Countercurrent Immunoelectrophoresis Research Articles

Investigation of Seroprevalence of Hydatidosis in High-risk Individuals in Sistan and Baluchestan Province, Southeast of Iran

Background: Hydatidosis is known as one of the most prevalent zoonotic diseases across the world. This complication is also endemic in Iran, followed by a higher risk of infection in rural areas. To our knowledge, there has been no study on the seroprevalence of hydatidosis in Sistan and Baluchistan Province, Southeast of Iran. The main objective of the current study was to examine the seroprevalence of hydatidosis and its risk factors in high-risk individuals (farmers and ranchers) living in Sistan and Baluchistan Province. Materials and Methods: This study included 500 serum samples, and the participants were requested to complete a researcher-made questionnaire. Subsequently, counter-current immunoelectrophoresis (CCIEP) and enzyme-linked immunosorbent assay (ELISA) methods were employed to analyze the anti-Echinococcus granulosus antibody. The analysis of the obtained data was conducted by logistic regression in SPSS software, version 22. Results: According to the results, four (0.8%) cases were found positive for anti-E. granulosus antibody by both CCIEP and ELISA tests. Seroprevalence of hydatidosis was more in rural people, compared to those in urban areas. It was also higher in illiterate people than in educated people. Nevertheless, seropositivity showed no significant differences with age, gender, occupational status, education level, place of residence, and contact with dogs (P>0.05). Conclusion: The prevalence rate of hydatidosis in Sistan and Baluchistan Province was similar to that in neighboring provinces. According to the findings, high-risk individuals offer remarkable information about the epidemiology of hydatidosis in Sistan and Baluchistan province in southeastern Iran. This could help to manage and prevent this infection.

Genetic Sequence of Drug-Resistant Haemophilus influenza Isolated from Respiratory Tract Infections

This work was done during the period from 1st May 2019 to 15th January 2020. By using the concentration of the inhibitor that was carried out for the Haemophilus influenza bacteria, isolates were identified that 50 samples of bacterial colonies of these bacteria, it was found that all isolates were resistant to ampicillin, cefuroxime, pipracilline, cefotoxone, erythromycin, and azithromycin. The counter-current immunoelectrophoresis (CCIE) test and the latex agglutination method have been employed for this purpose. These tests have aided in establishing a rapid diagnosis of H. influenzae type b infections in some cases, the positive isolates were identified for those infected with the bacteria were CCIE 7/10, 3/3, 0/5, 0/1, and 0/4, while the co-agglutination was 6/10, 3/3, 0/5, 0/1, and 0/4. The standard phenol/ chloroform extraction was used to purify DNA from the sputum’s pellet, followed by precipitation of ethanol. Each reaction set involved sterile water in place of DNA as a negative control. There is a change in the genetic sequence of drug-resistant isolates, CTT to CTA and TTT to TAT.

EFFICACY OF IMMUNOLOGICAL PROTOCOL TO APPRAISE THE DIAGNOSIS OF AMPHISTOME INFECTION IN LIVESTOCK

Parasites are a major cause of disease in domestic ruminants. More than half the livestock population live in misery and suffer vast economic losses due to parasites. Among the parasitic diseases, amphistomosis is one of the major parasitic diseases causing heavy economic losses to livestock industry. Livestock production can be increased by the reduction of losses due to disease and development of new immunological approaches. Immunodiagnosis of parasitic diseases requires highly sensitive and specific tests. In many cases the identification of helminth parasites concerns their epidemiology and it is important to distinguish between species and subspecies. Conventional techniques including serology and microscopy do not always meet these requirements. However, microscopy still remain the mainstay of several diagnostic laboratories. Development of diagnostic tools have opened new avenues for a vast improvement in parasitic disease detection. Anumber of new serology-based assays that are highly specific and sensitive have emerged, such as agar gel precipitation, agglutination, immunodiffusion (ID), counter current immunoelectrophoresis (CCIEP), FAST-ELISA, Dot-ELISA, Sandwich-ELISA, Plate-ELISA, SDS-PAGE, Western blotting, immunoblotting etc. These approaches have revealed potentiality in parasitic disease diagnosis such as amphistome infection with increased specificity and sensitivity. The use of immunodiagnostic approaches in the identification of paramphistome infection in livestock is discussed.

