Alternating 3 different influenza vaccines for swine in Europe for a broader antibody response and protection.

Heterologous prime-boost vaccination with experimental or commercial influenza vaccines has been successful in various animal species. In this study, we have examined the efficacy of alternating 3 different European commercial swine influenza A virus (swIAV) vaccines: the trivalent Respiporc® FLU3 (TIV), the bivalent GRIPORK® (BIV) and the monovalent Respiporc® FLUpan H1N1 (MOV). Five groups of 6 pigs each received 3 vaccinations at 4-6 week intervals in a homologous or heterologous prime-boost regimen. A sixth group served as a mock-vaccinated challenge control. Four weeks after the last vaccination, pigs were challenged intranasally with a European avian-like H1N1 (1C.2.1) swIAV, which was antigenically distinct from the vaccine strains. One heterologous prime-boost group (TIV-BIV-MOV) had higher hemagglutination inhibition (HI) and neuraminidase inhibition antibody responses against a panel of antigenically distinct H1N1, H1N2 and H3N2 IAVs than the other heterologous prime-boost group (BIV-TIV-MOV) and the homologous prime-boost groups (3xTIV; 3xBIV; 3xMOV). Group TIV-BIV-MOV had seroprotective HI titers (≥ 40) against 56% of the tested viruses compared to 33% in group BIV-TIV-MOV and 22-39% in the homologous prime-boost groups. Post-challenge, group TIV-BIV-MOV was the single group with significantly reduced virus titers in all respiratory samples compared to the challenge control group. Our results suggest that the use of different commercial swIAV vaccines for successive vaccinations may result in broader antibody responses and protection than the traditional, homologous prime-boost vaccination regimens. In addition, the order in which the different vaccines are administered seems to affect the breadth of the antibody response and protection.

Epidemiology of Newcastle disease in chickens of Ethiopia: a systematic review and meta-analysis.

The objective of this systematic review was to estimate the overall pooled prevalence of Newcastle disease in chickens in Ethiopia and identify the sources of heterogeneity among and within studies. The seroprevalence of Newcastle disease was estimated using a single-group meta-analysis. Attempts were also made to identify study-level variables that could explain the heterogeneity in the apparent seroprevalence of the Newcastle disease. The findings were based on 16 published articles and 33 district-level reports and were limited to studies performed during 2005-2017. Due to the presence of heterogeneity, pooled analysis from different districts was conducted using random-effects meta-analysis. The single-group summary of Newcastle disease seroprevalence in chickens was estimated to be 21.47% (19.54-23.4%) with a 95% confidence interval. Our results indicated high inter-study variability (Cochran's Q statistic = 196.2, true variance (τ2) = 0.36, inverse variance index (I2) = 90.0%, p < 0.001). Of all variables analysed, diagnostic techniques and regions were the most significant predictors (p ˂ 0.05) of heterogeneity. According to the diagnostic technique-based meta-analysis of random pooled prevalence, the haemagglutination inhibition test had the highest prevalence, followed by the enzyme-linked immunosorbent assay. In conclusion, the high-pooled prevalence estimates of the disease, combined with the scarcity of published data for the entire country of Ethiopia, indicate a significant data gap on the distribution of Newcastle disease in the country. While the high pooled prevalence tells the need for intervention to control the disease, there is also a need to assess the disease prevalence in all other parts of the country.

Pathology of Komarov vaccine in Hitchner B1 vaccinated and unvaccinated broilers.

