Immunosensor-based rapid quantitative detection of Newcastle disease virus antibodies using innovative gold immunochromatographic assay.

AIMS: A novel quantitative method for rapid Newcastle disease virus (NDV) antibody detection was developed based on an innovative gold immunochromatographic assay with a quantitative immunosensor. METHODS AND RESULTS: NDV antibody-detecting test strips containing a two-reaction system and double-test lines (T1, T2) were prepared. The test results were judged according to the signal ratio between the test and control lines as measured by the quantitative immunosensor. The minimum detection limit of the test strips for NDV antibodies was 22 titres. In addition, the assay was highly specific because only NDV antibodies produced visible test lines on the strip. The clinical application of the strips was tested by detecting NDV antibodies in 506 serum samples collected from chickens. The results showed a coincidence of 92·49% with those of the haemagglutination inhibition assay. CONCLUSIONS: The strips were successfully prepared and showed high specificity towards NDV, sensitivity and stability. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a new method for detection of NDV antibody and provides a reference basis for rapid and quantitative monitoring of NDV antibodies. This new method overcomes the limitation of the existing colloidal gold immunochromatography, which only produces qualitative or semi-quantitative results.

Seroprevalence and molecular detection of Newcastle disease virus in backyard chickens in Tripoli, Libya.

Background: Newcastle disease (ND) is a viral disease that affecting many avian species all over the world. Aim: ND has been successfully controlled by the vaccination of commercial poultry in Libya. However, there was a lack of information about the situation of ND in backyard chickens. Therefore, this study determined the prevalence of ND in backyard chickens in different locations of Tripoli. Methods: A total number of 280 cloacal swabs (190 in summer and 90 in winter) and 412 sera were collected from non-vaccinated backyard chicken flocks in different geographical locations within the area of Tripoli namely Qasr Ben Ghashier, Al-Sawani, Souq Al-Gomaa, Tajourah, Ein Zara, and Janzour. Cloacal swabs and sera were tested by real time polymerase chain reaction (PCR) and ELISA, respectively. Results: The prevalence of ND virus (NDV) infection in backyard chickens in different locations of Tripoli during summer and winter was 45% using real-time reverse transcription-PCR. Except in Qasr Ben Ghashier, the prevalence in summer season was significantly higher than in winter (X2 = 46.13, p ≥ 0.00001). ELISA test revealed 218 positive out of 412 tested samples with total prevalence of 53% across the city of Tripoli in all regions. Obviously, Qasr Ben Ghashier had significantly (X2 = 74.09, p ≥ 0.00001) the highest prevalence (82%) of NDV specific antibodies followed by Tajourah (68%). Conclusion: This study demonstrated the situation of ND in backyard chicken highlighting the necessity of a comprehensive vaccination plan for backyard chickens.

A comparative evaluation of serum biochemistry profile and antigenic relatedness among velogenic and mesogenic Avian avulavirus 1 infection in chickens and pigeons.

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects variety of avian species around the globe. Several AAvV 1 viruses of different genotypes have recently emerged with varying clinical impacts on their susceptible hosts. Although experimental infection with velogenic and mesogenic strains in chickens and pigeons is well-studied, nevertheless, there exists a paucity of data for comparative variations in serum biochemistry profile of susceptible hosts upon challenge with isolates of varying pathogenicities. With this background, a comparative assessment of a range of serum biochemical parameters was made following challenge with duck-originated velogenic strain (sub-genotype VIIi; MF437287) and pigeon-originated mesogenic strain (sub-genotype VIm; KU885949) in chickens and pigeons. For each of the isolate, commercial broiler chickens and wild pigeons were challenged (10-6.51 EID50/0.1 mL for sub-genotype VIIi and 10-6.87 EID50/0.1 mL sub-genotype Vim) separately via intranasal and intraocular route. Sera were collected on 0, 3rd, 5th, 7th, and 9th day post-infection (dpi), and processed for quantitative analysis of different biochemical parameters. By day 3 post-infection (pi), a substantial decrease (p < 0.0001) in serum alkaline phosphatase (ALP) was observed in chickens and pigeons challenged with velogenic isolate. On the other hand, from day 5 pi and onward, a significant increase (p < 0.001) in serum ALP and total protein concentration was observed exclusively in pigeons challenged with mesogenic isolate. For serum aspartate aminotransferase (AST), a significant increase (p < 0.05) in concentration was observed on day 3 pi which decreased from day 5 pi and onward in pigeons and chickens challenged with mesogenic isolate. Also, to reveal antigenic differences among homologous and heterologous vaccine and field-prevalent strains, cross-hemagglutination inhibition assay demonstrated antigenically diverse nature (R-value < 0.5) of both strains from vaccine strain (LaSota, genotype II). The study concludes antigenic differences among prevalent genotypes than vaccine strain and, although requires further studies to ascertain study outcomes, the serum biochemical profile may facilitate presumptive diagnosis of disease in their susceptible hosts.

