The beneficial effects of spraying of probiotic Bacillus and Lactobacillus bacteria on broiler chickens experimentally infected with avian influenza virus H9N2.

This study investigated the clinical, antiviral, and immunological effects of spraying Bacillus spp. and Lactobacillus spp. as a single or mixture probiotic compound on experimentally infected broiler chickens with AIV H9N2. Two hundred and forty 1-day-old broilers were randomly assigned to 6 groups as follows: Ctrl- (no challenge AIV; no spray probiotic), Ctrl+ (AIV challenged; no spray probiotic), AI+B (AIV challenged; daily spraying of probiotic Bacillus spp.), AI+L group (AIV challenged; daily spraying of probiotic Lactobacillus spp.), AIV+BL (AIV challenged; daily spraying of probiotic Bacillus spp. and Lactobacillus spp.), and G-DW (daily spraying of normal saline; no AIV challenged). The birds were reared for 35 d. On the 22nd day of age, broiler chickens were challenged by AIV H9N2. The probiotics were sprayed at 9×109 CFU/m2 daily for 35 d. Growth performance, clinical signs, virus shedding, macroscopic and microscopic lesions were evaluated at various days in all groups. Spraying with probiotics improved the body weight gain and food conversion ratio in the AI+B, AI+L, and AI+BL groups compared to the Ctrl+. The severity of clinical signs, gross lesions, pathological lesions and viral shedding in the probiotic treatment groups was lower than in the Ctrl+ group. The findings of this study suggest the daily spraying of Lactobacillus and Bacillus probiotics alone or in combination during the rearing period reduce clinical and nonclinical aspects of H9N2 virus infection; so, it can be effective as a preventive protocol for controlling the severity of AIV H9N2 infection in broilers.

Apoptosis in field and experimental cases of avian influenza H9N2 infection in broiler chickens in Iran.

Avian influenza virus subtype H9N2 is widely circulating around the globe affecting many species of animal including mammals and birds as well as human beings. The virus has pandemic potential due to segmented nature of the viral genome. Ultra-structural features of apoptosis in field and experimental infection of H9N2 avian influenza virus were studied. Freshly dead birds from affected broiler farms and experimentally infected broiler chickens with H9N2 subtypes were subjected to routine necropsy. Post-mortem findings in different organs were recorded. Appropriate specimens from the trachea were taken for electron microscopy studies. In electron microscopy study, frequent apoptotic bodies were observed in the epithelial cells of the trachea. Increase of antibody titer to H9N2 virus following challenge with the virus in experimental group indicates that the infectious cycle has been initiated in the affected birds.

High incidence of multidrug resistance and class 1 and 2 integrons in Escherichia coli isolated from broiler chickens in South of Iran.

The objective was to investigate the multidrug resistance and presence of class 1 and 2 integrons in 300 Escherichia coli isolates obtained from 20 broiler farms during three rearing periods (one-day-old chicks, thirty-day-old chickens, and one day before slaughter) in Fars, South Iran. Results showed that 81.00%, 82.00%, and 85.00% of isolates were multidrug-resistant on the first day, thirty-day-old chickens, and one day before slaughter, respectively. Multidrug-resistant E. coli isolates were further examined for the presence of class 1 and 2 integrons using PCR assay. The existence of class 1 integron-integrase gene (intI1) was confirmed in 68.40%, 72.70%, and 60.90% of multidrug-resistant isolates from stage 1, stage 2, and stage 3 of the rearing period, respectively. The frequency of class 2 integron-integrase gene (intI2) during the first to the third stage of sampling was 2.60%, 25.50%, and 30.40%. Also, sequence analysis of the cassette arrays within class 1 integron revealed the presence of the genes associated with resistance for trimethoprim (dfrA), streptomycin (aadA), erythromycin (ereA), and orfF genes. The results revealed that percentages of antimicrobial resistance in E. coli isolates were significantly higher in the middle and end stages of the rearing period. In conclusion, widespread dissemination of class 1 integrons in all three stages and rising trends of class 2 integrons existence in E. coli isolates during the rearing period of broiler chickens could exacerbate the spread of resistance factors among bacteria in the poultry industry. Future research is needed to clarify its implication for human health.

Role of cardiac hypoxia in the pathogenesis of sudden death syndrome in broiler chickens - A metabolic and molecular study.