Newcastle disease (ND) is a major problem of poultry production worldwide. Control is by biosecurity and vaccination. In this project, we studied the pathology of Komarov vaccine which is commonly used in many countries of Africa on the Hitchner B1 (HBI) vaccinated and unvaccinated broilers. Seventy-five Arbor Acres broilers were obtained at 1 day old. Twenty-five of the broilers were given HB1 vaccine at the hatchery and Komarov vaccine at 5 weeks of age (group HK). A second group of 25 broilers were given only Komarov vaccine at 5 weeks of age (group K). The third group remained as unvaccinated (UU). All the groups were observed for clinical signs and lesions. Depression, sneezing, coughing and noisy respiration were observed in group K broilers from day 2 post Komarov vaccination (PKV). Leg paralysis occurred in 6 broilers on day 8 PKV. The clinical signs were milder in the HK broilers. Only one broiler showed leg paralysis in this group on day 18 PKV. No mortality occurred in the three groups. The bursa, spleen and thymus showed mild to moderate enlargement, atrophy and depletion of lymphocytes on days 3, 5, 8 and 14 PKV in HK and K groups. The trachea and lungs were congested. The haemagglutination inhibition (HI) antibody titres in the K group were higher than those of HK and UU groups on days 7, 24 and 21 PKV. The above observations show that Komarov vaccine may cause no mortality in vaccinated and unvaccinated broilers and higher HI antibodies are produced in broilers that have not been vaccinated earlier.

Immunogenicity and protective efficacy of an EB66® cell culture-derived duck Tembusu virus vaccine.

The avian EB66® cell line, derived from duck embryonic stem cells, has been widely used for producing human and animal therapeutic proteins and vaccines. In current study we evaluated the potential use of EB66® cell line in a cell culture-derived duck Tembusu virus (DTMUV) vaccine development. After optimizing the growth conditions of DTMUV HB strain in EB66® cells, we successfully generated three batches of viruses with ELD50 titres of 105.9/0.1 ml, 105.3/0.1 ml and 105.5/0.1 ml, respectively, for using in the preparation of inactivated vaccines. The immunogenicity and protective efficacy of these EB66® cells-derived inactivated vaccines were examined in ducks. Results indicated that all three batches of vaccines induced haemagglutination-inhibition (HI) antibody response in immunized birds at 2 weeks after a single immunization. Immunized ducks and ducklings were protected against a virulent challenge at 4 weeks after a booster immunization. The duration of immunity was for 3-4 months after a booster immunization. These results demonstrated the feasibility of using EB66® cell line to grow up DTMUV for vaccine preparation. RESEARCH HIGHLIGHTS Duck Tembusu virus can be propagated in EB66® cells. EB66® cell-derived inactivated DTMUV vaccines are immunogenic and can provide protection against a virulent challenge. A long-lasting immunity is induced after a booster immunization.

Evaluation of an in-house indirect enzyme-linked immunosorbent assay of feline panleukopenia VP2 subunit antigen in comparison to hemagglutination inhibition assay to monitor tiger antibody levels by Bayesian approach.

BACKGROUND: Feline panleukopenia virus (FPV) is an etiologic pathogen of feline panleukopenia that infects all members of Felidae including tigers (Panthera tigris). Vaccinations against FPV among wild felid species have long been practiced in zoos worldwide. However, few studies have assessed the tiger immune response post-vaccination due to the absence of a serological diagnostic tool. To address these limitations, this study aimed to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the monitoring of tiger antibody levels against the feline panleukopenia vaccine by employing the synthesized subunit capsid protein VP2. An in-house horseradish peroxidase (HRP) conjugated rabbit anti-tiger immunoglobulin G (IgG) polyclonal antibody (HRP-anti-tiger IgG) was produced in this study and employed in the assay. It was then compared to a commercial HRP-conjugated goat anti-cat IgG (HRP-anti-cat IgG). Sensitivity and specificity were evaluated using the Bayesian model with preferential conditional dependence between HRP-conjugated antibody-based ELISAs and hemagglutination-inhibition (HI) tests. RESULTS: The posterior estimates for sensitivity and specificity of two indirect ELISA HRP-conjugated antibodies were higher than those of the HI test. The sensitivity and specificity of the indirect ELISA for HRP-anti-tiger IgG and HRP-anti-cat IgG were 86.5, 57.2 and 86.7%, 64.6%, respectively, while the results of the HI test were 79.1 and 54.1%. In applications, 89.6% (198/221) and 89.1% (197/221) of the tiger serum samples were determined to be seropositive by indirect ELISA testing against HRP-anti-tiger and HRP-anti-cat, respectively. CONCLUSION: To the best of our knowledge, the specific serology assays for the detection of the tiger IgG antibody have not yet been established. The HRP-anti-tiger IgG has been produced for the purpose of developing the specific immunoassays for tigers. Remarkably, an in-house indirect ELISA based on VP2 subunit antigen has been successfully developed in this study, providing a potentially valuable serological tool for the effective detection of tiger antibodies.

