Characterization of two pigeon paramyxovirus type 1 isolates in China

For over three decades, there has been a continuing panzootic caused by a virulent variant avian paramyxovirus type 1 strain, the so-called pigeon paramyxovirus type 1. It is found primarily in racing pigeons, but it has also spread to wild birds and poultry. In this study, two pigeon paramyxovirus type 1 strains, SD12 and BJ13, obtained from diseased pigeons in China, were characterized. Phylogenetic analysis based on complete sequences allowed characterization of both strains as genotype VI, class II. Further phylogenetic analysis of a 374-nucleotide section of the fusion gene showed that SD12 fell into lineage VIbii-d and BJ13 into VIbii-f. The deduced amino acid sequence of the cleavage site of the fusion protein confirmed that both isolates contained the virulent motif ¹¹²K/RRQKR↓F¹¹⁷ at the cleavage site. Nevertheless, the values of intracerebral pathogenicity indices showed the SD12 isolate to be a velogenic strain and BJ13 isolate to be a mesogenic strain. The SD12 isolate was further investigated via clinical observation, RNA detection, histopathology and viral serology in experimentally infected 3-week-old chickens. It showed a mild pathological phenotype in chickens, with viral replication restricted to a few tissues. The molecular mechanism for the SD12 isolate to have a virulent motif but low levels of virulence for chickens requires further study.     

Presence of Newcastle disease viruses of sub-genotypes Vc and VIn in backyard chickens and in apparently healthy wild birds from Mexico in 2017

Virulent Newcastle disease viruses (NDV) have been present in Mexico since 1946, and recently, multiple outbreaks have been reported in the country. Here, we characterized eleven NDV isolated from apparently healthy wild birds and backyard chickens in three different locations of Jalisco, Mexico in 2017. Total RNA from NDV was reverse-transcribed, and 1285 nucleotides, which includes 3/4 of the fusion gene, was amplified and sequenced using a long-read MinION sequencing method. The sequences were 99.99–100% identical to the corresponding region obtained using the Illumina MiSeq. Phylogenetic analysis using MinION sequences demonstrated that nine virulent NDV from wild birds belonged to sub-genotypes Vc and VIn, and two backyard chicken isolates were of sub-genotype Vc. The sub-genotype Vc viruses had nucleotide sequence identity that ranged from 97.7 to 98% to a virus of the same sub-genotype isolated from a chicken in Mexico in 2010. Three viruses from pigeons had 96.3–98.7% nucleotide identity to sub-genotype VIn pigeon viruses, commonly referred to as pigeon paramyxovirus, isolated in the USA during 2000–2016. This study demonstrates that viruses of sub-genotype Vc are still present in Mexico, and the detection of this sub-genotype in both chickens and wild birds suggests that transmission among these species may represent a biosecurity risk. This is the first detection and complete genome sequencing of genotype VI NDV from Mexico. In addition, the utilization of an optimized long-read sequencing method for rapid virulence and genotype identification using the Oxford nanopore MinION system is demonstrated.

     

Construction and characterization of an OmpH-deficient mutant of Pasteurella multocida strain X-73

A capsule-defective mutant strain PBA129 of Pasteurella multocida was constructed by electroporation of phagemid containing the coding region of the antisense RNA of the ompH gene into the wild type strain X-73 (serovar A:1) of P. multocida. The pathogenicity and protective potency of the mutant against homologous and heterologous challenge in mice and chickens were characterized. Greyish colonies of the mutant, indicating lower capsule thickness, on selective dextrose starch agar were observed under an obliquely transmitted light stereomicroscope and compared to iridescent colonies of the wild type strain X-73. Strain PBA129 had lower capsule thickness than the wild type strain as observed with an electron microscope. Strain PBA129 was apparently attenuated, as mice and chickens inoculated with the bacteria at 10⁸ CFU survived. Protection was observed in both mice and chickens inoculated with strain PBA129 upon challenge exposure to avian P. multocida strains X-73 and P-1059 (serovar A:3), respectively. In conclusion, the mutant strain PBA129 of P. multocida strain X-73 was completely attenuated, and it was possible to induce sufficient protection against avian P. multocida strains.     