Sudden death syndrome (SDS) is an economically important disorder in broiler chickens with unknown aetiology. The aim of the present study was to evaluate the metabolic and molecular alterations related to hypoxia in the myocardium of broiler chickens with SDS. Samples from the cardiac muscle of internal control broiler chickens (ICs) (n = 36) and chickens having died of SDS (n = 36) were obtained during the rearing period. The activities of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) and the concentration of lactate were measured in the cardiac tissue using available commercial kits. The expression of hypoxia-inducing factor 1α (HIF1α), glucose transporter 1 (GLUT1), pyruvate dehydrogenase kinase 4 (PDHK4) and monocarboxylate transporter 4 (MCT4) genes was determined in the myocardium by real-time PCR analysis. The results showed the elevation of lactate level and activities of LDH and CPK in the cardiac muscle of SDS-affected chickens compared with the IC birds (P < 0.05). The cardiac muscle expression of HIF1α, MCT4 and GLUT1 genes was increased, while the PDHK4 mRNA level was decreased in the SDS-affected group compared to those in the IC chickens (P < 0.05). Our results showed that metabolic remodelling associated with hypoxia in the cardiac tissues may have an important role in the pathogenesis of cardiac insufficiency and SDS in broiler chickens.

Effect of Newcastle disease and infectious bronchitis live vaccines on the immune system and production parameters of experimentally infected broiler chickens with H9N2 avian influenza.

H9N2 Avian influenza (AI) is an infectious disease which considered to have low pathogenic virulence, but in the case of coinfection with other pathogens it has the potential to become a major threat to the poultry industry. Infectious bronchitis (IB) and Newcastle diseases (ND) are other common problems to the poultry industry, which there are an extensive vaccination program against these viral pathogens. To investigate the effects of administration of infectious bronchitis and Newcastle disease live vaccines (IBLVs and NDLVs) in the presence of H9N2 AI infection on the immune system and some production parameters, 180 one-day-old broiler chicks were randomly allocated into six groups with different vaccination programs including H120 IBLV, 4/91 IBLV, B1 NDLV and LaSota NDLV. At the age of 20 days, all birds of the experimental groups except the negative control group, were inoculated intra-nasally (at dose of 106 EID50) with H9N2 AIV. After the inoculation, gross and microscopic lesions of the immune organs, serological changes and some production parameters were examined. The findings of this study showed that coinfection of H9N2 AI with NDLVs exacerbated the gross and microscopic injuries in the immune organs; especially the bursa of Fabricius. LaSota + AIV group had the most severe lesion in the bursa of Fabricius, spleen and thymus. Furthermore, the birds of LaSota + AIV group consumed the least amount of feed and water and their final body weight were significantly (P ≤ 0.05) lower in comparison with the other groups. Interestingly, in the context of this experiment both 4/91 and H120 IB live vaccines enhanced the HI antibody titers against H9N2 AIV, but the 4/91 showed the most significant (P ≤ 0.05) increase compared to the other experimental groups.

Gizzard Erosion Associated with Fowl Adenovirus Infection in Slaughtered Broiler Chickens in Iran.

Gizzard erosions have been noticed in slaughtered broiler chickens during inspection at a processing plant in Iran. The condition was detected in piled gizzards derived from seven commercial broiler farms brought to slaughter on the same day. In total, 48 gizzards with lesions underwent thorough pathologic and virologic investigation. Perforation, roughening, and discoloration of the koilin layer as well as inflammation of the mucosa were observed macroscopically. Histologic examination showed dissociation of and cellular debris in the koilin layer accompanied by a loss and degeneration of glandular epithelium with mild to marked infiltration of inflammatory cells in the mucosa, submucosa, and muscular layer. Fowl adenovirus serotypes 1 (FAdV-1), 11 (FAdV-11), and 8a (FAdV-8a) were found in 13, 12, and 1 gizzard(s), respectively. Therein included were two gizzards that showed mixed infections with FAdV-1 and FAdV-11. Detailed analysis of the hexon gene revealed that the Iranian FAdV-1 isolates could be divided into two subclusters, more closely related to either the European (CELO) or the Asian (Ote) FAdV-1 reference strains. The present study, for the first time, describes not only the appearance of gizzard erosion but also the isolation of FAdV-1 and FAdV-8a from broilers in Iran and offers insights on the epidemiology of FAdV infection in Iranian flocks.