Early migration pattern of Avibacterium paragallinarum in the nasal passage of experimentally infected chicken and Japanese quail by immunohistochemistry

ABSTRACTInfectious coryza (IC) is often a curse for poultry farmers when it occurs concurrently with several pathogens causing swollen head syndrome. The disease is caused by Avibacterium paragallinarum, which inflicts initial damage to the nasal and respiratory epithelium. This facilitates the progression of disease pathology across the nasal cavity, thereby providing a platform for multiplication of opportunistic microbes. In this study, we attempted to investigate the early entrance and migration pattern of A. paragallinarum in chicken and Japanese quail following experimental infection, by employing an in-house developed polyclonal antiserum against this pathogen. Antigenic-specificity of the raised antiserum was subsequently evaluated through immune-dot blot techniques and counter-current immunoelectrophoresis (CIE). The resultant antiserum characterized the antigen localization within formalin-fixed and partially decalcified nasal tissue sections though immunohistochemistry (IHC). Japanese quail showed prominent localization of the bacterial antigen at 12 h post-infection in anterior turbinates. However, the chicken exhibited a higher level of the bacterial pathogen with intense immuno-reactivity at 24 and 48 h post-inoculation. The decline in immunostaining intensity in the nasal tissue of chicken as well as Japanese quail by 72 h post-infection signifies either an attempt to resolve the infection by the resident immune cells across the nasal passage of the host, or its dissipation by certain inherent innate immune factors present across the nasal passage that are still unknown to us. In the present study, we used a moderately virulent pathogen (A. paragallinarum) that inflicted a mild to moderate degree of damage to histo-architecture of the nasal passage and provided a discernible migratory pattern with fewer alterations, along with provision toward unravelling basics of the immuno-pathogenetic mechanism. This knowledge will support efforts towards the development of a future mucosal nasal vaccine in birds affected with IC.

Practical Aspects of Introduction of Multiplex Bead Technology into the Routine Diagnostic Immunology Laboratory.

Multiplex bead assays, also known as addressable laser bead immunoassays (ALBIA) or Luminex® technology, have provided an alternative to enzyme-linked immunoassay, which is still the most widely utilized routine immunoassay for detection of specific autoantibodies. Our laboratory adopted the ALBIA technology early into its routine service. We report the performance and utility of measurement of three different autoantibody types tested using the FIDIS (BMD, Marne La Vallee, France) ALBIA system. The analytes discussed are thyroid antibodies (thyroglobulin [TG], thyroid peroxidase [TPO]), anti-neutrophil cytoplasmic antibodies (ANCA), and ribonucleo-protein (RNP) antibodies. In single antibody analysis, TPO antibody testing was superior to TG antibody in identifying patients with Graves' disease and Hashimoto's thyroiditis. However, testing only TPO antibody would result in missing 8.6% of Graves' and 11.9% of Hashimoto's thyroiditis patients, hence demonstrating an advantage for the multiplex TG plus TPO assay. With respect to ANCA, the FIDIS ALBIA produced an overall similar level of performance to our comparator method, the Phadia fluorescent enzyme linked immunoassay. Sensitivity of the ALBIA for RNP antibodies was low in comparison to countercurrent immunoelectrophoresis, but performance was improved by altering the cutoff value for the assay. ALBIA technology has many potential advantages in the routine laboratory, but as with any new assay, evaluation must be thorough and ongoing to ensure satisfactory clinical performance is obtained. Both false positive and false negative results have been reported in ALBIA studies. It may be necessary to re-evaluate assay performance and cutoff and consider further clinical correlation.

Standardization of dot-enzyme-linked immmunosorbent assay for the diagnosis of bovine visceral schistosomiasis.