Characterization of a novel VIIl sub-genotype of Newcastle disease virus circulating in Iran

Newcastle disease is an economically important and highly contagious disease affecting wild and domestic avian species. Despite extensive vaccination efforts within the poultry industry, Newcastle disease virus (NDV) outbreaks causing significant economic losses still occur. Rural chickens may act as a potential reservoir of NDVs for commercial poultry due to poor biosecurity and inadequate vaccination. The aim of this study was to investigate the phylogenetic relationship and molecular characterization of eight NDVs isolated from backyard poultry in Iran during 2011–2013. The complete coding sequence of fusion (F) and haemagglutinin-neuraminidase (HN) genes of eight NDVs were determined and compared with other published NDVs. Based on inter-population distances and phylogenetic topology between available NDV categories, Iranian isolates formed a novel VIIl sub-genotype distinct from previous groups designated in genotype VII. Furthermore, both F and HN genes of the Iranian isolates shared high nucleotide sequence similarity with viruses isolated in China. All viruses analysed contained a polybasic cleavage site motif (¹¹¹G/RRRQKR↓F¹¹⁷), indicating that all isolates could be categorized as a virulent pathotype. No mutation was observed in the neutralizing epitopes of the F protein. Analysis of amino acids associated with neutralizing antigenic sites within the HN protein revealed that all isolates exhibited a unique amino acid (Q) at position 347. These results emphasize the need for strengthening the biosecurity measures implemented on village flocks and practicing a mandatory vaccination programme for local poultry. Moreover, continuous monitoring of NDVs in different species of birds can help to gain more knowledge about the evolution of this virus and prevent future panzootics.     

Mycoplasma gallisepticum experimental infection and tissue distribution in chickens,sparrows and pigeons

The most effective approaches to control the spread of Mycoplasma gallisepticum include strict biosecurity measures, continuous surveillance and eradication of infected flocks. The rapid expansion of the poultry industry worldwide in restricted geographical areas and severe economic losses due to M. gallisepticum outbreaks make it crucial to identify and better control the vectors responsible for the transmission of the disease. In the present study we evaluated the susceptibility of sparrows and pigeons to M. gallisepticum and the tissue distribution of M. gallisepticum in these species as compared with chickens. This information will further define the role of these common avian species in M. gallisepticum transmission. Twenty-six chickens, pigeons, and sparrows were experimentally inoculated with a field strain of M. gallisepticum and were monitored for the development of clinical signs, seroconversion, productive infection by culture, and M. gallisepticum distribution in their tissues by immunohistochemistry. All M. gallisepticum-inoculated chickens showed mild respiratory signs, seroconverted (haemagglutination inhibition geometric mean titre = 494) and shed M. gallisepticum in their tracheas. M. gallisepticum antigens were observed at high levels by immunohistochemistry in their tracheas, conjunctivas, nasal turbinates, and air sacs. The pigeons and sparrows did not show clinical signs or seroconvert but M. gallisepticum was reisolated up to 7 days post inoculation from pigeons and intermittently from sparrows. M. gallisepticum antigens were observed at low level in the conjunctiva of some pigeons and sparrows, as well as in the trachea of some sparrows. We conclude that pigeons and sparrows are partially susceptible to M. gallisepticum infection but do not seroconvert or maintain a steady carrier state similar to chickens and that these species may play a role in M. gallisepticum transmission between poultry farms as mechanical vectors.     

