Agar Gel Precipitin Test Research Articles

Capsular polysaccharide determines the serotyping of Riemerella anatipestifer.

Riemerella anatipestifer (R. anatipestifer) is a Gram-negative pathogen that causes significant economic losses to the farming industry. The polysaccharides that determine serotype are well understood in other bacteria; they have not yet been fully characterized in R. anatipestifer. In this study, we unexpectedly discovered a strain, RCAD0392, that is capable of cross-agglutination. Whole genome sequencing revealed base mutations disrupting a gene on its capsular polysaccharide synthesis gene cluster. By constructing homologous gene deletion mutants in CH-2, we demonstrated that deletion of this gene leads to altered serological phenotypes. India ink and transmission electron microscopy experiments revealed the disappearance of capsule on the surface of the bacteria, indicating the association of the gene with capsule synthesis. In addition, agar-gel precipitin tests showed that lipopolysaccharide binds to antisera of multiple serotypes, while the capsular polysaccharide only binds to the corresponding antisera. This suggests that capsular polysaccharide is the specific antigen that determines the serotype of R. anatipestifer. IMPORTANCE Riemerella anatipestifer (R. anatipestifer) is one of the most important veterinary pathogens with at least 21 serotypes. However, the exact polysaccharide(s) that determine R. anatipestifer serotype is still unknown. This study has provided a preliminary exploration of the relationship between capsular polysaccharides and serotyping in R. anatipestifer and suggests possible directions for further investigation of the genetic basis of serotypes in this bacterium.

Host-feeding pattern of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in heterogeneous landscapes of South Andaman, Andaman and Nicobar Islands, India.

Mosquito foraging behavior is a determinant of host-vector contact and has an impact on the risk of arboviral epidemics. Therefore, blood-feeding patterns is a useful tool for assessing the role in pathogen transmission by vector mosquitoes. Competent vectors of dengue and chikungunya viz. Aedes aegypti and Aedes albopictus are widely prevalent in the Andaman and Nicobar archipelago. Considering the vector potential, medical importance of both these mosquito species and lack of information on host-feeding patterns, blood meal analysis of both these vector mosquitoes was undertaken. Biogents Sentinel traps were used for sampling blooded mosquitoes, for identifying the source of blood meal by agar gel-precipitin test. We identified vertebrate source of 147 and 104 blood meals in Ae. aegypti and Ae. albopictus from heterogeneous landscapes in South Andaman district. Results revealed that Ae. aegypti (88 %) and Ae. albopictus (49 %) fed on human and a small proportion on mammals and fowls, indicative of predominance of anthropophilism. Ae. aegypti predominantly fed on human blood (94.2 %-densely built urban, 89.8 %-low vegetation coverage, and 78.3 %-medium vegetation coverage). Anthropophilism in Ae. albopictus was maximal in densely built urban (90.5 %) and progressively decreased from low vegetation-vegetation/forested continuum (66.7, 36.4, and 8.7 %), indicating plasticity in feeding across these landscapes. Epidemiological significance of the findings is discussed.

Avian influenza virus sample types, collection, and handling.

Successful detection of avian influenza (AI) virus, viral antigen, nucleic acid, or antibody is dependent upon the collection of the appropriate sample type, the quality of the sample, and the proper storage and handling of the sample. The diagnostic tests to be performed should be considered prior to sample collection. Sera are acceptable samples for ELISA or agar gel precipitin tests, but not for real-time RT-PCR. Likewise, swabs and/or tissues are acceptable for real-time RT-PCR and virus isolation. The sample type will also depend on the type of birds that are being tested; although it is optimal to collect both oropharyngeal and cloacal swabs, oropharyngeal swabs should be collected from gallinaceous poultry and cloacal swabs should be collected from waterfowl. In addition to collecting the appropriate sample for the tests to be performed, selecting the right materials for sample collection (i.e., type of swab) is very important. This chapter outlines the collection of different specimen types and procedures for proper specimen handling.

A Sero-Epidemiological Survey of Infectious Bursal Disease in Scavenging Village Chickens in Enugu State, Nigeria

A serological survey was conducted to determine the prevalence of antibodies to Infectious Bursal Disease (IBD) virus using the Agar Gel Precipitin Test (AGPT) in unvaccinated scavenging adult village chickens in NsukkaLocal Government Area (L.G.A.) in South Eastern Nigeria. Of the 800 sera samples tested, 328 (41%) were positive for IBD virus antibodies. There was no significant difference (P>0.05) in seasonal distribution, being 43% in the wet and 39% in the dry seasons; and gender being 41.1% and 41%for cocks and hens respectively. The study thus confirmed the endemicity of IBD in local chickens. A national IBD control programme is recommended.

