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SUMMARY Physiological stress is known to promote economic losses by causing immunosuppression. Various factors induce stress, including non-optimal management and infection with various pathogens. The concentration of the stress hormone, corticosterone, is indicative of stress in birds. However, its measurement is difficult due to its elevation caused by catching, handling, bleeding, short half-life in the blood, and short secretion period in feces. We describe a novel method to assess corticosterone concentration by sampling feathers. The viruses studied were 2 immunosuppressive viruses, Marek's disease virus and chicken anemia virus, the respiratory viruses, influenza virus H9N2, Newcastle disease virus, and Infectious bronchitis virus. The study contributes novelty as virus-induced stress by feather examination was described sporadically in chickens previously, and feathers were used to detect corticosterone mainly in dead wild and captive birds. Also, the development of extraction method from feathers is novel by using PBS instead of methanol, and by preparing the feather homogenate with steroid displacement reagent. The study findings revealed that virus infections increased the corticosterone concentrations in feather tips to various extents, from 2× to 10× fold, demonstrating that birds endure stress in addition to the clinical and pathological effects induced by the virus infections.

During 2019, five carcasses of juvenile Egyptian fruit bats (Rousettus aegyptiacus) were submitted to the Kimron Veterinary Institute. These bats exhibited typical poxvirus like lesion plaques of different sizes on the skin, abdomen and the ventral side of the wings. Clinical and histopathological findings suggested a poxvirus infection. Infectious virus was isolated from skin swabs, skin tissue and tongue of the dead bats and was further confirmed to be a Poxvirus by molecular diagnosis using PCR with pan-chordopoxviruses primers. All the dead bats were found positive for two Poxvirus genes encoding a metalloproteinase and DNA dependent DNA polymerase. In this study, a novel real time quantitative PCR (qPCR) assay was established to further confirmed the presence of specific poxvirus viral DNA in all pathologically tested tissues. Moreover, according to sequence analysis, the virus was found to be highly similar to the recently discovered Israeli Rousettus aegyptiacus Pox Virus (IsrRAPXV).

Since 1997, G1-lineage H9N2 avian influenza viruses have been circulating in Asia and later on in the Middle East, and they have been associated to mild respiratory disease, drops in egg production and moderate mortality in chickens, in particular in the presence of concurrent infections. In this study, we investigated the importance of the G1-lineage H9N2 A/chicken/Israel/1163/2011 virus as a primary pathogen in layers, analyzing its tropism and binding affinity for the oviduct tissues, and investigating the long-term impact on egg production. Besides causing a mild respiratory infection, the virus replicated in the oviduct of 60% of the hens causing different degrees of salpingitis throughout the organ, in particular at the level of the infundibulum, where the detection of the virus was associated with severe heterophilic infiltrate, and necrosis of the epithelium. Binding affinity assays confirmed that the infundibulum was the most receptive region of the oviduct. The drop in egg production was at its peek at 2 weeks post-infection (pi) (60% decrease) and continued up to 80 days pi (35% decrease). On day 80 pi, non-laying birds showed egg yolk peritonitis, and histopathological analyses described profound alteration of the infundibulum architecture, duct ectasia and thinning of the epithelium, while the rest of the oviduct and ovary appeared normal. Our results show that this H9N2 virus is a primary pathogen in layer hens, and that its replication in the infundibulum is responsible for acute and chronic lesions that limits the effective functionality of the oviduct, compromising the commercial life of birds. Electronic supplementary material The online version of this article (10.1186/s13567-018-0575-1) contains supplementary material, which is available to authorized users.

Protection against diseases caused by the avian viruses, Marek's disease, Infectious laryngotracheitis, chicken anemia and turkey meningoencephalitis is achieved by live vaccines. The application quality is important to assure proper uptake in commercial flocks. We describe a novel evaluation method for the vaccination process by sequential monitoring the vaccine viruses in feathers. Feather collection is easy, non-invasive and non-lethal for the birds, therefore advantageous for monitoring purposes. To demonstrate the vaccine virus presence, an innovative assay of nested real-time amplification was approached because vaccine viruses presence in vivo is less abundant comparing to virulent wild-type isolates. The Marek's disease virus vaccine virus, Rispens/CVI988, in feathers of commercial flock was detected from 4 to 7 days and for at least 3 months post-vaccination, until the survey stopped. As the drinking water route was newly adopted for Infectious laryngotracheitis vaccination, one or two vaccine doses/bird were administered. The virus uptake was detected in feathers between 2 and 20 days-post-vaccination. With a doubled vaccine dose the positivity bird rate was higher. For the first time the chicken anemia vaccine virus presence in chicken feathers was demonstrated between 14 and 35 days-post-vaccination. No previous studies were available, thus in parallel to feathers the vaccine virus was demonstrated in the livers and spleens. The turkey meningoencephalitis vaccine virus uptake in turkey feather-pulps is even more innovative because this is the first turkey virus amplified from feather-pulps. The vaccine virus presence resemble the kinetics of the other 3 viruses, 3–21 days-post-vaccination. Detecting the specific antibodies following vaccination possessed a lower sensitivity than vaccine virus demonstration in feathers. In summary, the presented assay can be adopted for the quality evaluation of the vaccination process in poultry.

