The route of inoculation dictates the replication patterns of the infectious laryngotracheitis virus (ILTV) pathogenic strain and chicken embryo origin (CEO) vaccine

Infectious laryngotracheitis virus (ILTV) has a high proclivity to replicate in the larynx and trachea of chickens causing severe lesions. There is a lack of knowledge on the ability of ILTV to replicate in other respiratory associated tissues apart from in the trachea. The objective of this study was to investigate how tissues that first encounter the virus dictate further sites of viral replication during the lytic stage of infection. Replication patterns of the pathogenic strain 63140 and the chicken embryo origin (CEO) vaccine in the conjunctiva, the Harderian gland, nasal cavity and trachea were evaluated after ocular, oral, intranasal or intratracheal inoculation of specific pathogen-free chickens. Viral replication was assessed by detection of microscopic cytolytic lesions, detection of viral antigen and viral genome load. The route of viral entry greatly influenced virus replication of both strain 63140 and CEO vaccine in the conjunctiva and trachea, while replication in the nasal cavity was not affected. In the Harderian gland, independently of the route of viral entry, microscopic lesions characteristic of lytic replication were absent, whereas viral antigen and viral genomes for either virus were detected, suggesting that the Harderian gland may be a key site of antigen uptake. Findings from this study suggest that interactions of the virus with the epithelial-lymphoid tissues of the nasal cavity, conjunctiva and the Harderian gland dictate patterns of ILTV lytic replication.     

Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes

In comparison to secondary lymphoid organs, gut-associated lymphoid tissues such as isolated lymphoid follicles (ILF) and cryptopatches (CP) have been less intensively studied. To gain a better insight into processes regulating organization and function of these structures, which are believed to participate in immune responses and extrathymic T cell development, we characterized the lymphoid structures of the murine small intestine in more detail. The size and cellular composition of small intestinal lymphoid aggregations were analyzed in C57BL/6 and BALB/c wild-type and lymphotoxin (LT)-deficient mice, by flow cytometry, histology and automated multi-color immunofluorescence microscopy evaluating large coherent areas of the intestine. These evaluations demonstrate that aggregated lymphoid structures in the small intestine vary in size and cellular composition, with a majority of structures not matching the current definitions of CP or ILF. Accordingly, significant variations depending on species, age and mouse strain were observed. Furthermore, small bowel transplantation revealed a rapid exchange of B but not T cells between host and grafted tissue. Moreover, LT-deficient animals lack any intestinal lymphoid aggregations yet possess the complete panel of intraepithelial lymphocytes (IEL). In summary, our observations disclose intestinal lymphoid aggregations as dynamic structures with a great deal of inborn plasticity and demonstrate their dispensability for the generation of IEL.     

Seeding of the mucosal leukocytes in the HALT and trachea of White Leghorn chickens

Characterising the immune cells of the head-associated lymphoid tissues (HALT) and trachea during maturation in young birds is critical to understanding the immunological responses to avian respiratory diseases and vaccines. Selected mucosal leukocytes of the conjunctiva-associated lymphoid tissue (CALT), Harderian gland (HG), nasal-associated lymphoid tissue (NALT) and trachea from 4-, 6-, 8-, and 10-week-old chickens were enumerated and phenotyped. HG, NALT, and trachea cellularity increased as the birds aged with cell viability varying by tissue. The results showed that the T cell subset numbers, but not B cell numbers, increased in the mucosal tissues of chickens during aging.     

Early occurrence of apoptosis in lymphoid tissues from chickens infected with strains of Newcastle disease virus of varying virulence

Newcastle disease virus (NDV), the causative agent of Newcastle disease, is a prevalent problem in the poultry industry and often the cause of severe economic loss. There are many strains of the virus and these have varying virulence. The most virulent strains cause systemic lesions of lymphoid tissues, with necrosis and severe lymphoid depletion. Less virulent strains do not cause as much necrosis, but may predispose to secondary infection with other pathogens. Apoptosis or programmed cell death, has been demonstrated to play a role in the pathogenesis of other paramyxovirus infections, notably those caused by measles and canine distemper viruses. To investigate the role of apoptosis in lymphoid organs during NDV infection, immunohistochemistry for determination of expression of caspase-3, a marker of imminent apoptosis, was performed on formalin-fixed paraffin wax-embedded tissues (spleen, thymus, caecal tonsils and bursa of Fabricius) from 4-week-old chickens infected with NDV strains of varying virulence 2 days previously. The amount of apoptosis was proportional to the severity of the clinical disease elicited by the strains.     

