Hemagglutination Inhibition Test Research Articles

Characterization of Amino Acid Mutations of Newcastle Disease Virus (NDV) In Swan Geese (Anser cygnoides) In East Java, Indonesia

Background: Newcastle disease (ND) is a highly contagious viral disease affecting the poultry industry. The NDV is classified into three strains based on their relative virulence, namely velogenic or highly virulent, mesogenic or moderately virulent, and lentogenic or lowly virulent. The clinical manifestations of the disease vary depending on many factors, such as host susceptibility and the virulence of the NDV strain. Objective: This study aims to analyze the amino acid mutations of the NDV in unvaccinated swan goose (Anser cygnoides) from various locations in Java. Methods: Samples were collected through cloacal swabs and isolated by inoculation in Specific Pathogen-Free (SPF) embryonated eggs that were nine days old. Hemagglutination and hemagglutination inhibition tests were conducted to confirm that the isolated virus was NDV. The isolated virus was processed using reverse transcription-polymerase chain reaction (RT-PCR) with primers that amplified partial sequences of the fusion (F) gene, which was analyzed to determine the pathotype. Results: The results indicated the presence of mutations in several regions. The amino acid changes occurred in 17 variable sites (7.2%) between RefSeq/JF950510 and ND/SW1/2018, 12 variable sites (5.1%) between RefSeq/JF950510 and ND/SW2/2018, 13 variable sites (5.5%) between RefSeq/JF950510 and ND/SW3/2018, and 19 variable sites (8.1%) between RefSeq/JF950510 and ND/SW4/2018. The amino acid sequences of the cleavage site of the fusion (F) protein revealed that all isolates had low virulence. Conclusion: The results indicated that mutations in the region outside the cleavage site not were incapable of altering the virulence of the virus.

Level of influenza virus strain antibodies in healthy vaccinated people at the end of the COVID-19 pandemic

Introduction. Abnormally low global influenza detection rates were observed at the peak of ongoing COVID-19 infection. In the 2021–2022 season, influenza resumed circulation, and in December 2022 its incidence rate returned to the 2015–2016 season levels, which exceeded the highest value recorded in 2018 worldwide. There is a need to assess changes in pattern of population immunity after influenza vaccination during the global COVID-19 pandemic. The study was aimed at analyzing level of vaccine influenza virus strain antibodies in healthy people formed during the COVID-19 pandemic. Materials and methods. A total of 123 healthy volunteers immunized with influenza quadrivalent inactivated subunit adjuvanted vaccine and 47 unimmunized volunteers were enrolled in the study. Influenza virus strains antibodies were assessed by performing a hemagglutination inhibition (HI) test one month after vaccination. Results. A significantly increased seroprotection rate (SPR) (p 0.05) was show in main group that reached 78.9% against the A/Victoria/2570/2019(H1N1)pdm09 strain, 94.3% — against A/H3N2/Darwin strain /9/2021, 69.1% — against B/Phuket/3073/13 strain and 48.8% — against B/Austria/1359417/2021 strain one month after vaccination. The GMT ratio to influenza A(H1N1), B (Victoria) and B (Yamagata) viruses exceeded the required minimum value (p 0.05) and comprised 4.1 (CI 3.68–4.54), 3.80 (CI 3.33–4.33) and 4.76 (CI 3.87–5.47), respectively. The post-vaccination GMT ratio to influenza A(H3N2) virus was 2.45, which is lower than the required level of 2.50 (p 0.05). The seroconversion rate (SCR) for strains A/Victoria/2570/2019(H1N1)pdm09, B/Phuket/3073/2013 and B/Austria/1359417/2021 was 56.1%, 50.4% and 51.2%, respectively. The SCR of the influenza A(H3N2) virus like GMT ratio was minimal among the four strains (p 0.05) and comprised 38.2%. Conclusion. The vaccine-related immunogenic activity met the requirements for inactivated seasonal influenza vaccines by at least one criterion.

