Caecal Tonsils Research Articles

Effects of Titanium Dioxide Nanoparticles on Chick Embryo: Immunomodulatory, Hepatic and Biochemical Alterations.

The utilization of titanium dioxide nanoparticles (TiO2 NPs) has significantly increased across various industries. This study rigorously explored the impact of TiO2 NPs exposure on chicken embryos, focusing particularly on alterations in the immune system, liver functionality and key biochemical markers. The study involved three groups of 30 eggs each, subjected to increasing doses of TiO2 NPs: Group C (control), Group T1 (150µg/mL) and Group T2 (300µg/mL). After 48h of incubation, the eggs in Groups T1 and T2 each received an injection of 0.3mL of the TiO2 NPs solution. In contrast, the eggs in the control group (Group C) were injected with 0.3mL of saline solution. Histopathological changes were analysed using haematoxylin and eosin (H&E) staining, whereas amniotic fluid's biochemical properties were examined photometrically. The study also assessed the expression of immune genes (AvBD9, IL6 and IL8L2) through quantitative PCR. The evaluations included growth metrics, amniotic fluid biochemistry and histological analysis of the liver, caecal tonsil and bursa of Fabricius. The results revealed subcutaneous haemorrhage, significant reductions in total body weight and marked changes in biochemical markers, including urea, creatinine, alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), in the amniotic fluid of the groups treated with TiO2 NPs, compared to the control. Histological examinations indicated noticeable alterations in the liver, caecal tonsil and bursa of Fabricius following TiO2 NP exposure. These alterations were characterized by disruptions in cellular structures and variations in lymphocyte counts. Furthermore, a notable decrease in the expression of immunity genes, namely, AvBD9, IL8L2 and IL6, was observed in the TiO2 NP-treated groups compared to the control. The findings underscore the need for risk assessments of TiO2 NPs exposure due to its impact on development and immunity. Future research should explore its impact on neurodevelopment and degeneration.

Newcastle disease (ND) in laying birds in Asaba, Delta State: A Case Report

This is a case report of Newcastle disease in 52 weeks old laying birds with flock size of 2,500 birds and average weight of 1.7kg. The client presented history of continuous deaths and poor laying performance. The clinical signs observed in this case study were mortalities, low egg production, depression, nasal discharge, weakness, and diarrhoea (perineum matted with greenish faeces). Post-mortem investigation revealed haemorrhagic caecal tonsils, enteritis, slightly enlarged and friable liver with areas of discolouration and pin point haemorrhages on pro-ventricular glands. Blood samples were collected from 10 birds and the sera subjected to Haemagglutination and Heamagglutination Inhibition (HI) tests using known NDV (LaSota strain). Low Newcastle disease virus (NDV) antibody titres were detected from the sera samples suggesting that the birds were not protected against ND. The flock was managed by re-vaccination using NDV LaSota strain, followed by administration of a vitamins-mineral supplement, antibiotics, glucose and an analgesic and the treatment yielded favourable therapeutic results. The farmer was advised to apply strict biosecurity measures.

Immuno-pathogenesis study of local infectious bronchitis virus G1-1 lineage variant showed altered tissue tropism in experimental broiler chickens.

Infectious bronchitis (IB) is an acute contagious disease of poultry caused by infectious bronchitis virus (IBV). This study investigated the immunopathogenesis and tissue tropism of an Indian IBV field isolate (IBV/Chicken/India/IVRI/Rajasthan/01/2023) in experimental broiler chickens. This isolate belongs to the G1-1 lineage and is closely associated with the Mass genotype. 106.23 EID50/0.2 mL of the virus was administered intranasally and intraocularly to the IBV-challenge group, whereas uninoculated allantoic fluid was administered to the control group. Clinical signs, gross and histopathological lesions, immunohistochemistry (IHC), viral load, humoral responses, and the relative expression of immune response genes were evaluated at seven observation points. The infected group showed a significant reduction in weight gain from 3 dpi onwards, with clinical signs of varying severity from 3 to - 11 dpi. Gross lesions and microscopic changes were observed in the nasal turbinates, trachea, lungs, and kidneys, mainly representing epithelial degeneration and necrosis with mononuclear infiltrates. The caecal tonsils also showed microscopic lesions at 7-9 dpi. Absolute viral load estimation in the organs corroborated the lesion severity scores and IHC results. The expression of innate immune responses broadly demonstrated higher expression in the trachea and lungs of the IBV-infected group during the early phase of infection, whereas similar responses were observed in the kidneys and caecal tonsils during the later phases of infection. This study suggests that the given IBV isolate may cause significant production losses in broilers and exhibit tissue tropism for both respiratory and non-respiratory tissues, triggering varying innate and adaptive immune responses.