Latency characteristics in specific pathogen-free chickens 21 and 35 days after intra-tracheal inoculation with vaccine or field strains of infectious laryngotracheitis virus

ABSTRACT

Latency is an important feature of infectious laryngotracheitis virus (ILTV) yet is poorly understood. This study aimed to compare latency characteristics of vaccine (SA2) and field (CL9) strains of ILTV, establish an in vitro reactivation system and examine ILTV infection in peripheral blood mononuclear cells (PBMC) in specific pathogen-free chickens. Birds were inoculated with SA2 or CL9 ILTV and then bled and culled at 21 or 35 days post-inoculation (dpi). Swabs (conjunctiva, palatine cleft, trachea) and trigeminal ganglia (TG) were examined for ILTV DNA using PCR. Half of the TG, trachea and PBMC were co-cultivated with cell monolayers to assess in vitro reactivation of ILTV infection. ILTV DNA was detected in the trachea of approximately 50% of ILTV‐inoculated birds at both timepoints. At 21?dpi, ILTV was detected in the TG only in 29% and 17% of CL9- and SA2-infected birds, respectively. At 35?dpi, ILTV was detected in the TG only in 30% and 10% of CL9- and SA2-infected birds, respectively. Tracheal organ co-cultures from 30% and 70% of CL9- and SA2-infected birds, respectively, were negative for ILTV DNA at cull but yielded quantifiable DNA within 6 days post-explant (dpe). TG co-cultivation from 30% and 40% of CL9-and SA2-infected birds, respectively, had detectable ILTV DNA within 6 dpe. Latency characteristics did not substantially vary based on the strain of virus inoculated or between sampling timepoints. These results advance our understanding of ILTV latency and reactivation.

RESEARCH HIGHLIGHTS

• Following inoculation, latent ILTV infection was detected in a large proportion of chickens, irrespective of whether a field or vaccine strain was inoculated.

• In vitro reactivation of latent ILTV was readily detected in tracheal and trigeminal ganglia co-cultures using PCR.

• ILTV latency observed in SPF chickens at 21 days post-infection was not substantially different to 35 days post-infection.

     

Transcriptional response of immune genes in gills and the interbranchial lymphoid tissue of Atlantic salmon challenged with infectious salmon anaemia virus

Previously, it has been assumed that fish lack organized mucosa-associated lymphoid structures. Recently, an interbranchial lymphoid tissue (ILT) was described in salmonid gills at a site with substantial exposure to antigen.     

Long-term protection against a virulent field isolate of infectious laryngotracheitis virus induced by inactivated,recombinant, and modified live virus vaccines in commercial layers

Infectious laryngotracheitis (ILT) is an acute respiratory disease of chickens controlled through vaccination with live-modified attenuated vaccines, the chicken embryo origin (CEO) vaccines and the tissue-culture origin (TCO) vaccines. Recently, novel recombinant vaccines have been developed using herpesvirus of turkey (HVT) and fowl pox virus (FPV) as vectors to express ILTV immunogens for protection against ILT. The objective of this study was to assess the protection efficacy against ILT induced by recombinants, live-modified attenuated, and inactivated virus vaccines when administered alone or in combination. Commercial layer pullets were vaccinated with one or more vaccines and challenged at 35 (35 WCH) or 74 weeks of age (74 WCH). Protection was assessed by scoring clinical signs; and by determining the challenge viral load in the trachea at five days post-challenge. The FPV-LT vaccinated birds were not protected when challenged at 35 weeks; the HVT-LT and TCO vaccines in combination provided protection similar to that observed in chickens vaccinated with either HVT-LT or TCO vaccines when challenged at 35 weeks, whereas protection induced by vaccination with HVT-LT followed by TCO was superior in the 74 WCH group compared with the 35 WCH group. Birds given the inactivated ILT vaccine had fewer clinical signs and/or lower viral replication at 74 WCH when combined with TCO or HVT-LT, but not when given alone. Finally, the CEO-vaccinated birds had top protection as indicated by reduction of clinical signs and viral replication when challenged at 35 weeks (74 weeks not done). These results suggest that certain vaccine combinations may be successful to produce long-term protection up to 74 weeks of age against ILT.     