Aim:Bovine visceral schistosomiasis has been reported as an important disease entity as it affects animal health, productivity, causes economic losses due to liver condemnation, and produces a high morbidity. This study was conducted to standardize an easy, reliable dot-enzyme-linked immmunosorbent assay (ELISA) for the diagnosis of visceral schistosomiasis caused by Schistosoma spindale and to know the prevalence rate in and around Hyderabad.Materials and Methods:A dot-ELISA was standardized in the laboratory using whole worm antigen (WWA) and excretory-secretory antigen (ESA) of S. spindale. The standardized test was used for the diagnosis of bovine visceral schistosomiasis at field level. The sensitivity and specificity of the test was compared with counter current immunoelectrophoresis. In total, 288 sera (125 cattle and 163 buffalo) were screened by dot-ELISA.Results:The dot-ELISA detected 32.63% of infection (94/288) using WWA and 40.62% of infection (117/288) using ESA. In cattle, the prevalence rate was 32.80% (41/125) using WWA and 40.80% (51/125) of infection. Similarly, in buffaloes, the prevalence rate was 32.51% (53/163) using WWA and 40.49% (66/163) of infection using ESA. The overall sensitivity of dot-ELISA was 76.74% and 80.48% with WWA and ESA, respectively, and specificity was 73.3% and 78.57% in WWA and ESA, respectively.Conclusion:As ante-mortem diagnosis of visceral schistosomiasis is difficult in subclinical conditions, dot-ELISA can be used as a reliable immunodiagnostic test for diagnosis at field level.

Identification and Titration of Hydro Pericardium Syndrome Virus (HPSV) by using Modified Counter-Current Immuno-Electrophoresis (MCCIE)

A rapid, simple, and accurate Modified Counter Current Immuno-electrophoresis (MCCIE) technique was developed and compared with the Reverse Passive Heamagglutination Assay (RPHA) for the titration of HPS virus. The MCCIE test had 100% correlation with the conventional RPHA and correctly titrated 116 samples which were already titrated with RPHA. Instead of horizontal electrophoresis chamber used in counter current Immuno-electrophoresis test, a modification in Counter Current Immuno-Electrophoresis (CCIE) test was made by using 1% melted agarose gel filled in 8 cm long and 3 mm narrow U-shaped glass tubing for the detection as well as quantification & titration of HPS virus. Results of Liver samples of 116 poultry birds including 16 broilers (6 livers were from clinically positive), 50 Desi and 50 commercial layers (adult) obtained through modified counter current Immuno-electrophoresis (MCCIE) were found similar to those found though CCIE and titres obtained through MCCIE were found similar to RPHA tests. The titres in MCCIE test were expressed as appearance of hazy colored precipitation band in the twelve U-shaped tubes. All of the six clinically positive liver samples gave positive results with titres ranging from 1:32 to 1:128. Of these 6 positive, 3 samples had 1:32, 2 samples had 1:64 and 1 sample had 1:128 titres. While none of desi and commercial layer was found to be positive. The results indicated that MCCIE is simple and inexpensive test as compared to CCIE & RPHA to detect and titrate HPS virus in the infected poultry liver samples. All the HPS virus isolates from HPS infected broiler livers and adenovirus group-I obtained from NARC, Islamabad and VRI, Lahore showed MCCIE & RPHA titre (1:64) with specific HPS antibodies.

Entomological determinants of malaria transmission in an epidemic prone area of District Nuh (Haryana state), India.