Erosión de molleja asociada con infección por adenovirus del pollo en pollos de engorde procesados en Irán. Se han observado erosiones de molleja en pollos de engorde sacrificados durante la inspección en una planta de procesamiento en Irán. La condición se detectó en mollejas apiladas derivadas de siete granjas comerciales de pollos de engorde que fueron sacrificados el mismo día. En total, 48 mollejas con lesiones se sometieron a una exhaustiva investigación patológica y virológica. Se observó macroscópicamente perforación, rugosidad y la decoloración de la capa de queratina, así como inflamación de la mucosa. El examen histológico mostró disociación y restos celulares en la capa de queratina acompañada por una pérdida y degeneración del epitelio glandular con infiltración leve a marcada de células inflamatorias en la mucosa, la submucosa y la capa muscular. Se encontraron aviadenovirus del pollo de los serotipos 1 (FAdV-1), 11 (FAdV-11) y 8a (FAdV-8a) en trece, doce y una molleja (s), respectivamente. Se incluyeron dos mollejas que mostraban infecciones mixtas con FAdV-1 y FAdV-11. El análisis detallado del gene de la proteína del hexon reveló que los aislamientos iraníes del serotipo FAdV-1 se dividieron en dos subgrupos, más estrechamente relacionados con las cepas de referencia del serotipo 1 de Europa (CELO), o de Asia (Ote). El presente estudio describe por primera vez, no solo la aparición de la erosión de la molleja, sino también el aislamiento de FAdV-1 y FAdV-8a de los pollos de engorde en Irán y ofrece información sobre la epidemiología de la infección por FAdV en parvadas iraníes.

Respiratory and GIT tract immune responses of broiler chickens following experimental infection with Newcastle disease's virus.

Newcastle disease causes a lymphoproliferative response in the tracheal and intestinal mucosa of the infected birds. In this study, the Hitchner B1 and I-2 vaccine and challenging of ND field strains were used to evaluate the populations of T lymphocyte subsets infiltrated intestinal and tracheal, also to shed some light on cell-mediated immune response using enzyme-linked immunosorbent assay (ELISA) detecting chicken's serum interferon-γ. Three hundred-day-old broilers were randomly divided into four groups. Groups 1 and 2 received I-2 and B1 vaccines, respectively, while groups 3 and 4 were challenged-unvaccinated and unchallenged-unvaccinated groups. Blood samples were taken from five random chicks and were then tested with ELISA test. Three chicks of each group were euthanized after vaccine administration and also challenging with acute virus. Interferon-γ changes were significant in time (p < 0.001). Totally, there was no significant difference between I-2 and B1 groups. The number of CD3+, CD4+, and CD8+ cells of I-2 and B1 vaccinated group's intestine and the trachea samples was significantly increased compared with the negative control group (p < 0.001). The results indicated the significant increase in CD4+ and CD8+ in intestinal and tracheal tissues, while the level of interferon-γ of the vaccinated group was more than the unvaccinated one. Finding no significant differences between the vaccinated groups indicated the potential of both vaccines in producing CD4+ and CD8+ in the tracheal and intestinal tissues and the equality of interferon-γ production in the sera.

Anticoccidial effects of Artemisia annua ethanolic extract: prevention, simultaneous challenge-medication, and treatment.

The effect of Artemisia annua ethanolic extract (AE) as a potential source of herbal anticoccidial activity was investigated on experimental coccidiosis in chicken. One hundred ninety-two one-day-old chicks were divided in to 8 groups (n = 24) including AE prevention group, AE-treated group, simultaneously challenged AE-medicated group, challenged-untreated group (positive control), unchallenged-untreated group (negative control), salinomycine prevention group, salinomycine-treated group, and simultaneously challenged salinomycine-medicated group, in a completely randomized design. Oral challenge carried out by a suspension containing a mixture of 200,000 oocysts Eimeria acervulina, 30,000 oocysts Eimeria necatrix, and 20,000 oocysts Eimeria tenella on day 21 of age. Weight gain in AE prevention group significantly increased compared to positive control group (p < 0.05). Unlike salinomycine prevention group, the food conversion ratio (FCR) of AE prevention group was not significantly higher than negative control. Oocyst per gram (OPG) in simultaneously challenged AE-medicated group had no significant difference, while for 38% of the days, in simultaneously challenged salinomycine-medicated group significantly decreased (p < 0.05). The food intake of AE-treated group had no significant difference with salinomycine-treated group (p > 0.05). In half of the days of OPGs sampling, AE-treated group was reduced significantly compared to positive control group (p < 0.05). Collectively, the in vivo study of anticoccidial effects of AE in the prevention section was more effective than the treatment section, while the treatment section was more effective than the simultaneous section. We concluded that AE has a potential value to use as an herbal medicine for preventive measure in chicken coccidiosis.