Molecular characterization of new emerging sub-genotype VIIh Newcastle disease viruses in China

Newcastle disease (ND) has been enzootic in China for several decades since the first recognition of the disease in 1946 in China. Continuous surveillance revealed that the sub-genotype VIId Newcastle disease virus (NDV) has been predominantly responsible for most of ND outbreak in China in recent years. But in the present study, three virulent NDVs isolated from poultry in southern China were classified as sub-genotype VIIh, which is highly related to the viruses circulating in some Southeast Asia countries. Continuous isolation of genotype VIIh NDV strains in the region suggests its panzootic potential. This is the first report of the sub-genotype VIIh NDVs in domestic poultry in China. The complete genome length of the three isolates was 15,192 nucleotides, and the motif at the cleavage site of F protein was ¹¹²RRRRR/F¹¹⁷ or ¹¹²RRRKR/F¹¹⁷, which was typical of virulent NDV. Phylogenetic analysis based on the F gene revealed that the three viruses had close relationship with the sub-genotype VIIh virus isolated from wild bird in 2011 in China. These viruses might have formed a stable lineage in poultry during 2012–2016 and have the potential to cause enzootic in China. Our study revealed the genetic and phylogenetic characteristics of the three sub-genotype VIIh isolates, which could help us to better understand the epidemiological context of these viruses.

     

Genomic analysis of Newcastle disease virus strain NA-1 isolated from geese in China

Newcastle disease virus (NDV) has been thought to infect only domestic avian species, with waterfowl such as geese either not being infected, even by virulent strains, or developing only in apparent infection. In 1997, a new infectious disease producing high morbidity and mortality among geese broke out in many provinces of China, which was caused by a serotype I avian paramyxovirus (APMV-1)--NDV. To investigate how NDV spreads between chickens and geese, the complete genome of one NDV strain isolated from a goose was cloned and analyzed. The results indicate that there is conservation in NDV structural genetic evolution but that there are also considerable differences between goose and chicken NDV strains. Separate patterns of NDV evolution exist among wild bird species. Meanwhile, there is evidence indicating that the goose NDV may have evolved from chicken NDV strain Herts/33. In addition, the possibility was investigated that this new strain of NDV may bind to different sialic acid receptor binding sites than the normal NDV strains that have been investigated so far. This might provide clues to the evolution of the goose NDV.     

Comparative genomic analyses of Mycoplasma synoviae vaccine strain MS-H and its wild-type parent strain 86079/7NS: implications for the identification of virulence factors and applications in diagnosis of M. synoviae

ABSTRACT

Mycoplasma synoviae is an economically important avian pathogen worldwide, causing respiratory disease, infectious synovitis, airsacculitis and eggshell apex abnormalities in commercial chickens. Despite the widespread use of MS-H as a live attenuated vaccine over the past two decades, the precise molecular basis for loss of virulence in this vaccine is not yet fully understood. To address this, the whole genome sequence of the vaccine parent strain, 86079/7NS, was obtained and compared to that of the MS-H vaccine. Except for the vlhA expressed region, both genomes were nearly identical. Thirty-two single nucleotide polymorphisms (SNPs) were identified in MS-H, including 11 non-synonymous mutations that were predicted, by bioinformatics analysis, to have changed the secondary structure of the deduced proteins. One of these mutations caused truncation of the oppF-1 gene, which encodes the ATP-binding protein of an oligopeptide permease transporter. Overall, the attenuation of MS-H strain may be caused by the cumulative and complex effects of several mutations. The SNPs identified in MS-H were further analyzed by comparing the MS-H and 86079/7NS sequences with the strains WVU-1853 and MS53. In the genomic regions conserved between all strains, 30 SNPs were found to be unique to MS-H lineage. These results have provided a foundation for developing novel biomarkers for the detection of virulence in M. synoviae and also for designing new genotyping assays for discrimination of MS-H from field strains.     

Newcastle disease in the European Union 2000 to 2009

Newcastle disease (ND) is a devastating disease of poultry that has to some extent been neglected by those working in the field in the past 10 to 15 years while attention has been focused on the emergence and spread of highly pathogenic avian influenza caused by a H5N1 subtype virus. During 2000 to 2009 in the European Union (EU) member states, ND viruses virulent for chickens have been detected in wild birds, domesticated pigeons and poultry. Based on these isolations it appears that the epizootic in racing pigeons caused by the variant viruses termed pigeon avian paramyxovirus type 1, which form the genetic group 4b(VIb) first seen in Europe in 1981, continued during 2000 to 2009, and the virus is probably enzootic in racing pigeons in some EU countries. This virus appears to have spread regularly to wild birds, especially those of the Columbidae family, and has been the cause of significant outbreaks in poultry. Other avian paramyxovirus type 1 viruses responsible for ND outbreaks in the EU during 2000 to 2009 have been those from genetic groups 5b(VIIb) and 5d(VIId). There is evidence that the former may well represent spread from a wild bird source and these viruses have also been isolated from wild birds, while the latter represents continuing spread from the East. Future legislation or recommendations aimed at the control and eradication of ND will need to encompass these three sources of virulent ND viruses.     

