Pathogenicity of highly pathogenic avian influenza viruses of H5N1 subtype isolated in Thailand for different poultry species

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused several rounds of outbreaks in Thailand. In this study, we used 3 HPAI viruses isolated in Thailand in January 2004 from chicken, quail, and duck for genetic and pathogenetic studies. Sequence analysis of the entire genomes of these isolates revealed that they were genetically similar to each other. Chickens, quails, domestic ducks, and cross-bred ducks were inoculated with these isolates to evaluate their pathogenicity to different host species. A/chicken/Yamaguchi/7/04 (H5N1), an HPAI virus isolated in Japan, was also used in the chicken and quail studies for comparison. All four isolates were shown to be highly pathogenic to chickens and quails, with 100% mortality by 10(6) EID50 inoculants of the viruses. They caused sudden death in chickens and quails within 2-4 days after inoculation. The mean death times (MDT) of quails infected with the Thai isolates were shorter than those of chickens infected with the same isolates. Mortality against domestic and cross-bred ducks ranged from 50 to 75% by intranasal inoculation with the 10(6) EID50 viruses. Neurological symptoms were observed in most of the inoculated domestic ducks and appeared less severe in the cross-bred ducks. The MDTs of the ducks infected with the Thai isolates were 4.8-6 days post-inoculation. Most of the surviving ducks infected with the Thai isolates had sero-converted until 14 dpi. Our study illustrated the pathobiology of the Thai isolates against different poultry species and would provide useful information for improving control strategies against HPAI.     

Experimental infection of chickens, ducks and quails with the highly pathogenic H5N1 avian influenza virus

Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype have spread since 2003 in poultry and wild birds in Asia, Europe and Africa. In Korea, the highly pathogenic H5N1 avian influenza outbreaks took place in 2003/2004, 2006/2007 and 2008. As the 2006/2007 isolates differ phylogenetically from the 2003/2004 isolates, we assessed the clinical responses of chickens, ducks and quails to intranasal inoculation of the 2006/2007 index case virus, A/chicken/Korea/IS/06. All the chickens and quails died on 3 days and 3-6 days post-inoculation (DPI), respectively, whilst the ducks only showed signs of mild depression. The uninoculated chickens and quails placed soon after with the inoculated flock died on 5.3 and 7.5 DPI, respectively. Both oropharyngeal and cloacal swabs were taken for all three species during various time intervals after inoculation. It was found that oropharyngeal swabs showed higher viral titers than in cloacal swabs applicable to all three avian species. The chickens and quails shed the virus until they died (up to 3 to 6 days after inoculation, respectively) whilst the ducks shed the virus on 2-4 DPI. The postmortem tissues collected from the chickens and quails on day 3 and days 4-5 and from clinically normal ducks that were euthanized on day 4 contained the virus. However, the ducks had significantly lower viral titers than the chickens or quails. Thus, the three avian species varied significantly in their clinical signs, mortality, tissue virus titers, and duration of virus shedding. Our observations suggest that duck and quail farms should be monitored particularly closely for the presence of HPAIV so that further virus transmission to other avian or mammalian hosts can be prevented.     

Using mean infectious dose of high- and low-pathogenicity avian influenza viruses originating from wild duck and poultry as one measure of infectivity and adaptation to poultry

