Pathotypic and molecular characterization of a fowl adenovirus associated with inclusion body hepatitis in Saskatchewan chickens

Inclusion body hepatitis (IBH) is one of the major global disease problems, causing significant economic losses to poultry industry of the United States and Canada. The disease is characterized by its sudden onset and high mortalities. Amongst different serotypes of fowl adenoviruses (FAdVs) associated with IBH, serotype 8 of group I FAdV has been isolated from majority of IBH cases. In present studies, we isolated a FAdV from morbid liver of a 17-day-old broiler from a Saskatchewan broiler farm. This newly isolated virus was designated as IBHV(SK). However, based on the sequence analysis of the L1 region of the hexon gene, the IBHV(SK) may be classified as FAdV 8b strain 764. These studies describe for the first time the complete hexon gene sequence of FAdV serotype 8b. Experimental infection of 2-day-old (n = 48) and 2-wk-old (n = 56) chicks caused 83% and 43% mortalities, respectively. Determination of the complete hexon gene sequence of IBHV(SK) with establishment of a disease model in chickens will facilitate the development of type-specific diagnostic reagents and assays for the evaluation of potential experimental vaccines against pathogenic FAdV infections.     

Isolation and Molecular Characterization of Fowl Adenovirus and Avian Reovirus from Breeder Chickens in Japan in 2019–2021

Fowl adenoviruses (FAdVs) and avian reoviruses (ARVs) are ubiquitous in poultry farms and most of them are not pathogenic, yet often cause damage to chicks. A total of 104 chicken fecal samples were collected from 7 farms of breeder chickens (layers and broilers) in Japan from 2019 to 2021, and yielded 26 FAdV plus 14 ARV isolates. By sequencing, FAdV isolates were classified as FAdV-1, 5 and 8b. ARV isolates were classified as genotype II, IV and V. These results suggest that FAdVs and ARVs are resident in the breeder chicken farms in Japan.     

Ⅰ群禽腺病毒血清11型的分离鉴定及致病性试验

【目的】 了解江苏地区禽腺病毒（Fowl adenovirus,FAdV）的基因遗传演化情况及致病性,为FAdV的流行病学研究和疫病防控提供参考。【方法】 通过SPF鸡胚盲传、PCR鉴定、电镜观察、全基因组测序、序列比对与相似性分析判定病毒血清分型,并将分离株以5×10^5.33 EID₅₀剂量胸肌注射的方式感染10日龄SPF雏鸡进行动物回归试验。【结果】 PCR结果显示,该分离株为Ⅰ群FAdV阳性,在透射电镜下可见球型、无囊膜、具有腺病毒典型的二十面体结构,通过全基因组和Hexon基因进行序列分析表明该分离毒株为FAdV D种血清11型毒株,命名为JSNT-1株。将该毒株感染SPF雏鸡,死亡率为10%（1/10）,病死鸡剖检可见肝脏褪色变黄,出血肿胀,边缘钝圆;肾脏肿大出血,苍白等病变。通过实时荧光定量PCR试验可从口腔及泄殖腔中检测到排毒,且病毒在鸡体内多个组织器官均有分布。病理组织学结果显示,死亡鸡的肝细胞变性坏死,出现嗜碱性核内包涵体;肾小球上皮细胞变性坏死;心肌可见大量炎性细胞浸润。【结论】 分离株为FAdV血清11型,感染SPF鸡后临床发病不明显,致死率低,病死鸡能产生特征性的包涵体肝炎病变,且病毒可在鸡体内外进行复制。     

Application of high-resolution melting curve analysis for typing of fowl adenoviruses in field cases of inclusion body hepatitis