Laboratory Diagnosis of Transboundary Animal Diseases in Pakistan

Control of animal and poultry diseases is one of the primary objectives of government livestock departments in Pakistan. Main emphasis is given to the prevention and control of transboundary animal diseases (TADs), i.e. rinderpest, foot and mouth disease (FMD), Peste des petits ruminants (PPR) and avian influenza (AI) in the country. Recently (2007), the Office International des Epizooties has certified Pakistan as a rinderpest-free country but other TADs are still a threat to the livestock and poultry sectors in the country and the region at large. Keeping in view the significance of livestock and poultry industry for the livelihood of the farmers as well as the economy of the country, a network of diagnostic facilities has been established both at federal and provincial levels. The National Veterinary Laboratory (NVL) Islamabad has been designated as a referral laboratory for the diagnosis of rinderpest, FMD and PPR. Presence of these viruses is confirmed using ELISA. Diagnosis and monitoring of AI is the responsibility of National Reference Laboratory for Poultry Diseases Animal Health, Animal Sciences Institute, Islamabad where hemagglutination, agar-gel-precipitin test (AGPT), ELISA and PCR are employed to confirm the diseases. In each of the four provinces, the Veterinary Research Institute (VRI) plays a central role to investigate an outbreak and implement measures to contain infection. Besides VRIs, district diagnostic laboratories have also been established to provide basic diagnostic facilities to the farmers. Undiagnosed cases or samples collected from TADs are referred to either the concerned VRI or the NVL for further analysis. The Animal Sciences Institute (ASI) at the National Agricultural Research Centre and eight veterinary universities/faculties also play an important role in the development/improvement of new assays/biologics and dissemination of knowledge to other institutes in the country. The present status of TADs and recommendations for harmonizing the diagnostic tools for these diseases are discussed.

Susceptibility of North American ducks and gulls to H5N1 highly pathogenic avian influenza viruses.

Since 2002, H5N1 highly pathogenic avian influenza (HPA1) viruses have been associated with deaths in numerous wild avian species throughout Eurasia. We assessed the clinical response and extent and duration of viral shedding in 5 species of North American ducks and laughing gulls (Larus atricilla) after intranasal challenge with 2 Asian H5N1 HPAI viruses. Birds were challenged at approximately equal to 10 to 16 weeks of age, consistent with temporal peaks in virus prevalence and fall migration. All species were infected, but only wood ducks (Aix sponsa) and laughing gulls exhibited illness or died. Viral titers were higher in oropharyngeal swabs than in cloacal swabs. Duration of viral shedding (1-10 days) increased with severity of clinical disease. Both the hemagglutination-inhibition (HI) and agar gel precipitin (AGP) tests were able to detect postinoculation antibodies in surviving wood ducks and laughing gulls; the HI test was more sensitive than the AGP in the remaining 4 species.

Serological survey of racing pigeons for selected pathogens in Taiwan

To investigate the pathogens that racing pigeons in Taiwan are exposed to, a total of 3764 pigeons from 90 lofts were analysed by collection of blood samples in the period between October 2000 and September 2001. The haemagglutination inhibition (HI) test was performed to detect antibodies against Newcastle disease virus (NDV), type 2 avian paramyxovirus (APMV-2), and egg drop syndrome '76 virus (EDS-76V). The agar-gel precipitin (AGP) test was used to detect antibodies against fowl adenovirus (FAV), goose parvovirus (GPV), and avian reovirus (REO). The virus neutralisation (VN) test was applied to detect antibodies against the serotypes FAV-1 and FAV-8. A rapid serum agglutination test was applied for the detection of antibodies against Mycoplasma spp. Antibodies to several infectious agents were found, including NDV (43.3%), EDS-76V (19.2%), FAV (0.8%), REO (0.5%), APMV-2 (0.2%), Mycoplasma columbinum (10.3%), M. columborale (7.1%), M. synoviae (1.8%) and M. gallisepticum (1.3%). Antibodies against GPV, FAV-1, and FAV-8 were not detected in any serum sample. NDV seroprevalence was significantly higher in pigeons of more than one year of age than in pigeons younger than one year. ND or EDS-76 seroprevalence of pigeons vaccinated with ND vaccine or EDS-76 vaccine was significantly higher than that of pigeons that did not receive any vaccination.