Entomological investigations were carried out in highly malarious villages under Ujina PHC of District Nuh (Haryana state) which is an epidemic prone area in northwestern region of India. The study was aimed to have an in-depth understanding of the entomological parameters influencing malaria transmission in the study area. The seasonal prevalence and biological attributes of vector mosquitoes were investigated during 2015 and 2016. Indoor resting vector mosquitoes were collected from human dwellings/cattle sheds and morphologically identified. Anopheles culicifacies were categorized to sibling species by species-specific inversions in polytene chromosomes and An. stephensi to ecological races on the basis of ridge number on egg float. The blood meal source analysis and incrimination studies of vectors were done by counter-current immunoelectrophoresis and enzyme-linked immunosorbent assay, respectively. Insecticide susceptibility test on vectors was performed as per WHO guidelines. Seasonal abundance of An. culicifacies and An. stephensi in the study area showed variation; the peak densities of both the vectors were observed during monsoon months which correlated well with the average monthly rainfall data. Though both vectors were found to be primarily zoophagic, the human blood index of An. culicifacies (HBI = 0.17) was significantly higher than that of An. stephensi (HBI= 0.02). Analysis of sibling species composition of An. culicifacies population showed that it comprised almost of sibling species A (>98%) which is an established malaria vector. Anopheles culicifacies was incriminated for Plasmodium vivax and P. falciparum circumsporozoite (CS) antigen during monsoon months in 2015 and 2016. Assessment of insecticide susceptibility status of malaria vectors against 0.5% deltamethrin revealed that An. culicifacies is more susceptible (95% mortality) than An. stephensi (85% mortality). The results suggest that An. culicifacies (species A) is playing a major role in malaria transmission in the study area and is almost susceptible to deltamethrin. Timely two rounds of indoor residual spray of synthetic pyrethroid with proper dosage and good coverage would be helpful in reducing vector population and consequently the malaria incidence. In addition, personal protection measures by the community would supplement the major intervention tool (IRS) in decreasing the man-vector contact.

Adaptation of Capripox Virus Isolate from Goats in Heterologous Cells

An outbreak of goatpox was attended in district Durg and surrounding regions of Chhattisgarh state. Dried skin scabs were collected from 250 goats of different age groups showing clinical signs suggestive of pox. The prevalence rate was studied based on severity of clinical signs followed by confirmation with agar gel immuno diffusion (AGID) test. Positive scabs were further processed for virus isolation in embryonated chicken eggs via dropped chorio allantoic membrane (CAM) route followed by inoculation of CAM material in chinese hamster ovary and chicken embryo fibroblast cell cultures and propagated up to fifth passage level. Goatpox was reported with a prevalence rate of 74% using AGID. Distribution of disease in young animals, particularly in the kids (below 6 month) was more (45.4%) as compared to those between 6 to 24 months (31.35%) and 24 months and above (23.24%). Capripox virus showed cytopathic effect in chinese hamster ovary cells only after third passage and no cytopathic effect in chicken embryo fibroblast cells even up to fifth passage. Cytopathic effects appeared after 72 hrs in chinese hamster ovary cell and were characterized initially by rounding and clumping of cells, cytoplasmic vacuolation followed by cell detachment. Identity of capripox virus was confirmed positive in both the cultures from third to fifth passage by both AGID and countercurrent immuno electrophoresis (CIE) test. In conclusion, field isolate of capripox virus was successfully adapted and propagated on chinese hamster ovary cell following third passage.

Validation of an automated ELISA system for detection of antibodies to Aleutian mink disease virus using blood samples collected in filter paper strips.

BackgroundAleutian mink disease virus (AMDV) is the cause of a chronic immune complex disease, Aleutian disease (AD), which is common in mink-producing countries. In 2005, implementation of an AMDV eradication programme in Finland created a need for an automated high-throughput assay. The aim of this study was to validate an AMDV-VP2 -recombinant antigen ELISA, which we developed earlier, in an automated assay format for the detection of anti-AMDV antibodies in mink blood and to determine the accuracy of this test compared with the reference standard (counter-current immunoelectrophoresis, CIEP).MethodsA blood sampling method based on filter paper 12-strips (blood combs) and a device to introduce these strips to an ELISA plate for elution of the samples were developed. Blood and serum samples were collected from 761 mink from two farms with low (2%) and high (81%) seroprevalences of AMDV infection in 2008. ELISA sensitivity and specificity were estimated with a Bayesian 2-test 2-population model that allowed for conditional dependence between CIEP and ELISA. Agreement between the two tests was assessed with kappa statistic and proportion agreement.ResultsThe sensitivity and specificity of the automated ELISA system were estimated to be 96.2% and 98.4%, respectively. Agreement between CIEP and ELISA was high, with a kappa value of 0.976 and overall proportion agreement of 98.8%.ConclusionsThe automated ELISA system combined with blood comb sampling is an accurate test format for the detection of anti-AMDV antibodies in mink blood and offers several advantages, including improved blood sampling and data handling, fast sample throughput time, and reductions in costs and labour inputs.