Development of a recombinant NP protein based indirect ELISA for the detection of antibodies against Newcastle disease virus and differentiation of infected or recombinant vaccine immunized chickens.

Newcastle disease (ND) is a highly contagious and economically important disease in the poultry industry caused by avian avulavirus-1, historically known as Newcastle disease virus (NDV). Control of ND primarily relies on prophylactic vaccination of flocks, and many vaccines are available on the market, both conventional and more recently introduced new generation recombinant types. To assess the protection level achieved by vaccination ELISA tests are typically used, they also are to track an infection with field strains in non-vaccinated flocks. Special modifications of ELISA can be used as a screening tool to detect infection in flocks vaccinated with new generation vaccines. In this study, we have developed an ELISA test for the detection of antibodies against the nucleoprotein (NP) of NDV and for differentiation of chickens vaccinated with commercial and prototype in-house recombinant vector vaccines from those infected with field NDV strains. The NP gene of LaSota NDV strain expressed in a baculovirus vector was used as a coating antigen in the ELISA. The developed test was optimized, validated and compared to other serological tests. The sensitivity, specificity and accuracy of recombinant NP protein-based ELISA were respectively 96.1%, 96.3%, and 96.2%. Inter-rater (kappa) agreement between the NP-ELISA and the gold standard HI test was calculated to be 0.995. In our comparisons, commercially available ELISA tests revealed different specificities ranging from 95.5-100% and sensitivities at variance, ranging from 90.1 to 99.0%. A high level of maternally derived antibodies was measured in the serum of 1-day-old broilers in the NP-ELISA assay. These antibodies had disappeared and were undetected at 3, 5 and 6 weeks post-vaccination but birds became positive again at 2 weeks after control infection with a velogenic NDV strain. In SPF chickens, antibodies against NP protein were detected only after a challenge. The recombinant NP protein-based ELISA test is sensitive, specific and accurate when compared to the gold standard HI test and commercially available kits. Moreover, the method could be also used for the differentiation between vaccinated and infected birds.

In vivo cell-mediated immune, hemagglutination inhibition response, hematological and biochemical values in native vs. exotic chicken breeds.