Viral quantity and pathological changes in broilers experimentally infected by IRFIBV32 isolate of infectious bronchitis virus.

An Iranian isolate of avian infectious bronchitis virus IRFIBV32 was quantified in experimentally infected broilers using real-time reverse transcriptase polymerase chain reaction and histopathological changes was investigated. Thirty-six 3-week-old commercial broilers were inoculated by 10(5) ELD50/0.1 ml of the virus. On the various days post inoculation (dpi) different tissues were collected. The virus strongly started the replication in trachea at 1 dpi and reached to the maximum titer at 3 dpi. The highest IBV RNA level was shown in this organ. In lung, the virus was replicated with the titer lower than that of the trachea, but it rose up more at 5 dpi. The kidneys were the tissues with the least viral genome copy number, although the duration of the virus presence was considerable. The virus replicated in testes sooner than ovaries also disappeared sooner but the maximum viral yield in the ovaries was more. The virus titer in the studied tissues had an interesting fluctuation especially in caecal tonsils. Testes and ovaries were the organs that the virus could reactivate without using any chemical. The most severe lesions were observed in tracheae but they appeared in the lungs later. Lymphocyte infiltration in the kidneys was noted from 5 dpi even sooner than the lungs. There were no lesions in the caecal tonsils, testes and ovaries in spite of the virus replication in a high titer.

Effects of Artemisia annua extracts on sporulation of Eimeria oocysts.

The present study aimed to compare the effect of different Artemisia annua extracts on sporulation rate of mixed oocysts of Eimeria acervulina, Eimeria necatrix, and Eimeria tenella. Three types of A. annua extracts including petroleum ether (PE), ethanol 96° (E), and water (W) extracts were prepared. Artemisinin, a sesquiterpene lactone endoperoxide derived from the A. annua analysis of each extract was done by high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Fresh fecal samples containing three Eimeria species were floated and counted, and the oocysts were transferred into 50 tubes, each containing 10(5) oocysts per milliliter. Five tubes were control. Each of the other 45 tubes contained one of three doses (1 part per thousand (ppt), 2 ppt, and 5 ppt) and one of three extracts (PE, E, and W extracts) with five replications. The tubes were incubated for 48 h at 25-29 °C and aerated. Sporulation inhibition assay was used to evaluate the activity of extracts. The results showed that the E and PE extracts inhibit sporulation in 2 and 5 ppt concentrations, but the W extract stimulates it in all concentrations. The proportions of oocyst inhibition relative to control were 31 % (5 ppt) and 29 % (2 ppt) for PE and 34 % (5 ppt) and 46 % (2 ppt) for E extract. Furthermore, many oocysts in PE and E groups were wrinkled and contained abnormal sporocysts. The proportions of sporulation stimulation relative to control were 22 % (5 ppt), 24 % (2 ppt), and 27 % (1 ppt) in W extract. Our study is the first to demonstrate that all types of A. annua extracts do not necessarily have a similar activity, and the interaction of all contents and their relative concentrations is an important factor for sporulation stimulation or inhibition. It seems, some parts of unmetabolized excreted PE and E extracts could inhibit oocyst sporulation and eventually affect infection transmission.

Tetracycline Resistance Genes in Campylobacter jejuni and C. coli Isolated From Poultry Carcasses.