Objective Fowl adenoviruses (FAdVs) cause inclusion body hepatitis (IBH) in chickens. In this study, clinical cases of IBH from Australian broiler flocks were screened for the presence and genotype of FAdVs. Methods Twenty‐six IBH cases from commercial poultry farms were screened. Polymerase chain reaction (PCR) coupled with high‐resolution melt (HRM) curve analysis (PCR/HRM genotyping) was used to determine the presence and genotype of FAdVs. For comparison, field isolates were also assessed by virus microneutralisation and nucleotide sequence analysis of the hexon loop 1 (Hex L1) gene. PCR detection of chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) was also employed. Results FAdV‐8b and FAdV‐11 were identified in 13 cases each. In one case, FAdV‐1 was also identified. Cross‐neutralisation was observed between the FAdV‐11 field strain and the reference FAdV‐2 and 11 antisera, a result also seen with the type 2 and 11 reference FAdVs. Field strains 1 and 8b were neutralised only by their respective type antisera. The FAdV‐8b field strain was identical to the Australian FAdV vaccine strain (type 8b) in the Hex L1 region. The Hex L1 sequence of the FAdV‐11 field strain had the highest identity to FAdV‐11 (93.2%) and FAdV‐2 (92.7%) reference strains. In the five cases tested for CAV and IBDV, neither virus was detected. The evidence suggested the presence of sufficient antibodies against CAV and IBD in the parent flocks and there was no indication of immunosuppression caused by these viruses. Conclusion These results indicate that PCR/HRM genotyping is a reliable diagnostic method for FAdV identification and is more rapid than virus neutralisation and direct sequence analysis. Furthermore, they suggest that IBH in Australian broiler flocks is a primary disease resulting from two alternative FAdV strains from different species.     

Serologic and pathogenetic studies on avian reoviruses isolated in Japan

Eighty-nine avian reoviruses isolated from diseased and clinically normal chickens were classified serologically using antisera against five prototype strains. Eighty-three strains were classified into five serotypes; six strains were untypable. Most of the cytopathogenic strains that produced a clear cytopathic effect (CPE) in chicken embryo fibroblasts (CEFs) were highly pathogenic for chicken embryos (80% or more mortality via the allantoic sac) and for chicks (severe footpad swellings and tenosynovitis). These strains were classified into a single serotype represented by the TS-142 prototype strain. However, 10 strains that could not produce a clear CPE in CEFs showed very low pathogenicity for embryos and chicks, and these strains were serologically different from the TS-142 prototype strain. There was a strong relationship between pathogenicity and serotype. About 17% of the isolates were considered highly pathogenic.     

Study of vertical transmission of fowl adenoviruses

The vertical transmission of fowl adenoviruses (FAdVs) was studied by polymerase chain reaction (PCR) and virus isolation. Liver, spleen, kidney, and bursa of Fabricius were collected from 60 chicks 1 d old representing progenies hatched to 6 broiler breeder flocks in 6 geographically different premises in Ontario, Canada. The presence of FAdV DNA sequences was detected by PCR with the use of primers specific for the conserved pVI gene of FAdV-9 in 58 (24%) of the 240 samples tested. All samples from 1 flock were negative for FAdV sequences, and only a few samples were positive in 3 flocks, whereas 32% and 72% of the samples from the other 2 flocks were positive. Testing of 1 sample with primers designed to amplify the L1 region of the hexon protein gene and amino acid sequence analysis of the PCR product indicated that the sequences were similar to serotype-8a FAdV sequences. No fowl adenoviruses were isolated in chicken hepatoma cells from any of the 30 samples inoculated. These findings imply that vertical transmission and establishment of latent infection with FAdVs can occur in chickens.     

Emergence of fowl aviadenovirus C-4 in a backyard chicken flock in California

Fowl aviadenovirus (FAdV) species D and E are associated with inclusion body hepatitis (IBH); species C, serotype 4 (hereafter, FAdV4) is associated with hepatitis–hydropericardium syndrome (HHS) in young chickens. Outbreaks of HHS have led to significant losses in the poultry industry in several countries, predominantly in China. In April 2020, FAdV4 was detected in a remote backyard flock in California. In a mixed flock of chickens of various breeds and ages (6 mo to 2 y old), 7 of 30 were found dead within a week without premonitory signs. One additional bird died after the flock was relocated to fresh pasture, bringing the total mortality to 8 of 30 (27%). Postmortem examination of 3 birds revealed good body condition scores and active laying. One chicken had subtle hemorrhages throughout the liver, and the other 2 had diffusely dark mahogany livers. On histopathology, 2 chickens had hepatic necrosis with hepatocytes containing large, mostly basophilic, intranuclear inclusion bodies, identified by electron microscopy as 82.2-nm diameter adenoviral particles. Virus isolation and genomic sequencing performed on a liver sample revealed strains with 99.9% homology to FAdV4 isolates reported from China. To our knowledge, FAdV4 has not been reported in the United States to date. Furthermore, the chickens affected here were all adults and exhibited a variation of serotype 4 disease in which IBH was present but not hydropericardium.     

Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates

Outbreaks ofhydropericardium syndrome (HPS), caused by fowl adenovirus serotype 4 (FAdV-4), have occurred in Korea and caused severe economic loss due to mortality and weight loss. From these outbreaks, several adenoviruses were isolated and identified in samples from broilers, layers, breeders, and native Korean fowl. In pathologic examinations, hydropericardium and multifocal hepatic necrosis, with an intranuclear inclusion body in hepatocytes, were observed. Specific adenovirus particles were also observed in the nucleus of hepatocytes, by electron microscopic examination. Polymerase chain reaction (PCR) analysis of the hexon gene identified all of the isolates as FAdV, serotype 4 and genotype C. To reproduce FAdV-4 field cases, 8- and 52-week-old specific pathogen free (SPF) chicks were infected intramuscularly with the field isolate CBU070244. The mortality rate of infected chicks ranged from 10%-40%, and specific pathologic lesions, such as swollen livers and hydropericardium, were observed. Further studies to determine the prevalence of infection, and analysis of the economic impact to the poultry industry, are needed in the near future.     

我国主要流行的4种血清型禽腺病毒-I群感染SPF鸡的致病性比较研究

旨在对我国流行的4种血清型(4、8a、8b和11型)禽腺病毒-I群(fowl adenovirus, FAdVs)对鸡胚和鸡的致病性进行研究,选取12日龄SPF鸡胚和10日龄SPF鸡感染4种血清型FAdVs,对SPF鸡胚和鸡的死亡率及鸡胚胚体质量进行统计;对感染鸡的大体剖检变化、组织学变化进行观察和病毒载量进行研究。结果显示,FAdV-8b和FAdV-11感染SPF鸡胚后其死亡率高于45%,FAdV-8a感染后胚体质量降低最严重,FAdV-4感染后对胚体质量影响最小;FAdV-4感染SPF鸡死亡率高达53.3%,其他组鸡无死亡;除肝出现肿胀、变性、出血和炎性细胞浸润外,4种病毒感染SPF鸡后分别造成不同组织器官的严重损伤,FAdV-4感染出现心包积液、FAdV-8a感染肌胃出现糜烂、FAdV-8b感染导致腺胃肿胀和FAdV-11感染导致胰腺坏死,各损伤组织均出现实质细胞坏死和炎性细胞浸润;4种病毒感染SPF鸡均出现免疫器官的组织学损伤,其中,FAdV-8a、-8b 和-11感染鸡引起的免疫器官内淋巴细胞缺失和损伤较FAdV-4更为严重;组织病毒载量结果显示,FAdV-4感染鸡心和肾病毒载量最高,FAdV-8b和FAdV-11感染鸡胰腺病毒载量高于FAdV-4感染组,且FAdV-4感染鸡心、肝和肾病毒载量在感染后第5天高于第3、7天,除FAdV-8a感染组外,病毒载量与各组织损伤情况相一致。综上表明,4种血清型FAdVs中,FAdV-11对SPF鸡胚的致死性最强,FAdVs对SPF鸡的致死率最高,尽管FAdV-8a、-8b 和-11不致死SPF鸡,但对SPF鸡胚、SPF鸡的消化系统和免疫系统损伤较为严重,这将会造成鸡胚孵化率降低、鸡生长发育缓慢和易于继发其他病原感染。     

Pathologic study of specific-pathogen-free chicks and hens inoculated with adenovirus isolated from hydropericardium syndrome.

The mortality and pathology caused by serotype 4 adenovirus, isolated from chickens with hydropericardium syndrome (HPS) in Japan, was investigated in specific-pathogen-free (SPF) chickens. One-day-old to 15-mo-old SPF chickens were inoculated intramuscularly, orally, and intranasally with liver homogenates from HPS chickens or isolated serotype 4 adenovirus. There were no clinical signs before death. The mortality rate in all groups of 1-day-old chicks was 100%, irrespective of the inoculum or inoculation route. Four-week-old chickens inoculated with liver homogenate also had a 100% mortality rate. Five-week-old chickens inoculated with cell culture of HPS adenovirus had a 40% mortality rate. The mortality rates of 7-mo-old hens inoculated with liver homogenates intramuscularly and orally were 75% and 25%, respectively. In 15-mo-old hens inoculated with liver homogenates intramuscularly, the mortality rate was 70%. Gross lesions were hydropericardium and swelling and congestion of the liver with occasional petechial hemorrhages. Histologically, the liver had diffuse or multifocal hepatic necrosis and hemorrhage with intranuclear inclusion bodies noted within hepatocytes. In the spleen, macrophages containing erythrocytes and yellow pigment were prominent in the red pulp. In the lung, a moderate diffuse macrophage infiltration was noted throughout the lung parenchyma, and these macrophages contained yellow pigment. In the pancreas of the chicks inoculated at 1 day old, there was multifocal necrosis of glands with intranuclear inclusion bodies. Intranuclear inclusion bodies were seen also in the gizzard, proventriculus, duodenum, cecum, kidney, and lung of the chicks inoculated at 1 day old. Immunohistochemically, the intranuclear inclusion bodies of various organs showed positive reactions against group I avian adenovirus. Adenovirus was recovered from the liver of chickens with HPS. This study indicates that HPS adenovirus is able to reproduce HPS lesions and mortality in SPF chicks and even adult chickens and that it is a highly pathogenic strain.     