Molecular And Serological Detection Of Newcastle Disease Virus In Live-Bird Markets, Jos, Plateau State In Nigeria

Newcastle disease (ND) is a highly infectious viral disease of birds caused by the Newcastle disease virus (NDV) and it has been reported in domestic birds in Nigeria. Waterfowls and village poultry in live bird markets (LBM) acts as reservoirs, potentially reintroducing the virus to commercial poultry. This study aims at molecular and serological detection of NDV at live bird markets in Plateau State, Nigeria. A cross sectional analysis involved 309 pooled cloacal and tracheal swabs over three months were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), RT-qPCR, virus isolation and haemagglutination and haemagglutinationinhibition test (HA &HI). Virus isolation was attempted in 9 to 11 days old specific antibody negative (SAN) embryonated chicken eggs and fifteen samples showed haemagglutination. Subsequent tests confimed nine of these as NDV positive through haemagglutination and haemagglutination inhibition (HI) tests. Conventional RT-PCR and RT-qPCR further validated five of the nine NDV positive isolates. The phylogenetic analysis of partial F gene nucleotide sequences revealed that all three isolates belonged to class II genotype XIV.2_XIVb. This finding underscores the persistent threat of NDV to local poultry, necessitating comprehensive virological surveillance to understand, isolate and characterize the virus in Nigeria. Therefore, monitoring for emerging lineages and sub-lineages in Nigeria birds is crucial for safeguarding commercial poultry production.

Effects of astragalus extract, levamisole and ascorbic acid on humoral immunity in chickens vaccinated with newcastle disease vaccines

This study was designed to assess the effects of Astragalus extract, levamisole and ascorbic acid on the humoral immune response of chickens vaccinated with live and inactivated Newcastle Disease (ND) vaccines. Sixty day-old Cobb500 broiler chicks were used for the study. At day old, the maternally derived antibodies (MDA) to ND virus was determined and the chicks were then housed in a bio-secured pen with water and feed given ad-lib. On the sixth day, the MDA decay was determined thereafter, the chicks were shared into 4 treatment groups. Group A (Astragalus extract); Group B (levamisole); Group C (ascorbic acid) and Group D (Control) of 15 chicks each. Response to ND vaccinations was determined through bleeding to obtain sera for haemagglutination inhibition test at 7 and 14 days except for the second booster with inactivated Komarov vaccine where it was done at 7, 14 and 21 days post vaccination respectively. Antibody titres in the chicks 7 days post first dose of vaccination with La Sota was high in all the treatment groups above the control with 100 % of the chicks having protective antibody titre of ≥4 log2 until day 30 when the antibody titres in all the groups dropped drastically following the second dose of live La Sota vaccination as the first booster vaccine. However, following the second booster with inactivated Komarov the antibody titres increased in all the treatment groups in comparison to the control especially in groups B and C with GMTs of 5.8 ± 0.19 log2 and 6.1 ± 0.27 log2 respectively. We observed that ascorbic acid and Levamisole may have humoral immuno-stimulating effects on vaccinated chickens through yet to be fully explored mechanism. It is recommended that ascorbic acid or levamisole could be used during vaccinations as immuno-stimulating agents to enhance humoral antibody response in vaccinated flocks.

Assessment of Different Newcastle Disease Virus Antigens and Inactivators of Binary Ethylene Amine and Formalin for the Hemagglutination Inhibition Assay.

Newcastle disease is a severe viral threat to the global poultry industry due to its high prevalence and rapid transmission. Evaluating vaccination timing and effectiveness is crucial, often accomplished through the hemagglutination inhibition (HI) assay. This test relies on the virus's agglutination ability in certain animals, utilizing various inactivated antigens. Our study aimed to assess multiple Newcastle viral antigens ( LaSota, clone, thermo-resistant strain, B1, and V4 ) inactivated by binary ethylene amine (BEA) and formalin, seeking the best antigen and inactivator for the HI assay. We prepared the different ND antigens include; LaSota, Clone, thermo resistant, B1, V4 and the mixture of the antigens then inactivated them using BEA and formalin. The hemagglutination (HA) assay determined mean titers, comparing BEA and formalin inactivation. These antigens were also subjected to the HI test using 112 serum samples from different commercial poultry flocks to assess their performance. BEA-inactivated antigens exhibited significantly higher mean titers in the HA assay than formalin-inactivated antigens. In the evaluation of different antigens in the HI test, the mean titer of antigen B1 followed by clone and LaSota displayed a higher mean titer than others. In conclusion, this study recommends using Hitchner pathotype antigens, specifically the B1 vaccine, for Newcastle disease HI testing. BEA is the preferred inactivator, preserving antigen structure particularly the structure of hemagglutinin antigen while minimizing risks. These findings can enhance serological testing accuracy, contributing to more effective disease control and prevention in the poultry industry.