Assessment of Newcastle Disease Cases in Maiduguri Based on Gross Necropsy Findings from 2013 to 2023: A Retrospective Study

This study was conducted to evaluate gross pathologies of Newcastle disease in Maiduguri metropolis observed during necropsy. The study utilized poultry necropsy records from University of Maiduguri Veterinary Teaching Hospital (UMVTH) and Senator Ali Modu Sherriff Veterinary Hospital, Maiduguri (2013-2023). 1,175 (15.774%) out of the 7,449 carcasses examined were ND positive. The yearly trend of ND cases showed a slight decline (r= -0.177) but not significant (P>0.05). Cases based on chicken types differ significantly (P<0.05). It occurred in 73.957% broilers, 11.915% layers, 4.522% noilers, 0.937% local chickens and 0.426% pigeons. Turkeys and cocks had 2.043% and 1.957%, respectively (P>0.05), and 0.085% peacock, 0.085% duck and 0.085 brahma (P>0.05). the proportion of poultry types reared in Maiduguri vary significantly (P<0.05). There were 70.154% broilers, 23.834% layers, and the least were 0.004% duck, 0.003% brahma and 0.002% peacock (P>0.05). Cases of ND occurred in birds aged 1 to >20 weeks, but occurrence was more frequent in birds aged 3 to 4 weeks. 6.383% of 1,175 cases were complicated by diseases such as colibacillosis (41.333%), which varied significantly (P<0.05) form salmonellosis (18.667%), coccidiosis (14.667%), IBD (13.333%), and necrotic enteritis (10.667%) that had comparable values (P>0.05), and IBD + colibacillosis had 1.333% (P<0.05). Digestive organs (intestine, proventriculus, liver) had 1,519, lymphoid organs (spleen, caecal tonsils) had 943, respiratory organs (lung, trachea) had 333 and urinary organ (kidney) had 38 occurrences of gross lesion (P<0.05). This report, therefore, confirms the endemicity of ND in poultry in Maiduguri metropolis.

Effect of Selenium-Yeast supplementation on gut microbiome, intestinal histomorphology and immunity in broilers

Selenium-yeast was mixed with the basal diet of treatment groups of birds at the dose rate of 0.15 mg/kg diet in treatment-I (T-I), 0.225 mg/kg in treatment-II (T-II) and 0.30 mg/kg in treatment-III (T-III) group. The body weight and weekly weight gain increased and, feed conversion ratio improved significantly (p<0.05) in the T-III group as compared to control group. The number of goblet cells, lymphatic nodules in the intestine was more in treated birds. The villi height and crypts depth and their ratio were significantly highest in T-III group. Bursal morphology significantly improved and the number of Hassall’s corpuscle, macrophages and plasma cells were more frequent in treated birds. Among the lymphoglandular organs, the maximum IL-10 positive cells were found in the caecum and Bursa of Fabricius (BF). Among the groups, T-III group showed weak to nil IL-10 reaction. When compared between the days, BF and spleen showed better IL-10 positive reaction on 7th day but in caecal tonsil and thymus, the better reaction was on 35th day. The expression of IL-10 in the lymphoglandular organs was significantly (p<0.05) lower and T-III had minimum expression. T-II and T-III groups were better than T-I and control groups for enhancing humoral immunity against Newcastle disease virus, reduction of gut Escherichia coli, Salmonella and increased gut Bifidobacterium spp. In conclusion, supplementation of Se-yeast to the poultry diet at the dose rate 0.30 mg/kg improved the growth performance and guts microbial health, and enhanced the immunity of poultry birds.

Selection of suitable reference genes for normalization of RT-qPCR in three tissues of Northern bobwhite (Colinus virginianus) infected with eyeworm (Oxyspirura petrowi).