Study of complement activation, C3 and interleukin-6 levels in burn patients and their role as prognostic markers

Purpose: The management of burn patients is always challenging for the clinician due to high risk of bacterial sepsis, multi-organ failure and death. Our objective was to study complement activation, C3 and interleukin-6 (IL-6) levels in burn patients and evaluate their role as prognostic markers. Materials and Methods: A total of 63 burn patients and 60 healthy controls were included in this study. Blood was collected from patients within 24 h and at 7th day of injury. Complement activation was determined by crossed electrophoresis and counter-current immunoelectrophoresis. C3 levels were measured using a single radial immunodiffusion. IL-6 was detected by ELISA. Results: All patients showed initial complement activation. Mean C3 levels showed an inverse correlation with the severity of burn. Patients with ≥20% burns had lower C3 than the controls (P < 0.001) and those with <20% burns (P < 0.001). Patients with ≥40% burns had activated complement and low C3 in 2nd week; they subsequently developed infection. Complement was inactive and C3 levels recovered in patients with <40% burns. The non-survivors showed significantly lower C3 than the survivors (P < 0.05) in 2nd samples. Patients who developed infection had C3 significantly lower than those who remained free of infection (P < 0.05). All patients showed initial elevation in IL-6 levels. Patients with ≥60% burns had significantly higher IL-6 than controls (P < 0.001) and those with <60% burns (P < 0.001). Non-survivors had higher IL-6 than survivors in both samples (P < 0.001). Patients who developed infection showed significantly higher IL-6 in 2nd samples than those without infection (P < 0.001). Conclusions: Complement activation, C3 and IL-6 levels correlated well with the severity of injury and development of infection in burn patients. These parameters can be used to predict the onset of infection, septicaemia and mortality in burn patients.

Diagnosing Aleutian mink disease infection by a new fully automated ELISA or by counter current immunoelectrophoresis: A comparison of sensitivity and specificity

Aleutian disease (AD) is a severe disease characterized by hypergammaglobulinemia causing multiple symptoms such as acute renal failure, arteritis, reduced reproductive performance and pneumonia in mink. AD is caused by the parvovirus Aleutian mink disease virus (ADV) and diagnosed primarily based on ADV serology sometimes supplemented by organ PCR analysis. In Denmark, approximately 3.5–4 million serum samples are tested every year for the presence of anti ADV antibodies as part of a national eradication program. The present study compares the diagnostic performance of the two most commonly used assays for serological screening for Aleutian disease: counter current immunoelectrophoresis (CIEP) and ELISA. In total, 3810 mink were sampled in doublets and analyzed by CIEP and a newly developed fully automated ELISA. The results show that the two assays have a comparable diagnostic performance with the ELISA having a higher sensitivity but lower specificity than the CIEP assay. The ELISA has been approved by the Danish authorities for diagnosing Aleutian disease in mink.

English

CONTEXT/BACKGROUND: The management of polytraumatized patients is always challenging to the clinician due to high risk of sepsis, multi-organ failure and death. AIM: Our objective was to study Complement activation, C3 and IL-6 levels in trauma patients so that clinical outcome can be predicted for proper management of injury. SETTINGS AND DESIGN: This case-control study was conducted in the Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh. MATERIALS AND METHODS: 44 trauma patients and 40 healthy controls, selected on a random basis, were included in this study. Blood was collected within 24 hours and at 7th day of injury. Complement activation was determined by Countercurrent Immunoelectrophoresis and Crossed Electrophoresis. C3 levels were measured using Single Radial Immunodiffusion. IL-6 was detected by ELISA. STATISTICAL ANALYSIS USED: Unpaired t test and Chi-square test were used to analyze the data statistically. Probability values less than 0.05 were considered significant. RESULTS: All patients showed complement activation within 24 hours of injury. Patients with Injury Severity Score (ISS) ≥15 had lower C3 as compared to controls (p < 0.001) and those with ISS <15 (p < 0.001). Patients with ISS ≥30 continued to have activated complement and low C3 in 2 nd week; they subsequently developed infection. Complement was inactive and C3 levels recovered in patients with ISS <30. Non-survivors showed lower C3 than survivors (p < 0.05). Patients with infection had lower C3 than those who remained free of infection (p < 0.05). All patients with ISS ≥15 showed initial elevation in IL-6 levels. Patients with ISS ≥45 had higher IL-6 than controls (p < 0.001) and those with ISS <45 (p < 0.05). Non-survivors had higher IL-6 than survivors (p < 0.05). Patients who developed infection showed higher IL-6 than those without infection (p < 0.001). CONCLUSIONS: Complement activation, C3 and IL-6 levels correlated well with severity of injury and development of infection. Serial estimation of complement activation status, C3 levels and IL-6 levels are useful tools in monitoring the clinical progress of trauma patients as these parameters vary significantly with respect to the severity of injury and the development of local and systemic infection in such patients. Hence, they can be used to decide timely the course of therapeutic interventions, predict the onset of infection/septicemia and mortality in polytraumatized patients.

