Full genome sequence-based comparative study of wild-type and vaccine strains of infectious laryngotracheitis virus from Italy

Infectious laryngotracheitis (ILT) is an acute and highly contagious respiratory disease of chickens caused by an alphaherpesvirus, infectious laryngotracheitis virus (ILTV). Recently, full genome sequences of wild-type and vaccine strains have been determined worldwide, but none was from Europe. The aim of this study was to determine and analyse the complete genome sequences of five ILTV strains. Sequences were also compared to reveal the similarity of strains across time and to discriminate between wild-type and vaccine strains. Genomes of three ILTV field isolates from outbreaks occurred in Italy in 1980,2007 and 2011, and two commercial chicken embryo origin (CEO) vaccines were sequenced using the 454 Life Sciences technology. The comparison with the Serva genome showed that 35 open reading frames (ORFs) differed across the five genomes. Overall, 54 single nucleotide polymorphisms (SNPs) and 27 amino acid differences in 19 ORFs and two insertions in the UL52 and ORFC genes were identified. Similarity among the field strains and between the field and the vaccine strains ranged from 99.96% to 99.99%. Phylogenetic analysis revealed a close relationship among them, as well. This study generated data on genomic variation among Italian ILTV strains revealing that, even though the genetic variability of the genome is well conserved across time and between wild-type and vaccine strains, some mutations may help in differentiating among them and maybe involved in ILTV virulence/attenuation. The results of this study can contribute to the understanding of the molecular bases of ILTV pathogenicity and provide genetic markers to differentiate between wild-type and vaccine strains

Pathogenic and transmission potential of wildtype and chicken embryo origin (CEO) vaccine revertant infectious laryngotracheitis virus

Infectious laryngotracheitis (ILT) is an infectious upper respiratory tract disease that impacts the poultry industry worldwide. ILT is caused by an alphaherpesvirus commonly referred to as infectious laryngotracheitis virus (ILTV). Vaccination with live attenuated vaccines is practiced regularly for the control of ILT. However, extensive and improper use of live attenuated vaccines is related to vaccine viruses reverting to virulence. An increase in mortality and pathogenicity has been attributed to these vaccine revertant viruses. Recent studies characterized Canadian ILTV strains originating from ILT outbreaks as related to live attenuated vaccine virus revertants. However, information is scarce on the pathogenicity and transmission potential of these Canadian isolates. Hence, in this study, the pathogenicity and transmission potential of two wildtype ILTVs and a chicken embryo origin (CEO) vaccine revertant ILTV of Canadian origin were evaluated. To this end, 3-week-old specific pathogen-free chickens were experimentally infected with each of the ILTV isolates and compared to uninfected controls. Additionally, naïve chickens were exposed to the experimentally infected chickens to mimic naturally occurring infection. Pathogenicity of each of these ILTV isolates was evaluated by the severity of clinical signs, weight loss, mortality, and lesions observed at the necropsy. The transmission potential was evaluated by quantification of ILTV genome loads in oropharyngeal and cloacal swabs and tissue samples of the experimentally infected and contact-exposed chickens, as well as in the capacity to produce ILT in contact-exposed chickens. We observed that the CEO vaccine revertant ILTV isolate induced severe disease in comparison to the two wildtype ILTV isolates used in this study. According to ILTV genome load data, CEO vaccine revertant ILTV isolate was successfully transmitted to naïve contact-exposed chickens in comparison to the tested wildtype ILTV isolates. Overall, the Canadian origin CEO vaccine revertant ILTV isolate possesses higher virulence, and dissemination potential, when compared to the wildtype ILTV isolates used in this study. These findings have serious implications in ILT control in chickens