Antigenic variation between Newcastle disease viruses of goose and chicken origin

Newcastle disease virus (NDV) is believed to infect only domestic avian species, with waterfowl such as geese either not being infected, even by virulent strains, or developing only inapparent infection. Since the late 1990s, a new infectious disease producing high morbidity and mortality among geese broke out in many provinces of China. This disease was caused by a serotype I avian paramyxovirus known as (APMV-1)—NDV. To investigate how NDV spreads between chickens and geese, the serological similarities of NDV strains (goose-origin NDV/NA-1 and chicken-origin NDV/F48E9, F48E8) were assessed by cross-neutralization assays both in vivo and in vitro. The results indicated that antigenic variation had occurred between NDV/NA-1 strains and NDV/F48E9, F48E8 strains. Notably, NDV/NA-1 effectively protected vaccinated birds from morbidity and mortality against NDV/NA-1 strain challenge and significantly reduced virus shedding from the vaccinated birds when compared with F48E9-vaccinated birds. This might provide clues to the evolution of the goose NDV.     

Co-circulation of paramyxo- and influenza viruses in pigeons in Egypt

In recent years, avian influenza virus (AIV) and Newcastle disease virus (NDV) have caused large-scale outbreaks in many countries, including Egypt. The culling and vaccination strategies have failed to control both viruses in Egypt. In this study, we investigated the outbreaks of nervous manifestations and deaths in pigeons between 2013 and 2015. The H5N1 subtype of the highly pathogenic avian influenza virus and pigeon paramyxovirus-1, an antigenic variant of NDV, were found to be the cause; AIV and pigeon paramyxovirus-1 were isolated from 61.3% (19/31) and 67.8% (21/31) of tested pigeons, respectively. Co-infection with both viruses was detected in 51.6% of pigeons (16/31). The AIV sequences showed PQGEKRRKKR/GLF motif at the haemagglutinin gene cleavage site, which is typical of the highly pathogenic H5N1 subtype. The phylogenetic tree showed that the highly pathogenic avian influenza belonged to clade 2.2.1.2. The NDV sequences carried one of the three motifs, ¹¹²GKQGRL¹¹⁷, ¹¹²KRQKRF¹¹⁷ or ¹¹²RRQKRF¹¹⁷, at the fusion protein cleavage site and were classified as genotypes I, VI and II in NDV-class II, respectively. This indicated that different genotypes of NDV can circulate simultaneously among pigeons. Further analysis revealed the clustering of some sequences in sub-genotypes Ia and VIb.2. To the best of our knowledge, these sub-genotypes have not been previously reported from pigeons in Egypt. Our results should serve as a base for future studies on both viruses in Egypt.     

Serological prevalence, genetic identification, and characterization of the first strains of avian hepatitis E virus from chickens in Korea

Avian hepatitis E virus (avian HEV) is associated with hepatitis-splenomegaly (HS) syndrome or big liver and spleen disease in chickens. At least three genotypes of avian HEV have been identified from chickens worldwide. A total of 297 serum samples collected from chickens in 35 flocks in Korea were tested for avian HEV antibody with an enzyme-linked immunosorbent assay. The results showed that approximately 57?% of chicken flocks and 28?% of chickens from Korea were positive for antibodies to avian HEV. Thirteen pooled fecal samples from chickens were tested for avian HEV RNA by RT-PCR, and three fecal samples were positive. The partial helicase and capsid genes of the Korean avian HEV isolates were determined, and sequence analyses revealed that the Korean avian HEV isolates were clustered together and closely related to the genotype 1 avian HEV from Australia. The complete genomic sequence of a Korean avian HEV strain HH-F9 from a broiler breeder was determined, and shown to be 6,653 nt in length, excluding the poly (A) tail, which is 1 nt shorter than the prototype avian HEV from chicken with HS syndrome in the United States. Compared to the full-length sequences of other 5 known avian HEV strains worldwide, the Korean avian HEV shared approximately 83-97?% nucleotide sequence identity. The finding that Korean avian HEV belongs to genotype 1 avian HEV which was previously identified only from chickens in Australia has significant implication in understanding the global epidemiology of avian HEV.     