The appearance of viral antigen and antibody in the trachea of naive and vaccinated chickens infected with infectious laryngotracheitis virus

In chickens vaccinated with SA-2 infectious laryngotracheitis (ILT) virus, viral antigen could no longer be detected in tracheal washings from day 7 post infection (pi). Total specific antibody was detected in tracheal washings from day 5 pi, IgA antibody appeared at day 6 pi, but neutralising antibody could not be detected until day 14. In the serum of vaccinated chickens, total antibody appeared on day 5 pi and neutralising antibody on day 7. However, no IgA antibody could be detected in serum. There was a substantial increase in the numbers of IgA- and IgG-synthesising cells in the trachea by day 3 pi, with a marked increase in the numbers of IgA-positive cells at day 7 pi. Following challenge with virulent CSW-1 ILT virus, no virus could be detected in the trachea of vaccinated chickens. There was also no evidence of an anamnestic antibody response in the trachea or in serum up to day 10 post challenge, and there was no significant change in the numbers of IgA- or IgG-synthesising cells in the tracheas of vaccinated chickens up to day 7 post challenge.     

Impairment of B and T cell maturation in gut associated lymphoid tissues due to malnutrition during lactation

Previously we found that malnutrition during lactation in rats produces an impairment in the immune response to cholera toxin. In this report we found that malnutrition during lactation provokes in 28-day-old rats an increase of Thy1⁺ cμ⁺ cells in gut associated lymphoid tissues concomitantly with a decrease of sIgA⁺ B cells. No differences were found in the percentages of the IgM⁺B cell populations. Furthermore, no differences were found in the Peyer's patch (PP) and mesenteric lymph node (MLN) T cell subsets in weaning rats when compared to controls. However, after 1 week of refeeding a higher percentage of the Thy1⁺ cμ⁻ subset together with a lower percentage of CD5⁺, CD4⁺, and CD8⁺ T cells, were found in malnourished rats when compared to controls. The above results may indicate that B-cell maturation is delayed in malnourished rats at two stages of differentiation: (a) in the passage of pre-B cells (Thy1⁺ cμ⁺) to immature B cells (sμ⁺), and (b) in the switch from sμ⁺ B cells to s⁺ B cells. The decrease of CD5⁺, CD4⁺, and CD8⁺T cells together with and increase of the Thy1⁺ cμ⁻ subset in gutassociated lymphoid tissues (GALT) may indicate that T-cell maturation is also delayed. Results obtained at weaning may be due to an engraftment by maternal milk-derived lymphocytes in the pups.     

Evaluation of vaccination against infectious laryngotracheitis (ILT) with recombinant herpesvirus of turkey (rHVT-LT) and chicken embryo origin (CEO) vaccines applied alone or in combination

ABSTRACT

The chicken embryo origin (CEO) infectious laryngotracheitis (ILT) live attenuated vaccines, although capable of protecting against disease and reducing challenge virus replication, can regain virulence. Recombinant ILT vaccines do not regain virulence but are partially successful at blocking challenge virus replication. The objective of this study was to evaluate the effect of rHVT-LT vaccination on CEO replication and how this vaccination strategy enhances protection and limits challenge virus transmission to naïve contact chickens. The rHVT-LT vaccine was administered at 1 day of age subcutaneously and the CEO vaccine was administered at 6 weeks of age via eye-drop or drinking water. CEO vaccine replication post vaccination, challenge virus replication and transmission post challenge were evaluated. After vaccination, only the group that received the CEO via eye-drop developed transient conjunctivitis. A significant decrease in CEO replication was detected for the rHVT-LT?+?CEO groups as compared to groups that received CEO alone. After challenge, reduction in clinical signs and challenge virus replication were observed in all vaccinated groups. However, among the vaccinated groups, the rHVT-LT group presented higher clinical signs and challenge virus replication. Transmission of the challenge virus to naïve contact chickens was only observed in the rHVT-LT vaccinated group of chickens. Overall, this study found that priming with rHVT-LT reduced CEO virus replication and the addition of a CEO vaccination provided a more robust protection than rHVT alone. Therefore, rHVT-LT?+?CEO vaccination strategy constitutes an alternative approach to gain better control of the disease.     