Birds (364) of both sexes, 11-wk-old, belonging to 2 native (Brown Nicobari and Ghagus) breeds and 1 exotic breed (Dahlem Red) were evaluated for cell-mediated immune response (CMI) by phytohemagglutinin-P (PHA-P), hemagglutination inhibition (HI) assay against Newcastle disease virus (NDV) antigen (LaSota stock virus), flow cytometric analysis of CD8+ cytotoxic T lymphocytes (CTLs), and hematology and biochemical assays. The cutaneous basophil hypersensitivity response PHA-P% increase in wattle thickness (mm) was highest in Ghagus (431.14 ± 22.56) which differed significantly with that of Brown Nicobari (269.1 ± 22.66) and Dahlem Red (218.42 ± 22.30). Sex-wise observation showed that females are having significantly higher response than males. Hemagglutination inhibition test was performed to determine the serum antibodies against Newcastle disease (ND) virus. Brown Nicobari showed highest HI antibody titer than Ghagus and Dahlem Red to similar vaccination program after booster NDV dose. Flow cytometry assay revealed significantly higher CTLs proliferation in Brown Nicobari than Ghagus and Dahlem Red. Moreover, CTLs were found to be higher in control group than the treatment group. Other hematological parameters (103/µL) significant difference was found in white blood cell count between Dahlem Red (38.41 ± 1.03) with that of Brown Nicobari (35.28 ± 1.04) and Ghagus (34.57 ± 1.04) in treatment groups. Same trend was observed in the Lymphocyte treatment group. However, in Granulocyte treatment group, Brown Nicobari (11.04 ± 0.35) was found to be significantly different from Dahlem Red (8.68 ± 0.34) and Ghagus (9.27 ± 0.35). Correlations between body weight at 11 wk of age and CMI, HI, cytotoxic T cell were -0.093, 0.047, and -0.036, respectively. Egg weight was found to be positively correlated with that of chick weight. Serum biochemical values showed that Dahlem Red was having significantly higher creatinine levels compared to Ghagus. Triglycerides level was also significantly higher in Ghagus compared to Dahlem Red. No significant breed effect was observed for alkaline phosphate, aspartate transaminase, and alanine transaminase. Cholesterol and total serum protein levels were significantly higher in Dahlem Red compared to Brown Nicobari.

Development of a novel Newcastle disease virus (NDV) neutralization test based on recombinant NDV expressing enhanced green fluorescent protein.

BACKGROUND: Newcastle disease is one of the most important infectious diseases of poultry, caused by Newcastle disease virus (NDV). This virus is distributed worldwide and it can cause severe economic losses in the poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. Protection against NDV in chickens has been associated with development of humoral response. Although hemagglutination inhibition (HI) assay and ELISA do not corroborate the presence of neutralizing antibodies (nAbs); they are used to measure protection and immune response against NDV. METHODS: In this study, we established a system to recover a recombinant NDV (rLS1) from a cloned cDNA, which is able to accept exogenous genes in desired positions. An enhanced green fluorescent protein (eGFP) gene was engineered in the first position of the NDV genome and we generated a recombinant NDV carrying eGFP. This NDV- eGFP reporter virus was used to develop an eGFP-based neutralization test (eGFP-NT), in which nAbs titers were expressed as the reciprocal of the highest dilution that expressed the eGFP. RESULTS: The eGFP-NT gave conclusive results in 24 h without using any additional staining procedure. A total of 57 serum samples were assayed by conventional neutralization (NT) and eGFP-NT. Additionally, HI and a commercial ELISA kit were evaluated with the same set of samples. Although HI (R 2 = 0.816) and ELISA (R 2 = 0.791) showed substantial correlation with conventional NT, eGFP-NT showed higher correlation (R 2 = 0.994), indicating that eGFP-NT is more accurate method to quantify nAbs. CONCLUSIONS: Overall, the neutralization test developed here is a simple, rapid and reliable method for quantitation of NDV specific nAbs. It is suitable for vaccine studies and diagnostics.

Longitudinal Study of Avian Influenza and Newcastle Disease in Village Poultry, Mali, 2009-2011.