The Northern bobwhite (Colinus virginianus) is an economically important, and popular game bird in North America. Northern bobwhites have experiencing declines of > 3.5% annually in recent decades due to several factors. The eyeworm Oxyspirura petrowi is a nematode parasite frequently found in the eyes of bobwhites. Although reported frequently in wild bobwhites, there is no research to understand the host-parasite mechanism. Hence, it is important to investigate mechanisms of eyeworm invasion and immune modulation in bobwhite. Cytokine gene expression using RT-PCR is widely used to identify the innate immune response of a host to an infection. In this study, we evaluated ten reference genes (HMBS, RPL19, RPL32, RPS7, RPS8, TATA, SDHA, YWHAZ, GAPDH, and ACTB) for their stability across three tissues (liver, spleen, and caecal tonsils) of control and O. petrowi infected Northern bobwhites. Primer efficiency and reference genes stability were assessed using GeNorm, NormFinder, and BestKeeper. Expression of these reference genes with respect to O. petrowi infection in bobwhites showed RPL32 and HMBS were the most stable genes in the liver, HMBS and SDHA were the most stable genes in the spleen, and HMBS and YWHAZ were equally stable reference genes in the caecal tonsils. Based on the geometric mean of all three analyses, our results indicate that the combination of RPL32 and HMBS for the liver, HMBS and SDHA for the spleen, and YWHAZ and HMBS for caecal tonsils might be used as reference genes for normalization in gene expression investigations on Northern bobwhites.

Effect of enrofloxacin on clinical parameters and mucociliary system of broilers challenged with H9N2 avian influenza/infectious bronchitis viruses.

Effect of antibacterials on mucociliary system and clinical outcome of chickens with mixed viral respiratory conditions is not properly addressed. We evaluated enrofloxacin effects on clinical parameters and mucociliary system of broilers challenged with H9N2/IB viruses. Broilers (105), at the age of 25 days, were randomly allocated into three groups: Group 1 (negative control), no treatment; Group 2 (positive control [PC]) challenged by intranasal and intraocular route. Group 3 (antibiotic [AB]-treated) challenged and also received enrofloxacin started after manifestation of clinical signs (day 2 post-challenge [pc]) and continued for 5 days. Administration of AB was not associated with appreciable changes in body weight, feed conversion ratio (FCR) or the severity of clinical signs although it slightly reduced mortality rate as compared to PC group (p>0.05). Virus shedding period and number of virus positive tracheal and caecal tonsil samples were also statistically similar between PC and AB groups. In necropsy, the most profound effect of AB was decreased pleuropneumonia severity score on day 12 pc. Histopathological lesion scores were statistically the same between PC and AB groups. However, the administration of AB increased the number of tracheal goblet cells, with no effect on ciliostasis. We found a weak positive effect of enrofloxacin administration in H9N2/IB-infected chickens. Considering the risks of AB treatment in broiler chickens, the results of this small-scale study do not encourage the benefit of enrofloxacin use in these viral diseases.

Distribution and spatiotemporal development of organised lymphoid tissues in the chicken intestinal tract

Chickens exhibit a distinct immune architecture characterised by the absence of draining lymph nodes and the presence of a well-developed mucosa-associated lymphoid tissue. The structure and spatiotemporal development of chicken lymphoid tissues in the intestine are poorly documented. The macroscopically indistinct structure of chicken Peyer's patches has impeded studies into their development. The generation of CSF1R-eGFP reporter transgenic chickens enables visualisation of the development, organisation and extent of chicken lymphoid tissues by unique macroscopic views. CSF1R-eGFP reporter transgenic chickens were used to investigate the distribution and spatiotemporal development of PP and caecal tonsils in embryonic day 18 to 8-week-old chickens. Peyer's patch anlagen are present at ED18 with a similar frequency and distribution pattern observed in 2- and 8-week-old chickens. These findings can support in ovo and post-hatch mucosal vaccination strategies and the development of vaccine delivery systems targeted to the specialized epithelium overlying the Peyer's patches.

4G mobile phone radiation alters some immunogenic and vascular gene expressions, and gross and microscopic and biochemical parameters in the chick embryo model.