Impact of early feed restriction and continued supplementation with coated organic acid and essential oils on sustainability of laying performance, egg quality, fertility, hatchability, immunity status, and gut microbiota of Japanese quail hens

Abstract Three hundred and sixty, 42-day-old Japanese quail (JQ) hens were randomly divided into six experimental groups with 5 replicates/group. Each replicate consisted of 12 birds (8 females and 4 males). During the growing period, chickens were fed restricted (FR) during only the second week of age at days 7, 9, 11, and 13. Three levels of FR were applied: 0.0 (control), 12.5 (12.5FR), and 25.0% (25FR) of the ad libitum intake of chickens measured on the previous day (groups 1, 2 and 3). In the other three groups (4, 5 and 6) the control, 12.5FR and 25.0FR diets were supplemented with coated organic acid + essential oils (COAWEO ) at 100 g/ton feed. The trial lasted from 6 to 18 weeks of age. Feed intake was significantly reduced by 12.5% FR compared with the control group. FR improved all external and internal egg quality of JQ hens except for egg weight, while albumen % decreased in the group fed 12.5FR diet. Coated organic acid with essential oils did not affect all JQ hens’ external and internal egg quality. Feed restriction did not affect fertility, pipped and dead chicks, and hatchability, as total and fertile egg percentage. COAWEO increased fertility and hatchability percentage. FR did not affect blood serum biochemistry and immunity characteristics except for high density lipoprotein (HDL) and hemagglutination inhibition test for infectious bursal disease virus (HI IBDV). Coated organic acid with essential oils did not affect quail blood characteristics but significantly decreased total lipids and increased IL6 at 18 weeks of age. Dietary COAWEO decreased E. coli, Clostridia, and Salmonella while increasing Lactobacillus counts. It can be concluded that 25.0% FR supplemented with COAWEO considerably improved the sustainability of productive performance of JQ hens without adverse effects on egg production, quality, fertility and hatchability traits, serum biochemistry and immune markers, and gut microbiota.

Evaluation of the Newcastle disease virus genotype VII–mismatched vaccines in SPF chickens: A challenge efficacy study

Newcastle disease virus (NDV) strains, while falling under a single serotype, are classified into distinct genotypes. Genotype VII virulent NDVs pose a significant threat to poultry due to their association with high mortality rates and economic losses. This study aimed to evaluate the efficacy of three commercial live vaccines based on genotype II against genotype VII virulent NDV (vNDV) in specific pathogen-free (SPF) chickens. Forty one-day-old chickens were randomly divided into four groups (n = 10) and inoculated with one dose of each ND pneumotropic vaccine—B1, Clone.12IR, and La Sota—or received phosphate-buffered saline (PBS) as a control at eight days of age via eye drop. At 28 days of age (20th post-vaccination days), chickens were intramuscularly challenged with genotype VII virulent NDV (≥ 105 LD50). Serum samples were collected at 28 days of age (challenge day), 7 and 14 post-challenge days to measure NDV antibodies via the hemagglutination inhibition (HI) test. Cloacal and oropharyngeal swabs were taken on the 3rd, 5th, 7th, and 10th post-challenge days to evaluate virus shedding. Vaccinated groups exhibited significantly higher antibody titers and greater protection levels compared to the control group (P≤ 0.001). While HI antibody titer was not different at 28 and 35 days of age between vaccinated chickens, the Clone.12IR groups showed higher HI antibody titer compared to B1 at day 42 of age (9.43 vs. 7.42; P≤ 0.002). La Sota and Clone.12IR vaccines demonstrated superior protection against mortality compared to the B1 vaccine (90 %, 80% vs. 60 %, respectively) with 6.0 and 2.67 odds ratio of survivability. All three mismatched vaccines effectively curbed the shedding of virulent genotype VII NDV, with 0 % to 11 % positive cloacal samples up to the 3rd post-challenge day. These findings demonstrate that in the experimental setting, the administration of mismatched ND vaccines, particularly La Sota and Clone.12IR, confer protection against genotype VII virulent NDV and control viral shedding, which can help to develop effective vaccination strategies to mitigate the impact of vNDV outbreaks in the poultry farms.

