Infectious Laryngotracheitis Virus Research Articles

Molecular Characterization and Cluster Analysis of Field Isolates of Avian Infectious Laryngotracheitis Virus from Argentina.

Avian infectious laryngotracheitis (ILT) is a worldwide infectious disease that causes important economic losses in the poultry industry. Although it is known that ILT virus (ILTV) is present in Argentina, there is no information about the circulating strains. With the aim to characterize them, seven different genomic regions (thymidine kinase, glycoproteins D, G, B, C, and J, and infected cell polypeptide 4) were partially sequenced and compared between field samples. The gJ sequence resulted to be the most informative segment, it allowed the differentiation among field sample strains, and also, between wild and vaccine viruses. Specific changes in selected nucleotidic positions led to the definition of five distinct haplotypes. Tests for detection of clustering were run to test the null hypothesis that ILTV haplotypes were randomly distributed in time in Argentina and in space in the most densely populated poultry region of this country, Entre Rios. From this study, it was possible to identify a 46 km radius cluster in which higher proportions of haplotypes 4 and 5 were observed, next to a provincial route in Entre Rios and a significant decline of haplotype 5 between 2009 and 2011. Results here provide an update on the molecular epidemiology of ILT in Argentina, including data on specific genome segments that may be used for rapid characterization of the virus in the field. Ultimately, results will contribute to the surveillance of ILT in the country.

A multiplex real-time RT-PCR for simultaneous detection of four most common avian respiratory viruses

A one-step multiplex real-time reverse transcription-PCR (rRT-PCR) assay was developed for simultaneous detection and quantification of four avian respiratory viruses: avian influenza virus (AIV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV). In comparison with the singleplex rRT-PCR, the specificity, the sensitivity and the reproducibility of the new assay were evaluated and validated using 70 clinical samples. The optimal cutoff point, the corresponding limit of quantification (LoQ) and the limit of detection (LoD) were statistical established based on receiver operating characteristic (ROC) curve analysis. The results showed that the multiplex assay presents higher sensitivity and specificity. Correlation coefficients (R2) and amplification efficiencies (E) of all singleplex and multiplex rRT-PCR reactions are within the acceptable range. The 95% LoDs of multiplex assay were in the range [3–19] copies genomic/ µl, and its corresponding cutoff cycles were in the range [34.16–36.59]. No competitive inhibition for the detection of the four targets and no specific amplification or cross reactivity with other tested viruses was observed. Excellent results were attained in the inter-assay and intra-assay reproducibility evaluation. All identified samples by the multiplex rRT-PCR assay proved to be 100% concordant with the results of the singleplex assays. The results achieved showed that the multiplex assay is very suitable as a routine laboratory test for rapid and specific detection and quantification of co-infections in field samples.

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome.

Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome.IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease.

Serological evidence for eight globally important poultry viruses in Trinidad & Tobago

Viruses affecting poultry cause significant levels of disease leading to severe economic losses among poultry farmers worldwide. The Americas region continues to be vulnerable to the spread of poultry viruses across the continents and Caribbean island chains. In Trinidad and Tobago (T&T) there is limited information on the viruses circulating in poultry. Many flock are vulnerable to infection and there are occasional outbreaks of disease resulting in high levels of morbidity and mortality. This study aims to identify important viruses of poultry circulating in T&T through a broad-based surveillance approach. Serum samples from 29 layer farms in Trinidad and 14 layer farms in Tobago were collected from the eldest laying hens. Samples were tested from unvaccinated birds for antibodies by enzyme-linked immunosorbent assay (ELISA) against Avian influenza virus (AIV), Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Infectious laryngotracheitis virus (ILTV), Avian pneumovirus (APV), Infectious bursal disease virus (IBDV), Fowl adenovirus Gp1 (FADV) and Egg drop syndrome virus (EDSV). In Trinidad, the estimated true seroprevalence levels of antibodies were 0% (CI 95%: 0–0%) for AIV, 100% (CI 95%: 97–100%) for IBV, 79.8% (CI 95%: 70.6–86.9%) for NDV, 1% (CI 95%: 0–2.6%) for ILTV, 67.55% (CI 95%: 62.3–72.4%) for APV, 94.93% (CI 95%: 88.0–98.6%) for IBDV, 100% (CI 95%: 99.7–100%) for FADV and 67.8% (CI 95%: 62.4–72.8%) for EDSV. In Tobago, seroprevalence levels were 0% (CI 95%: 0–0%) for AIV, 100% (CI 95%: 95.6–100%) for IBV, 80.5% (CI 95%: 70.1–88.5%) for NDV, 29.9% (CI 95%: 20.8–40.6%) for ILTV, 100% (CI 95%: 97.7–100%) for APV, 97.1% (CI95%: 89.9–100%) for IBDV, 100% (CI 95%: 97.5–100%) for FADV and 100% (CI 95%: 99–100%) for EDSV. The results reveal strong evidence for the circulation of IBV, NDV, APV, IBDV, FADV and EDSV in layer poultry on both islands, as well as ILTV in Tobago.