Genetic characterization of Trichomonas gallinae (Rivolta, 1878) in companion birds in Japan and the genotypical relationship in the Asia region

Background/purposeAvian trichomonosis is a parasitic infection that affects a wide range of avian species, including free-ranging and pet birds worldwide, and Trichomonas gallinae has been considered as the only causative agent for decades. The sequence of the 5.8S ribosomal RNA with internal transcribed spacer (ITS) regions was widely used for identifying genotypes and determining inter-specific and intra-specific diversity. Moreover, the sequence of Fe-hydrogenase (FeHyd) was proposed as the second genetic marker for providing improved resolution of strain subtyping discrimination. Though the correlation between genetic variability and strain virulence is controversial, FeHyd analyses seemed to be useful to investigate the host or geographic origin of isolates. This study aimed to investigate the genetic characteristics of avian Trichomonas spp.MethodsForty-seven oral swabs and crop lavage fluids were collected from 9 avian genera, which were diagnosed as Trichomonas-positive by microscopy in animal hospitals in Japan, were analyzed.ResultsGenetic analysis of clonal isolates revealed the prevalence of the single genotype, ITS-OBT-Tg-1, by ITS region analysis, while two different subtypes, A2 and novel A3, were suggested by FeHyd gene analysis among Japanese companion birds. Phylogenetic analyses of available ITS sequences obtained from the Asia region (China, Iran, Iraq, and Saudi Arabia) were also preformed, revealing endemic ITS-OBT-Tg-1, ITS-OBT-Tg-2, ITS-OBT-Ttl-1, genotype III, and Saudi Arabia's unique lineages. Furthermore, ITS-OBT-Tg-2 predominance in these countries indicates different strains origination from Japan.ConclusionThis is the first report of the genetic characterization of T. gallinae in Japan with discovery of novel subtype A3.     

RT-PCR-RFLP用于麻疹病毒疫苗株和H1基因型的分析

目的 建立RT-PCR-RFLP方法 用于天津地区2002-2008年流行的麻疹野病毒基因型研究.方法 采集疑似麻疹患者的尿标本和咽拭子,传代细胞分离病毒.提取病毒液中的RNA,用一步RT-PCR法扩增麻疹病毒核蛋白(nucleoprotein,N)基因C端594个核苷酸片段,扩增产物经Bcn I酶切后琼脂糖凝胶电泳进行限制性片段多态性分析(RFLP),同时与序列分析进行对比验证.根据结果 构建基因系统发生树进行亲缘关系和遗传距离分析.结果 2002-2008年189份标本,分离获得69株麻疹病毒,N基因C端RT-PCR检测全部阳性,RFLP分析H1a是优势流行亚型,占98.55%(68/69),仅1株(1.45%)为H1b基因亚型,分型结果 与序列测定完全一致.系统发生树分析显示H1a亚型毒株分为2个亚枝,变异范围为0.2%～3.8%,存在不同病毒株引起的传播链共循环.结论 基于Bcn I限制性内切酶的RT-PCR-RFLP方法 能够特异性区分A基因型、H1a、H1b基因亚型,具有快速、简便、准确和经济的优点,更适用于国内大规模麻疹病毒的监测.     

