSV in white clover in Korea.