Development of an xTAG-multiplex PCR array for the detection of four avian respiratory viruses

Acute respiratory tract infections are of paramount importance in the poultry industry. We developed an xTAG bead assay for the simultaneous detection and discrimination of avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis virus (ILTV). The assay lacked nonspecific reactions with other common avian viruses and the limit of detection was 6.75 × 102– 3.52 × 103copies/μL. We examined 60 clinical specimens and found 18 positive for respiratory viruses. Our result demonstrated that xTAG-multiplex PCR method is a high-throughput, rapid, specific and sensitive assay for use in epidemiological studies and clinical detection of avian respiratory pathogens.

Experimental infection on the locally isolated avian infectious laryngotracheitis virus

The aim of this study was to evaluate virulence of local isolated avian infectious laryngotracheitis virus in experimentally infected chicken. Forty chickens 10 weeks old were used for the experimental infection with the locally isolated infectious laryngotracheitis virus. Chickens were divided into three groups, the first group consisted from 20 chickens infected with isolated infectious laryngotracheitis virus (2×104.16 TCID 50/50 µl) via eyes and mouth drops (one drop for each). The second group consisted of 10 chickens (non-infected) in contact with infected group inoculated with maintenance media (Minimum essential medium) on their eyes, to observe if the infected group can spread the virus. The third group consisted from 10 chickens (non-infected) were left as a control group separated from other groups, inoculated with maintenance media (Minimum essential medium) on their eyes. Clinical signs and mortality were examined daily up to 12 days post infection. The main clinical signs were depression coughing and gasping with mild conjunctivitis and no mortality. Enzyme linked immunosorbent assay (ELISA) test was conducted on the collected sera of chickens before and after experimental infection with isolated virus. The results of ELISA test was negative for all groups of chickens before experiment and positive results for infected group with titer approximately ranging from (2534-7910); Measure of central tendency and dispersion were used with mean (4874.75) and stander error (355.96\ 13.6%); while negative results for contact group and control group. Eighteen chickens (10 weeks old) separately were divided into three groups (infected, contact and control) treated as mention above and were used for histopathological examination; the chickens were killed, two in each group at 24 hr., 48 hr. and 72 hr. post infection. The histopathological changes on trachea and larynx were intracellur inclusion bodies formation detected at 72hr., post infection for infected group only.

Molecular Characterization and Pathological Changes of Infectious Laryngotracheitis Virus in Chicken Farms at Ismailia

