Conventional Serological Methods Research Articles

Pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to H5N1 influenza viruses

The standard serological methods present limitations for the measurement of immunity against H5N1 influenza strains. The hemagglutination inhibition (HI) assay lacks sensitivity and requires standardization, while the viral micro-neutralization (MN) assay needs handling of live virus. We produced pseudoparticles expressing hemagglutinin from clades 1 or 2 H5N1 in order to measure neutralizing antibodies in human sera after prime-boost vaccination with plain or MF59-adjuvanted H5N1 clade 1 subunit vaccines. Titers measured by pseudoparticle neutralization (PPN) assay significantly correlated with those measured by HI, single radial haemolysis or MN, with a PPN titer of 1:357 corresponding to an MN titer of 1:80. Notably, results from the PPN assay, confirm that MF59-H5N1 vaccine induces potent and long-lasting neutralizing antibody responses not only against the vaccine strain, but also against several heterologous clade 2 strains. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of H5N1 vaccine immunogenicity.

A low density oligonucleotide microarray for the detection of viral and atypical bacterial respiratory pathogens

Acute respiratory tract infections are a major cause of morbidity and mortality worldwide and exert a considerable economic burden on healthcare systems. Acute respiratory tract infections of the upper and lower respiratory tract are caused by a wide variety of viral and bacterial pathogens, which require comprehensive laboratory investigations. Conventional serological and immunofluorescence-based diagnostic methods for acute respiratory tract infections lack sensitivity when compared to polymerase chain reaction (PCR)-based approaches and the development of new diagnostic methodologies is required, to provide accurate, sensitive and rapid diagnoses. In the present study, a PCR-based low density oligonucleotide microarray was developed for the detection of 16 viral and two atypical bacterial pathogens. The performance of this DNA microarray-based analysis exhibited comparable sensitivities and specificities to multiplex real-time reverse transcription polymerase chain reactions (rtPCRs) confirming the potential diagnostic utility of the method. In contrast to routine multiplex PCR, the microarray incorporates an intrinsic redundancy as multiple and non-identical probes per target on the array allow direct intra-assay confirmation of positives. This study demonstrates that microarray technology provides a viable alternative to conventional serological-based approaches and multiplex PCR for pathogen identification in acute respiratory tract infections.

Historic milestones in the evolution of the crossmatch

Abstract The introduction of the serologic crossmatch—first proposed by Hektoen (1907) and first performed by Ottenberg (1908)—made it possible to transfuse blood without fear of unpredictable and potentially disastrous acute hemolytic reactions, most of which were attributable to direct agglutinating (IgM) anti-A, anti-B, or anti-A,B. Previously transfused or previously pregnant recipients continued to experience sporadic hemolytic transfusion reactions as a result of “incomplete” (IgG) blood group antibodies. Coombs’ introduction of the antiglobulin test (1945) made it possible to detect “incomplete” (IgG) antibodies and to develop laboratory methods to identify and transfuse serologically compatible RBCs. During the past 50 years, the antibody screen has evolved to be more effective than the crossmatch for detecting the presence of potential serologic incompatibility and has, in fact, replaced the crossmatch as the key step in pretransfusion compatibility testing. The antibody screen has become the serologic surrogate for the crossmatch and, currently, the computer crossmatch is becoming the electronic surrogate for the crossmatch. In historical perspective, the past 100 years witnessed the rise and fall of the cross-match. Today, hemolytic transfusion reactions are more likely to be the result of misidentifying the intended transfusion recipient than of failure of routine compatibility testing to detect serologic incompatibility. The introduction of the serologic crossmatch—100 years ago—had a major positive impact on all aspects of clinical medicine. As a result of these advances, we are quickly approaching the limits of improving the safety and efficacy of blood transfusions by conventional serologic methods. Future improvements are more likely to be the result of applications of molecular genotyping—which could not have become available at a more perfect time. When Immunohematology publishes its 50th-anniversary issue, the improvements in transfusion science are more likely to be based on molecular methods than on conventional blood bank serology. Immunohematology 2009;25:147–151.