Newcastle disease (ND) is endemic in West Africa, which has also experienced outbreaks of highly pathogenic avian influenza (AI) H5N1 since 2006. We aimed to estimate the prevalence and incidence of AI and ND in village poultry in Mali and to identify associated risk factors. A longitudinal serologic study was conducted between November 2009 and February 2011 using ELISA commercial kits to detect antibodies. Sera (5963) were collected from 4890 different poultry. AI was rare, with a seroprevalence of 2.9% (95% confidence interval [CI] 2.3-3.5) and a seroincidence rate of 0.7 birds per 100 bird-months at risk (95% CI 0.4-1.0). AI antibodies were short lived, with a seroreversion rate of 25.4 birds per 100 bird-months at risk (95% CI 19.0-31.7). Risk factors for AI were limited: temporal variation occurred, but proximity to a water body was a risk factor only when large populations of wild waterbirds were present. ND was very common, with seroprevalence of 68.9% (95% CI 61.9-76.0) and a seroincidence rate of 15.9 birds per 100 bird-months at risk (95% CI 11.9-19.8). ND seroreversion rate was 6.2 birds per 100 bird-months at risk (95% CI 3.6-8.9). Regarding risk factors for ND, temporal variations occurred, and ND was more likely to be present in the Sudanian agro-ecological zone than in the Sahelian zone, in chickens than in other species, in flocks with higher numbers of Guinea fowl, and in flocks that had access to a waterbody. Control efforts would benefit from further increasing the ND vaccination coverage of village poultry, although this was already quite high (54.9%) for an African country. Seroconversion seemed satisfactory in vaccinated poultry, since 90.0% (95% CI 87.6-92.4) of these had ND antibodies. Further research should investigate the apparent lack of an epidemiologic role of domestic ducks for AI in Mali (unlike in Southeast Asia) and the potential role of Guinea fowl as a reservoir for ND.

Atrophy of the lymphoid organs and suppression of antibody response caused by velogenic Newcastle disease virus infection in chickens.

This project was undertaken to study the immunosuppressive capabilities of velogenic viscerotropic pathotype of Newcastle disease virus (VVNDV) infection in cockerels. Two hundred six-week-old cockerels were divided into four groups. Groups B/VUC and C/VC were vaccinated with LaSota in drinking water at 6 weeks of age. Groups C/VC and D/UC were challenged with VVNDV at 8 weeks of age. Three days post challenge (PC), the cockerels in group D/UC came down with clinical signs which included depression and greenish diarrhoea. Total mortality was 74.6 %. The C/VC cockerels showed no clinical signs. But both challenged groups showed significant weight loss, significant loss of total serum proteins, globulin and albumen (P < 0.05). These losses were more severe in the D/UC than in the C/VC. There was severe atrophy of the bursa, spleen and thymus in both groups. Histopathology showed severe necrosis and depletion of the lymphocytes in the three lymphoid organs. However, the lesions were more severe in the D/UC than in C/VC cockerels. On day 28, PC groups B/VUC, C/VIC and D/UIC were revaccinated with LaSota. The haemagglutination inhibition antibody response on days 35, 42 and 49 PC was very low in groups C/VIC and D/UIC when compared with B/VUC cockerels. These observations show that VVNDV infection both clinical and subclinical can cause immunosuppression and vaccine failure due to severe destruction of the lymphocytes in the lymphoid organs. This will be a serious problem for poultry production in those countries where the disease is enzootic.

Modification of lily polysaccharide by selenylation and the immune-enhancing activity.

Lily polysaccharide (LP) was extracted, purified and selenizingly modified by HNO3-Na2SeO3 method according to L9(3(4)) orthogonal design. Nine selenizing LPs, sLP1-sLP9, were obtained and their immune-enhancing activities were compared taking unmodified LP as control. The results in vitro test showed that sLP6 presented the strongest activity in promoting lymphocytes proliferation in single and synergetic with PHA, and the relative expression level of IL-2, IL-6 and IFN-γ mRNA of chicken peripheral lymphocytes. The results in vivo test showed that sLP6 could promote lymphocytes proliferation and enhance the serum antibody titers and serum IL-2, IL-6, IFN-γ contents more significantly than LP in chickens vaccinated with Newcastle Disease (ND) vaccine. These results indicate that polysaccharide selenizing can significantly enhance the immune-enhancing activity of LP and the optimal modification conditions are 400 mg of Na2SeO3 per 500 mg of LP, the reaction temperature of 70 °C and the reaction time of 6 h.

Antiviral effect of sulfated Chuanmingshen violaceum polysaccharide in chickens infected with virulent Newcastle disease virus.