Novel reassortant H10N7 avian influenza viruses isolated from chickens in Eastern China

BackgroundSince 2004, the H10N7 subtype avian influenza virus (AIV) has caused sporadic human infections with variable clinical symptoms world-wide. However, there is limited information pertaining to the molecular characteristics of H10N7 AIVs in China.ObjectiveTo more fully characterize the genetic relationships between three novel H10N7 strains isolated from chickens in Eastern China and the strains isolated from birds throughout Asia, and to determine the pathogenicity of the H10N7 isolates in vivo.Study designAll eight gene segments from the Chinese H10N7 strains were sequenced and compared with AIV strains available in GenBank. The virulence of the three isolates was determined in chickens and mice.ResultsThree H10N7 subtype avian influenza viruses were isolated from chickens in live poultry markets in Eastern China in 2014: (1) A/chicken/Zhejiang/2C66/2014(H10N7) (ZJ-2C66), (2) A/chicken/Zhejiang/2CP2/2014(H10N7) (ZJ-2CP2), and (3) A/chicken/Zhejiang/2CP8/2014(H10N7) (ZJ-2CP8). Phylogenetic analysis indicated that the viruses contained genetic material from H10, H2, H7, and H3 AIV strains that were circulating at the same time. The reassortant H10N7 viruses were found to be minimally pathogenic in chickens and moderately pathogenic in mice. The viruses were able to replicate in mice without prior adaptation.ConclusionThese results suggest that H10N7 surveillance in poultry should be used as an early warning system for avian influenza outbreaks. The novel strains identified here may post a threat to human health in the future if they continue to circulate.     

Pathogenicity of two Toxoplasma gondii strains in chickens of different ages infected via intraperitoneal injection

This experiment was conducted to investigate the pathogenicity of Toxoplasma gondii in broilers of different ages. Chickens at the ages of 7, 14, 21 and 28 days were injected intraperitoneally with 1 × 10⁸ tachyzoites of RH and JS strains of T. gondii, respectively. The clinical signs and death of chickens were recorded daily post inoculation. Serum samples were collected at days 0, 4, 11, 18, 25, 32, 39, 46 and 53 post infection to screen T. gondii circulating antigens (TCA) and T. gondii circulating antibodies (TCAb). The results showed that T. gondii infection of 7-day-old chickens caused death, even though the mortality rate of the JS strain (100%) was significantly higher than that of the RH strain (70%). Chickens at 14 days old showed only mild clinical signs, but no death. Neither clinical signs nor death were recorded in 21-day-old and 28-day-old chickens. TCA and TCAb became positive at days 4 and 11, respectively. Both the TCA and the TCAb of groups 21 days old (RH strain) and 28 days old (both RH and JS strains) decreased to a negative level earlier than the other experimental groups. Specific T. gondii DNA was detected by polymerase chain reaction in chickens that survived in the 7-day-old group (RH strain) and in all infected chickens of groups 14 days old and 21 days old injected with both strains. In the groups injected at 28 days old, three samples (RH strain) and one sample (JS strain) were found negative. The results indicated that the age of the chicken was an important factor affecting the pathogenicity of T. gondii and that these two strains of T. gondii displayed different virulence for chickens.     

Effect of cyclophosphamide on infections produced by Escherichia coli of high and low virulence in chickens

The effect of cyclophosphamide (Cy) on infections caused by Escheri-chia coli strains of high (Expt 1) and low (Expt 2) virulence was examined in 4-week-old specified-pathogen-free chickens. In Expt 1 the mortalities of Cy-treated and non-treated chickens given 5 x 10(7 )cfu of a strain of E. coli of high virulence were both 100%. In the groups given 5 x 10(5) cfu, the mortality of Cy-treated chickens was 90% and that of non-treated chickens was 10%. In Expt 2 the groups given 1 x 10(9) cfu of an E. coli strain of low virulence showed a morttality of 30% when treated with Cy and 0% when non-treated. The chickens given 5 x 10(7) or 5 x 10(5) cfu showed no mortality, clinical signs or histological lesions. Cy-treated chickens showed severe hypoplasia of granulopoiesis in the bone marrow. Haematological examination of Cy-treated chickens revealed leukopenia, especially lymphopenia, and thrombocytopenia. This study suggests that Cy treatment may enhance infection caused by E. coli strain of high virulence and manifest signs of infection caused by E. coli strain of low virulence in the chickens.