肝脏粘膜相关淋巴瘤——附3例报道及文献复习

报道３例肝脏粘膜相关淋巴瘤，起源于汇管区淋巴细胞。具有诊断性淋巴上皮病变，分别为中心细胞样细胞型（２例）及淋巴浆细胞型（１例），经免疫组化证实为单克隆Ｂ细胞性。其临床特点为：生长缓慢，局部受累，很少转移，低度恶性。以手术切除为主，预后较好。本文对其发病机理进行了探讨。     

Multi-tissue single-cell analysis deconstructs the complex programs of mouse natural killer and type 1 innate lymphoid cells in tissues and circulation

1. Download : Download high-res image (296KB)
2. Download : Download full-size image

     

Collision of EBV‐associated gastric carcinoma and primary gastric extranodal marginal zone lymphoma of mucosa‐associated lymphoid tissue in the remnant stomach

Reported herein is a case of EBV‐associated gastric carcinoma with primary gastric extranodal marginal zone lymphoma of mucosa‐associated lymphoid tissue (MALT lymphoma). A 69‐year‐old Japanese man was found to have an ulcer lesion in his stomach on endoscopy, and a biopsy indicated malignancy. He underwent gastrectomy. Microscopically the tumor had features typical of lymphoepithelioma‐like carcinoma. The neoplastic epithelial cells proliferated in a trabecular fashion. On in situ hybridization for EBV‐encoded RNA, positive signals were observed in most neoplastic epithelial cells. Numerous lymphocytes surrounded the neoplastic epithelial cells. In the stroma, numerous lymphocytes with mild atypia were positive for CD20 and CD79a. In addition, monoclonal proliferation of B cells was confirmed on polymerase chain reaction for IgH. These findings supported MALT lymphoma. The coexistence of EBV‐associated gastric carcinoma and MALT lymphoma is extremely rare.     

Heterogenous Na+, K+-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissues

Na⁺, K⁺-ATPase expression in the epithelia of rabbit gut-associated lymphoid tissue was measured using indirect immunofluorescence and confocal laser scanning microscopy. All four major sites of aggregated lymphoid tissue, i. e. Peyer's patch, sacculus rotundus, caecal patch and appendix, were studied. Na⁺, K⁺-ATPase expression was localized to the basolateral surface of cells of the follicle-associated epithelium (FAE) and adjacent villous or surface epithelia (non-FAE), where increased expression during enterocyte migration was evident. In the FAE, expression of Na⁺, K⁺-ATPase appeared to be lower in the specialized M cells than in enterocytic-type cells, although expression in both cell types was lower than in adjacent non-FAE. Quantification of immunofluorescent staining of Na⁺, K⁺-ATPase by confocal laser scanning imaging showed a reduction of expression in the FAE to approximately 20–60% relative to that in the adjacent non-FAE. These results are consistent with a primary role of the FAE in mucosal immunity with minimal involvement in active solute absorption.     

Contributions of the S2 spike ectodomain to attachment and host range of infectious bronchitis virus