Diagnostic Approach for Differentiating Infected from Vaccinated Poultry on the Basis of Antibodies to NS1, the Nonstructural Protein of Influenza A Virus

Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in Escherichia coli or to chemically synthesized NS1 peptides. Immune sera were obtained from chickens and turkeys inoculated with live AI virus, inactivated purified vaccines, or inactivated commercial vaccines. Seroconversion to positivity for antibodies to the NS1 protein was achieved in birds experimentally infected with multiple subtypes of influenza A virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated gradient-purified vaccines had no seroconversion to positivity for antibodies to the NS1 protein, and animals vaccinated with commercial vaccines had low, but detectable, levels of NS1 antibodies. The use of a second ELISA with diluted sera identified a diagnostic test that results in seropositivity for antibodies to the NS1 protein only in infected birds. For the field application phase of this study, serum samples were collected from vaccinated and infected poultry, diluted, and screened for anti-NS1 antibodies. Field sera from poultry that received commercial AI vaccines were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test, but they were negative by the NS1 ELISA. Conversely, diluted field sera from AI-infected poultry were positive for both AGP and NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industries.

Host-feeding pattern of Culex quinquefasciatus Say and Mansonia annulifera (Theobald) (Diptera: Culicidae), the major vectors of filariasis in a rural area of south India.

Culex quinquefasciatus and Mansonia annulifera are abundant in the rural areas of Kuttanadu, Kerala, India. Bloodmeal identification for individuals of these species collected in this region was determined by the agar-gel precipitin test. A total of 2,328 blood smears from Cx. quinquefasciatus (1,148) and Ma. annulifera (1,180) was tested. Results showed that Cx. quinquefasciatus and Ma. annulifera were highly anthropophilic and that human feeding accounted for 74 and 66% of the total bloodmeals tested. Feeding on cattle accounted for only 1.5 and 2.1% of Cx. quinquefasciatus and Ma. annulifera bloodmeals, respectively. This study showed the high anthropophilic feeding rates of Cx. quinquefasciatus and Ma. annulifera collected from an endemic belt of Malayan filariasis, where epidemiological studies revealed the coexistence of Bancroftian and Malayan filariasis.

Susceptibility of laughing gulls (Larus atricilla) to H5N1 and H5N3 highly pathogenic avian influenza viruses.

This investigation detailed the clinical disease, gross and histologic lesions, and distribution of viral antigen in juvenile laughing gulls (Larus atricilla) intranasally inoculated with either the A/tern/South Africa/61 (H5N3) (tern/SA) influenza virus or the A/chicken/Hong Kong/220/97 (H5N1) (chicken/HK) influenza virus, which are both highly pathogenic for chickens. Neither morbidity nor mortality was observed in gulls inoculated with either virus within the 14-day investigative period. Gross lesions resultant from infection with either virus were only mild, with the tern/SA virus causing decreased lucency of the air sacs (2/6), splenomegaly (2/6), and pancreatic mottling (1/6) and the chicken/HK virus causing only decreased lucency of the air sacs (2/8) and conjunctival edema (2/8). Histologic lesions in the tern/SA-inoculated gulls included a mild to moderate heterophilic to lymphoplasmacytic airsacculitis (6/6), mild to moderate interstitial pneumonia (3/6), and moderate necrotizing pancreatitis and hepatitis at 14 days postinoculation (DPI) (2/6). Immunohistochemical demonstration of viral antigen occurred only in association with lesions in the liver and pancreas. In contrast, viral antigen was not demonstrated in any tissues from the chicken/HK-inoculated gulls, and inflammatory lesions were confined to the air sac (3/8) and lungs (3/8). Both viruses were isolated at low titers (<10(1.68) mean embryo lethal dose) from oropharyngeal and cloacal swabs up to 7 days postinoculation (DPI), from the lung and kidney of one of two tern/SA-inoculated gulls at 14 DPI, and from the lung of one of two chicken/HK-inoculated gulls at 7 DPI. Antibodies to influenza viruses as determined with the agar gel precipitin test at 14 DPI were detected only in the two tern/SA-inoculated gulls and not in the two chicken/HK-inoculated gulls.