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.

Study of the underlying mechanisms and consequences of pathogenicity differences between two in vitro selected G1-H9N2 clones originating from a single isolate.

The G1-H9N2 avian influenza virus (AIV) has caused significant economic losses in the commercial poultry industry due to reduced egg production and increased mortality. The field observations have shown that H9N2 viruses circulate and naturally mix with other pathogens and these simultaneous infections can exacerbate disease. To avoid an incorrect virus characterization, due to co-infection, isolates were purified by in vitro plaque assays. Two plaque purified G1-H9N2 clones, selected on different cell types, named MDCK-and CEF-clone in regards to the cell culture used, were studied in vivo, revealing two different virulence phenotypes. Subsequently, the underlying mechanisms were studied. Specifically, the phenotypical outcome of SPF bird infection by the two clones resulted in completely different clinical outcomes. These differences in clinical outcome were used to study the factors behind this output in more detail. Further studies demonstrated that the more severe disease outcome associated with the MDCK-clone involves a strong induction of pro-inflammatory cytokines and a lack of type I interferon production, whereas the mild disease outcome associated with the CEF-clone is related to a greater antiviral cytokine response. The immunosuppressive effect of the MDCK-clone on splenocytes was further demonstrated via ChIFN-γ lack production after ex vivo mitogenic stimulation. Genome sequencing of the two clones identified only four amino acid differences including three in the HA sequence (HA-E198A, HA-R234L, HA-E502D-H9 numbering) and one in the NA sequence (NA-V33M). In the present study, valuable insights on the mechanisms responsible for AI pathogenicity and molecular mechanisms of H9N2 infections in chicken were obtained while highlighting the impact of the cells viruses are grown on their virulence.

Antimicrobial and biochemical characterization of a C-type lectin isolated from pearl spot (Etroplus suratensis).

The present study reveals purification and characterization of a C-type lectin from the serum of pearl spot, Etroplus suratensis (Es-Lec). The Es-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 75 kDa in SDS-PAGE. The surface morphology of purified Es-Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 2.958 min was appeared in high performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis expresses a single peak at 31.8372̊ and MALDI-TOF peaks which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Es-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy and haemagglutination inhibition was also done. In addition, purified Es-Lec showed the broad spectrum of antibacterial activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila. Antibiofilm potential of purified Es-Lec against selected Gram-negative bacteria exhibited the disruption of biofilm architecture at the concentration of 50 µg ml-1 and also it exhibited antiviral and anticancer activity.

Protection afforded by avian influenza vaccination programmes consisting of a novel RNA particle and an inactivated avian influenza vaccine against a highly pathogenic avian influenza virus challenge in layer chickens up to 18 weeks post-vaccination.

The efficacies of an oil adjuvanted-inactivated reverse genetics-derived H5 avian influenza virus (AIV) vaccine and an alphavirus replicon RNA particle (RP) AIV vaccine were evaluated in commercial Leghorn chickens. Challenge utilized A/turkey/MN/12582/2015, an isolate representing the U.S. H5N2 Clade 2.3.4.4 responsible for the 2015 highly pathogenic avian influenza (HPAI) epornitic in commercial poultry the United States. As part of a long-term, 36-week study, chickens were challenged at seven weeks of age after receiving a single vaccination, at 18 weeks of age following a vaccine prime-single boost, and at 36 weeks of age after a prime- double-boost. All vaccine programmes reduced virus oropharyngeal and cloacal shedding and mortality compared to the non-vaccinated control birds; however, chickens receiving at least one administration of the RP vaccine generally had diminished viral shedding especially from the cloacal swabbings. A detectable serum antibody response and protection were observed through 18 weeks post-vaccination. Our data suggest that, in conjunction with a comprehensive eradication, enhanced biosecurity and controlled marketing plan, vaccination programmes of commercial layer chickens with novel RP vaccines may represent an important tool for preventing HPAI-related mortalities and decreasing viral load during a catastrophic influenza outbreak. RESEARCH HIGHLIGHTS Immunization of poultry following a vaccination schedule consisting of inactivated and RNA particle vaccines offered significant protection against lethal disease following HPAIV challenge. Virus shedding was significantly (P < 0.05) reduced in chickens vaccinated with either inactivated and/or recombinant vaccines. Serum antibody titres were not a reliable indicator of protection. An inactivated vaccine containing 384 HAU of the homologous antigen was unable to induce complete protection.