Prevalence and incrimination of Anopheles fluviatilis species S (Diptera: Culicidae) in a malaria endemic forest area of Chhattisgarh state, central India

BackgroundChhattisgarh state in central India is highly endemic for malaria and contributes about 13% of annually reported malaria cases in the country with predominance of P. falciparum. Entomological investigations were carried out in a tribal forested area of district Bastar located in the southern part of Chhattisgarh state to record the prevalence of sibling species of Anopheles fluviatilis and An. culicifacies complexes. The vector species complexes were investigated at sibling species level for their biology in terms of resting and feeding behavior and malaria transmission potential.MethodsIndoor resting vector mosquitoes collected during 2010–2011 were identified to sibling species by cytotaxonomy and polymerase chain reaction (PCR) assay. The blood meal source analysis and incrimination studies were done at sibling species level by counter current immunoelectrophoresis and enzyme linked immunosorbent assay (ELISA) respectively.ResultsAnalysis of sibling species composition revealed predominance of An. fluviatilis species S in the study area, which was found to be highly anthropophagic and rested in human dwellings whereas the sympatric species T was primarily zoophagic. Incrimination studies showed high sporozoite rate in species S, thereby confirming its vectorial efficiency. An. culicifacies was encountered in low numbers and comprised species B and C in almost equal proportion. Both these species were found to be exclusively zoophagic.ConclusionThe observations made strongly suggest that species S of Fluviatilis Complex is the principal vector of malaria in certain forest areas of district Bastar, Chhattisgarh state and should be the target species for vector control operation. Vector control strategies based on biological characteristics of Fluviatilis S will lead to substantial decline in malaria incidence in such areas.

Immunodetection of coproantigens for the diagnosis of amphistomosis in naturally infected Indian Water Buffalo, Bubalus bubalis

The infection of gastrointestinal helminths in livestock is routinely diagnosed by microscopical examination of faecal samples for the presence of ova/eggs but this approach becomes ineffective for the seasonally egg producing trematodes. Therefore, an alternative approach to detect the coproantigens of liver and rumen amphistomes, Gigantocotyle explanatum and Gastrothylax crumenifer respectively, infecting Indian water buffalo Bubalus bubalis, was undertaken using ELISA, immunodot and countercurrent immunoelectrophoresis (CCIEP). The hyperimmune polyclonal antisera were separately raised in rabbits against excretory/secretory (ES) antigens of both the flukes under study. An overall 70% buffalo faecal samples were tested positive for G. crumenifer and 75% for G. explanatum in Aligarh region. The ELISA results reflected higher infection intensity among individual buffaloes that was also observed at necropsy. Using the respective homologous hyperimmune antiserum, 55% buffaloes tested positive for G. crumenifer and 65% positive for G. explanatum in immunodot assay. Further, the faecal samples with high absorbance values in ELISA and strong immunodot reaction tested positive in CCIEP. The analysis of CCIEP result revealed two and one precipitin bands in G. crumenifer and G. explanatum respectively, indicating prominent antigenic differences in the coproantigens of these two parasites. Taken together, it is suggested that polyclonal antibodies could be conveniently used for the detection of coproantigens by ELISA and immunodot methods, particularly during the non-egg producing phase of the seasonally regulated reproductive cycle of the rumen amphistome G. crumenifer. It is concluded that the coproantigen detection is a good alternative over conventional method for the diagnosis of amphistomosis in livestock; however, further studies are required on a larger sample size of field buffaloes to augment the reproducibility of the present results.