Etiological diagnostic methods and research progress of forest encephalitis

Forest encephalitis is a natural focal disease transmitted through the bite of hard ticks, and its pathogen is the tick-borne encephalitis virus from the Flaviviridae family. The mortality rate of forest encephalitis is relatively high, making laboratory testing significant in diagnosing this disease. This article elaborates on the etiological diagnostic methods and recent research progress in forest encephalitis. Laboratory tests for forest encephalitis mainly include routine examinations, serological tests, virus isolation, and molecular biological testing. The detection of serum-specific IgM antibodies against the forest encephalitis virus is of great importance for early diagnosis, and specific IgG antibodies serve as a "gold standard" for differentiation from other diseases. Techniques such as enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay for detecting specific IgM antibodies in serum and/or cerebrospinal fluid, the serum hemagglutination inhibition test or serum complement fixation test, and the double serum hemagglutination inhibition test or complement fixation test all contribute to the early diagnosis. The development of molecular testing methods is rapid, and techniques such as metabolomics, digital PCR, and matrix metalloproteinases are also applied in the early diagnosis of forest encephalitis.

Immunogenicity of inactivated H5 avian influenza vaccine used in commercial laying pullet in Tehran province

Highly pathogenic avian influenza (HPAI) is a viral disease caused by some H5 and H7 subtypes of influenza virus type A in most species of birds, especially poultry. HPAI viruses are amongst the most challenging viruses that threaten both human and animal health. Consequently, various strategies such as the use of vaccines have been proposed to control the disease. After a catastrophic pandemic and the failure of conventional methods (elimination and extermination) in Iran, multiple vaccines has been used to control the disease. This study investigates the immunogenicity of two recombinant inactivated commercial vaccines of H5N1 and H5N3 subtypes in laying pullet flocks in Tehran Province, Iran. From 32 halls in 6 breeding units of laying pullets, 3200 sera and 800 tracheal and cloacal swabs were collected. After collecting the samples, Serum neutralisation (SN) and hemagglutination inhibition (HI) tests were conducted on sera to determine the serum titers of H5 specific antibody obtained from vaccine inoculation in three steps: before, after first vaccination and after the second vaccination (booster). SN and HI tests were carried out by the alpha and beta methods on the pooled samples by the vaccine type (as antigen for HI and SN), and the results were compared. The PCR was performed on the tracheal and cloacal swab samples to possibly detect the HA (H5)virus in the studied flocks. The HI test results showed that both vaccines had a Serum antibody titre above 5 (log2) after two vaccination rounds, indicating a desirable immunogenic response. The SN test results also showed a neutralisation index above 104.5 for both vaccines, indicating more than 50% reduction in antigenicity of the virus. The PCR results were negative. This study was the first investigation of immunogenicity following two-time vaccination against H5 subtype vaccines in Iranian poultry flocks, indicated suitable antibody titer against the influenza virus in vaccinated flocks.

THE EFFECT OF NATURAL GUARD ADMINISTRATION IN DRINKING WATER ON ND-ANTIBODY TITERS IN BROILERS

The broiler is the largest sector producing protein sources in Indonesia. Poultry farms are susceptible to viral diseases, including the Newcastle Disease Virus (NDV). Viral diseases can be prevented by vaccination to increase antibody titters in broiler chickens. Induction of antibody titters can be increased by providing additional ingredients such as immune system stimulants known as immunomodulators. One material that can be used as an immunomodulator is the NATURALGUARD (NG). This research was conducted to determine the effect of NG supplementation in drinking water on ND antibody titters in broilers. This study used a completely randomized design with four treatments, namely P0 as a control without NG treatment, and P1, P2, and P3 were given NG treatment at doses of 1, 2, and 4 g/L, respectively adlibitum for 30 days starting at three days of age. Blood samples were taken on day one before treatment, 15 and 30 days after treatment for the hemagglutination inhibition (HI) test. The results showed that maternal antibodies against ND on the first day were protective but not uniform; the highest mean HI 4 log 2 titter was at P0 (512 ± 280.43) and the lowest at P2 (384 ± 140.21). The decrease in antibodies occurred at the age of 18 days to the highest at P0 and P3, namely 16 ± 0, and the highest at P2 (16.66 ± 4.13), and at the age of 33 days, the lowest titter was at P1 (4 ± 0) and the highest was at P2 and P3 (10±6.57). It can be concluded that NATURALGUARD supplementation shows an immunomodulating effect in broiler chickens that have not been re-vaccinated with Newcastle Disease through a slower regulation of decreasing antibody titters at NG supplementation doses of 2 and 4 g/L.