Novel protein microarray for the detection of avian influenza virus antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes.

Avian influenza virus (AIV) can cause serious zoonotic disease, thereby threatening the poultry industry and human health. An efficient and rapid detection approach is crucial to prevent and control the spread of avian influenza. In this study, a novel protein microarray was developed. Haemagglutinin proteins of H5 and H7 subtypes and nucleoprotein (NP) were purified and spotted onto the initiator-integrated poly-(dimethylsiloxane) as antigens. Monoclonal antibodies with inhibition effect were screened and utilized for the synchronous detection of three avian influenza antibodies in different species. In the protein microarray, the cut-off values were 40%, 50% and 30% inhibition for H5 antibody detection; 50%, 50% and 20% for NP antibody detection; 40%, 50% and 40% for H7 antibody detection in chicken, peacock and duck sera, respectively. The 95 serum samples were detected by microarray, and results were compared with the findings of AIV antibody test enzyme-linked immunosorbent assay (ELISA) or haemagglutination inhibition (HI) test. NP antibody detection in the microarray showed 100% (55/55) agreement ratio in chicken using ELISA. Compared with HI, H5 antibody detection in the microarray showed 100% (95/95) agreement ratio in chicken, peacock and duck, whilst those of H7 displayed 98.18% (54/55) agreement in chicken, 100% (20/20) in peacock and 90% (18/20) in duck. In conclusion, this novel protein microarray is a high-throughput and specific method for the detection of AIV antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes. It can be applied to the serological diagnosis and epidemiological investigation of AIV. RESEARCH HIGHLIGHTS A novel protein microarray method has been developed. The microarray can detect AIV antibodies and distinguish between H5 and H7 subtypes. The study lays the foundation for simultaneous identification of multiple pathogens.

Continuing evolution of H6N2 influenza a virus in South African chickens and the implications for diagnosis and control.

BACKGROUND: The threat of poultry-origin H6 avian influenza viruses to human health emphasizes the importance of monitoring their evolution. South Africa's H6N2 epidemic in chickens began in 2001 and two co-circulating antigenic sub-lineages of H6N2 could be distinguished from the outset. The true incidence and prevalence of H6N2 in the country has been difficult to determine, partly due to the continued use of an inactivated whole virus H6N2 vaccine and the inability to distinguish vaccinated from non-vaccinated birds on serology tests. In the present study, the complete genomes of 12 H6N2 viruses isolated from various farming systems between September 2015 and February 2019 in three major chicken-producing regions were analysed and a serological experiment was used to demonstrate the effects of antigenic mismatch in diagnostic tests. RESULTS: Genetic drift in H6N2 continued and antigenic diversity in sub-lineage I is increasing; no sub-lineage II viruses were detected. Reassortment patterns indicated epidemiological connections between provinces as well as different farming systems, but there was no reassortment with wild bird or ostrich influenza viruses. The sequence mismatch between the official antigens used for routine hemagglutination inhibition (HI) testing and circulating field strains has increased steadily, and we demonstrated that H6N2 field infections are likely to be missed. More concerning, sub-lineage I H6N2 viruses acquired three of the nine HA mutations associated with human receptor-binding preference (A13S, V187D and A193N) since 2002. Most sub-lineage I viruses isolated since 2015 acquired the K702R mutation in PB2 associated with the ability to infect humans, whereas prior to 2015 most viruses in sub-lineages I and II contained the avian lysine marker. All strains had an unusual HA0 motif of PQVETRGIF or PQVGTRGIF. CONCLUSIONS: The H6N2 viruses in South African chickens are mutating and reassorting amongst themselves but have remained a genetically pure lineage since they emerged more than 18 years ago. Greater efforts must be made by government and industry in the continuous isolation and characterization of field strains for use as HI antigens, new vaccine seed strains and to monitor the zoonotic threat of H6N2 viruses.