The mean infectious doses of selected avian influenza virus (AIV) isolates, determined in domestic poultry under experimental conditions, were shown to be both host-dependent and virus strain-dependent and could be considered one measure of the infectivity and adaptation to a specific host. As such, the mean infectious dose could serve as a quantitative predictor for which strains of AIV, given the right conditions, would be more likely transmitted to and maintained in a given species or subsequently cause an AI outbreak in the given species. The intranasal (IN) mean bird infectious doses (BID50) were determined for 11 high-pathogenicity AIV (HPAIV) isolates of turkey and chicken origin for white leghorn (WL) chickens, and for low-pathogenicity AIV (LPAIV) isolates of chicken (n = 1) and wild mallards (n = 2) for turkeys, and WL and white Plymouth rock (WPR) chickens, domestic ducks and geese, and Japanese quail. The BID50 for HPAIV isolates for WL chickens ranged from 10(1.2) to 10(4.7) mean embryo infectious dose (EID50) (median = 10(2.9)). For chicken-origin HPAIV isolates, the BID50 in WL chickens ranged from 10(1.2) to 10(3.0) EID50 (median = 10(2.6)), whereas for HPAIV isolates of turkey origin, the BID50 in WL chickens was higher, ranging from 10(2.8) to 10(4.7) EID50 (median = 10(3.9)). The BID50 of 10(4.7) was for a turkey-origin HPAIV virus that was not transmitted to chickens on the same farm, suggesting that, under the specific conditions present on that farm, there was insufficient infectivity, adaptation, or exposure to that virus population for sustained chicken transmission. Although the upper BID50 limit for predicting infectivity and sustainable transmissibility for a specific species is unknown, a BID50 < 10(4.7) was suggestive of such transmissibility. For the LPAIVs, there was a trend for domestic ducks and geese and Japanese quail to have the greatest susceptible and for WL chickens to be the most resistant, but turkeys were susceptible to two LPAIV tested when used at moderate challenge doses. This suggests domestic ducks and geese, turkeys, and Japanese quail could serve as bridging species for LPAIVs from wild waterfowl to chickens and other gallinaceous poultry. These data do provide support for the commonly held and intuitive belief that mixing of poultry species during rearing and in outdoor production systems is a major risk factor for interspecies transmission of AIVs and for the emergence of new AIV strains capable of causing AI outbreaks because these situations present a more diverse host population to circumvent the natural host dependency or host range of circulating viruses.     

Infectivity and transmissibility of H9N2 avian influenza virus in chickens and wild terrestrial birds

Genetic changes in avian influenza viruses influence their infectivity, virulence and transmission. Recently we identified a novel genotype of H9N2 viruses in widespread circulation in poultry in Pakistan that contained polymerases (PB2, PB1 and PA) and non-structural (NS) gene segments identical to highly pathogenic H7N3 viruses. Here, we investigated the potential of these viruses to cause disease and assessed the transmission capability of the virus within and between poultry and wild terrestrial avian species. Groups of broilers, layers, jungle fowl, quail, sparrows or crows were infected with a representative strain (A/chicken/UDL-01/08) of this H9N2 virus and then mixed with naïve birds of the same breed or species, or different species to examine transmission. With the exception of crows, all directly inoculated and contact birds showed clinical signs, varying in severity with quail showing the most pronounced clinical signs. Virus shedding was detected in all infected birds, with quail showing the greatest levels of virus secretion, but only very low levels of virus were found in directly infected crow samples. Efficient virus intra-species transmission was observed within each group with the exception of crows in which no evidence of transmission was seen. Interspecies transmission was examined between chickens and sparrows and vice versa and efficient transmission was seen in either direction. These results highlight the ease of spread of this group of H9N2 viruses between domesticated poultry and sparrows and show that sparrows need to be considered as a high risk species for transmitting H9N2 viruses between premises.     

Pathogenicity and transmission of reticuloendotheliosis virus isolated from endangered prairie chickens

The pathogenicity and transmission of a field isolate of reticuloendotheliosis virus (REV) was studied using an experimental model in Japanese quail. Oncogenicity was also evaluated after inoculations in chickens and turkeys. The original REV (designated APC-566) was isolated from Attwater's prairie chickens (Tympanuchus cupido attwateri), an endangered wild avian species of the southern United States. The transmissibility of the REV isolate was studied in young naive Japanese quail in contact with experimentally infected quail. Vertical transmission was not detected by virus isolation and indirect immunofluorescence. Seroconversion was detected in few contact quails, suggesting horizontal transmission. The APC-566 isolate induced tumors beginning at 6 wk of age in quails infected as embryos. Most of the tumors detected in Japanese quail were lymphosarcomas, and 81% of these neoplasias contained CD3+ cells by immunoperoxidase. REV APC-566 was also oncogenic in chickens and turkeys infected at 1 day of age, with tumors appearing as early as 58 days after infection in chickens and at 13 wk of age in turkeys. This study was conducted in part as an attempt to understand the potential for pathogenicity and transmission of REV isolated from endangered avian species.     