Serological Monitoring of Pandemic Influenza Virus Markers in the Russian Federation in 2021–2023

State Scientific Center of Virology and Biotechnology “Vector” has been monitoring highly pathogenic influenza since 2005.The aim of this work was to track the markers of highly pathogenic influenza in the blood sera of people who had a contact with infected and/or deceased birds, as well as of residents from regions where emergence of new variants of influenza A virus is most likely to occur.Materials and methods. Sera were studied using hemagglutination inhibition test (HI test). HI-positive sera were subjected to virus neutralization reaction.Results and discussion. In 2021, 2076 blood serum samples from 19 regions of Russia were collected. Only 7 samples demonstrated significant titers in HI test with A/H5N8 viruses. In 2022, 1620 blood serum samples from 23 regions were obtained; 25 of them were positive for influenza А/H5N8 and А/H5N1 viruses. In 2023 (January-August), 3335 serum samples from 31 regions of the Russian Federation were collected. 28 samples were positive for influenza А/H5N8 and А/H5N1 viruses. Furthermore, we monitored blood sera for low-pathogenic A/H9N2 virus. The number of positive samples in 2021 was lower than 1 % (13 out of 2076); in 2022, it reached 5 % (81 out of 1620); in 2023, the share was lower than 1 % (31 out of 3335). The data obtained suggest indirectly that currently there is no stable circulation of zoonotic influenza A/H5N8 and A/H5N1 viruses in Russia. Influenza viruses A/H9N2 have widely spread in many countries of the world and actively participate in evolution of highly pathogenic influenza A/H5Nx viruses. The Russian Federation demonstrates a gradual increase in the number of blood serum samples with antibodies to A/H9N2 virus.

Cytokine expression, protection and shedding reduction induced by the combination of lipopolysaccharide with Montanoid ISA71 in oil-based Newcastle disease vaccine

Oil-based inactivated ND vaccines are a commonly used control strategy for this endemic disease in Egypt. One of the major limitations of these inactivated vaccines is the time taken to develop a protective response in vaccinated birds. In the present study, we aimed to formulate an inactivated oil-based ND vaccine incorporated with lipopolysaccharide (LPS) that stimulates the early onset innate response to inactivated vaccines via proinflammatory cytokine production. Five groups of 21-day old SPF chicks were reared in isolators and were treated as follows: G1: Montanoid ISA71 adjuvanted NDV vaccinated group, G2: LPS and Montanoid ISA71 adjuvanted NDV vaccinated group, G3: LPS and Montanoid ISA71 with phosphate buffer saline received group and two non-vaccinated control groups. NDV specific antibodies and cell mediated immune responses were evaluated by hemagglutination inhibition and lymphocyte proliferation tests, respectively. Transcriptional responses of the TLR4, IFN-γ and IL-2 genes were analyzed in peripheral blood mononuclear cells (PBMCs) following vaccination by qRT-PCR. Protection % was determined after challenge with a lethal strain of NDV 106 EID50/0.5 ml. Viral shedding was assessed on oropharyngeal swabs by qRT-PCR and infectivity titration on SPF-ECE. The results revealed that the incorporation of LPS with ISA71 in the oil-based ND vaccine induced a synergistic response confirmed by significant humoral and lymphoproliferative responses with a significant increase in Th1 cytokine transcripts. The simultaneous use of both adjuvants in G2 demonstrated complete protection and a significant reduction in viral shedding compared to the ISA71-adjuvated ND vaccine in G1, which conferred 90 % protection.

Molecular and serological detection of Newcastle disease virus in live-bird markets, Jos, Plateau State Nigeria.