Infectious laryngotracheitis (ILT) is an economically important disease of chickens caused by type 1gallidherpesvirus (infectious laryngotracheitis virus, ILTV). Samples from lungs, trachea, liver and spleen were taken, representing 20 farms at Ismailia Province, for molecular studies and histopathological examination on Infectious Laryngotracheitis virus. The samples were obtained from broiler Saso chicken (50 days old) and breeders (65-100 day old) with variable morbidities and mortalities. Four pooled samples from the 20 farms were examined for characterization and identification of ILT virus by PCR. Electrophoretic patterns showed a specific band at 688bp positive in two samples while it was negative in the other two samples. Samples were genetically characterized as Gallid herpesvirus 1 with similarity to ILTV/Brazil/2007/USP-65 ICP4 by 98%Gen bank acc. no: gb|FJ477379.1|.The charactrestic histopathological lesions were found in the lung and trachea. The lung showed severe congestion emphysema and collapse. The trachea revealed degeneration and sloughing of tracheal epithelium with hyperemia, edema and leuckocytic infiltrations in the lamina propria and submucosa. The detected virus in this study was genetically characterized as Gallid herpes virus 1 with similarity to ILTV/Brazil/2007/USP ICP4 by 98%. For the prevention on latent infection need to improve recombinant vaccines. The PCR study proved the occurance of ILTV in the respiratory disease complex (RDC) at Ismailia poultry flocks. Recombinant vaccine improvement is recommended as prophylactive measures for the latent infections among chickens.

Evaluation of factors influencing the development of late Marek’s disease virus-induced immunosuppression: virus pathotype and host sex

ABSTRACTMarek’s disease virus (MDV) is a herpesvirus that induces lymphoma and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is complex and can be divided into two phases: early-MDV-IS associated with cytolytic infection in the lymphoid organs in chickens lacking maternal antibodies against MDV (MAbs) and late-MDV-IS that appears later in the pathogenesis and occurs even in chickens bearing MAbs. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model evaluates late-MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccines against challenge with ILT virus. In the present study, we have used this model to investigate the role of two factors (MDV pathotype and host sex) on the development of late-MDV-IS. Five MDV strains representing three different pathotypes: virulent (vMDV; 617A, GA), very virulent (vvMDV; Md5), and very virulent plus (vv+MDV; 648A, 686), were evaluated. Only vv+ strains were able to induce late-MDV-IS. An immunosuppression rank (IS-rank) was established based on the ability of MDV to reduce the efficacy of chicken embryo origin vaccine (values go from 0 to 100, with 100 being the highest immunosuppressive ability). The IS-rank of the evaluated MDV strains ranged from 5.97 (GA) to 20.8 (617A) in the vMDV strains, 5.97 to 16.24 in the vvMDV strain Md5, and 39.08 to 68.2 in the vv+ strains 648A and 686. In this study both male and female chickens were equally susceptible to MDV-IS by vv+MDV 686. Our findings suggest that late-MDV-IS is a unique feature of vv+ strains.

Multiple pathogen biomarker detection using an encoded bead array in droplet PCR

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host.

To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination.

Impairment of infectious laryngotracheitis virus replication by deletion of the UL[-1] gene.

Infectious laryngotracheitis virus (ILTV) encodes several unique genes, including a pair of unique nuclear proteins UL0 and UL[-1] that are expressed during replication in cell culture. Although the UL0 gene has been shown to be dispensable for replication, the role of UL[-1] has not been elucidated. In this study a deletion mutant of ILTV lacking the UL[-1] gene was constructed using homologous recombination. The coding sequences of the gene were replaced with the gene for enhanced green fluorescent protein and the cytomegalovirus major immediate early promoter element. The progeny virus carrying the reporter gene was readily identified using fluorescent microscopy, but was unable to propagate in the permissive cells in the absence of wild type ILTV. Even after plaque purification and fluorescent associated cell sorting the recombinant virus deficient in UL[-1] gene could not be successfully isolated. Our findings suggest that the UL[-1] gene has an important role in ILTV replication.

Examining responses of chicken embryonic neural stem cell to infectious laryngotracheitis virus infections

Infectious laryngotracheitis virus (ILTV) infection causes economic losses in the poultry industry. Outbreaks of the infection in commercial poultry are controlled by using attenuated live vaccines. The genomic and antigenic characteristics between virulent and vaccine strains of ILTV are very similar, thus the differential diagnosis of ILTV strains is targeted in controlling programs. Here, the chicken embryonic neural stem (cENS) cell serves as a host model to study virulence characteristics of ILTVs. The susceptibility of cENS cells to different ILTV strains infection was investigated by analyzing the impact of infection on cell death, virus latency, and determining the host cell responses. We found that cENS cells are highly susceptible to vaccine strain ILTV infection. Upon infection, the strain showed faster replication kinetics in cell culture and marked cytopathic effects with affecting the expression of cellular neuropeptide Y (NPY). Virulent strain was able to enter cENS cells, but no infectious virus was produced at third passage. The establishment of latency state was confirmed by molecular detection of viral ICP4 gene and virus reactivation assay. In contrast to vaccine strain, transcription level of cellular NPY was also upregulated significantly following virulent strain infection. The difference expression pattern of NPY in ILTV strains-infected cells is most closely with the presence of CPE in culture. The neural stem cell system may provide the most relevant tool for differential diagnosis of ILTV strains.