The risks to human health have grown over the past 10 years due to the excessive use of mobile phones. The study was designed to determine the harmful effects of 4G mobile phone radiation on the expression of immunogenic and vascular genes and gross, microscopic and biochemical alterations in the development of chicken embryos. Sixty individuals in the exposure group were subjected to mobile phones with a specific absorption rate of 1.4W/kg and a frequency of 2100MHz positioned at a distance of 12cm in the incubator for 60min/night for 14 days. The histopathological examination involved hematoxylin and eosin staining, whereas cresyl violet staining was used to evaluate the condition and number of neurons in the brain. The biochemical parameters of amniotic fluid were analysed using the photometry method, and the expression of VEGF-A and immunity genes (AvBD9, IL6) was measured using the real-time PCR (qPCR) technique. Compared to the control, the exposure group's body weight and length significantly decreased (p<0.05). Subcutaneous bleeding was seen in the exposure group. Urea, creatinine, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase levels were all significantly higher than in the control group (p<0.05). The exposed group showed pathological lesions in the liver and degenerated neurons with lightly stained nuclei in the cerebral cortex. Hyperchromatic neurons were significantly higher in the exposure group (58.8±2.28) compared to the control (6.6±0.44) (p<0.05). 4G exposure reduced lymphocyte count in the caecal tonsil (86.8±5.38) compared to the control (147.2±9.06) (p<0.05). Vascular gene mRNA expression was higher, but immune gene expression was lower in the exposed group. Exposure to mobile phone radiation may result in gross, microscopic and biochemical changes, as well as alterations in gene expression that could hinder embryonic development.

Colonization of the Gastrointestinal Tract of Chicks with Different Bacterial Microbiota Profiles.

This study aimed to investigate the consequences of early-life microbiota transplantation using different caecal content sources in broiler chicks. We hypothesized that chicks receiving at-hatch microbiota from organic hens would harbour a distinct microbiota from chicks receiving industry-raised broiler microbiota after six weeks of age. Three hundred Cobb broilers eggs were randomly assigned to one of four groups according to the caecal content received: organic laying hens (Organic); autoclaved caecal content of organic laying hens (Autoclaved); conventionally grown broilers (Conventional); and sterile saline (Control). caecal microbiota transplantation was given by gavage on day 1. Ten birds/group were euthanized on days 2, 7, 14, 28, and 42. The caecal tonsils and contents were collected for cytokines and microbiota analyses. The microbiota from chicks receiving live inocula resembled the donors' microbiota from day seven until day 42. The microbiota composition from the chickens who received the Organic inoculum remained markedly different. Starting on day 7, the Organic group had higher richness. Simpson and Shannon's indices were higher in the Conventional group on days 2 and 7. Chickens in the Conventional group presented higher production of IL-1β and IL-6 in plasma on days 2 and 28, increased IL-6 expression in the caecal tonsils at days 7 and 42, and increased IL-12 expression on day 7. However, the Conventional group was infected with Eimeria spp., which likely caused inflammation. In conclusion, microbiota transplantation using different microbiota profiles persistently colonized newly hatched broiler chicks. Future studies evaluating the importance of microbiota composition during infections with common enteropathogens are necessary. This study also highlights the need for a strict screening protocol for pathogens in the donors' intestinal content.

Studies on immunopathological changes induced by commercial IBD live vaccines in poultry birds

Intermediate plus live strain infectious bursal disease virus (IBDV) vaccines are used to control IBDV endemic infections in India. In the present study, immunopathological changes induced by commercial infectious bursal disease live vaccines with different immunization regimes were compared. A total of days old 108 Cobb broiler chicks were randomly divided into five groups with 24 chicks each in groups I, II, III and 18 chicks each in group IV and V. Group I served as control I (no immunization) and group II and III chicks were immunized with a single dose of vaccines 1 and 2 on 17th day of age (DOA), respectively. The group IV and V chicks were immunized with vaccines 1 and 2, respectively with primary dose on 17th DOA followed by booster dose on 24th DOA. Both intermediate plus live vaccines produced gross and histopathological lesions in lymphoid organs (bursa of Fabricius, thymus, spleen and caecal tonsils). Increased CD4 + , CD8 + T cells in affected bursa of Fabricius was evidenced by immunohistochemistry. Further, up-regulation in relative mRNA expression of IFN-γ, IL-1β and IL-6 were observed in bursa of Fabricius of treated birds, with maximum alteration particularly on 14th day post single immunization and 7th day post booster immunization. The findings suggest that single immunization regime on the 17th day of age showed immunization equivalent to booster immunization with lesser lesions, therefore, may be practiced and promoted in the field conditions for the better economic returns and animal welfare.