Novel reassortant H5N6 highly pathogenic influenza A viruses in Vietnamese quail outbreaks

Avian influenza A H5N6 virus is a highly contagious infectious agent that affects domestic poultry and humans in South Asian countries. Vietnam may be an evolutionary hotspot for influenza viruses and therefore could serve as a source of pandemic strains. In 2015, two novel reassortant H5N6 influenza viruses designated as A/quail/Vietnam/CVVI01/2015 and A/quail/Vietnam/CVVI03/2015 were isolated from dead quails during avian influenza outbreaks in central Vietnam, and the whole genome sequences were analyzed. The genetic analysis indicated that hemagglutinin, neuraminidase, and polymerase basic protein 2 genes of the two H5N6 viruses are most closely related to an H5N2 virus (A/chicken/Zhejiang/727079/2014) and H10N6 virus (A/chicken/Jiangxi/12782/2014) from China and an H6N6 virus (A/duck/Yamagata/061004/2014) from Japan. The HA gene of the isolates belongs to clade 2.3.4.4, which caused human fatalities in China during 2014–2016. The five other internal genes showed high identity to an H5N2 virus (A/chicken/Heilongjiang/S7/2014) from China. A whole-genome phylogenetic analysis revealed that these two outbreak strains are novel H6N6-like PB2 gene reassortants that are most closely related to influenza virus strain A/environment/Guangdong/ZS558/2015, which was detected in a live poultry market in China. This report describes the first detection of novel H5N6 reassortants in poultry during an outbreak as well as genetic characterization of these strains to better understand the antigenic evolution of influenza viruses.     

Phylogenetic analysis of subgroup J avian leucosis virus from broiler and native chickens in Taiwan during 2000-2002

Subgroup J avian leucosis virus (ALV-J) causes great economic losses in the poultry industry. One in 3 grandparent farms was closed due to ALV-J infection in 1998 in Taiwan. The remaining 2 farms were forced to import breeding chicks from different breeding companies afterwards. We report on the ALV-J infection status among these breeders, their progeny and Taiwan native chickens during 2000-2002. The weekly mortality for the male line among the infected breeders was higher than that for the female line. Sixty-three percent (5/8) of the broiler flocks were infected with ALV-J. The surface (SU) portion of the env gene from the ALV-J field isolates was cloned and sequenced. The phylogenetic results show that all of the isolates fell into 2 clusters. Unexpectedly, the isolates from the same breeds fell into different clusters, with a cluster including isolates from different breeding companies. ALV-Js from native chickens crossbred with imported chickens were placed into the same clusters as those from the imported breeds. The high similarities observed in different ALV-J isolates suggest that different ALV-Js were mixed in the pedigree generations in different breeding lines.     

禽Ⅰ型副黏病毒各种禽源分离株对4种禽类的致病性

选取从临床感染禽Ⅰ型副黏病毒（APMV-1）的鸡、鹅、鸽、鹌鹑、珍珠鸡、孔雀、画眉鸟等7种禽类病例分离到的9个代表性毒株，分别对鸡、鹌鹑、鹅和鸽进行了人工感染试验。结果，除鸽源毒株gxp22对鸡和鹅无致病力外，其他8个分离毒株对鸡、鹌鹑和鹅都有较强的致病力，死亡率为60％～100％，试验鸡表现的症状和病理变化特征最明显，鹅的比较明显，鹌鹑的则最不明显；3个鸽源分离株对鸽的致病力都很强，死亡率均为100％。所有毒株对4种禽类的致病性与其临床特征相符。研究结果表明，试验所用的9个分离株除鸽源分离株gxp22外，均为泛嗜性的新城疫强毒株。     

Immunopathology of Marek's disease in quails: presence of antinuclear antibody and immune complex.

Antinuclear antibody (ANA), a marker for autoimmune reactions, was detected in the sera of quails with Marek's disease (MD). The autoantibody was detected 3 weeks after infection in quails infected with chicken Marek's disease virus and 4 weeks after infection in quails infected with quail Marek's disease virus. The ANA titers were low and ranged from 10 to 40. A speckled type of nuclear fluorescence was the characteristic staining feature. In addition to the presence of ANA, immune complexes (IC) were also detected in the kidney glomeruli of quail infected with Marek's disease virus. Initially about 25-30% of the glomeruli in the kidneys of infected quails had IC deposits. In subsequent periods, the amount of IC deposit and the number of glomeruli showing IC also increased considerably. The findings of the present study suggested autoimmunity may play a pathogenic role in MD.     