A minor study on avian metapneumovirus

Avian metapneumovirus (aMPV) is an important pathogen that causes respiratory diseases and reproductive failure in various avian species. The disease was first reported in South Africa in the late 1970s. Later, the disease has been reported from all countries around the world, except for Australia, and has caused economic losses in poultry industry worldwide due to the transient drop in eggs and meat production. In 1998 there was an outbreak in Sweden but due to good biosecurity and effective vaccine control program the disease has been under control since then. In South America the industry of egg-laying hens struggles with many respiratory diseases, such as aMPV, Infectious bronchitis, Laryngotracheitis and Newcastle disease. This study is a field project on the Avian metapnuemovirus and took place in the state of São Paulo, Brazil. The study contributed to a bigger research project at the University of Sao Paulo under the department of Preventive Veterinary and Animal Health where the veterinarians are trying to control the problems with respiratory diseases in Brazilian poultry production. The scope of this study was to detect aMPV in egg-laying hens with respiratory disease and reproductive failure and further sequence the viral RNA and compare it to previously found viral strains. This information is important to obtain accurate vaccination-programs for control of the virus. Organ-samples and blood-samples were collected from five different farms in Bastos, a town in the state of São Paulo with extensive egg-production. Reverse transcriptase PCR was performed to detect RNA of aMPV in the organs of the sampled birds. One out of 66 pools of samples was positive. The sequencing of the RNA was unsuccessful and no phylogenetic tree comparing this strain to other known viral strains of aMPV could be designed. The blood-samples were tested with two types of ELISAs, one indirect ELISA and one blocking ELISA, for antibodies against aMPV. Antibodies could be detected in 85 % of the birds with the SVANOVIR-kit; and in 87 % of the birds using the IDEXX-kit. A majority of the birds had antibody-titers corresponding to previous infection. At the farm where the PCR-positive sample was collected, the birds had not seroconverted in spite of reported vaccination against aMPV one month earlier. The result of this study is consistent with previous conclusions from other studies; that RNA of aMPV is difficult to detect and that vaccine prevention of the disease is sometimes unsuccessful

Avian influenza and co-infections : investigation of the interactions in the poultry models

The purpose of this study was to assess the burden of co-infections in the field and to better understand the possible synergism between pathogens in a laboratory setting. We focussed on E. coli (O78) and low pathogenic avian influenza virus (LPAIV, H6N1) in turkey model and infected the birds via the aerosol route to reproduce respiratory disease. Viral shedding and lesions were more severe and persisted longer during coinfection indicating possible enhancement of pathogenesis for LPAIV by E. coli and vice versa. These findings all endorse our conclusions that E. coli and LPAIV exercise an additive pathogenic effect in the reproduction of respiratory disease if given simultaneously or spaced by three days between the viral and the bacterial challenges to susceptible turkeys. In parallel, we studied avian respiratory agents circulating in the field in Pakistani farms. There, we focussed on co-infections as well, targeting viruses only as a first study. We observed frequent LPAIV H9 (G1 lineage) and Newcastle disease virus (genotype VII) coinfections in the field

Studies on the immunopathogenesis, diagnosis and control of infectious bronchitis and avian metapneumoviruses in chicken

This thesis describes field and experimental investigations on various aspects of the immunopathogenesis, diagnosis and vaccination of infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) in the chicken. The immunopathogenesis of an economically important variant IBV (IS/885/00 like) seen in the Middle East and North Africa was examined in one day old specific pathogen free (SPF) and commercial broiler chicks (Chapter 3). The virus caused respiratory distress, depression and diarrhoea in both SPF and broiler chicks but the severity was milder in the latter. Mild head swelling was observed in one infected broiler chick at 15 days post infection (dpi) and virus with 100% nucleotide level similarity to the inoculum was detected by reverse transcription polymerase chain reaction (RT-PCR) and virus isolation (VI). In the IS/885/00-like infected SPF chicks, cystic oviducts were found in two female chicks. IBV with 99% part S1 sequence similarity to the initial inoculum was isolated from the cystic fluid. The protection provided by current commercial vaccines against variant IBV IS/885/00 like and IS/1494/00 like was investigated in day old commercial broiler chicks (Chapter 4). Protection was evaluated based on the clinical signs, gross lesions, tracheal ciliary scores and virus detection by RT PCR. It was found that administering combined live H120 and CR88 vaccines simultaneously at day old, followed by CR88 vaccine at 14 days old gave more than 80% ciliary protection against both of the Middle East isolates. Cellular and local immune responses in the trachea following vaccination of day old broiler chicks with different live IBV vaccines were evaluated (Chapter 5). In addition, protection conferred against virulent IBV was also examined. All vaccination programmes were able to induce measurable levels of CD4+, CD8+ and IgA bearing B cells in the trachea following vaccination when compared to unvaccinated birds. Expression levels of CD4+ and CD8+ cells varied between the vaccinated groups. Vaccines containing Mass2 combined with 793B2 produced good protection against challenge with virulent IBV QX compared to vaccines containing Mass (Mass1 or Mass2) alone or Mass1 with D274 or CR88. All vaccination programmes produced more than 80% protection against homologous (M41 and 793B) challenge. In Chapter 6, IBVs with high nucleotide level similarity to IS/885/00 like and IS/1494/06 like strains were detected by RT PCR in a broiler flock exhibiting high mortality and respiratory distress in Libya. For the first time, these findings have highlighted the circulation of variant IBVs in the Eastern part of Libya. Humoral and cellular immune responses and protection studies in SPF chicks that received live Newcastle disease virus (NDV), aMPV and IBV vaccines in single, dual or triple combinations were examined (Chapter 7). Protection against virulent IBV or aMPV was not affected when the vaccines were given either singly or in combination. There were no significant differences in the mean antibody titres of the NDV-vaccinated groups and they remained above the protective titre. The mean titres of antibodies against aMPV were suppressed when aMPV vaccine was given with other live vaccines but the aMPV-vaccinated groups were fully protected when challenged with virulent aMPV. The mean titres of antibodies were similar in the IBV-vaccinated groups and all IBV-vaccinated groups gave almost 100% protection against M41 challenge. Between the vaccinated groups, there were no significant differences in the mean numbers of CD4+, CD8+ and IgA-bearing B cells, reflecting similar levels of tracheal cellular and IgA responses irrespective of single, dual or triple vaccine applications. Despite the aMPV humoral antibody suppression, the efficacy of the live vaccines was not compromised when they were given simultaneously to young SPF chicks. Comparative studies in day old SPF chicks using both aMPV subtype A or B, separately or in combination, were evaluated (Chapter 8). There were significant differences in the degree of the clinical signs induced by the single subtypes A, B or A+B given together, with most severe signs observed in the latter two groups. By RT-PCR or VI, subtype B virus persisted longer than subtype A. Even though similar titres of the viruses were used, birds given subtype B alone or in combination showed a greater increase in antibody titres than those given A. These findings demonstrate that for the two strains used, subtype B was more pathogenic than subtype A and was excreted and persisted in the tissues for longer. The use of Flinders Technology Associates (FTA) cards for detection of several avian pathogens has been previously reported. To date, no information has been published on the use of FTA cards for detection of aMPV. In Chapter 9, the feasibility of using FTA cards for the molecular detection of aMPV subtype A and B by RT-PCR was investigated. Findings showed that FTA cards are suitable for collecting and transporting aMPV-positive samples, providing a reliable and hazard-free source of RNA for molecular characterization