Fowl adenoviruse-4 infection induces strong innate immune responses in chicken

Fowl adenovirus (FAdV), as the causative agent of hepatitis-hydropericardium syndrome (HHS), poses a significant threat to the poultry industry in China in recent years. In this study, we investigated the immunopathogenesis of a FAdV-4 strain HN/151025 in 60-day-old chickens. The virus was highly virulent in chickens, with a broader tissue tropism in chickens, causing 60 % mortality. Postmortem findings of dead chickens showed mild HHS and liver degeneration and necrosis. Importantly, FAdV-4 infection induced significant upregulation of genes encoding most toll-like receptors, some cytokines (interleukin-1β, 2, 6, 8, and 18, and interferon-γ), most of avian β-defensins, myeloid differentiation primary response protein 88, p38 mitogen-activated protein kinases, and inducible nitric oxide synthase, in tissues of infected chicken, especially in spleen and bursa of Fabricius. There was also a significant positive correlation between FAdV-4 genome load and the mRNA expression levels of most of these factors in specific infected tissues. The results indicated the potential role of these proteins in host immune response against FAdV-4 infection. However, overexpression of these proteins might contribute to tissue damage of FAdV-4 infected chickens, and eventually lead to chicken death.     

A paramyxovirus of serotype 3 isolated from African and Australian finches

Two virus isolates were obtained from exotic finches (Ortygospiza atricollis and Poephila cincta) suffering from apathy, diarrhea, conjunctivitis, and dysphagia. The isolates were identified as paramyxoviruses based on their multiplication characteristics in embryonating chicken eggs, chicken embryo fibroblasts, and chicken embryo kidney cell cultures, on morphology upon electron microscopy, and on other biological properties. Both isolates were serologically related to the reference strain of the paramyxovirus serotype 3. Intravenous infection of 42-day-old chicks resulted in no clinical signs, but intracerebral infection of 1-day-old chicks resulted in mortality and intracerebral pathogenicity indices of 0.25 to 0.35. Of five finches from various species inoculated with isolate 840/85, three remained clinically healthy through 6 weeks, but two died: one (Poephila cincta) 5 days postinoculation after showing nervous distress, and the other (Amandava amandava) suddenly 42 days postinoculation.     

Molecular characterization of Malaysian fowl adenovirus (FAdV) serotype 8b species E and pathogenicity of the virus in specific-pathogen-free chicken

BackgroundInclusion body hepatitis (IBH) is an economically important viral disease primarily affecting broiler and breeder chickens. All 12 serotypes of fowl adenovirus (FAdV) can cause IBH.ObjectivesTo characterize FAdV isolates based on phylogenetic analysis, and to study the pathogenicity of FAdV-8b in specific-pathogen-free (SPF) chickens following virus inoculation via oral and intramuscular (IM) routes.MethodsSuspected organ samples were subjected to virus isolation and polymerase chain reaction (PCR) for FAdV detection. Hexon gene sequencing and phylogenetic analysis were performed on FAdV-positive samples for serotype identification. One FAdV-8b isolate, UPM/FAdV/420/2017, was selected for fiber gene characterization and pathogenicity study and was inoculated in SPF chickens via oral and IM routes.ResultsThe hexon gene phylogenetic analysis revealed that all isolates belonged to FAdV-8b. The fiber gene-based phylogenetic analysis of isolate UPM/FAdV/420/2017 supported the grouping of that isolate into FAdV species E. Pathogenicity study revealed that, chickens infected with UPM/FAdV/420/2017 via the IM route had higher clinical score values, higher percent mortality, higher degree of the liver lesions, higher antibody response (p < 0.05), and higher virus shedding amounts (p < 0.05) than those infected via the oral route. The highest virus copy numbers were detected in liver and gizzard.ConclusionsFAdV-8b is the dominant FAdV serotype in Malaysia, and pathogenicity study of the FAdV-8b isolate UPM/FAdV/420/2017 indicated its ability to induce IBH in young SPF chickens when infected via oral or IM routes.     