BACKGROUND: Campylobacter is one of the leading bacterial species causing foodborne illnesses in humans. Antimicrobial agents have been extensively used for treatment of Campylobacter infections; but in the recent years, both animal and human isolates of this bacterium have shown resistance to several antibiotics such as tetracycline. OBJECTIVES: The aim of this study was to investigate the presence of genetic determinants of tetracycline resistance in Campylobacter spp. recovered from poultry carcasses in Shiraz, Iran. MATERIALS AND METHODS: Eighty-three thermophilic Campylobacter spp. Isolates were first identified based on multiplex polymerase chain reaction (PCR) and then screened for presence of tetracycline resistance genes (tet (A), tet (B), tet (O) and te (S)) by PCR. RESULTS: The overall prevalence of Campylobacter jejuni and C. coli among the examined isolates was 51.8% and 48.2%, respectively. Tetracycline resistance genes of tet (B) and tet (S) were not seen among these Campylobacter spp. Isolates, whereas the most common tet gene identified was tet (O), found in 83.1% (69/83) of all the isolates. The tet (O) gene sequence comparison between C. jejuni and C. coli showed 100% similarity and these sequences (JX853721and JX853722) were also identical to the homologous sequences of other strains of Campylobacter spp. existing in the GenBank databases. In addition, tet (A) was found in 18% (15/83) of Campylobacter spp. isolates. To our knowledge, this represents the first report of tet (A) in Campylobacter spp. There was 100% homology between the sequences of tet (A) from this study (JX891463 and JX891464) and the tet (A) sequences mentioned for other bacteria in the GenBank databases. CONCLUSIONS: The high prevalence of tet (O) resistance gene along with new detection of tet (A) resistance gene in Campylobacter spp. isolated from poultry carcasses revealed an extensive tetracycline resistance among Campylobacter isolates from poultry in Iran. It emphasized the need for cautious use of tetracycline in poultry production to decrease the extension of tetracycline-resistant Campylobacter spp.

Rapid detection of Escherichia coli gyrA and parC mutants in one-day-old broiler chicks in Iran.

Vertical and consequently horizontal transmission of quinolone and fluoroquinolone resistant Escherichia coli clones following hatch in chickens enables a massive amplification of these clones into a large population. The aim of this study was to determine the antibiotic resistance and susceptibility of Iranian E. coli isolates (n=105) from one-day-old chicks to fluoroquinolones and the relation of this resistance with mutations in gyrA and parC genes using PCR-RFLP. For the first time, EcoRV restriction enzyme was used for rapid mutation screening in parC (Ser80Ile). The results showed that the low level of Minimum Inhibitory Concentration (MIC) for ciprofloxacin (0.25-4µg ml-1) and enrofloxacin (0.25-4µg ml-1) corresponded to a single mutation in gyrA, while intermediary to high level of MIC for ciprofloxacin (8 --> 64 µg ml-1) and enrofloxacin (16 --> 64 µg ml-1) were related to 2 mutations in gyrA or 3 mutations, 2 in gyrA and 1 in parC. There was a strong positive correlation (R = 0.93, P < 0.001) between MIC levels of enrofloxacin and ciprofloxacin among these isolates. The article concludes by stressing that the rising incidence of enrofloxacin resistant E. coli isolates from chicken sources may increase the potential risk of ciprofloxacin resistant E. coli acquisition by humans.

Changes of several acute phase factors in broiler chickens in response to infectious bronchitis virus infection.

This study was performed to clarify the acute phase response following infectious bronchitis virus inoculation. Ninety clinically healthy 1-d-old Ross chicks were randomly assigned into 2 groups: control (n = 20) and infected group (n = 70). At the age of 20 d, all birds in the infected group were challenged intranasally with allantoic fluid containing 10(5) embryo lethal dose (ELD50)/0.1 mL of the infectious bronchitis virus. Blood samples were collected from 20 clinically healthy and 70 infected chicks at prior and 1, 2, 3, 5, 7, 11, 13, 15, and 20 d postinoculation. On d 1, 7, and 11 postinoculation 4 chickens from the experimental group and 2 chickens from the control group were randomly selected. Their trachea, lungs, and cecal tonsil were collected for virus detection and quantitation by real-time reverse-transcription PCR assay. In the serum the acute phase proteins (haptoglobin and serum amyloid A), pro-inflammatory cytokines (interferon-γ and tumor necrosis factor-α), and serum sialic acid (total, TSA; lipid-bound, LBSA; and protein-bound, PBSA) concentrations were measured using validated standard procedures. All variables were significantly higher in the infected birds after virus inoculation compared with the healthy group (P < 0.05). There were positive correlations between all variables in the infected group. Correlation coefficients were significantly positive between haptoglobin and interferon-γ, LBSA and TSA, and TSA and LBSA (P < 0.05). There were positive correlations among viral RNA and all studied variables; however, these correlations were not statistically significant (P > 0.05).