Molecular epidemiology of infectious bronchitis coronavirus in southern African poultry from 2011 to 2020

Dissertation (MSc (Production Animal Studies))--University of Pretoria, 2023.Infectious bronchitis virus (IBV) (family Gammacoronavirus) is an ever-evolving avian pathogen that causes major economic losses within the poultry industry worldwide. Antigenic variations allow the virus to evade vaccine induced immunity and produce new variants at an alarming rate. Epidemiological studies of IBV are imperative to selection of vaccines. Field isolates (n=385) grown in SPF embryonated chicken eggs at Deltamune (Pty) Laboratory, that originated in commercial flocks were collected from Botswana, Eswatini, Namibia and South Africa’s Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, North West, and Western Cape provinces from 2011-2020, were analysed in this study. A 745 bp region of the spike protein gene was amplified and sequenced, and phylogenetic analysis was performed. Twenty-four (6,2 %) samples contained mixed sequences from viral coinfections and were not evaluated further. In the remaining 364 isolates, 7 genetic lineages were identified. 184 (50,5%) viruses were identified as GI-19 (QX) strains and 78 (21,4%) as the GI-1 (Mass/H120) strain. 39 (10,7%) samples were identified as the GI-13 (4/91) lineage detected between 2011 and 2019, 29 (8,0%) as the GVI-1 (TC07-2) lineage detected from 2010-2020, 19 (5,2%) as the GI-23 (Var II) lineage from 2019-2020 with single isolates in 2010 and 2015, and 13 (3,6%) as the GI-11 (UFMG/G - Brazil) lineage detected from 2011-2020. Two (0.5%) viruses were closely related to GIV-1 (DE/072/92) lineages but were only isolated in 2011 and 2013. Three viruses from GI-1 and GI-13 may have been recombinants, but further analysis is required to confirm this. Overall, this study reveals the co-circulation of diverse IBV field and vaccine-derived genotypes in southern African poultry flocks for the first time.This study was funded by the University of Pretoria under NRF-DSI SARChI no. N00705/114612Production Animal StudiesMSc (Production Animal Studies)UnrestrictedFaculty of Veterinary Scienc

An Assessment of the Risk Associated with the Movement of Pullets Out of the Pullet Barn In a Control Area during a Highly Pathogenic Avian Influenza Outbreak in the United States

Cardona, Carol; Bonney, Peter; Culhane, Marie; Goldsmith, Timothy; Halvorson, David; Malladi, Sasidhar; Ssematimba, Amos; Walz, Emily; Umber, Jamie. (2019). An Assessment of the Risk Associated with the Movement of Pullets Out of the Pullet Barn In a Control Area during a Highly Pathogenic Avian Influenza Outbreak in the United States. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/206602