Effect of chicken infectious bronchitis vaccine on morphogenesis and differentiation of cells in caecal tonsils

The study of the chickens’ immune system morphofunctional state allows assessing critical periods of their development and the body as a whole, as well as the effectiveness of vaccine prevention methods. The purpose of this study was to identify morphological and immunohistochemical changes in the caecal tonsils of chickens aged 8, 20, 40, 90, 110 days for vaccine prevention of infectious bronchitis. During the study, the following research methods were used: cytological, histological, immunohistochemical, morphometric, light-optical, statistical. Histological preparations of caecal tonsils of poultry aged 8, 20, 40, 90, 110 days of vaccinated and unvaccinated groups were analysed and studied. Up to 20 days of age, no lymphoid nodules were detected in the caecal tonsils of chickens, both vaccinated and non-vaccinated groups. Histological and morphometric studies were conducted, which showed that vaccination of poultry accelerates the development of lymphoid formations in the early stages of the postnatal period of ontogenesis, especially in chickens aged 20 days, which is manifested by an increase in the number and size of lymphoid nodules of caecal tonsils relative to the control group. Immunohistochemical studies have established that the placement and accumulation of T-lymphocytes with markers CD4+ , CD8+ , as well as B-lymphocytes (CD20+ ) and CD45RA+ cells in caecal tonsils, namely in lymphoid nodules and diffuse accumulation of lymphoid cells, depend on the age of chickens and multiplicity of vaccinations. It was proved that in chickens aged 8 days, the number of T-lymphocyte subpopulations with surface markers CD4+ , CD8+ prevailed over CD45RA+ and CD20+ . With the increase in the number of immunizations (after three-fold vaccination against infectious bronchitis of chickens), immunocytometric studies indicated that in vaccinated chickens aged 40 and 90 days, there was a clear increase in the number of mature B-lymphocytes by 1.58 and 1.37 times, respectively. Considering the fact that the number of CD8+ lymphocytes in vaccinated chickens aged 40 days was 1.49 times greater than the number of CD4+ cells, this led to a sharp decrease in the immunoregulatory index of the caecal tonsils, which must be factored in when carrying out preventive vaccinations

Mucosal and systemic lymphoid immune responses against Clostridium perfringens strains with variable virulence in the production of necrotic enteritis in broiler chickens

ABSTRACT Necrotic enteritis (NE), caused by Clostridium perfringens, is an economically important disease of chickens. Although NE pathogenesis is moderately well studied, the host immune responses against C. perfringens are poorly understood. The present study used an experimental NE model to characterize lymphoid immune responses in the caecal tonsils (CT), bursa of Fabricius, Harderian gland (HG) and spleen tissues of broiler chickens infected with four netB+ C. perfringens strains (CP1, CP5, CP18, and CP26), of which CP18 and CP26 strains also carried the tpeL gene. The gross and histopathological lesions in chickens revealed CP5 to be avirulent, while CP1, CP18, and CP26 strains were virulent with CP26 being “very virulent”. Gene expression analysis showed that, while the virulent strains induced a significantly upregulated expression of pro-inflammatory IL-1β gene in CT, the CP26-infected birds had significantly higher CT transcription of IFNγ and IL-6 pro-inflammatory genes compared to CP5-infected or uninfected chickens. Furthermore, CP26 infection also led to significantly increased bursal and HG expression of the anti-inflammatory/regulatory genes, IL-10 or TGFβ, compared to control, CP5 and CP1 groups. Additionally, the splenic pro- and anti-inflammatory transcriptional changes were observed only in the CP26-infected chickens. An antibody-mediated response, as characterized by increased IL-4 and/or IL-13 transcription and elevated IgM levels in birds infected with virulent strains, particularly in the CP26-infected group compared to uninfected controls, was also evident. Collectively, our findings suggest that lymphoid immune responses during NE in chickens are spatially regulated such that the inflammatory responses against C. perfringens depend on the virulence of the strain.