Newcastle disease (ND) is a highly infectious viral disease of birds caused by the Newcastle disease virus (NDV) and it has been reported in domestic birds in Nigeria. Waterfowls and village poultry in live bird markets (LBM) acts as reservoirs, potentially reintroducing the virus to commercial poultry. This study aims at molecular and serological detection of NDV at live bird markets in Plateau State, Nigeria. A cross sectional analysis involved 309 pooled cloacal and tracheal swabs over three months were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR), RT-qPCR, virus isolation and haemagglutination and haemagglutination-inhibition test (HA &HI). Virus isolation was attempted in 9 to 11 days old specific antibody negative (SAN) embryonated chicken eggs and fifteen samples showed haemagglutination. Subsequent tests confimed nine of these as NDV positive through haemagglutination and haemagglutination inhibition (HI) tests. ConventionalRT-PCR and RT-qPCR further validated five of the nine NDV positive isolates. The phylogenetic analysis of partial F gene nucleotide sequences revealed that all three isolates belonged to class II genotype XIV.2_XIVb. This finding underscores the persistent threat of NDV to local poultry, necessitating comprehensive virological surveillance to understand, isolate and characterize the virus in Nigeria. Therefore, monitoring for emerging lineages and sub-lineages in Nigeria birds is crucial for safeguarding commercial poultry production.

A novel lateral flow immunochromatographic assay using a recombinant VP2 antigen for total antibody detection of canine parvovirus-2.

Canine parvovirus-2 (CPV-2) is a viral disease of dogs causing acute hemorrhagic gastroenteritis and myocarditis with high morbidity and mortality rates. The infection is still widespread all over the world. Vaccines developed against infection have great importance in preventing infection. However, it is difficult to recommend a practical vaccination program without knowing the antibody level of a puppy. Despite widespread vaccination, difficulties in detecting the maternal antibodies in puppies remain the main cause of vaccination failure. The hemagglutination inhibition (HAI) test is the gold standard to determine the immune status of dogs for canine parvovirus 2, but the HAI test has several disadvantages such as the need for fresh porcine blood, well-equipped laboratory, and long incubation periods. In this study, for the first time we developed a colloidal gold-based competitive lateral flow assay (cLFA) system for the rapid detection of total antibodies in canine serum using CPV-2b-VP2 derived from field isolates. The recombinantly expressed capsid protein of CPV-2 in the prokaryotic expression system was used as a labeled molecule in cLFA. We carried out studies on our cLFA system using the standard antibody solution and the clinical samples from vaccinated puppy serum. We compared the results of the LFAs with the HAI test. Competitive lateral flow assay results showed good correlation with the gold standard method, the HAI test. In the developed platform, the limit of detection of the standard antibody was determined to be 375 ng mL-1, while the cut-off level of antibodies was observed to be 1 : 40 HAI titer in clinical samples. Our reported system will be a strong alternative for CPV-2 antibody-based detection applications.

Characterization of the dominant strain (G-VII) of Newcastle disease viruses isolated from commercial chickens in Bangladesh during recent outbreaks.

Newcastle disease virus genotype VII (NDV-GVII), an extremely infectious pathogen, has been causing severe economic consequences for the chicken industry. The current study aimed to isolate and characterize NDV-GVII from commercial chickens in Bangladesh during a recent outbreak. From clinically suspected chickens from 70 commercial poultry farms, a total of 420 samples (trachea, lungs, and brain tissue) were collected. The samples were cultivated in 9-10 day-old seronegative embryonated chicken eggs (ECEs) after evaluating them using the rapid Newcastle disease virus (NDV) antigen detection kit. The hemagglutination (HA) inhibition test, agar gel immune diffusion (AGID) test, molecular detection by reverse transcription-polymerase chain reaction (RT-PCR), and phylogenetic studies using gene sequences of fusion (F) protein. The HA pattern of isolated NDV was determined using different avian and mammalian red blood cells (RBCs). The pathogenicity of the isolated virus was evaluated using mean death time (MDT), intravenous pathogenicity index (IVPI), and intracerebral pathogenicity index (ICPI). The study found 87 NDV samples positive using the rapid NDV Ag detection kit and then 60 positives for virus isolation in ECEs. All 60 isolates were positive for NDV by HI, AGID, and RT-PCR. Phylogenetic tree analysis indicated that recent NDV isolates belong to genotype VII and exhibit a similarity of 99.7%-98.5% with isolates from Bangladesh, Iran, and India. The new isolates, identified as velogenic strains of NDV, possess an F protein cleavage site with 112-R-T-K-R-F-117 amino acid motifs. The isolated NDV showed diversified HA activity while using RBCs from birds and mammals. The results of ICPI, IVPI, and MDT indicated that the recent NDV isolates were very virulent. This study concluded that NDV-GVII is prevalent in commercial poultry farms in Bangladesh.