Discovery and characterization of Plum pox virus isolates in Montenegro

In 2006, twenty extensive and intensive plum orchards, surrounding Nik s i C in Montenegro, were screened for the presence of symptoms of Plum pox virus (PPV) infection. Mild to severe symptoms were found in 15 orchards, usually only on some trees. In total 19 samples were collected and 17 proved to be positive by DAS-ELISA (Bioreba). Of these positives, 16 were selected for molecular identification. PPV isolates have been assigned to two major (PPV-D and PPV-M) and two minor (PPV-EA, PPVC) groups. Recently, a fifth group (PPV-REC) has been established, which corresponds to naturally-occurring recombinants between PPV-M and PPV-D isolates. All PPV-REC isolates share the same recombination breakpoint on the NIb polymerase gene and therefore conventional CP gene-based molecular and serological methods are not able to discriminate between PPV-M and PPV-REC isolates. Subgroup characterization was performed using two different PCR systems: one positioned downstream of the recombinant crossover (using the PPV-M and -D specific primer pairs P4/P3-M and P4/P3-D respectively; Dallot et al. , in press); and the other upstream of the recombination breakpoint (using the primer pairs CIf/CI-M (PPV-M specific) and CIf/CI-D (PPV-D specific); Glasa et al. , 2002). The results showed the presence of PPV-D type isolates in 4 samples. These samples were also negative for PPV-M when tested by TAS-ELISA (Agritest). The remaining 12 samples were PPV-M positive by TAS-ELISA. Of these, seven proved to be of PPV-REC type by RT-PCR. However, the isolate type could not be determined for the remaining five samples using the previously described method. Therefore they were further analyzed using primer pairs mM5/mM3, mD5/mD3 and mD5/ mM3 ( P ubr et al. , 2004), situated around the recombination breakpoint. All samples gave a positive result only with primer pair mD/mM3 and were therefore also identified as the PPV-REC type. Although plum production is important in Montenegro, data about PPV infection is scarce. Sharka symptoms were observed by Miju s kovi C (2002), but until now the presence of PPV was never confirmed. As a result, this is the first report confirming PPV as being present in Montenegro.

Combining Residual Insecticide Spraying Campaigns with Targeted Detection and Specific Chemotherapy for Trypanosoma cruzi Infection in Children

Combining Residual Insecticide Spraying Campaigns with Targeted Detection and Specific Chemotherapy for Trypanosoma cruzi Infection in Children

Evaluation of BioSewoomTM HLA-A, -B, -C PCR/SSP Kit

Human leukocyte antigen (HLA) typing based on polymerase chain reaction (PCR) is rapidly replacing the conventional serological method. This study was intended to evaluate BioSewoom HLA-A, -B, -C PCR/SSP kit (BioSewoom SSP) which had recently been developed in Korea. A total of 158 samples from domestic (21) and international (137) HLA proficiency testing (PT) were genotyped with BioSewoom SSP, and its results were compared to consensus results. For comparison with INNO-LiPA HLA-A, -B, -C Typing Kit (INNO-LiPA, Innogenetics, Belgium), 20 samples of Koreans were genotyped with both kits for each HLA-A, -B, -C locus. Among the 21 samples of domestic PT, BioSewoom SSP showed ambiguities as follows: 4 samples (19.0%) in HLA-A, 6 (28.6%) in HLA-B, and 1 (4.8%) in HLA-C. The ambiguities could be resolved by considering the allele distribution of Koreans. Among the 137 samples from international PT, BioSewoom SSP also showed ambiguities as follows: 12 samples (8.8%) in HLA-A, 26 (19.0%) in HLA-B and 6 (4.4%) in HLA-C. Considering the allele distribution of Koreans, the serologic equivalents obtained from BioSewoom SSP showed a full agreement with those of INNO-LiPA in all the loci tested. Twelve (0.007%) among 1,760 PCR reactions from the 21 samples of domestic PT and 20 patient samples produced faint nonspecific bands, but it was negligible. PCR failure of internal control just barely occurred (15 PCR reactions, 0.009%), but it had no bearing on allele assignment. The performance of BioSewoom SSP was comparable to that of INNO-LiPA. All the ambiguities could be resolved by considering the allele distribution of Koreans. It is concluded that BioSewoom SSP has good performance to be used in routine HLA laboratories.