禽流感病毒番鸭分离株对不同禽类致病性的研究

将禽流感病毒番鸭分离株ZM的感染性鸡胚尿囊液静脉或肌肉接种不同禽类,同时设鸡源JC株对照,以了解毒株的致病特点。结果显示ZM株与同期分离JC株致病能力相当,两毒株均对鸡、鹅、番鸭、鹌鹑和鹧鸪显示了很强的致病能力,致死率50％-100％,死亡禽类的肝、肾、肺、脾、胰腺等多个组织器官均存在严重的损伤。鸽子,肉鸭和绍鸭(除zM株致死一只绍鸭外)均未发生死亡,外观健康。     

微山湖野生日本鸣鹑与家鹑亲缘程度的研究

在迁飞滞留季节捕获的微山湖地区野生日本鸣鹑中随机抽取36只，检测编码脏器、肌肉酶型的10个结构基因座上的频率分布，搜集国内外20个家鹑和野生日本鸣鹑群体的相同资料，以模糊集合方法对21个群体进行亲缘关系聚类。研究发现：就起源系统而言，微山湖野生日本鸣鹑比日本列岛各地的野生日本鸣鹑更接近家养鹌鹑群体。这一研究结果表明，当代关于鹌鹑原始驯化地域的论断，值得重新斟酌。     

Pathogenic properties of Newcastle disease virus isolates in the Sudan.

Six Newcastle disease virus (NDV) isolates were obtained from disease outbreaks on different poultry farms in the Sudan between 1988 and 1991. The pathogenic properties of these isolates were studied in comparison to those of strain Herts 33/56. All the isolates were similar in that they killed chicken embryos quickly, in mean death time (MDT) and embryo lethal dose 50 per cent (ELD50), had higher intracerebral pathogenicity indices (ICPI), and produced viscerotropic lesions in the infected chickens. The field isolates had the characteristics of the velogenic viscerotopic strains of NDV. The pathogenesis of infection caused by one of the isolates was studied. The virus was first detected in different organs and in oral and cloacal swabs on the third day after infection.     

An occurrence of Newcastle disease in pigeons: Virological and serological studies on the isolatesFoyers de maladie de Newcastle chez le pigeon: Etudes virologiques et serologiques sur des souches isolees

The antigenic and pathogenetic relationship between pigeon Newcastle disease virus (NDV) isolates during outbreaks of 1982 in Italy and reference pathogen and non-pathogen NDV-strains were investigated.The pigeon-isolates were slow-eluters and showed a thermostability at 56°C of over 30 min. They proved to be lentogenic as measured by the mean-death-time in chicken-embryos, and between lentogenic and mesogenic as measured by the Hanson test. They failed to produce plaques in chicken-embryo-fibroblasts and showed high pathogenicity for experimentally infected pigeons, low-pathogenicity for quails and were not pathogenic for chickens. They were antigenically different from the LaSota strain as measured by the cross-HI-test and induced considerable seroconversion in inoculated animals. The existence of a lentogenic neurotropic pigeon-pathogenic strain was considered.     

Experimental studies on Marek's disease in Japanese quail (Coturnix coturnix japonica)

Day-old quails experimentally infected with Marek's disease (MD) virus of quail origin developed lymphoid tumors. The severity of the disease increased considerably with serial passage. Tumor transplants could be made with cells derived from gross tumors in skeletal muscles, spleen cells, and blood from MD-affected quails. After five to six serial transplants, the tumor could not be transplanted further. Marek's disease tumor-associated surface antigen (MATSA) was demonstrated in lymphoid cells of spleen and peripheral blood lymphocytes of MD-affected quails. The MATSA of quail differed from the MATSA of chicken. Chickens were susceptible to MD virus isolated and propagated in quails.     

Investigation of Marek’s disease virus from chickens in central Ethiopia

Marek’s disease (MD) is a lymphoproliferative and neuropathic disease of domestic chickens and less commonly, turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpesvirus. In Ethiopia, MD is believed to be introduced with importation of exotic and crossbred to improve the poultry production and has been reported to be a potential threat to the poultry sector both in backyard and commercial farming systems. This study was aimed at isolation and molecular analysis of MD virus isolates circulating in chicken population in the central part of Ethiopia where commercial farms are populated. From September 2013 to January 2014, clinical and post-mortem examination were conducted on diseased chickens suspected of MD virus infection. Representative spleen and feather follicle samples were collected following sterile procedure, and infectious virus isolation was performed using primary chicken fibroblast cell culture. Cell culture inoculated with suspension of pathological samples developed characteristic MD virus cytopathic effect of rounding of the cells and small plaques. Further analysis of the virus was conducted by conventional PCR amplifying the ICP4 gene fragment from eleven tissue samples using MD virus specific primers. PCR products were further sequenced and analyzed. Nucleotide sequence similarity search of the local isolates resulted a high degree of sequence similarity with Gallid Herpes virus type 2 strain (Marek’s disease virus type 1, JN034558). To our knowledge, the present study is the first report conducted on virus isolation and molecular characterization of MD virus isolates circulated in Ethiopia. Eleven ICP4-like gene fragment (318 bp) sequences generated in the present study were uploaded in the public database (KU842366–76). Further research on virus isolation, genetic characterization, and infection dynamics is recommended targeting chickens of all age groups reared in different agro-ecological zones under different production system.