Clinical Diagnostic Immunology in A Clinical Microbiology Laboratory

This review unravels the novel approaches, innovations and applications of immunology in the diagnosis of infections in a clinical microbiology laboratory. Many assays or tests in immunology are used in the diagnosis of infections. The availability of monoclonal antibodies has brought rapid progress to this area and they are used , for example , to identify specific microorganisms in culture or in tissues and fluids from infected patients. Examples of common diagnostic applications of antibody- antigen reactions discussed here includes agglutination tests, complement tests, complement fixation tests, ELISA, cytotoxic tests, radioimmunoassay tests, haemagglutination inhibition tests, hypersensitivity tests, phagocytic cell activity test, migration inhibition test, lymphocyte transformation test , lymphocyte fixation test , skin tests, delayed hypersensitivity tests and many others. The development of monoclonal antibody technology and the subsequent availability of unlimited quantities of pure antibody has improved the selectivity of these assays. The development of simple and highly sensitive methods for detection of nucleic acids is being applied to diagnosis of infection using nucleic acid hybridization and this provides a valuable additional aid to immunoassays in the clinical microbiology diagnostic laboratory.

Effect of Vaccination on Distribution and Immune Response of Avian Influenza Virus H9N2 in Coturnix coturnix.

Influenza viruses can multiply in quails and be transmitted to other animal species. As vaccination reduces virus shedding in chickens, the effect of the killed H9N2 avian influenza virus (AIV) on tissue distribution and virus shedding was evaluated in quails. One hundred 20-day-old quails were divided into six equal groups, kept in separate pens, and fed ad libitum. Before vaccination, blood samples were randomly collected from the wing veins. Four groups were vaccinated with the inactivated H9N2 Razi Institute vaccine at 21 days subcutaneously at the back of neck. Three weeks later, two groups were re-vaccinated. Two weeks later, at the age of 56 days, three groups were challenged with 100 μL of allantoic fluid containing 105 EID50 H9N2 through the oculonasal route. Blood samples were collected from quails at 42, 56, 63, and 70 days from each group to determine AIV antibodies by the hemagglutination inhibition test. Three quails were randomly selected and euthanized from each group on days 1, 3, and 6 post-inoculation (PI). Tissue samples were collected, and the RT-PCR test was performed. No clinical signs or gross lesions existed in any of the groups during the experiment. However, the virus was detected in different tissues on the first, third, and sixth days after the challenge in unvaccinated challenged birds. Virus detection was significantly more frequent in the quails vaccinated once and challenged than in the twice-vaccinated challenged group (P≤0.05). On the third day of PI, the virus was detected in some organs of the challenged groups. On the sixth day of PI, the virus was detected only in the lungs of two unvaccinated and once-vaccinated challenged birds. It was concluded that the vaccination of quails against AIV H9 is necessary to protect them from clinical signs, as well as respiratory tract and intestine replication. Two-time vaccination significantly protects the respiratory and intestine tracts, compared to one-time vaccination (P≤0.05).

Antiviral efficacies of neem (Azadirachta indica J.) and scent (Ocimum sanctum L.) leaf meals against Newcastle and infectious bursal disease in broiler chickens

The effects of neem (Azadirachta indica) and scent (Ocimum santum) leaf meals fed singly or in combination on the growth response, haemato-biochemical parameters and their antiviral potencies in broiler chickens were investigated. Two hundred 1-day old Ross 308 unsexed broiler chicks were randomly allotted to 5 treatments, consisting of 5 replicates with 8 birds each. Treatments 1 and 2 were basal diets with no leaf meals, treatments 3, 4 and 5 had the basal diets plus 3% neem leaf meal (NLM), 3% scent leaf meal (SLM) and the blend of 2% NLM + 2% SLM, respectively. Birds in treatments 2-5 were unvaccinated against Newcastle disease and Infectious bursal disease while birds in treatment 1 were vaccinated. On days 21 and 42, blood samples were randomly collected from 2 birds per replicate for haemato-biochemical assay, haemagglutination inhibition and agar gel precipitation tests. Data were subjected to descriptive statistics and ANOVA at p<0.05. At the starter phase, an improved feed conversion ratio (p<0.05) was recorded in birds fed the blend at the starter (1.83) and finisher (1.92) phases and the overall performance (1.88) compared to other treatment groups. The least platelet counts (11.08 ×104/µL) were recorded in birds placed on the blend of 2% neem leaf meal + 2% scent leaf meal while the 3% scent leaf meal group recorded the lowest lymphocyte counts (53.60%). At the finisher phase, lymphocyte counts of birds that received the blend of 2% neem leaf meal + 2% scent leaf meal (63.00%) were higher than the other treatments. In conclusion, birds fed diets supplemented with 3% neem and 3% scent leaf meals singly had haemagglutination inhibition titre values and conferred immunity above the protective threshold against Newcastle disease and produced antibodies against Infectious bursal disease. Birds fed the blend of 2% neem + 2% scent leaf meal diets had improved overall performance.