Serosurvey for Newcastle disease and avian influenza A virus antibodies in great cormorants from France.

Inland great cormorants (Phalacrocorax carbo) culled in France were examined in the winter of 1997-98 and 1998-99 for antibodies to Newcastle disease (ND) and influenza A strains H5 and H7 by the hemagglutination inhibition test. Antibodies to influenza A group antigen were tested by agar gel precipitin test. Ten of 53 adult individuals were seropositive for ND virus. All sera were negative for influenza A antibodies. It is speculated that ND occurred in the sampled population.

Application of the agar gel precipitin test to detect antibodies to Salmonella enterica serovar enteritidis in serum and egg yolks from infected hens

Serological surveillance can be an important component for egg quality assurance programs geared toward controlling problems with Salmonella enterica serovar Enteritidis (S. enteritidis) within a flock. Serum is the primary sample source for the procedures, although egg yolk antibody assays have become popularity in recent years. However, these assays tend to be labor intensive, requiring procedures for extracting antibodies from the yolk followed by assaying the samples. We describe an adaptation of the agar gel precipitin (AGP) test for use in detecting antibodies to S. enteritidis deposited in egg yolks of infected hens. Yolk or sera from infected birds were administered to wells cut into seven-well clusters in an agar gel plate, and detection antigen was added to the center well. The agar gels were incubated for 24 h and then examined for the presence of precipitin lines formed by the interaction of antibody with antigen. Three different antigens were tested: S. enteritidis flagella, SEF14 (a 14-kDa fimbrial protein produced ostensibly by S. enteritidis), and a sodium deoxycholate extract of whole S. enteritidis organism. Flagella and the organism extract detected antibodies to S. enteritidis in the yolk and sera, whereas SEF14 was not reactive. Positive reactions were observed in serum 1 wk postchallenge, whereas in yolks, this was further delayed by 1 wk. The sensitivity of the test was slightly less than the standard microagglutination assay, although specificity was slightly higher, as indicated by results from sera and yolks from birds infected with Salmonella enterica serovar Typhimurium. Simplicity and low labor requirements of the assay would allow for the potential testing of several hundred egg samples within a day, which would make up for test shortcomings due to sensitivity. The AGP test could be an important tool for individuals using serological testing to monitor the S. enteritidis situation within their flocks or as a rapid screen for vaccine responses. The assay could also be used in tandem with other AGP tests to screen for the presence of multiple avian pathogens.

Efficacy of Recombinant Fowl Poxvirus Vaccine in Protecting Chickens against a Highly Pathogenic Mexican-Origin H5N2 Avian Influenza Virus

Internationally and nationally, governments and the poultry industries have used various strategies to control avian influenza (AI), ranging from a minimum of living with mildly pathogenic AI virus (AIV) infections to the other extreme of implementing a total quarantine-slaughter approach for eradication of highly pathogenic (HP) forms of the disease. However, recent economic considerations in various countries have prompted a broader reevaluation of vaccination as one of several tools to be used in AI control programs, including H5 and H7 HP AI. In the current study, 1-day-old chickens were immunized with a recombinant fowl poxvirus vaccine containing a hemagglutinin gene insert (Vector-HA) from an H5 AIV. Vector-HA- and negative control (vector-control)-vaccinated chicks were challenged with a HP H5N2 AIV isolated from chickens in Mexico. All immunized chickens were antibody negative on the agar gel precipitin test, indicating that vaccination would not interfere with routine AI serologic surveillance programs in the United States. However, in the hemagglutinin-inhibition test, a few immunized chickens (8%) had low serologic titers. Protection against illness (90-100%) and death (90-100%) was provided by the vector-HA vaccine from 3 wk of age to the end of the 20-wk study. The number of chickens shedding the challenge AIV from their enteric tracts was significantly reduced (50-75%) and the quantity of challenge AIV shed from respiratory and enteric tracts was significantly reduced (10(1)-10(2.1) mean embryo lethal dose/ml) in most vector-HA vaccine groups when compared with vector-control groups. Furthermore, vector-HA vaccination reduced in contact transmission of HP AI challenge virus to both vector-HA- and vector-control-vaccinated chickens. These findings indicate the recombinant fowl poxvirus vaccine can be a useful tool in an AI control program by preventing illness and death in chickens and reducing intestinal and respiratory shedding of H5 AIV. However, for an AI control program to be successful, enhanced biosecurity and surveillance must be practiced, and the vaccine's use must be controlled by an industry and/or government task force.