Newcastle disease virus (NDV) belonging to the Paramyxovirinae subfamily is one of the most devastating pathogens in poultry. Although vaccines are widely applied to control the infection, outbreaks of Newcastle disease (ND) repeatedly happen. Currently, there are no alternative control measures available for ND. In the present study, we found that sulfated Chuanmingshen violaceum polysaccharide (sCVPS) were potent inhibitors of NDV in specific pathogen free chickens infected with a virulent strain. With sCVPS treatment, the survival rate increased by almost 20% and virus titers in test organs, including brain, lung, spleen and thymus, were significantly decreased. The sCVPS also exhibited the ability to prevent viral transmission by reducing the amount of virus shed in saliva and feces. Higher concentrations of interferon α and γ in serum were detected in chickens treated with sCVPS, indicating that one of the antiviral mechanisms may be attributed to the property of immunoenhancement. Histopathological examination showed that sCVPS could alleviate the tissue lesions caused by NDV infection. These results suggest that sCVPS are expected to be a new alternative control measure for NDV infection and further studies could be carried out to evaluate the antiviral activity of sCVPS against other paramyxoviruses.

[Comparative research into sensitivity and specificity of immune-enzyme analysis with chemiluminescence and colorimetric detection for detecting antigens and antibodies to avian influenza viruses and newcastle disease].

The goal of this work was to demonstrate the results of the development of the enzyme-linked immunosorbent tests with chemiluminescence detection and colorimetric detection of specific viral antigens and antibodies for identifying the avian influenza and the Newcastle disease viruses: high sensitivity and specificity of the immuno- chemiluminescence assay, which are 10-50 times higher than those of the ELISA colorimetric method. The high effectiveness of the results and the automation of the process of laboratory testing (using a luminometer) allow these methods to be recommended for including in primary screening tests for these infectious diseases.

Development of a duplex Fluorescent Microsphere Immunoassay (FMIA) for the detection of antibody responses to influenza A and newcastle disease viruses.

Highly pathogenic avian influenza virus (HPAI) and virulent forms of avian paramyxovirus-1 (APMV-1) cause serious illnesses in domestic poultry, both of which are reportable to the World Organization of Animal Health (OIE). The clinical presentation of avian influenza (AI) and APMV-1 infections are difficult to differentiate, emphasizing the importance of rapid and sensitive serologic assays that are able to distinguish them. Currently, a variety of serological assays are used for the serologic diagnosis of both diseases, but these assays are not used in multiplex formats. In this study, development of a duplex fluorescent microsphere immunoassay (FMIA) based on Luminex xMAP Technology is described. The assay employs MagPlex magnetic microspheres that are covalently coated with recombinant avian influenza virus nucleoprotein and APMV-1 nucleocapsid antigens produced in a baculovirus insect cell expression system. The assay is able to detect AIV antibodies against all existing hemagglutinin (H1-H16) subtypes and simultaneously detect antibodies against APMV-1. In the process of this assay development different bead coupling conditions were compared. The assay has the capability of detecting serum antibodies from chickens and turkeys and optimization was accomplished by using 2462 chicken and 446 turkey field and experimental sera and had a comparable detection capability with currently used assays in the laboratory. Assay threshold values were calculated with Receiver Operating Characteristic Analysis (ROC) in non-parametric analysis due to a highly skewed data distribution; this analysis resulted in AIV nucleoprotein relative diagnostic sensitivity and specificity of 99.7%, and 97.3% respectively. The APMV-1 nucleocapsid relative diagnostic sensitivity and specificity were 95.4%, and 98.5% respectively.

The adjuvanticity of Ganoderma lucidum polysaccharide for Newcastle disease vaccine.