Identification and differentiation of canine Mycoplasma isolates by 16S–23S rDNA PCR-RFLP

Conventional serological methods for the identification of canine mycoplasma isolates depend on the availability of a panel of species-specific diagnostic antisera and are not always reliable in terms of specificity. To enable simultaneous identification of field isolates, PCR-RFLP analysis of the 16S–23S rRNA intergenic spacer region was used to characterize the type strains of the 12 currently described canine mycoplasmas of the Genus Mycoplasma which represent the “classic” non-hemotropic species. The use of 16S–23S rDNA PCR in the first step of this analysis revealed specific size differences of amplicons which allowed to classify these 12 canine Mycoplasma species into three groups. Depending on the length of the amplicon, subsequent RFLP analysis of PCR products using two restriction endonucleases in a single digest (ApoI/DdeI or TaqI/VspI) generated unique banding patterns. For further evaluation of the 16S–23S rDNA PCR-RFLP assay system as identification and differentiation tool, a total of 262 field isolates collected from the canine genital tract were tested. PCR-RFLP results for 251 field isolates correlated with traditional serological test results. The remaining 11 isolates had an RFLP pattern distinct from the type strains included in this study and were identified by 16S rDNA sequencing as closely related to M. sp. HRC689. The PCR-RFLP assay established in this study enabled a rapid, accurate and easily performed identification and differentiation of all 12 currently described non-hemotropic canine Mycoplasma species.

Evaluation of Different PCR Assays for Early Detection of Acute and Relapsing Brucellosis in Humans in Comparison with Conventional Methods

Human brucellosis is a significant public health problem in many Mediterranean countries including Greece. The conventional serological methods, as well as blood cultures, have serious limitations, especially in chronic, relapsing, and focal forms of the disease. Four different PCR assays were applied in 4,926 buffy coat, whole-blood, and serum samples received from 200 patients admitted with brucellosis to the Infectious Diseases Hospital, Thessaloniki, Greece, for the rapid diagnosis of acute infection and relapses and compared to blood culture and serological tests (i.e., Wright's seroagglutination test, Coombs' antibrucella test, and the complement fixation test). The four PCR assays had excellent sensitivity and specificity and were able to detect all of the cases of acute disease. Buffy coat and whole blood were the optimal specimens. All four PCR assays were negative in all follow-up samples from 183 patients who had completed a successful treatment and were positive in all follow-up samples from 17 patients who had relapses in the first year after therapy, including the times of the relapses. In conclusion, PCR is a very useful tool for the rapid diagnosis of acute brucellosis and a good marker for the posttreatment follow-up and the early detection of relapses.

MLST-v, multilocus sequence typing based on virulence genes, for molecular typing of Salmonella enterica subsp. enterica serovars

Salmonella enterica subsp . enterica is one of the main causative agents of food-borne disease in man, and can also be the cause of serious systemic illness. Organisms belonging to this genus have traditionally been classified on the basis of the antigenic properties of the cell-surface lipopolysaccharide and of the phase 1 and phase 2 flagellar proteins. Primary isolation, biochemical identification, and serotyping are laborious and time consuming. Molecular identification based on suitable marker genes could be an attractive alternative to conventional bacteriological and serological methods. We have assessed the applicability of two housekeeping genes, gyrB, atpD, in combination with the flagellin genes fliC and fljB in multilocus sequence typing of Salmonella. Sequencing and comparative analysis of sequence data was performed on multiple strains from Austria, the United Kingdom, and Switzerland, representing all subspecies and 22 of the more prevalent non-typhoid S. enterica subsp. enterica serovars. A combination of these four marker genes allowed for a clear differentiation of all the strains analysed, indicating their applicability in molecular typing. The term MLST-v, for multilocus sequence typing based on virulence genes, is proposed to distinguish this approach from MLST based solely on housekeeping genes. An assortative recombination of the fliC gene was found in seven of the analysed serovars indicating multiple phylogenetic origin of these serovars.

Infection by Leishmania infantum in cats: Epidemiological study in Spain

More than 40 cases of feline leishmaniasis have been reported in the scientific literature. The influence of some immunodepressive conditions of viral origin, such as leukemia and feline immunodeficiency, are still unknown. The purpose of this study was to assess the prevalence of Leishmania infection in cats and possible relations with these viral infections. Markers of Leishmania infection were searched in 183 cats from Southern Spain by IFAT, PCR, Giemsa stain and culture, with a follow-up of positive cats. Seropositivity was 60.0% (Ab titer ≥10) and 28.3% of animals presented Ab titers ≥40. Around 25.7% of the cats studied were parasitemic and some of them remained positive for months. Combining both data, 70.6% of the feline population was, or could be, infected. We observed a negative association between seropositivity to Leishmania and infection by FeLV. Hence, production of antibodies against the parasite appears to be compromised in cats with leukemia, which have a prevalence of 36% in our study. In contrast, we found no association with feline immunodeficiency. The results makes us doubt the value of conventional serological methods to detect active Leishmania infection in cats.