Pathogenicity and Diagnosis of H5N2 Mexican Avian Influenza Viruses in Chickens

Chickens were inoculated with one of five H5N2 Mexican-origin avian influenza virus (AIV) isolates to determine their pathogenicity for chickens and to determine the ability of routine virologic and serologic tests to detect infections. In laboratory infections, three AIVs, H5/94, M5/94, and J12/94, produced sporadic illness and death and were categorized as mildly pathogenic. Q1/95 produced illness and death in all inoculated chickens and was categorized as highly lethal and highly pathogenic (HP). P11/94B commonly produced clinical illness, but deaths were infrequent. During the presence of clinical signs, oropharyngeal swabs were superior for isolation of AIV, but cloacal swabs were more successful after disappearance of clinical signs. Agar gel precipitin (AGP) serologic test was superior for detecting AIV infection during the clinical phase, but AGP and hemagglutinin inhibition tests were equally effective in detecting infections after recovery from clinical illness. Passage of P11/94B parent stock and selected 14-day-embryo-passed AIVs in adult hens resulted in emergence of some HP AIV derivatives. The hemagglutinin of Q1/95 and P11/ 94B parent stock and derivative AIVs had an identical proteolytic cleavage site of.... Pro-Gln-Arg-Lys-Arg-Lys-Thr-Arg-Gly, consistent with AIVs of high pathogenicity. However, no consistent differences were identified in the sequence of the hemagglutinin gene to explain the discrepancy in lethality patterns of the P11/94B AIVs. This suggests that genes other than the hemagglutinin impact the full expression of high lethality of Mexican-origin AIV infections in chickens.

Isolation of serotype 1 Marek's disease viruses from vaccinated Australian flocks

Field samples were received for Marek's disease virus (MDV) isolation from clinically affected flocks from several regions of Eastern Australia. Lymphocytes were fractionated in Ficoll-Paque and passaged once or twice in chicken embryo kidney cultures. Serotype-specific virus was detected in infected cultures by indirect immunofluorescence with monoclonal antibodies. Serotype 1 MDV was isolated from 10 flocks. In samples from 5 of these flocks, serotype 2 and 3 vaccine viruses were isolated from the same specimen. In a parallel study, plasmas obtained during lymphocyte isolation were tested for antibodies to MDVs by agar gel precipitin (AGP) tests using serotype 1 and 3 antigen extracts. No correlation was observed between the rate of virus isolation and AGP positivity. The AGP test was incapable of discriminating between the different MDV serotypes.

A Highly Sensitive, Broad-Spectrum Infectivity Assay for Infectious Bursal Disease Virus

In order to develop an infectivity assay for infectious bursal disease virus (IBDV), an enzyme-linked immunosorbent assay (ELISA) for detecting IBDV antigens was developed using a rabbit antiserum to IBDV. The ELISA detected both serotypes 1 and 2 of IBDV, purified virus proteins at a concentration of about 0.1 ng/well and about 10(4) to 10(5) infectious units/well, and was about 10(3) times more sensitive than an agar gel precipitin test. By using the ELISA for detecting IBDV antigens in the cultured fluids, infectivity titration could be determined within 5 days of cultivation in seven of the 10 virus-cell combinations when chicken embryo fibroblasts (CEFs), BGM-70 cells, and LSCC-BK3 cells were used. IBDV antigens were not detected in three combinations remaining after 7 days of cultivation. When 18 IBDV strains, including highly virulent, classically virulent, and attenuated strains, were tested for growth in four types of cells using the ELISA, eight strains in CEFs, seven strains in chicken kidney cells, five strains in BGM-70 cells, and 17 strains in LSCC-BK3 cells were detected. These results indicated that LSCC-BK3 cells had the broadest spectrum for IBDV. When 26 field bursal homogenates were tested for infectious IBDV using LSCC-BK3 cells, all 19 field IBDV isolates that were detected by immunostaining of the cells were also detected by the ELISA. These results indicate that this infectivity assay for IBDV using LSCC-BK3 cells and the ELISA has a high sensitivity and a broad spectrum for IBDV and is especially useful for large-scale applications.