Impact of crossing Fayoumi and Leghorn chicken breeds on immune response against Newcastle disease virus vaccines.

This study was conducted to evaluate the immune response against Newcastle disease (ND) virus vaccines (live attenuated and inactivated) in purebred Lohman Selected Leghorn (LSL), Fayoumi, male Fayomi × female LSL (FL crossbred), male LSL × female Fayomi (LF reciprocal crossbred) chickens. One-hundred-day-old chicks of each genetic type were assigned to five equal replicates. The log geometric means of the hemagglutination inhibition (HI) antibody titers were calculated. The FL crossbred chickens had a significantly higher HI antibody titer at day 26 of age when compared with the LSL chickens (P = 0.039). The Fayoumi and FL crossbred chickens had significantly higher HI antibody titers at day 45 of age (2.35 and 2.23, respectively) when compared with the LSL chickens (P = 0.031). In the same way, the purebred Fayoumi and FL crossbred chickens had significantly higher HI antibody titers at 60 and 75 days of age (P = 0.009 and 0.041, respectively) when compared with the purebred LSL and LF crossbred chickens. The LSL chickens showed a significantly higher (P < 0.05) correlation estimate between HI titer to the ND vaccine on day 75 of age and body weight at week 12 of age. When challenged with the virulent ND virus, the hazard ratio (HR) for mortality rates in purebred LSL and LF crossbred chickens were significantly (HR = 3.52 and 2.07; P = 0.001 and 0.049, respectively) higher than the Fayoumi chickens. In conclusion, purebred Fayoumi and FL crossbred chickens showed superior antibody titers against live attenuated and inactivated ND virus vaccines. Hence, Fayoumi breed may be incorporated in the crossbreeding programs to improve the genetic resistance to ND.

Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks.

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any 'gold standard'. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability.

Efficacy of vaccination with La Sota strain vaccine to control Newcastle disease in village chickens in Nepal.

The efficacy of vaccination with Newcastle disease (ND) La Sota and R2B (Mukteswar) modified live strain vaccines was determined by experimental challenge and with ND La Sota vaccine under field conditions in Nepal. Booster vaccination with ND La Sota vaccine after a primary vaccination with ND La Sota vaccine, induced a geometric mean titre (GMT) of 5.0 log2 haemagglutination inhibition (HI) units, compared to a GMT of 6.0 log2 HI units following booster vaccination with R2B vaccine 1 month after primary vaccination with ND La Sota vaccine. Both vaccines provided 100% protection against challenge with a local field ND strain. Furthermore, booster vaccination with ND La Sota vaccine induced protective levels of antibody after field use in villages in Jhapa, and no outbreaks of ND occurred during the study period. The ND La Sota modified live vaccine is immunogenic and efficacious and is a suitable vaccine for use in vaccination programmes in village chickens in the rural areas of Nepal.

Evaluation of a conserved HA274-288 epitope to detect antibodies to highly pathogenic avian influenza virus H5N1 in Indonesian commercial poultry.