The spike protein is the major viral attachment protein of the avian coronavirus infectious bronchitis virus (IBV) and ultimately determines viral tropism. The S1 subunit of the spike is assumed to be required for virus attachment. However, we have previously shown that this domain of the embryo- and cell culture adapted Beaudette strain, in contrast to that of the virulent M41 strain, is not sufficient for binding to chicken trachea (Wickramasinghe et al., 2011). In the present study, we demonstrated that the lack of binding of Beaudette S1 was not due to absence of virus receptors on this tissue nor due to the production of S1 from mammalian cells, as S1 proteins expressed from chicken cells also lacked the ability to bind IBV-susceptible embryonic tissue. Subsequently, we addressed the contribution of the S2 subunit of the spike in IBV attachment. Recombinant IBV Beaudette spike ectodomains, comprising the entire S1 domain and the S2 ectodomain, were expressed and analyzed for binding to susceptible embryonic chorio-allantoic membrane (CAM) in our previously developed spike histochemistry assay. We observed that extension of the S1 domain with the S2 subunit of the Beaudette spike was sufficient to gain binding to CAM. A previously suggested heparin sulfate binding site in Beaudette S2 was not required for the observed binding to CAM, while sialic acids on the host tissues were essential for the attachment. To further elucidate the role of S2 the spike ectodomains of virulent IBV M41 and chimeras of M41 and Beaudette were analyzed for their binding to CAM, chicken trachea and mammalian cell lines. While the M41 spike ectodomain showed increased attachment to both CAM and chicken trachea, no binding to mammalian cells was observed. In contrast, Beaudette spike ectodomain had relatively weak ability to bind to chicken trachea, but displayed marked extended host range to mammalian cells. Binding patterns of chimeric spike ectodomains to these tissues and cells indicate that S2 subunits most likely do not contain an additional independent receptor-binding site. Rather, the interplay between S1 and S2 subunits of spikes from the same viral origin might finally determine the avidity and specificity of virus attachment and thus viral host range.     

A novel variant of the infectious bronchitis virus resulting from recombination events in Italy and Spain

Infectious bronchitis is considered to be one of the most devastating diseases in poultry. Control of its spread is typically attempted through biosecurity measures and extensive vaccination. However, the remarkable genetic and antigenic variability of the virus, which originate from both mutations and recombination events, represents an unsolved challenge for this disease. The present study reports on the emergence and spread of recombinant clusters detected in Italy and Spain between 2012 and 2014. A total of 36 Spanish and Italian infectious bronchitis virus (IBV) field strains were investigated and genetically characterized using phylogenetic, molecular, recombination and selection pressure analyses of the complete S1 gene. Based on the partial S1 sequencing, 27 IBV strains originating from Spain and nine from Italy were initially classified as being closely related to the Guandong/Xindadi (XDN) genotype. Phylogenetic analysis of the complete S1 gene revealed that the XDN strains formed a homogeneous clade with the Spanish IBV isolates within the QX genotype, whereas there was higher variability within the Italian strains. Recombination analysis determined that these strains belonged to four groups, which originated from independent recombination events between the QX and 793B IBV genotypes. Our data support the hypothesis of two different scenarios: firstly, in Spain, the large and homogeneous clade probably originated from a single offspring of the recombinant founder, which became dominant and spread throughout the country. Secondly, the nine Italian recombinants, which are characterized by three different recombination patterns, probably represent less fitted strains, because they were less viable with respect to their recombinant parents.     

Chicken infectious anaemia virus infections in chickens in northern Vietnam: epidemiological features and genetic characterization of the causative agent

ABSTRACT

Since the first report of chicken infectious anaemia virus (CIAV) in Vietnam in 2013, there have not been many studies focused on the detection of CIAV or the molecular characteristics of the virus. This study attempted to investigate the presence of CIAV in northern Vietnam by molecular-based methods. Regarding the spatial distribution of CIAV, the PCR-based results showed that CIAV was detected in 47 out of 64 farms (73.4%) and in all 10 investigated provinces. Of the 119 samples assayed by PCR, 74 (62.2%) tested positive for CIAV DNA. By arranging the samples into different categories, it was found that CIAV was detected at high rates (above 50%) based on all 4 evaluated criteria as follows: production type of chicken, housing system, flock size and age group. Different housing systems were significantly associated with the detection rates of CIAV (P?=?0.003). By genetic analyses, all of the Vietnamese CIAVs were found to (i) lack substitutions related to attenuation substitutions, (ii) group separately from vaccine-like CIAVs and (iii) belong to genogroups G2 and G3 of CIAV. Because of the wide distribution of CIAV and because the virus was confirmed not to be vaccine-like viruses, it is suggested that further studies be conducted on the clinical form of chicken infectious anaemia, as well as the immunosuppressive effect of CIAV on chickens in Vietnam.

RESEARCH HIGHLIGHTS

• Wide distribution of chicken infectious anaemia virus (CIAV) in northern Vietnam.

• Vietnamese CIAVs belong to genogroups G2 and G3 of CIAV.