Newcastle disease virus vectored infectious laryngotracheitis vaccines protect commercial broiler chickens in the presence of maternally derived antibodies

Newcastle disease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoproteins B and D have previously been demonstrated to confer complete clinical protection against virulent ILTV and NDV challenges in naive chickens. We extended this study to assess whether maternally derived antibody (MDA) against NDV and ILTV would interfere with protection in vaccinated broiler chickens. Chickens with a mean NDV MDA hemagglutination inhibition (HI) titer of 6.4 (log2) and detectable ILTV neutralization (VN) antibodies at hatch were vaccinated with rLS/ILTV-gB or rLS/ILTV-gD at 1 or 10day of age (DOA) or on both days. Groups of birds vaccinated with the commercial ILT vaccines (FP-LT and CEO) or sham inoculated were also included in this study. All vaccinated birds were challenged with virulent ILTV strain at 21 DOA. By that time, NDV HI titers declined to 2.6 (log2) in unvaccinated birds, whereas the HI titers in NDV vectored vaccine groups increased to 3.5–6.3 (log2). At standard dosages, both vaccine candidates conferred significant clinical protection; however, the protection elicited by the rLS/ILTV-gD was superior to that of rLS/ILTV-gB. Recombinant rLS/ILTV-gD reduced ILTV shedding from tracheal and ocular tissues by approximately 3 log10 TCID50. Notably, there was no improvement in protection after booster vaccination at 10 DOA. Overall results indicate that the presence of maternal antibodies to NDV and ILTV did not significantly interfere with the ability of the NDV LaSota strain-vectored ILTV gB and gD vaccine candidates to elicit protective immunity against infectious laryngotracheitis.

Molecular Survey of Respiratory and Immunosuppressive Pathogens Associated with Low Pathogenic Avian Influenza H9N2 Subtype and Virulent Newcastle Disease Viruses in Commercial Chicken Flocks.

The study was carried out in 48 poultry flocks to elucidate the roles of various complicating pathogens involved along with Newcastle disease (ND)/ low pathogenic avian influenza (LPAI) outbreaks. Necropsy was conducted and samples were collected for the isolation of Newcastle disease virus (NDV), Influenza A virus, infectious bronchitis virus (IBV), pathogenic bacteria; molecular detection of infectious laryngotracheitis virus (ILTV), fowl adeno virus (FAV), chicken anaemia virus (CAV), Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). The isolation results confirmed that 18/48 flocks (37%) were positive for the presence of hemagglutinating agents. Out of 18 hemagglutination (HA) positive flocks, 11 flocks (61%) were positive for both avian influenza virus (AIV) and NDV; 4 flocks (22%) were positive for NDV; and 3 flocks (17%) were positive for AIV. Sequence analysis of hemagglutinin and neuraminidase genes of AIV revealed that all were belonging to LPAI-H9N2 subtype. Sequence analysis of F gene of NDV revealed that they belong to virulent type. The PCR results confirmed the presence of three to seven etiological agents (CAV, FAV, ILTV, MG, MS and avian pathogenic E. coli along with LPAI/NDV from all the 18 HA-positive flocks. The detection rate of triple, quadruple, quintuple, sextuple and sevenfold infections was 17% (3 flocks), 28% (5 flocks), 11%, (2 flocks) 28% (5 flocks) and 17% (3 flocks), respectively. In conclusion, the disease complex involved more than one pathogen, primarily resulting from the interplay between LPAI-H9N2 and NDV; subsequently this could be exacerbated by co-infection with other agents which may cause exacerbated outbreaks that may otherwise go undetected in field.