A peptide enzyme linked immunosorbent assay (ELISA) based on an epitope in the haemagglutinin (HA) of avian influenza virus H5N1, amino acid positions 274-288 (HA274-288) was evaluated for detection of H5N1-specific antibodies. An optimized ELISA based on the tetrameric form of the HA274-288 epitope designated MP15 gave low background with non-immune chicken sera and detected vaccinated and infected birds. The HA274-288 epitope was highly conserved in Indonesian H5N1 strains and antibody responses were detected in the majority of the vaccinated chickens regardless of the H5N1 strain used for vaccination. The HA274-288 epitope was also conserved in the majority of H5N1 strains from the neighbouring Asian region, and other H5 subtypes potentially allowing for a wider use of the MP15 ELISA in H5N1 vaccinated and infected flocks. The MP15 ELISA results correlated significantly with haemagglutination inhibition (HI) test results and test sensitivity and specificity were 87% and 92%, respectively. The MP15 ELISA titres were significantly higher than the HI titres in all immune sera allowing for sera to be tested at a single dilution of 1:400 which is of advantage in routine surveillance. The study indicated that the MP15 ELISA is potentially useful for serological detection of H5N1 vaccinated or infected poultry and to have some advantages over the standard HI test for routine monitoring of flocks' immunity after vaccination.

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.

Visual detection of goose haemorrhagic polyomavirus in geese and ducks by loop-mediated isothermal amplification.

Goose haemorrhagic polyomavirus (GHPV) is an aetiological agent of haemorrhagic nephritis and enteritis of geese occurring in geese (Anser anser). GHPV may also infect Muscovy ducks (Carina mochata) and mule ducks. Early detection of GHPV is important to isolate the infected birds from the rest of the flock thus limiting infection transmission. The current diagnosis of haemorrhagic nephritis and enteritis of geese is based on virus isolation, histopathological examination, haemagglutination inhibition assay, ELISA and polymerase chain reaction (PCR). Recently, real-time PCR assay was developed which considerably improved detection of GHPV. In spite of many advantages, these methods are still time-consuming and inaccessible for laboratories with limited access to ELISA plate readers or PCR thermocyclers. The aim of our study was to develop loop-mediated isothermal amplification (LAMP) that may be conducted in a water bath. Two pairs of specific primers complementary to VP1 gene of GHPV were designed. The results of GHPV LAMP were recorded under ultraviolet light. Our study showed LAMP was able to specifically amplify VP1 fragment of a GHPV without cross-reactivity with other pathogens of geese and ducks. LAMP detected as little as 1.5 pg of DNA extracted from a GHPV standard strain (150 pg/µl). The optimized LAMP was used to examine 18 field specimens collected from dead and clinically diseased geese and ducks aged from 1 to 12 weeks. The positive signal for GHPV was detected in three out of 18 (16.6%) specimens. These results were reproducible and consistent with those of four real-time PCR. To the best of our knowledge this is the first report on LAMP application for the GHPV detection.

Serological Surveillance of Wild Waterfowl in Northern Australia for Avian Influenza Virus Shows Variations in Prevalence and a Cyclical Periodicity of Infection.

The virological surveillance of 3582 wild waterfowl in northern Australia from 2004 to 2009 for avian influenza virus (AIV) found an apparent prevalence (AP) of 1% (31 of 2989 cloacal swabs; 95% CI: 0.71%-1.47%) using a Taqman Type A real-time reverse transcription polymerase chain reaction test and no viral isolations from 593 swabs tested by the embryonating chicken egg culture method. From serological testing using a nucleoprotein competitive enzyme-linked immunosorbent assay for AIV antibody, 1131 of 3645 sera had ≥ 40% inhibition, indicating an apparent seroprevalence of 31% (95% CI: 29.5%-32.6%). This value suggests that the low AP from virological testing does not reflect the dynamics of AIV infection in these populations. Spatiotemporal and species variations in seroprevalence were found at wetland sampling sites, with consistently higher values at Kununurra in Western Australia (AP  =  39%, 95% CI: 36.9%-41.4%) compared to other locations. At Kununurra, seroprevalence values had a two-year cyclical periodicity and suggest this location is a hotspot of AIV activity. From hemagglutination inhibition (HI) testing using multiple subtype antigens, the highest AP of HI reactions were to H6 and H5 subtypes. The phenomenon of cyclic periodicity in NP seroprevalence at Kununurra is hypothesized as being related to the prevalent H6 subtype that may have either become predominant or cycled back into a mostly AIV naïve flock. The inclusion of serological testing provided insight into the dynamics of AIV infection in wild birds such as species risk profiles and spatiotemporal patterns, important epidemiological information for a risk-based approach to surveillance.