The adjuvant activity of GLP was investigated in vitro and in vivo. In vitro experiment, the effects of GLP on chicken peripheral lymphocytes proliferation were compared by MTT assay. The results showed that GLP could significantly enhance lymphocytes proliferation singly or synergistically with ConA. The interferon-gamma (IFN-γ) mRNA levels of chicken peripheral lymphocytes stimulated by GLP synergistically with ConA were measured using fluorescent quantitative PCR. The results showed that GLP could promote interferon-γ mRNA levels in peripheral lymphocytes. In vivo experiment, 175 14-day-old chickens were randomly divided into 7 groups. The chickens except blank control (BC) group were vaccinated with Newcastle disease vaccine, repeated vaccination at 28 days old. At the same time of the first vaccination, the chickens in experimental groups were orally administrated with 5 different doses of GLP respectively, whereas vaccination control (VC) and BC groups were treated with physiological saline, once a day for three successive days. On Day 7, 14, 21 and 28 after the first vaccination, the peripheral lymphocytes proliferation and serum ND antibody titer were determined. The results showed that GLP could significantly promote lymphocyte proliferation and enhance serum antibody titer. The results indicated that GLP may be a novel immunomodulator.

A liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay for the detection of antibodies against Newcastle disease virus in serum of free-ranging pigeons.

A competitive liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay (LPB-ConA-ELISA) was developed in the current study. The assay used ConA as a capture reagent, and the sera of specific pathogen-free chickens immunized with nonpurified Newcastle disease virus (NDV) suspension as detector antibodies, to detect and quantify specific antiviral antibodies in serum samples from free-ranging pigeons. The comparison between the LPB-ConA-ELISA and the hemagglutination inhibition (HI) test for the detection of antibodies in serum samples from 107 pigeons showed significant correlation between the assays (r = 0.875), a high sensitivity (100%), specificity (95.8%), accuracy (96.3%) for the ELISA, and good agreement (κ = 0.83) between the 2 assays. The results of this study suggest that the LPB-ConA-ELISA could be a useful alternative to HI test in the serodiagnosis of NDV in pigeons, or other species of birds.

Development and field application of a competitive enzyme-linked immunosorbent assay for detection of Newcastle disease virus antibodies in chickens and ducks.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using a baculovirus-expressed recombinant nucleocapsid protein antigen (rNDV-N) and an rNDV-N-specific monoclonal antibody (5B3) was developed for the detection of Newcastle disease virus (NDV) antibodies, and its diagnostic performance was evaluated. The specificity and sensitivity of the C-ELISA was found to be 98.4 and 98.9%, respectively, for chickens, and 98.2 and 97.9% for ducks. However, the C-ELISA showed weak cross-reaction with hyperimmune antisera to some other avian paramyxovirus serotypes. In all experimentally vaccinated chickens, seroconversion rates at 7 d postinoculation were 100 and 40% when measured by C-ELISA and hemagglutination inhibition (HI), respectively. In field trials, the C-ELISA showed positive results in 98.9% of HI-positive sera and 40.8% of HI-negative sera from NDV-vaccinated chickens (n = 705). In domestic ducks (n = 158) from NDV-positive duck farms (n = 8), the positive rates according to C-ELISA were significantly higher than those according to the HI test. At the same time, 98.1% of ducks (n = 209) from NDV-negative duck farms (n = 11) were also negative by C-ELISA. Our results indicate that C-ELISA could be a useful alternative to HI testing for detecting NDV antibodies in different avian species such as chickens and ducks.

A survey of avian paramyxovirus type 1 infections among backyard poultry in New Zealand.