A survey for selected avian viral pathogens in backyard chicken farms in Finland

ABSTRACTBackyard poultry are regaining popularity in Europe and increased interest in the health and management of non-commercial farms has resulted. Furthermore, commercial poultry farm owners have become concerned about the risk represented by contagious avian diseases that nearby backyard poultry could transmit. Fifty-one voluntary backyard chicken farms were visited between October 2012 and January 2013. Blood samples and individual cloacal swabs were collected from 457 chickens. In 44 farms (86%), one or more of the tested chickens had antibodies against avian encephalomyelitis and chicken infectious anaemia viruses, 24 farms (47%) had chickens seropositive for infectious bronchitis virus, 10 farms (20%) had chickens seropositive for infectious bursal disease virus, six farms (12%) had chickens seropositive for infectious laryngotracheitis virus and two farms (5.4%) had chickens seropositive for avian influenza virus. No farms had chickens seropositive for Newcastle disease virus. Of the 51 farms, five (10%) had chickens positive for coronavirus reverse transcription polymerase chain reaction. A phylogenetic analysis showed that all backyard chicken coronaviruses collected were QX type infectious bronchitis viruses. All chickens tested for avian influenza and Newcastle disease viruses using real time reverse transcription polymerase chain reaction were negative. To our knowledge, there is no evidence to date to suggest that these diseases would have been transmitted between commercial and non-commercial flocks.

Detection of infectious bronchitis virus 793B, avian metapneumovirus, Mycoplasma gallisepticum and Mycoplasma synoviae in poultry in Ethiopia

A survey was conducted into respiratory infectious diseases of poultry on a chicken breeder farm run by the Ethiopian Institute of Agricultural Research (EIAR), located in Debre Zeit, Ethiopia. Oropharyngeal swabs were collected from 117 randomly selected birds, and blood was taken from a subset of 73 of these birds. A combination of serological and molecular methods was used for detection of pathogens. For the first time in Ethiopia, we report the detection of variant infectious bronchitis virus (793B genotype), avian metapneumovirus subtype B and Mycoplasma synoviae in poultry. Mycoplasma gallisepticum was also found to be present; however, infectious laryngotracheitis virus was not detected by PCR. Newcastle disease virus (NDV) was not detected by PCR, but variable levels of anti-NDV HI antibody titres shows possible exposure to virulent strains or poor vaccine take, or both. For the burgeoning-intensive industry in Ethiopia, this study highlights several circulating infectious respiratory pathogens that can impact on poultry welfare and productivity.

Epidemiology of Avian Infectious Laryngotracheitis with Special Focus to South America: an update

ABSTRACT Avian Infectious laryngotracheitis (AILT) is a respiratory tract disease of great importance because it causes significant economic losses in the poultry industry around the world. It is caused by a Gallid herpesvirus type 1, a member of the genus Iltovirus. The target system for Avian Infectious Laryngotracheitis virus (AILTV) infections is the respiratory system, and the main organ in which the virus remains latent is the trigeminal ganglia. However, the virus has demonstrated tropism for other organs besides the respiratory tract. The main transmission routes are ocular and respiratory. Infected birds with clinical symptoms are main sources of transmission, but birds with latent infections, litter, and contaminated fomites may also transmit the virus. Clinical signs usually appear 6-12 days after natural exposure and may be moderate or severe. The causative agent of this disease can be propagated in chorioallantoic membrane (CAM) of developing chicken embryos and replicate in mature chicken kidney cells, as well as in a variety of epithelial chick embryo cells, such as kidneys, liver and lungs. There are several procedures for the diagnosis of ILT such as the observation of clinical signs, the detection of gross and histopathological lesions, and the use of molecular techniques, including RFLP, polymerase chain reaction (PCR), real-time PCR, and loop-mediated isothermal amplification. Vaccination with different types of vaccine provides a good expectation on disease control, such as vaccines produced in chicken-embryo-origin (CEO), tissue-culture-origin (TCO), and recombinant vaccines. However, in endemic areas, biosecurity measures and best management practices are important for the control of the disease. It is distributed worldwide and, in South America, it has been reported in Brazil, Peru, Ecuador, Bolivia, and Argentina causing great economic losses.