HBV, HCV, and TTV detection by in situ polymerase chain reaction could reveal occult infection in hepatocellular carcinoma: comparison with blood markers

Objective: To report a retrospective analysis on the presence of hepatitis B virus (HBV), hepatitis C virus (HCV), and transfusion transmitted virus (TTV) sequences in formalin fixed, paraffin embedded liver...

Studies on the Human Leukocyte Antigens in Patients with Polycystic Ovary Syndrome in a Japanese Population – Possible Susceptibility of HLA‐A11 and ‐DRB1*0403 to Patient Population with Polycystic Ovary Syndrome

The objective of this study was to identify the human leukocyte antigen (HLA) alleles that confer susceptibility or resistance to polycystic ovary syndrome (PCOS) in the Japanese population. HLA-A, -B and -C antigens were determined in 56 patients with PCOS using conventional serological method. HLA-DRB1 genotypes were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in 68 patients with PCOS. As a control population, 237 healthy individuals (males and females) were examined concerning HLA-A, -B and -C antigens, and 292 individuals were examined concerning HLA-DRB1 genotypes. The rate of possession of each antigen was compared between the two populations. The rate of possession of the HLA-A11 and HLA-DRB1*0403 in the patients with PCOS was significantly higher compared with that in the control group. The rate of possession of HLA-B39 in the patients with PCOS was significantly lower compared with that in the general population group. Human leukocyte antigen systems appear to be linked to PCOS.

Assessment of real-time PCR for diagnosis ofMycoplasma pneumoniaepneumonia in pediatric patients

We developed a real-time PCR to detect Mycoplasma pneumoniae with a primer set designed for the 16S rRNA gene. Clinical samples (n=937) were collected from children with community-acquired pneumonia between April 2002 and March 2004 at 12 Japanese medical institutions. Sensitivity of real-time PCR was calculated as 10 colony-forming units per reaction tube using a pMP01 plasmid carrying a 225-bp target DNA fragment of the 16S rRNA gene in M. pneumoniae M129, a standard strain. Results, obtained within 2 h, were compared with those of conventional culture and serologic methods. Of all cases tested, 151 (16.4%) and 129 (13.8%) were positive for M. pneumoniae by real-time PCR and by culture, respectively. Among the 151 cases, almost all of those tested serologically by passive agglutination showed a rise in M. pneumoniae antibody titre between acute and convalescent sera. We conclude that this real-time PCR can identify M. pneumoniae rapidly and fulfills the need for rapid identification, high sensitivity, and high specificity.

Clinical Effects of Unrelated-Donor or Related-Donor Hematopoietic Stem Cell Transplantation for Leukemia.

Patients and Methods: From February 2003 to May 2005, 118 patients with leukemia received URD-HSCT or RD-HSCT at our institution. 43 cases received URD-HSCT and 75 cases received RD-HSCT. The basic conditioning regimens were TBI (total-body irradiation) + CY (CTX) protocol or modified BuCY (hydroxyurea, busulfan, Ara-C and CTX) protocol. Fludarabine, cytosine arabinoside, etoposide and etc. were added to the basic conditioning regimens when treated patients with refractory leukemia or in NR. The 43 patients who were treated with URD-HSCT, included 43 patients matched at 6/6 HLA antigens based on conventional serologic typing methods, 11 at 5/6 HLA antigens, 1 at 4/6 HLA antigens and 1 at 3/6 HLA antigens. Among the 75 patients with RD-HSCT, 69 patients were matched at 6/6 HLA antigens serologically, 6 were matched at 5/6 HLA antigens. The basic protocol GVHD prophylaxis is CsA (cyclosporine) and methotrexate (MTX). Low-dose antithymocytes globulin (ATG) was added to the basic protocol when patients treated with URD-HSCT, and mycophenolate mofetil (MMF) and low-dose ATG added to the basic protocol were used in some patients with URD-HSCT.Results and Conclusion: The patients treated with URD-HSCT or RD-HSCT have no significant differences in sex, age, disease type and pretransplantation condition. The overall survival was 57.5±0.83% and 58.7±10.5% in patients undergoing URD-HSCT and RD-HSCT, and the 3 year disease-free survival rate was 51.6±8.5% and 49.8±8.3%, respectively. The overall survival and 3 year disease-free survival differed little between unrelated and related transplantation(P>0.05). The relapse rate of leukemia between URD-HSCT and RD-HSCT were 9.3% and 18.8%, respectively, and significant difference was noted (P<0.05). II–IV° acute GVHD (aGVHD) occurred in 58.17% of the patients receiving URD- HSCT and in 33.00% of those with RD-HSCT, especially the incidence of III–IV° severe aGVHD in the two group was 16.0% and 9.0%, respectively, and there were significant differences, too. The mortality caused by GVHD in patients with URD-HSCT was 12.0% and that in RD-HSCT was 9.0%, and it differed little. The incidence of chronic GVHD (cGVHD) in patients who could be followed up after URD-HSCT was 51.15% and that in RD-HSCT was 53.85%, there was no significant difference. The mortality directly caused by infection in the two group was 7% and 4%, respectively, and there was no significant difference. Two patients with URD-HSCT died of venous occlusive disease (VOD), one hemorrhagic cystitis (HC) and one graft rejection, but there was no similar condition in RD-HSCT.