Phylogenetic analysis of the VP2 gene of Aleutian mink disease parvoviruses isolated from 2009 to 2011 in China

Aleutian mink disease parvovirus (AMDV) is a non-enveloped virus with a single-stranded DNA genome that causes a fatal, usually persistent immune complex disease in minks. In this study, a total of 18,654 serum samples were collected from minks that were farmed in China from 2009 to 2011. After testing by counter-current immunoelectrophoresis (CIE), the seroprevalence of AMDV was found to be 68.67 %. The results show that there is a serious epidemic among Chinese minks used for breeding. To gain detailed information regarding the molecular epidemiology of AMDV in China, nine strains of AMDV were isolated from mink samples that were collected from four of the primary mink farming areas in China. The full-length capsid protein VP2 gene from each strain was sequenced after PCR amplification, and a phylogenetic analysis was performed on the VP2 gene sequence, including the VP2 genes from the other 10 AMDV strains available in the GenBank database, which were submitted from the 1970s to 2009. The phylogenetic analysis showed that the AMDV isolates were divided into five independent clades. The Chinese AMDV strains were distributed among all five groups and showed a high level of genetic diversity. Over 50 % of the Chinese AMDV strains were classified into two clades that consisted only of isolates from China and that were distinct from AMDV strains found in other countries. This finding indicated that both local and imported ADMV species are prevalent in the Chinese mink farming population.

Identification of Klebsiella Pneumoniae by Capsular Polysaccharide Polyclonal Antibodies

The aim of this study is to develop simple and rapid diagnostic method which can be utilized routinely for detection of Klebsiella pneumoniae. This study was done in India at DRDE, Gwalior. We have collected standard K.pneumoniae strain 3296 from Yamaguchi University, Japan and total fifty-nine clinical isolates of Klebsiella species from Armed Force Medical College, Pune, and Patel chest hospital, New Delhi. Bacteria were grown on trypticase soy broth and two Newzealand wistar white rabbit were immunized subcutaneously with purified capsular polysaccharide (CPS) of K.pneumoniae. Specificity of hyper immune sera raised against CPS was tested by counter current immunoelectrophoresis. We have generated four monoclonal antibodies, out of which two IgM producing clones (KP-1 & KP-2) and hyper immune sera against CPS antigen were used for agglutination test. KP-1, KP-2 and CPS hyperimmune sera showed different reactions. Out of the twenty biochemically confirmed K.pneumoniae clinical isolates, hyperimmune sera to CPS detected sixteen as positive whereas KP-1 reacted to eleven and KP-2 to thirteen of these isolates. One clinical Klebsiella species biochemically negative for K.pneumoniae and other Enterobacteriaceae organisms were negative by agglutination test to all the three antibody reagents. K.aerogenes, however, reacted to KP-2 and CPS hyperimmune sera. Standard K.pneumoniae was positive to all the three antibodies. The rapidity and ease of performance merited the hyper immune sera to CPS and IgM monoclonal antibody based agglutination test as the preliminary identification system for K.pneumoniae.

Comparison of three immunodiagnostic tests for experimental Heterophyes heterophyes infection in dogs