Presence of DNA extracellular traps but not MUC5AC and MUC5B mucin in mucoid plugs/casts of infectious laryngotracheitis virus (ILTV) infected tracheas of chickens

Although it has been speculated that the tracheal obstructions and asphyxiation during acute infectious laryngotracheitis (ILT) are due to mucoid plugs/casts formed by mucus hypersecretion, there are no reports demonstrating this. Hence, in the present study, we first examined if the main respiratory mucins, MUC5AC and MUC5B, are expressed in the mucosae of larynx, trachea and bronchi of mock-inoculated and ILTV infected chickens. Second, the tracheas with plugs/casts were stained for mucins (MUC5AC and MUC5B) and nuclear material (traps). MUC5AC and MUC5B were produced by the mucosae of larynx, trachea and bronchi of mock-inoculated chickens. Interestingly, MUC5AC and MUC5B were exclusively present in the dorsal tracheal region of the cranial and middle part of trachea of mock-inoculated chickens. In ILTV infected chickens, the tracheal lumen diameter was almost 40% reduced and was associated with a strongly increased tracheal mucosal thickness. MUC5AC and MUC5B were scarcely observed in larynx, trachea and bronchi, and in tracheal plugs/casts of ILTV infected birds. Surprisingly, DNA fibrous structures were observed in connection with nuclei of 10.0±7.3% cells, present in tracheal plugs/casts. Upon inoculation of isolated blood heterophils with ILTV, DNA fibrous structures were observed in 2.0±0.1% nuclei of ILTV inoculated blood heterophils at 24hours post inoculation (hpi). In conclusion, the tracheal obstructions and suffocation of ILTV infected chickens are due to a strong thickening of the mucosa (inflammation) resulting in a reduced tracheal lumen diameter and the presence of mucoid plugs/casts containing stretched long DNA-fibrous structures (traps) but not MUC5AC and MUC5B mucins.

Early infection with Marek’s disease virus can jeopardize protection conferred by laryngotracheitis vaccines: a method to study MDV-induced immunosuppression

ABSTRACTMarek’s disease virus (MDV) is a herpesvirus that induces lymphomas and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is divided into two phases: early-MDV-IS occurring mainly in chickens lacking maternal antibodies (MAb) against MDV and associated with lymphoid organ atrophy; and late-MDV-IS occurring once MDV enters latency and during tumour development. Our objectives were to document the impact of late-MDV-IS on commercial poultry (meat-type chickens bearing MAb against MDV and that were vaccinated or unvaccinated against MD) and to optimize a model to study late-MDV-IS under laboratory conditions. The impact of late-MDV-IS was evaluated by assessing the effect of early infection (day of age) with a very virulent plus MDV (vv+MDV) on the efficacy of chicken-embryo-origin (CEO) infectious laryngotracheitis (ILT) virus vaccine against ILT challenge. The CEO ILT vaccine was administered in water at 14 days of age and ILT virus (ILTV) challenge was done intratracheally at 30 days of age. Development of ILT was monitored by daily evaluation of clinical signs, development of gross and histological lesions in trachea, and quantification of ILTV transcripts in trachea. Infection with vv+MDV strain 648A resulted in total abrogation of protection conferred by the CEO vaccine against ILTV challenge even in chickens vaccinated at 1 day of age with either HVT, HVT+SB-1, or CVI988. Chickens exposed to vv+MDV prior to vaccination with CEO ILTV vaccine had similar (P < 0.05) clinical scores, gross lesions, histopathologic lesion scores, and load of ILTV transcripts in trachea after ILTV challenge, as chickens that were not vaccinated with CEO ILTV vaccine.

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system.

BackgroundEimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens.ResultsIn this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella. Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV (vvVP2) and glycoprotein I from ILTV (gI) were cloned downstream of 5’Et-Actin or 5’Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5’Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens.ConclusionsE. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1756-2) contains supplementary material, which is available to authorized users.