USE OF AN IRON SUPEROXIDE DISMUTASE EXCRETED BY TRYPANOSOMA CRUZI IN THE DIAGNOSIS OF CHAGAS DISEASE: SEROPREVALENCE IN RURAL ZONES OF THE STATE OF QUERETARO, MEXICO

Four iron superoxide dismutase (SODI, SODII, SODIII, and SODIV) activities with pI values of 6.9, 6.8, 5.25, and 3.8, respectively, were isolated from epimastigote forms of the Maracay strain of Trypanosoma cruzi cultivated at 28 degrees C in Grace's medium supplemented with 10% heat-inactivated fetal bovine serum. The activity of SODe (pI 3.8), which coincides with that of SODIV, was detected in Grace's medium without serum in which T. cruzi epimastigotes were cultivated for 24 hours at 28 degrees C. SODe, which was excreted into the medium by the parasite, was immunogenic and antibodies to SODe were detected in serum to a dilution of 1:2,500 by Western blot. The role of SODe is related to the establishment of the parasite within the host, and its high immunogenicity and specificity make it a useful molecular marker in diagnosing infection with this parasite. To validate a Western blot result using SODe as a antigen fraction, 1,029 sera of individuals from 11 municipalities in the state of Queretaro, Mexico were analyzed. Sampling was done randomly and results were compared with those for the same sera with three conventional serologic methods: an enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination (IHA), and an indirect immunofluorescence assay (IFA) to detect antibodies to T. cruzi SODe. Samples that were positive by these three techniques were also positive by the Western blot method. The seroprevalence values for SODe (8.16% by ELISA and Western blot) in Queretaro were considerably higher than those reported in regions of Mexico considered to be endemic for Chagas disease. These results support the use of SODe in the serodiagnosis of Chagas disease.

A monoclonal antibody-based ELISA allows discrimination between responses induced by bovine herpesvirus subtypes 1 (BoHV-1.1) and 2 (BoHV-1.2)

Bovine herpesvirus type 1 (BoHV-1) has distinct subtypes according to genomic characterization. Immune responses induced by BoHV-1 subtype 1 (BoHV-1.1) are not distinguishable from those induced by BoHV-1 subtype 2 (BoHV-1.2) through conventional serological methods. In the present report, an enzyme linked immunosorbent assay is described that allows discrimination between immune responses in cattle immunized with either subtype, based on a monoclonal antibody that recognizes specifically the amino-terminal region of glycoprotein C (gC) on BoHV-1.1 strains, thus not reacting with BoHV-1.2a. The test displayed a sensitivity of 92%, specificity of 90% and a good correlation with serum neutralization tests on samples from BoHV-1.1-immunized calves ( κ = 0.799). The test may be useful to provide new insights into the roles played by each of these two subtypes in the epidemiology of BoHV-1 infections.

The application of new techniques to the improved detection of persistently infected cattle after vaccination and contact exposure to foot-and-mouth disease

Detection of antibodies to the non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) was compared with conventional serological and virological methods and with RT-PCR for the identification of FMDV carrier animals obtained after experimental contact challenge of vaccinated cattle. Transmission from carriers to sentinels was also monitored. Twenty FMDV vaccinated and five unvaccinated cattle were challenged by direct contact with five donor cattle excreting FMDV and monitored until 28 days post challenge-exposure [1]. Twelve vaccinated and three unvaccinated animals were retained up to 24 weeks post exposure to FMDV in order to monitor viral persistence, transmission and antibody responses. In nine vaccinated animals, infection persisted beyond 28 days post exposure, virus being detected more frequently and for longer in oesophagopharyngeal samples from these animals when examined by RT-PCR rather than by virus isolation. Although recovery of FMDV RNA became increasingly sporadic over time, the number of RNA copies detected in positive samples declined only slowly. Two naïve sentinel cattle housed with the persistently infected animals between 93 and 168 days after the latter had been challenge-exposed to FMDV did not become infected. There were differences in the ability of commercially available serological tests to detect antibodies to FMDV non-structural proteins (NSP) in vaccinated and subsequently challenged cattle. Although no single test could identify all of the vaccinated cattle that became persistently infected, the most poorly recognised animals were those with the least evidence of virus replication based on other tests. The potential of the detection of antibodies to the 2B NSP of FMDV for diagnosing persistent FMDV infection was demonstrated.