血清4型禽腺病毒强毒株GY株的分离鉴定和致病性研究

从山东某疑似感染禽腺病毒的发病鸡群中采集病料并分离到一株禽腺病毒,克隆纯化后进行PCR检测和序列分析,鉴定该毒株为血清4型禽腺病毒,命名为GY株。用LMH细胞繁殖的GY株病毒滴度可以达到107.5 TCID₅₀/0.1mL,并建立了纯净稳定的种子批。动物致病性试验结果显示,攻毒鸡只8/10死亡,剖检均出现心包积液,肝脏肿大、出血或坏死,表明该毒株为强毒株。就攻毒途径、攻毒剂量等方面研究发现,GY株经胸部肌肉注射后发病率和发病时间相对颈部皮下注射途径更有优势,确定攻毒途径为肌肉注射;通过比较不同剂量(5×10^3.0~5×10^6.0 TCID50)GY株病毒攻击SPF鸡的致病性,将攻毒剂量定为5×10^5.0 TCID₅₀。研究表明,GY株可作为禽腺病毒4型攻毒用强毒株。     

Characterization of fowl adenoviruses isolated in Ontario and Quebec, Canada

Fowl adenoviruses (FAdV) are generally considered ubiquitous, but certain serotypes and strains are known to be associated with primary diseases, such as inclusion body hepatitis (IBH). Fifty-two FAdV isolates were collected from the provinces of Ontario and Quebec over a 4-year period. These 2 provinces have the largest poultry industries in Canada. Except for one virus, which originated from a guinea fowl, all other viruses were isolated from chicken samples. Most of these were from broilers, although some were from broiler breeders, and one was from layer pullets. Thirty-four isolates were from clinical IBH cases with the final laboratory diagnosis of IBH; however, for 18 isolates, the varied case diagnosis was seemingly unrelated to FAdV. All IBH-associated viruses had deoxyribonucleic acid (DNA) profiles compatible with FAdV species E (28 cases) or species D (6 cases), and the DNA fragment profiles of 26 species E viruses were indicative of serotype 8. Two viruses were serotype 6, as confirmed by virus neutralization. All species D viruses had a DNA profile similar to that of FAdV-2. The number of serotype 8 virus isolations has increased over the years, and by 2001 serotype 8 had become the dominant serotype in Ontario, and continues to be so. Moreover, this virus (FAdV-8) has shown a strong association with IBH.     

Partial sequence of the DNA-dependent DNA polymerase gene of fowl adenoviruses: a reference panel for a general diagnostic PCR in poultry

Adenoviruses are frequent infectious agents in different poultry species. The traditional, serological typing of new isolates by virus neutralisation tests is now in transition to be replaced by PCR and sequencing. The first PCRs, recommended for the detection of adenoviruses, had been designed to target the gene of the major capsid protein, the hexon. In birds, members of three different genera of the family Adenoviridae may occur. Accordingly, three specific hexon PCRs had to be elaborated for the detection of adenoviruses in poultry. A significantly more sensitive PCR, targeting the viral DNA-dependent DNA polymerase gene, has been described recently. This method proved to be an efficient alternative for the general detection of adenoviruses irrespective of their genus affiliation. Fowl adenoviruses (FAdVs), isolated from chicken to date, comprise twelve serotypes classified into five virus species (FAdV-A to E). The polymerase gene sequence has been determined yet only from three FAdV types representing three species. In the present work, the panel of polymerase gene sequences was completed with those of the rest of FAdVs. The newly determined sequences will facilitate the identification of new FAdV isolates as an existing species or as a putative new FAdV. Once the polymerase sequence is known, more specific PCRs for the amplification of the hexon and other genes can be designed and performed according to the preliminary species classification.     