Pathogenesis and tissue distribution of avian infectious bronchitis virus isolate IRFIBV32 (793/B serotype) in experimentally infected broiler chickens.

Infectious bronchitis (IB) is one of the most important viral diseases of poultry. The aim of this study was to investigate the distribution of avian infectious bronchitis virus isolate IRFIBV32 (793/B serotype) in experimentally infected chicken. Ninety-one-day-old commercial broilers were divided randomly into two groups (seventy in the experimental and twenty in the control group). Chicks in the experimental group were inoculated intranasally with 10(5) ELD50/0.1 mL of the virus at three weeks of age. The samples from various tissues were collected at1, 2, 3, 5, 7, 11, 13, 15, and 20 days postinoculation. Chickens exhibited mild respiratory signs and depression. Viral RNA was detected in the kidney, lung and tracheas on days 1 to 13 PI, in the oviduct between, days 3 and 13, in testes between days 1 and 11 PI, and in the caecal tonsil consistently up to day 20 PI. The most remarkable clinical signs and virus detection appeared on day 1 PI. Data indicated that the number of infected chickens and viral RNA detection from tissues was reduced with increasing antibody titer on day 20 PI. The results demonstrated that the IRFIBV32 virus has wide tissue distribution for respiratory, urogenital, and digestive systems.

Risk factors for detection of bronchial casts, most frequently seen in endemic H9N2 avian influenza infection, in poultry flocks in Iran.

During the last decade, low pathogenic avian influenza (H9N2 subtype) outbreaks with high mortality have been reported in broiler chicken in various countries especially in some Asian and Middle East countries including Iran. Tubular bronchial cast extending to the lower bronchi (BC) is one of the most frequently observed post-mortem lesions in affected broiler chicken during H9N2 low pathogenic avian influenza virus outbreaks. This study was conducted to find out risk factors for high mortality in chickens suffering from respiratory symptoms and showing BC in post-mortem examination. History and general information of the flocks as well as vaccination programs, mortality rate and necropsy findings such as formation of BC in airways were collected for 563 broiler flocks in central part of Fars province, South-West of Iran, during 2001-2006. Results showed that overall mortality rate was 13.52% (95%CI: 12.2-14.8), decreasing from 18% in 2000 to 11% in 2006 (P<0.01). Corresponding measures for BC were 22% (95%CI: 18-26) with no significant decreasing trend during the study period (P=0.14). Multivariable logistic regression analysis showed that infectious bronchitis (IB) vaccination, flock size and geographical location were significantly associated with the observation of BC (P<0.05). Odds of BC observation in flocks with history of IB vaccination was seven times higher than flocks without vaccination, and in small flocks was nearly half of the large flocks. In conclusion, IB vaccine can be one of the candidate risk factors for enhancing the virulence of low pathogenic H9N2 virus in the fields.

Natural cases and an experimental study of H9N2 avian influenza in commercial broiler chickens of Iran.

Since 1998, an epidemic of avian influenza has occurred in the Iranian poultry industry. The agent was pathotyped as non-highly pathogenic and subtyped as an H9N2 avian influenza virus. Therefore it did not require eradication. However, frequent incidences of high mortality were observed commonly on broiler farms. No other species of bird were affected. The circulation of the virus and mixed infection with other respiratory pathogens, particularly infectious bronchitis virus and Mycoplasma gallisepticum, were incriminated in the high mortality on poultry farms and resulting great economic losses. Clinical signs in both field and experimental studies included swelling of the periorbital tissues and sinuses, nasal and ocular discharge, and severe respiratory distress. However, in the experimental study, the mortality rate was much lower than in the natural outbreak. Gross lesions identified included extensive congestion of the respiratory tissues, and exudation with cast formation in the tracheal bifurcation, which extended to the secondary bronchi. Severe necrotizing tracheatis was the predominate histological lesion. Ultrastructurally, orthomyxovirus-like particles were identified in the inoculum used for the experimental study. An inactivated H9N2 avian influenza vaccine prevented mortality in experimentally challenged chickens.