AIMS: To determine the presence and the pathotype of avian paramyxovirus type 1 (APMV-1), as well as the prevalence of APMV-1 antibodies, among backyard flocks of poultry in selected New Zealand locations. METHODS: Archival pooled (n = 162) tracheal and cloacal swabs collected from backyard poultry were tested for the presence of APMV-1 RNA by real-time and conventional reverse transcription (RT)-PCR assays. Archival blood samples (n = 240) from a subpopulation of the same birds were tested for the presence of the APMV-1 antibody using a commercial ELISA assay. The archival samples were collected from geographical areas close to bodies of water, in the Bay of Plenty or Wairarapa regions of the North Island of New Zealand, with the high likelihood of interactions between wild waterfowl and domestic poultry. RESULTS: Avian paramyxovirus type 1 RNA was not detected in any of the swabs tested. Antibodies to APMV-1 were detected on 18/19 farms, in 71/240 (29.5%) blood samples tested. The percentage of seropositive birds varied between seropositive farms and ranged from 8.3 to 100%. The percentage of seropositive birds on each farm was not statistically correlated with the flock size, the number of birds sampled, the number of farmed waterfowl, or with the distance to the closest lake/river. However, all chickens from the farm with the highest number of farmed ducks were seropositive for APMV-1. CONCLUSIONS: Lack of detection of APMV-1 in any of the samples indicates that APMV-1 was not circulating among the poultry at the time of sampling. However, detection of APMV-1 antibodies in a proportion of birds on each farm indicates that infection with APMV-1, or antigenically related APMV, is common among backyard poultry. CLINICAL RELEVANCE: On-going proactive surveillance and characterisation of circulating APMV-1 is important for monitoring changes in circulating genotypes of APMV-1 and for understanding the regional ecology of these viruses for the purpose of planning appropriate disease control and prevention strategies. Our data suggest that backyard flocks should be considered as potential reservoirs of APMV. Chickens from backyard farms with multiple bird species may provide good targets for surveillance purposes.

Oral exposure of broiler breeder hens to extra thyroxine modulates early adaptive immune responses in progeny chicks.

Based on the findings of a recent study suggesting a decreased cold-induced ascites incidence in broiler progeny from hyperthyroid (HYPER) breeder hens, and a controversy on the effects of hyperthyroidism on immunocompetence, the present study was conducted to determine the probable adverse effect of induced maternal hyperthyroidism on immune function in progeny chicks. Breeder hens (n = 88) were randomly allotted to the control or HYPER groups and received common or thyroxine (T4)-added (1 mg/L) water, respectively. The hens were artificially inseminated, and hatching eggs (n = 924) were incubated. Thereafter, the male hatchlings (n = 288) were reared for 42 d, and several cellular and humoral immune responses were evaluated at standard or low ambient temperature. Prevaccination antibody titers to Newcastle disease, infectious bronchitis, and infectious bursal disease virus were higher in HYPER chicks during 1 wk of age, although not different in their dams. For primary response to SRBC administered at 7 d of age, HYPER chicks recorded higher total, IgM (d 14), and IgG (d 21) anti-SRBC antibody titers. Higher cutaneous basophilic hypersensitivity response in HYPER chicks (d 10) was not observed at 35 d of age. Carbon clearance assay showed no difference, but in vitro lymphoproliferative response to concanavalin A was higher in 19-d-old HYPER chicks, independent of temperature treatment. An increase in lymphocyte percentage coincided with a decreased heterophil percentage and heterophil to lymphocyte ratio (d 14) in the HYPER group. The weight of lymphoid organs in progeny was not influenced by the oral exposure of dams to extra T4. Independent of T4 treatment, cold exposure was generally associated with decreased immune functions at early stages. The data suggested that oral exposure of broiler breeder hens to 1 mg/L of T4 not only had no adverse effect on immune function, but also modulated early adaptive immune responses in progeny chicks for which the causal mechanisms remain to be unraveled.

Mobilization of immunoglobulin (Ig)-containing plasma cells in Harderian gland, cecal tonsil and trachea of broilers vaccinated with Newcastle Disease Vaccine.