The aim of this study was to compare the performance of three in-house diagnostic tests, i.e. counter current immunoelectrophoresis (CCIE), intradermal (ID) and indirect fluorescent immunoassay (IFI), for the diagnosis of Heterophyes infection. One hundred and twenty puppies were randomly divided into eight groups ( n = 15/group). Heterophyes heterophyes infections were established in these puppies by administering a dose of 50 H. heterophyes encysted metacercariae/puppy by gavage. Forty puppies of similar age and sex were divided into eight groups, of five puppies each and were used as negative controls. Sera pooled from separate infected and uninfected groups were tested against H. heterophyes antigens, weekly for 8 weeks post-infection (PI). The ID assay detected infected puppies sooner than any of the serological tests. Sero-conversion was first detected 2 weeks PI by ID assay and 1 week later by CCIE and IFI assays. ID test performed well till the end of the experiment (sensitivity and specificity: 100% and 90%, respectively). Both IFI and CCIE assays had a sensitivity of 40% and 20%, respectively for early detection of antibody, which was less sensitive than ID but both assays were more specific (100%) than the ID assay. The lowest agreement was between ID and IFI tests (40.3%), whilst the highest was observed between CCIE and IFI tests (67.2%). Of the three evaluated methods, the ID test could be recommended for scientific and laboratory diagnosis of heterophyosis in naturally infected animals. However, since none of the investigated method are optimal (i.e, offers 100% specificity and sensitivity), the choice of test employed must depend on the aim of the study.

Capsular serotype K1 or K2, rather than magA and rmpA, is a major virulence determinant for Klebsiella pneumoniae liver abscess in Singapore and Taiwan.

Capsular serotypes, magA, and rmpA have been documented in high prevalence for Klebsiella pneumoniae liver abscess. To investigate the regional difference and the correlation of capsular serotype, magA, and rmpA with virulence, 73 isolates were collected in Singapore and Taiwan. Capsular serotypes were determined by countercurrent immunoelectrophoresis, the presence of magA and rmpA was determined by PCR, and virulence was determined by phagocytosis and mouse inoculation. Isolates from Singapore were similar to those from Taiwan in genomic heterogeneity, prevalence of serotype, and the presence of magA and rmpA. The most common serotype was K1 (34/73; 46.6%), followed by K2 (15/73; 20.5%). magA was restricted to serotype K1. All K1 or K2 isolates and 66.7% (16/24) of isolates that were neither serotype K1 nor serotype K2 (non-K1/K2) carried rmpA. Serotype K1 or K2 isolates demonstrated significantly more phagocytic resistance and virulence than did rmpA-positive and -negative groups of non-K1/K2 isolates. In the non-K1/K2 group, the virulence profiles of rmpA-positive strains from Taiwan and Singapore were different by phagocytosis assay and in the mouse model, indicating that factors other than rmpA contributed to virulence. The characteristics of K. pneumoniae liver abscess in Singapore and Taiwan are similar. Capsular serotype K1 or K2 plays a more important role than magA and rmpA in determining virulence in K. pneumoniae liver abscess.

Hepatitis B surface antigenaemia and alpha‐foetoprotein detection from dried blood spots: applications to field‐based studies and to clinical care in hepatitis B virus endemic areas

In many resource-limited regions with endemic hepatitis B virus (HBV), there is limited infrastructure to collect, process, transport, and store blood samples for identification of persons with chronic HBV infection or with hepatocellular carcinoma (HCC). We describe the application of a simple technique using commercially available kits for detection of HBV surface antigen (HBsAg) and alpha-foetoprotein (AFP) in dried blood spots (DBS) collected on filter paper. Study participants included subjects with and without chronic HBV infection and subjects with HCC or cirrhosis. Three to five blood drops were dried on filter paper. Dried blood (equivalent to 20 muL) was eluted and tested for HBsAg by Determine(TM) HBsAg and for AFP by counter-current immuno-electrophoresis and radio-immunoassay (RIA). The primary analysis focused on comparison of DBS results to serum testing results as the gold standard. The sensitivity of DBS for detecting chronic HBV infection was 96% (98-98) with specificity of 100% (CI 99-100). Sensitivity of DBS in detecting AFP compared with serum RIA was 73% (60-86) with specificity of 90% (81-98). Both HBsAg and AFP recovery were unaffected when DBS were left at room temperature (30-33 degrees C) and under humid conditions for up to 28 days prior to elution. We conclude that DBS can be reliably used as an economical and logical alternative for detection of HBsAg in chronically infected patients and for AFP-based diagnosis of HCC in clinical situations which preclude adequate collection and processing of blood samples. Both research-oriented field studies and routine clinical care may benefit from application of these techniques in resource-limited settings.