Vaccine Efficacy Against a New Avian Influenza (H9N2) Field Isolate from the Middle East (Serology and Challenge Studies).

Avian influenza subtype H9N2 is endemic in many countries in the Middle East. The reported prevalence of infection was variable between countries and ranged from 28.7% in Tunisia to 71% in Jordan. Several commercial killed whole-virus vaccine products are used as monovalent or bivalent mixed with Newcastle disease virus. Recently, we have noticed that many of the vaccinated broiler flocks did not show a production advantage over nonvaccinated flocks in the field. A new avian influenza field virus (H9N2) was isolated from these vaccinated and infected broiler flocks in 2013. This virus had 89.1% similarity of its hemagglutinin (HA) gene to the classical virus used for manufacturing the classical vaccine. Inactivated autogenous vaccine was manufactured from this new field isolate to investigate its serological response and protection in specific-pathogen-free (SPF) and breeder-male chickens compared to the classical vaccine. Oropharyngeal virus shedding of vaccinated breeder-male chickens was evaluated at 3, 9, 10, and 14 days postchallenge (DPC). Percentage of chickens shedding the virus at 3 DPC was 64%, 50%, and 64% in the classical vaccine group, autogenous vaccine group, and the control challenged group, respectively. At 7 DPC percentage of virus shedding was 42%, 7%, and 64% in the classical vaccine group, autogenous vaccine group, and the control challenged group, respectively. At 10 DPC only 9% of classical vaccine group was shedding the virus and there was no virus shedding in any of the groups at 14 DPC. There was statistical significance difference (P < 0.05) in shedding only at 7 DPC between the autogenous vaccine group and the other two groups. At 42 days of age (14 DPC), average body weight was 2.720, 2.745, 2.290, and 2.760 kg for the classical vaccine group, autogenous vaccine group, control challenged group, and control unchallenged group, respectively. Only the control challenged group had significantly (P < 0.05) lower average body weight. In another experiment, vaccinated SPF chicks had hemagglutination inhibition (HI) geometric mean titers (GMTs), with classical antigen, of 8.7 and 3.1 log 2 for classical and autogenous vaccine groups, respectively. When the autogenous antigen was used for HI, GMTs were 6.0 and 8.1 log 2, respectively. Both vaccines protected against body weight suppression after challenge. However, autogenous vaccine elicited significantly higher HI titer and reduced viral shedding at 7 DPC. In conclusion, it is important to revise the vaccine virus strains used in each region to protect against and control infection from new field strains. Further field experiments are needed to demonstrate the efficacy of new vaccines under field conditions.

Virus interference between H7N2 low pathogenic avian influenza virus and lentogenic Newcastle disease virus in experimental co-infections in chickens and turkeys.

Low pathogenicity avian influenza virus (LPAIV) and lentogenic Newcastle disease virus (lNDV) are commonly reported causes of respiratory disease in poultry worldwide with similar clinical and pathobiological presentation. Co-infections do occur but are not easily detected, and the impact of co-infections on pathobiology is unknown. In this study chickens and turkeys were infected with a lNDV vaccine strain (LaSota) and a H7N2 LPAIV (A/turkey/VA/SEP-67/2002) simultaneously or sequentially three days apart. No clinical signs were observed in chickens co-infected with the lNDV and LPAIV or in chickens infected with the viruses individually. However, the pattern of virus shed was different with co-infected chickens, which excreted lower titers of lNDV and LPAIV at 2 and 3 days post inoculation (dpi) and higher titers at subsequent time points. All turkeys inoculated with the LPAIV, whether or not they were exposed to lNDV, presented mild clinical signs. Co-infection effects were more pronounced in turkeys than in chickens with reduction in the number of birds shedding virus and in virus titers, especially when LPAIV was followed by lNDV. In conclusion, co-infection of chickens or turkeys with lNDV and LPAIV affected the replication dynamics of these viruses but did not affect clinical signs. The effect on virus replication was different depending on the species and on the time of infection. These results suggest that infection with a heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated, which decreases with time.