Salmonella enterica serovars in absence of ttrA and pduA genes enhance the cell immune response during chick infections

Salmonella spp. is one of the major foodborne pathogens responsible for causing economic losses to the poultry industry and bringing consequences for public health as well. Both the pathogen survival ability in the intestinal environment during inflammation as well as their relationship with the host immune system, play a key role during infections in poultry. The objective of this study was to quantify the presence of the macrophages and CD4+/CD8+ cells populations using the immunohistochemistry technique, in commercial lineages of chickens experimentally infected by wild-type and mutant strains of Salmonella Enteritidis and Salmonella Typhimurium lacking ttrA and pduA genes. Salmonella Enteritidis ∆ttrA∆pduA triggered a higher percentage of the stained area than the wild-type, with exception of light laying hens. Salmonella Typhimurium wild-type strain and Salmonella Typhimurium ∆ttrA∆pduA infections lead to a similar pattern in which, at 1 and 14 dpi, the caecal tonsils and ileum of birds showed a more expressive stained area compared to 3 and 7 dpi. In all lineages studied, prominent infiltration of macrophages in comparison with CD4+ and CD8+ cells was observed. Overall, animals infected by the mutant strain displayed a positively stained area higher than the wild-type. Deletions in both ttrA and pduA genes resulted in a more intense infiltration of macrophages and CD4+ and CD8+ cells in the host birds, suggesting no pathogen attenuation, even in different strains of Salmonella.

Using T cell lymphokines of hyperimmunized chickens with Salmonella pullorum to protect layer hens against Salmonella pullorum infection

Salmonella pullorum (SP) is well adapted to cause an acute systemic infection in hens with a high mortality rate. This study aimed to assess the efficacy and safety of a salmonella-immune lymphokine (SILK) produced by hyperimmunized pullets with Salmonella pullorum during the layer hens' production stage to boost their immune response against Salmonella pullorum infection. In this study, two groups of 25 pullets were used to produce lymphokines; the first group received three doses of the Salmonella pullorum vaccine at 12, 14, and 16 weeks, while the second group only received phosphate buffer saline (PBS), which served as the control. At 18 weeks, non-immune lymphokines (NILK) were isolated from the T cells of the second group, and salmonella-immune lymphokines (SILK) were isolated from the T cells of the first group. Then, 100-layer hens (ISSA Brown), at 30 weeks old, which entered the production stage, were separated into four groups, each with 25 chickens. G1: infected with SP and treated with SILK. G2: infected with SP and treated with NILK. G3: untreated and SP-challenged G4: no treatment or challenge. The current study aimed to describe the host humoral immune responses to infection in serum samples and bacterial shedding in hens challenged with SP during 31-33 weeks by qPCR techniques. Internal organ bacterial loads were estimated to evaluate the persistence of bacteria. The results show that the spleen, liver, and caecal tonsils tested were positive for bacteria in both groups (G2, G3), proving that Salmonella was not eliminated from the birds and suggesting that internal organ colonization bacteria may act as a reservoir for ongoing bacterial shedding with low IgG titer in compared to G1 with extremely low persistence and high IgG titer in weeks 31, 32, and 33. The current investigation shows that using SILK provides layer hens with better homogenous protection against SP and less internal organ persistence.

Molecular Detection of Newcastle Disease Virus from Field Outbreaks in Assam, India

The present study was carried out to detect the circulating Newcastle disease virus (NDV) from various field outbreaks in Assam during 2018-2021. Nine hundred ninety-two clinical and post mortem samples were collected from the backyard and commercial poultry and subjected to molecular detection by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). NDV could be detected in 445 (n=757, 58.78%) tissue samples and 53 (n=235, 22.55%) cloacal swabs collected from suspected poultry cases by RT-PCR. The findings of our study suggests that the sample of choice for detection of NDV from tissue samples was trachea (n=85, 92.94%), followed by spleen (n=96, 90.62%), lung (n=92, 86.95%), caecal tonsils (n=96, 77.08%), proventriculus (n=74, 60.81%), brain (n=92, 48.91%), intestines (n=73, 31.50%), kidney (n=52, 23.07%), and cloacal swab (n=235, 22.55%) by RT-PCR. Hence, the collection of specific tissue samples from field outbreaks for the detection of NDV is of paramount importance. In addition, RT-PCR can be considered a highly sensitive detection method for rapid and confirmative diagnosis of Newcastle disease virus.