Efficacy of Three Live Vaccines against Highly Virulent Infectious Bursal Disease Virus in Chickens with or without Maternal Antibodies

Since 1990, highly virulent infectious bursal disease virus (IBDV), which induces high mortality, has been infecting even vaccinated flocks in Japan. We report the efficacy of three live vaccines that are available in Japan. Two mildly attenuated strains (A and B) and one intermediate strain (C) were each tested both in specific-pathogen-free (SPF) chickens and in commercial chickens that have maternal antibodies against IBDV. Chickens were vaccinated at 20 days old and challenged with highly virulent IBDV 10 days post-vaccination. Protection was determined 7 days after challenge by measuring bursa/body weight ratios, histopathological lesions, and antibody responses to IBDV. All three lie vaccines conferred protection to SPF chickens. However, only vaccine C protected 100% of vaccinated commercial chickens against highly virulent IBDV; Vaccines A and B respectively protected three-fourths and none of vaccinated commercial chickens from severe bursal lesions. Vaccines A, B, and C and highly virulent IBDV induced bursal lesions in 3%, 0%, 23%, and 61% of inoculated commercial chickens, respectively. These results suggest that serological determination of the optimum vaccination time for each flock is required to effectively control highly virulent IBDV in the field. The optimum vaccination timing could be approximated by titrating the maternal IBDV antibodies of 1-day-old chicks by an enzyme-linked immunosorbent assay or by an agar gel precipitin test.

Entwicklung humoraler präzipitierender Antikörper gegen Campylobacter spp. beim Huhn

Development of humoral precipitating antibodies against Campylobacter spp. in chickens. The development of precipitating antibodies in chickens was examined by two-dimensional immunodiffusion test after immunization with a formol inactivated vaccine and after subcutaneous and oral application of different live campylobacter serovars. The supernatant of bacterial cells after sonication and centrifugation was used as an antigen in the agar-gel precipitin test. Antisera against different campylobacter serovars showed a high percentage of cross-reactions. In chickens immunized with an inactivated vaccine at an age of 1, 2, 3, 4 and 7 weeks, precipitating antibodies could be demonstrated for the first time at 7 days p.i. Except for 1-week-old birds, sera from the other groups reacted positively at 14 days p.o. After subcutaneous duplication of live organisms to 4-week-old chickens, antibodies could already be demonstrated at 4 days p.i. later in part of the experimental groups. No interrelation could be detected between antibody titers, measured by enzyme-linked immunoabsorbent assay (ELISA), from precipitating sera, as well as from those from non-precipitating sera. Precipitating antibodies and antibody titers in the ELISA were examined in sera from groups of birds infected at an age of 1, 2, 3, 4 and 7 weeks. During the Campylobacter excretion period, a distinct peak of antibody titers occurred in 1- and 7-week old birds, whereas other groups showed a steady increase in titers. Precipitating antibodies were only found in 1- and 2-week-old chickens.

A rapid quantitative method for detecting infectious bursal disease virus using polystyrene latex microspheres

A monoclonal antibody (mAb) to infectious bursal disease virus (IBDV) was bound to polystyrene latex microspheres. The microspheres agglutinated with extracts of bursae and sera from chickens infected with all strains or isolates of IBDV tested. Agglutination appeared within a 10-min reaction time. The assay could detect a 10 3.7 to 10 4.5 mean embryo infective dose (EID 50) of the virus in 0.01 ml and the titer of the assay was 10- to 40-times higher than that of the agar gel precipitin test.

Effect of Route of Administration on the Efficacy of a Recombinant Fowlpox Virus against H5N2 Avian Influenza

A recombinant fowlpox vaccine virus containing the H5 hemagglutinin gene of avian influenza virus was administered to susceptible chickens via wing-web puncture, eye drop, instillation into the nares, and drinking water. Even though there was a negligible hemagglutination-inhibition (HI) serologic response, all 10 chickens vaccinated by wing-web puncture remained without obvious signs of disease and survived challenge with a highly pathogenic strain of H5N2 avian influenza virus. All unvaccinated chickens and those vaccinated by nasal and drinking-water routes died following challenge. Eight of 10 chickens vaccinated with the recombinant by eyedrop died. All vaccinates were negative on the agar gel precipitin (AGP) test, and only one chicken had a positive HI titer before challenge. All chickens that survived challenge had high levels of HI antibody and were positive on the AGP test, indicating that they were infected by the challenge virus.