     

Evaluation of interspecific hybrids of the chicken, guinea fowl, and Japanese quail for innate resistance to coccidia

Experimental chicken/guinea fowl hybrids, guinea fowl, and chickens were orally inoculated with Eimeria acervulina or E. tenella, which are specific for chickens, or with E. grenieri, which is specific for guinea fowl. No intact oocysts were found in feces within 24 hr of inoculation, suggesting that excystation occurred in the normal and abnormal hosts. No oocysts were found in the feces of hybrids during a 9-day postinoculation period. The guinea fowl passed oocysts of guinea fowl coccidia (E. grenieri) but not those of chicken coccidia, and the chickens passed oocysts of chicken coccidia (E. acervulina and E. tenella) but not those of guinea fowl coccidia. Some asexual development (schizogony) occurred in hybrids inoculated with E. tenella, but sexual development (gametogony) did not. In contrast, quail/chicken hybrids became infected with oocysts of chicken coccidia (E. acervulina, E. tenella, and E. maxima) and quail coccidia (E. bateri) and passed a few oocysts during the normal patent period; control chickens and quails became heavily infected with oocysts of chicken and quail coccidia, respectively.     

Isolation of ureaplasmas from poultry and experimental infection in chickens

Ureaplasmas were isolated from the oropharynxes of 47 of 247 (19 per cent) Leghorn chickens (Gallus gallus domesticus) and from five Japanese bantams but none was isolated from the oropharynx or cloaca of other poultry comprising 10 Japanese game, 75 common quails (Coturnix coturnix japonica), 17 turkeys (Meleagris gallopavo) and 10 guinea fowls (Numida galeata). In apparently healthy chickens, ureaplasmas were found at various sites, including the conjunctiva, nasal cavity, oropharynx, upper and lower tracheas, but not from the air sac, lungs, yolk, oviduct, urine or cloaca. All the isolates were antigenically similar but had no serological relation to those isolated from man, monkey, cattle, goat, sheep, dog and cat. In chickens experimentally infected with an avian ureaplasma, the organisms infected the oropharynx and nasal cavity but none of the birds inoculated demonstrated any clinical signs or macroscopic lesions.     

Cryptosporidium infections in birds and mammals and attempted cross-transmission studies

Infections by Cryptosporidium were detected in association with clinical disease in 11 humans (Homo sapiens), 19 calves (Bos taurus), nine common quail (Coturnix coturnix), six mallard ducks (Anas platyrhynchos), five ring-necked pheasant (Phasianus colchicus) and a single budgerigar (Melopsittacus undulatus). Infections in mammals were accompanied by transient diarrhoea and anorexia, whereas infected birds exhibited clinical signs of respiratory distress. Repeated cross-transmission studies revealed apparent strain differences or differences in the host specificity of several mammalian and avian isolates for homologous vertebrate classes only. Oocysts from humans and calves were infective to mice, pigs or lambs, but not to chickens, whereas oocysts from quail and pheasant were infective to chickens, but not to mice.     

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.     

Pathogenicity of infectious bronchitis virus isolates from Ontario chickens

Infectious bronchitis (IB) is one of the important viral diseases of chickens, and in spite of regular vaccination, IB is a continuous problem in Canadian poultry operations. In an earlier study using sentinel chickens we determined the incidence of infectious bronchitis virus (IBV) in Ontario commercial layer flocks. The objective of this study was to determine the pathogenicity of 5 nonvaccine-related IBV isolates recovered from the sentinel birds. The clinical signs, gross, and histological lesions in specific pathogen-free chickens indicated that all 5 isolates caused mild lesions in the respiratory tract. An important finding of this study was the significantly lower average daily weight gain among virus-inoculated groups of chickens during the acute phase of infection. Based on sequences of part of the S1 gene IBV-ON2, IBV-ON3, and IBV-ON5 formed a cluster and they were closely related to strain CU-82792. IBV-ON4 had 98.7% identity with the strain PA/1220/9, a nephropathogenic variant.