Gizzard erosions in broiler chickens in Sweden caused by fowl adenovirus serotype 1 (FAdV-1): investigation of outbreaks, including whole-genome sequencing of an isolate

ABSTRACT The present paper describes the investigation of the first outbreaks of adenoviral gizzard erosions (AGE) in Sweden, in five broiler flocks. The investigation included whole viral genome sequencing and investigation of genomic organization and sequence relationships with other adenoviruses. All five flocks had a history of decreased growth and uneven size of birds since 9-10 days of age. Macroscopically, lesions consistent with AGE (detached koilin layers, discolouration, bleeding, erosions) were identified in gizzards in all five flocks. In four flocks histology was performed, and degeneration and inflammation of the koilin layer and gizzard mucosa were identified in all four. In one flock, intranuclear inclusion bodies typical for fowl adenovirus (FAdV) were detected in trapped epithelial cells in the koilin layer. In four flocks in situ hybridization was performed, and cells positive for FAdV serotype 1 (FAdV-1) were demonstrated in the koilin layer and gizzard mucosa. FAdV species A (FAdV-A) was detected in gizzard, liver, caecal tonsils and bursa of Fabricius by polymerase chain reaction (PCR) and sequencing. Ten out of ten examined parent flocks of the affected chickens were seropositive for FAdV, indicating former or on-going infection. However, FAdV was not detected in embryos from seropositive parent flocks and thus vertical transmission was not demonstrated. The entire nucleotide sequence of one sample was determined and found to be 43,856 base pairs (bp) in length. The genome sequence and organization were found to be similar to that of the reference apathogenic avian adenovirus “chicken embryo lethal orphan” (CELO). RESEARCH HIGHLIGHTS AGE in Swedish broilers: necropsy, histopathology, ISH, PCR, whole-genome sequencing. Whole FAdV-genome analysed: 43,856 bp, found to be most similar to CELO (U46933.1). Multiple point mutations, site insertions and deletions identified compared to CELO. Paper adds knowledge about European disease situation and pathogenic FAdV-strains.

Detection of Newcastle disease virus and assessment of associated relative risk in backyard and commercial poultry in Kerala, India.

BackgroundNewcastle disease (ND) is an economically important viral disease affecting the poultry industry. In Kerala, a state in South India, incidences of ND in commercial and backyard poultry have been reported. But a systematic statewide study on the prevalence of the disease has not been carried out.ObjectivesA cross‐sectional survey was performed to detect the presence of Newcastle disease virus (NDV) in suspect cases and among apparently healthy commercial flocks and backyard poultry, in the state and to identify risk factors for NDV infection.MethodsReal‐time reverse transcription‐PCR (RT‐PCR) was used to detect the M gene of NDV in choanal swabs and tissue samples collected from live and dead birds, respectively and the results were statistically analysed.ResultsThe predominant clinical signs of the examined birds included mild respiratory signs, huddling together and greenish diarrhoea. Nervous signs in the form of torticollis were noticed in birds in some of the affected flocks. On necropsy, many birds had haemorrhages in the proventriculus and caecal tonsils which were suggestive of ND. Of the 2079 samples tested, 167 (8.0%) were positive for the NDV M‐gene by RT‐PCR. Among 893 samples collected from diseased flocks, 129 (14.5%), were positive for M gene with pairwise relative risk (RR) of 15.6 as compared to apparently healthy flocks where 6 out of 650 (0.9%) samples were positive. All positive samples were from poultry; none of the ducks, pigeons, turkey and wild birds were positive. Commercial broilers were at higher risk of infection than commercial layers (RR: 4.5) and backyard poultry (RR: 4.9). Similarly, birds reared under intensive housing conditions were at a higher risk of being infected as compared to those reared under semi‐intensive (RR: 6.7) or backyard housing (RR: 2.1). Multivariable analysis indicated that significantly higher risk of infection exists during migratory season and during ND outbreaks occurring nearby. Further, lower risk was observed with flock vaccination and backyard or semi‐intensive housing when compared to intensive housing. When the M gene positive samples were tested by RT‐PCR to determine whether the detected NDV were mesogenic/velogenic, 7 (4.2%) were positive.ConclusionsIn Kerala, NDV is endemic in poultry with birds reared commercially under intensive rearing systems being affected the most. The outcome of this study also provides a link between epidemiologic knowledge and the development of successful disease control measures. Statistical analysis suggests that wild bird migration season and presence of migratory birds influences the prevalence of the virus in the State. Further studies are needed to genotype and sub‐genotype the detected viruses and to generate baseline data on the prevalence of NDV strains, design better detection strategies, and determine patterns of NDV transmission across domestic poultry and wild bird populations in Kerala.