Immunohistochemical studies of Harderian gland, cecal tonsil and trachea of various groups of broiler chickens and the response of Baby Chick Ranikhet Disease Vaccines (BCRDV) on the mobilization of Igs-cells during postnatal development of organs was investigated in the Dept. of Anatomy and Histology, Bangladesh Agricultural University, Mymensingh. In this study twelve chickens were grouped into vaccinated broilers (D14 and D28) which had received vaccines first at D3 of age and a booster dose given at D13; and non-vaccinated broilers (D1) which had not been vaccinated. In this study, it was observed that the frequency and distribution of Igs-positive cells were higher at D14 and at D28 rather than D1. Among Igs-positive cells, the IgG-positive cells were significantly higher than IgM and IgA-positive cells in the Harderian gland of D14 and D28 groups of chickens, however, in day-old chickens, the frequency of IgM-positive cells in this gland were greater. In the cecal tonsil, the frequency and distribution of IgG-positive cells were significantly higher than IgA- and IgM-positive cells both at D14 and D28 ages of chicken. On the other hand, in day-old chickens, the frequency and distribution of IgA-positive cells were insignificantly greater, followed by IgM and IgG-positive cells. In the trachea, few immunoglobulin-containing plasma cells were distributed in the subepithelial layer. IgM-positive cells were higher followed by IgG and IgA-positive cells in the trachea in D14 and D28 groups of chickens. In the same organ, IgG-positive plasma cells were greater than IgA and IgM-positive cells at one-day old. When the data for Harderian gland, cecal tonsil and trachea were compared statistically, it was observed that Igs-positive cells were statistically more common in cecal tonsils in day old chickens, and with the advancement of age, Igs-positive cells were found more in the Harderian gland. In conclusion, with the advancement of age in chickens the Harderian gland uptake is a function of the cecal tonsil due to its functional atrophy.

Prevalence of avian paramyxovirus 1 and avian influenza virus in double-crested Cormorants (Phalacrocorax auritus) in eastern North America.

Although it is well established that wild birds, such as cormorants, carry virulent avian paramyxovirus serotype 1 (APMV-1; causative agent of Newcastle disease) and avian influenza virus (AIV), the prevalence of these viruses among Double-crested Cormorants (Phalacrocorax auritus) in the Great Lakes region of North America has not been rigorously studied. We determined the prevalences of APMV-1 and AIV in Double-crested Cormorants from the interior population of eastern North America. From 2009 to 2011, oropharyngeal and cloacal swabs and serum samples were collected from 1,957 individual Double-crested Cormorants, ranging from chicks to breeding adults, on breeding colony sites in Michigan, Wisconsin, and Mississippi, USA, and Ontario, Canada, as well as on the wintering grounds of migratory populations in Mississippi, USA. Prevalence of antibodies to APMV-1 in after-hatch year birds was consistently high across all three years, ranging from 86.3% to 91.6%. Antibody prevalences in chicks were much lower: 1.7, 15.3, and 16.4% in 2009, 2010, and 2011, respectively. Virulent APMV-1 was detected in six chicks sampled in 2010 in Ontario, Canada. Only one adult was positive for AIV-specific antibodies and five individuals were positive for AIV matrix protein, but the latter were negative for H5 and H7 AIV subtypes. We provide further evidence that Double-crested Cormorants play an important role in the maintenance and circulation of APMV-1 in the wild, but are unlikely to be involved in the circulation of AIV.

Immune response to Newcastle disease virus vaccination in a wild passerine.

We studied the immune response of wild House Sparrows (Passer domesticus) experimentally challenged with different doses of inactivated Newcastle disease virus (NDV) vaccine. We evaluated within-individual cell-mediated and humoral responses in birds kept in outdoor aviaries, over a 6-wk period. Nonbreeding adult House Sparrows developed a significant humoral response to NDV experimental vaccination within 1 wk postchallenge, as measured by hemagglutination inhibition assay; values increased until week 4 and persisted until week 6. Differences among treatments appeared by week 1, with individuals challenged with the highest dose (0.2 mL) eliciting a higher humoral response than the rest (n = 18). By week 4, all individuals vaccinated displayed an increased humoral response, with individuals challenged with the highest dose remaining significantly above responses of individuals vaccinated with the middle dose (0.1 mL, n = 14), but not the lowest dose (0.05 mL, n = 15). The middle and lowest dose responded similarly and significantly different from controls (n = 23). Differences persisted through week 6 postchallenge. Cell-mediated responses were independent of the experimental treatment. All individuals experienced a rise in granulocyte concentration, whereas lymphocyte and monocyte concentrations decreased, most likely as a result of captivity. Adult wild House Sparrows immunochallenged with inactivated NDV vaccine developed a specific humoral response, highlighting the utility of this technique in immunologic and evolutionary ecology studies in wild birds.