Pathogenesis of two strains of avian paramyxovirus serotype 2, Yucaipa and Bangor, in chickens and turkeys

Nine serologic types of avian paramyxovirus (APMV) have been recognized. Newcastle disease virus (APMV-1) is the most extensively characterized virus, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two strains of APMV-2, Yucaipa and Bangor, in 9-day-old embryonated chicken eggs, 1-day-old specific-pathogen-free (SPF) chicks, and 4-wk-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was more than 168 hr for both strains, and their intracerebral pathogenicity index (ICPI) was zero, indicating that these viruses are nonpathogenic in chickens. When inoculated intracerebrally in 1-day-old chicks, neither strain caused disease or replicated detectably in the brain. This suggests that the zero ICPI value of APMV-2 reflects the inability of the virus to grow in neural cells. Groups of twelve 4-wk-old SPF chickens and turkeys were inoculated oculonasally with either strain, and three birds per group were euthanatized on days 2, 4, 6, and 14 postinoculation for analysis. There were no overt clinical signs of illnesses, although all birds seroconverted by day 6. The viruses were isolated predominantly from the respiratory and alimentary tracts. Immunohistochemistry studies also showed the presence of a large amount of viral antigens in epithelial linings of respiratory and alimentary tracts. There also was evidence of systemic spread even though the cleavage site of the viral fusion glycoprotein does not contain the canonical furin protease cleavage site.     

Experimental avian paramyxovirus serotype-3 infection in chickens and turkeys

Avian paramyxoviruses (APMV) are divided into nine serotypes. Newcastle disease virus (APMV-1) is the most extensively characterized, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two divergent strains of APMV-3, Netherlands and Wisconsin, in (i) 9-day-old embryonated chicken eggs, (ii) 1-day-old specific pathogen free (SPF) chicks and turkeys, and (iii) 2-week-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was 112 h for APMV-3 strain Netherlands and > 168 h for strain Wisconsin. The intracerebral pathogenicity index in 1-day-old chicks for strain Netherlands was 0.39 and for strain Wisconsin was zero. Thus, both strains are lentogenic. Both the strains replicated well in brain tissue when inoculated intracerebrally in 1-day-old SPF chicks, but without causing death. Mild respiratory disease signs were observed in 1-day-old chickens and turkeys when inoculated through oculonasal route with either strain. There were no overt signs of illness in 2-weeks-old chickens and turkeys by either strain, although all the birds seroconverted after infection. The viruses were isolated predominantly from brain, lungs, spleens, trachea, pancreas and kidney. Immunohistochemistry studies also showed the presence of large amount of viral antigens in both epithelial and sub-epithelial lining of respiratory and alimentary tracts. Our result suggests systemic spread of APMV-3 even though the viral fusion glycoprotein does not contain the canonical furin proteases cleavage site. Furthermore, there was little or no disease despite systemic viral spread and abundant viral replication in all the tissues tested.     

Genomic identification and characterization of avian adenoviruses associated with inclusion body hepatitis.

Four pathogenic avian adenovirus isolates associated with inclusion body hepatitis and mortality in commercial broiler chicks and chickens were characterized and identified. These group I avian adenovirus isolates were classified as group E (serotypes 6, 7, 8, and 9) avian adenoviruses on the basis of the restriction enzyme patterns of their viral DNA. Isolate 3718 was neutralized by a serotype 6 reference avian adenovirus antiserum and isolates 8193, 8380, and 8565 were all neutralized by a serotype 8 reference avian adenovirus antiserum by virus neutralization assays. Infectivity and virulence such as mortality, hemorrhages, enlarged green livers with intranuclear inclusion bodies, stunting, intestinal sloughing, and poor feathering were observed in specific-pathogen-free chicken embryos and were identical for all four isolates when embryos were inoculated via the yolk sac and/or chorioallantoic membrane. Complete mortality was observed within 72 hr postinoculation in specific-pathogen-free (SPF) chickens inoculated intramuscularly for all four avian adenovirus isolates.     

Immunogenicity of an Escherichia coli multivalent pilus vaccine in chickens

Immunogenicity of an oil-emulsified Escherichia coli multivalent pilus vaccine was evaluated in 4-week-old chickens. The vaccine contained 180 micrograms of pilus protein from each of serotypes O1 and O78 and 170 micrograms of pilus protein from serotype O2. Chickens were vaccinated twice subcutaneously at 4 and 6 weeks old and challenged via the posterior thoracic air sac with E. coli serotype O1, O2, or O78 2 weeks after the last vaccination. Unvaccinated challenged chickens suffered 8% to 26% mortality; no vaccinated chickens died. Vaccinated chickens had very mild gross lesions in the air sacs, livers, and pericardial sacs and eliminated E. coli more efficiently than the unvaccinated challenged chickens. The results showed that a multivalent pilus vaccine protects chickens against active respiratory infection.