Pathological Diagnosis and Genomic Characterization of ICP4 Gene of lnfectious Laryngotracheitis Virus (ILTV) Isolates in Clinically Infected Chicken in Tamil Nadu, India

Background: Infectious laryngotracheitis (ILT) is an economically important viral respiratory disease in poultry. Recently, re-emergence of Infectious laryngotracheitis virus (ILTV) has been reported in several countries including India. The current study aimed to evaluate the poultry flocks of Tamil Nadu with circulating GaHV-1 and to elucidate the origin of the virus involved in the outbreak. Methods: In this study, a molecular based survey on the overall occurrence of natural cases of Infectious laryngo-tracheitis in poultry flocks from Tamil Nadu, India were performed. Pathological findings in respiratory and secondary lymphoid organs like caecal tonsils and harderian gland was carried out. The PCR technique targeting Infected Cell Protein-4 (ICP4) gene along with molecular characterization was performed. Result: The overall prevalence rate in the outbreak was 42.86% with highest incidence in layer flocks (62.85%) than the broiler flocks (22.85%). The highest susceptible age groups were between 20-30 weeks old. Tracheal pathology revealed epithelial sloughing, syncytial cell formation, eosinophilic intranuclear inclusion bodies and heterophilic exudation microscopically. Partial genome sequencing and phylogenetic analysis of ICP4 gene revealed high genetic homology between field isolates and the virulent strains from Turkey, Germany, China and Brazil. In the present study, along with pathological findings, a rapid and sensitive PCR assay was used for detection of ILT virus specific ICP4 gene in commercial poultry farms in the region.

Age-dependent changes of gut-associated lymphoid tissue in one to four-month-old turkeys: A histological study

Gut-associated lymphoid tissue (GALT) has a pivotal role in the health of birds as the first line of defense against foodborne pathogens. On the other hand, this part of immune system is important for successful development of vaccines. Due to scarcity of knowledge on GALT of turkey as a major industrial species, this study aimed to evaluate histological features of GALT in this species. A total of 40 clinically healthy BUT6 turkeys from both sexes at the age of 1, 2, 3, and 4 months were included in the study. Samples were immediately removed after slaughter from different parts of the gut, including pharyngeal tonsil, cervical oesophagus, oesophageal tonsil, proventriculus, pyloric tonsil, ileum, Meckel’s diverticulum, caecal tonsil, the middle and apex of caecum, rectum, and cloaca. After fixation and routine procedures followed by hematoxylin-eosin staining, samples were studied histologically under light microscope. In addition, photomicrographs were taken from sections and were analysed for histomorphometric parameters using Zeiss Axio vision rel.4.8 software. According to the results, in one-month-old turkeys, lymphatic tissues in pharyngeal tonsil, pyloric tonsil, proventriculus, caecal tonsil, middle, and apex of caecum, Meckel’s diverticulum, and cloaca were completely developed, while in some other parts such as the cervical oesophagus and the rectum lymphatic structures were absent. In the ileum, the Peyer’s patch was observed as an aggregated structure, although the diffuse lymphatic tissue has properly evolved. In two months old turkeys, the dense lymphatic tissue including primary and secondary lymphatic follicles, was present in cervical oesophagus, Peyer’s patch, rectum, and cloaca. Many histomorphometric parameters of lymphoid tissue also developed age-dependently. In four months old birds the dense lymphatic tissue was obvious in all examined specimens. Moreover, no signs of tissue atrophy were seen at the age of four months (prepuberty) in the GALT specimens. In conclusion, the GALT of turkeys was histologically quite similar to those of other birds and these structures showed an age-dependent development from 1 to 4 months of age.