Molecular basis for para‐Bombay phenotypes in Chinese persons, including a novel nonfunctional FUT1 allele

The para-Bombay phenotype is characterized by H-deficient or H-partially deficient red blood cells (RBCs) in persons who secrete ABH antigens in their saliva. The studies that determined the genotypes for two Chinese individuals with the para-Bombay phenotype are described. RBC phenotypes were characterized by conventional serologic methods. Exons 6 and 7 of the ABO gene were amplified, as well as the entire coding region for FUT1 and FUT2, with four independence polymerase chain reactions (PCRs) from genomic DNA. PCR products were excised, purified from agarose gels, and sequenced directly. Mutations of FUT1 were identified by TOPO cloning sequencing. For both individuals, RBC ABO genotypes correlated with ABH substances in their saliva. One individual (a patient) had two heterozygous mutations of FUT1 by direct DNA sequencing, namely, a C-->T heterozygous mutation at position 293(C293T) and AG heterozygous deletion (CAGAGAG-->CAGAG) at position 547 to 552. These two mutations were confirmed to be compound heterozygotes; that is, each mutation was determined to be on a separate homologous chromosome by TOPO cloning sequencing. The FUT2 genotype was Se(357)Se(357). The other individual (a blood donor) had an AG deletion at position 547 to 552 homozygous allele in FUT1. The FUT2 genotype was Se(357)Se(357,385). C293T mutation can cause Thr/Met at amino acid position 98. AG deletion at position 547 to 552 caused a reading frameshift and a premature stop codon. A novel nonfunctional FUT1 allele C293T was identified in a person with the para-Bombay phenotype. This rare H-deficient phenotype may result from different nonfunctional alleles.

Genetic subtyping of influenza A viruses using RT-PCR with a single set of primers based on conserved sequences within the HA2 coding region

Influenza A viruses are subtyped conventionally according to the antigenic characteristics of the external glycoproteins, haemagglutinin (HA) and neuraminidase (NA). To date 15 HA and 9 NA subtypes have been described. There is a need to develop fast, accurate and reliable methods to identify influenza virus subtypes, which may be associated with disease outbreaks. An RT-PCR is described using a single primer pair based on a conserved region of the HA2 gene that can detect all 15 HA influenza A subtypes. The assay was validated initially using a panel of 12 known standard prototype strains of influenza virus representing 6 HA subtypes and subsequently in a blind study using a panel of 30 strains. Selected viruses represented all known HA subtypes derived from avian, swine and human hosts separated both geographically and with time Sequence analysis of RT-PCR product showed complete correlation with results obtained using conventional serological methods. It is concluded that this RT-PCR is a reliable, robust and reproducible tool for the rapid identification of a wide range of all the HA subtypes of influenza A viruses.

Disease Problems Affecting Fish in Tropical Environments

ABSTRACT Aquaculture produce in Asian countries contributes a major share to total global fish production. However, disease due to biotic and abiotic factors results in considerable losses to the industry. The most frequently encountered bacterial agents associated with fish diseases in tropical environments are Vibrio sp. in marine and brackish-water systems and motile aeromonads in freshwater systems. Virulence mechanisms of these bacterial species have been widely studied. Though a few viral agents such as rhabdovirus, reo-like virus, infectious pancreatic necrosis virus, picornavirus, and irido-like virus have been recorded in different parts of Asia, such reports are scanty. Among fungal pathogens, Aphanomyces invaderis is the most virulent and has been reported to be associated with epizootic ulcerative syndrome. Other fungal agents such as Achlya sp. and Saprolegnia sp. have been recorded in various disease conditions. In addition to various conventional microbiological and serological methods, nucleic acid-based methods are being widely adopted for diagnosis of different fish diseases. Few successful vaccines have been developed to protect against disease conditions, and some of the better known ones are against vibriosis and furunculosis. Many other vaccines are still in the experimental stages. Early diagnosis of disease and development of successful vaccines are important for the future development of aquaculture.