Evaluation of hemagglutination inhibition test for canine respiratory coronavirus antibodies and seroprevalence in Korean dogs

Canine respiratory coronavirus (CRCoV) is a significant pathogen that causes respiratory diseases in dogs, collectively known as a canine infectious respiratory disease. The virus is highly contagious and exhibits high seroprevalence worldwide. Currently, bovine coronavirus (BCoV) enzyme-linked immunosorbent assay (ELISA) kits are used to detect CRCoV antibodies. However, BCoV-ELISA kits cannot differentiate between infections caused by BCoV and those caused by CRCoV. In this study, we evaluated the hemagglutination inhibition (HI) test for CRCoV by comparing it with the virus neutralization (VN) test. Subsequently, we evaluated the seroprevalence of CRCoV in 383 dog serum samples collected from South Korea utilizing the HI test. The HI test for CRCoV showed a strong correlation with the VN test (R = 0.83, p < 0.001). The analysis of seroprevalence revealed that 52.2% (95% confidence interval [CI], 47.2%–57.1%) of the Korean dog serum samples were positive. The seroprevalence exhibited varied with age, with a positivity rate of 43.9% in dogs under 1 year of age and 66.7% in dogs aged 3 to 5 years (odds ratio [OR], 2.54; 95% CI, 1.43–4.59). In conclusion, the HI test to monitor CRCoV antibody proved to be closely related to the VN test. Furthermore, over half of the dogs in Korea tested positive for CRCoV antibodies. These findings contribute to a better understanding of the sero-epidemiology of CRCoV.

ANTIBODY TITERS AGAINST NEWCASTLE DISEASE AND INFECTIOUS BRONCHITIS VIRUSES IN BROILER BREEDERS RAISED UNDERFLOOR VERSUS CAGE SYSTEM

The experiment was evaluated to find out the serum antibody titer of broiler breeders raised under different rearing systems (cage vs. floor) against Newcastle disease virus and infectious bronchitis disease virus. Two hundred and forty-day-old broiler breeders were equally divided into six groups Floor (V+CH), (V+Not CH) and (Not V+CH) and cage (V+CH), (V+Not CH), and (Not V+CH). Birds were vaccinated with Mukteswar, La Sota, and Massachusetts M41 in 1st week, 3rd week, and 4th week. Blood /serum samples were collected at 42 days of age. Collected data were analyzed by using a complete randomized design. The geometric mean titer (GMT) for Newcastle disease virus (NDV) of broiler breeders reared under floor versus cage housing system was measured by using the hem agglutination inhibition test and the defensive potentiality of vaccines was measured by determining the survivability rate of chickens by challenge infection. Birds vaccinated and virus-challenged showed a protective antibody titer against Newcastle disease virus in both housing systems. Significantly higher GMT was recorded for the non-vaccinated group having virus-challenged in cage female (9.76 ±0.34) and floor female (9.94 ±0.23). Protective antibody titer was calculated for broiler breeders who were regularly vaccinated. There were no significant differences (P≥0.05) observed in the cage versus floor housing system. Higher serum antibody titer in caged females (9.22±0.36) and floor (8.98±0.32) was calculated due to infection challenges. Morbidity and mortality were significantly affected by the cage and floor-rearing system. Frequencies of diseased birds and mortality were found higher on the floor compared to cages in the non-vaccinated group. Despite the regular vaccination sick female broiler breeders were found higher compared to cage broiler breeders. In the non-vaccinated group floor reared birds have a higher percentage of morbidity and mortality compared to cages. In conclusion cage and floor-rearing systems have no adverse effect on serum antibody titer and survivability of broiler breeders.