Vaccination is frequently used as a control method for the H9 subtype of low pathogenicity avian influenza virus (AIV), which is widespread in Asia and the Middle East. One of the most important factors for selecting an effective vaccine strain is the antigenic match between the hemagglutinin protein of the vaccine and the strain circulating in the field. To demonstrate the antigenic relationships among H9 AIVs, with a focus on Israeli H9 isolates, antigenic cartography was used to develop a map of H9 AIVs. Based on their antigenic diversity, three isolates from Israel were selected for vaccination-challenge studies: 1) the current vaccine virus, A/chicken/Israel/215/2007 H9N2 (Ck/215); 2) A/chicken/Israel/1163/2011 H9N2 (Ck/1163); and 3) A/ostrich/Israel/1436/2003 (Os/1436). A 50% infective dose (ID50) model was used to determine the effect of the vaccines on susceptibility to infection by using a standardized dose of vaccine. Sera collected immediately prior to challenge showed that Ck/215 was the most immunogenic, followed by Ck/1163 and Os/1436. A significant difference in ID50 was only observed with Ck/215 homologous challenge, where the ID50 was increased by 2 log 10 per bird. The ID50 for Ck/1163 was the same, regardless of vaccine, including sham vaccination. The ID50 for Os/1436 was above the maximum possible dose and therefore could not be established.

Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus, ILTV. The live vaccine is applied worldwide by drinking water or by the respiratory route, and by the vent application in Israel. No system of direct evaluation of the efficacy of vaccination exists today, except of antibody elicitation, which is an indirect indication of vaccination intake and might happen due to environment exposure. We suggest for the first time an assay for evaluating the accuracy of the vaccination process by spotting the spread of the live vaccine systemically, namely by virus detection in the feather shafts of the vaccinated birds. The feathers are particularly beneficial as they are easy to collect, non-lethal for the bird, therefore advantageous for monitoring purposes. Moreover, the continuous survey of the vaccine virus unveiled the different kinetics of viremia by the different vaccination routes; while after the vent vaccination the systemic viremia peaks during the first week afterwards, after two consecutive vaccine administration by drinking water with 6 day interval, the vireamia peaks only after the second administration. A robust amplification was needed because the vaccine ILTV was present in the bird in minute quantities compared to the wild-type virus. For the vaccine virus identification in feather shafts a nested real-time PCR for the TK ILTV gene was developed. The sensitivity of detection of the nested rtPCR was greater by 1000 compared to conventional nested PCR and 10 times that real-time PCR.

Thus far, no human MERS-CoV infections have been reported from Israel. Evidence for the circulation of MERS-CoV in dromedaries has been reported from almost all the countries of the Middle East, except Israel. Therefore, we aimed to analyze MERS-CoV infection in Israeli camelids, sampled between 2012 and 2017. A total of 411 camels, 102 alpacas and 19 llamas' sera were tested for the presence of antibodies to MERS-CoV. Our findings indicate a lower MERS-CoV seropositivity among Israeli dromedaries than in the surrounding countries, and for the first time naturally infected llamas were identified. In addition, nasal swabs of 661 camels, alpacas and lamas, obtained from January 2015 to December 2017, were tested for the presence of MERS-CoV RNA. All nasal swabs were negative, indicating no evidence for MERS-CoV active circulation in these camelids during that time period. Keywords: MERS coronavirus, Antibodies, Israel, Dromedary camels, Llamas, Alpacas

The emergence in humans of the A(H1N1)pdm09 influenza virus, a complex reassortant virus of swine origin, highlighted the importance of worldwide influenza virus surveillance in swine. To date, large-scale surveillance studies have been reported for southern China and North America, but such data have not yet been described for Europe. We report the first large-scale genomic characterization of 290 swine influenza viruses collected from 14 European countries between 2009 and 2013. A total of 23 distinct genotypes were identified, with the 7 most common comprising 82% of the incidence. Contrasting epidemiological dynamics were observed for two of these genotypes, H1 hu N2 and H3N2, with the former showing multiple long-lived geographically isolated lineages, while the latter had short-lived geographically diffuse lineages. At least 32 human-swine transmission events have resulted in A(H1N1)pdm09 becoming established at a mean frequency of 8% across European countries. Notably, swine in the United Kingdom have largely had a replacement of the endemic Eurasian avian virus-like (“avian-like”) genotypes with A(H1N1)pdm09-derived genotypes. The high number of reassortant genotypes observed in European swine, combined with the identification of a genotype similar to the A(H3N2)v genotype in North America, underlines the importance of continued swine surveillance in Europe for the purposes of maintaining public health. This report further reveals that the emergences and drivers of virus evolution in swine differ at the global level. IMPORTANCE The influenza A(H1N1)pdm09 virus contains a reassortant genome with segments derived from separate virus lineages that evolved in different regions of the world. In particular, its neuraminidase and matrix segments were derived from the Eurasian avian virus-like (“avian-like”) lineage that emerged in European swine in the 1970s. However, while large-scale genomic characterization of swine has been reported for southern China and North America, no equivalent study has yet been reported for Europe. Surveillance of swine herds across Europe between 2009 and 2013 revealed that the A(H1N1)pdm09 virus is established in European swine, increasing the number of circulating lineages in the region and increasing the possibility of the emergence of a genotype with human pandemic potential. It also has implications for veterinary health, making prevention through vaccination more challenging. The identification of a genotype similar to the A(H3N2)v genotype, causing zoonoses at North American agricultural fairs, underlines the importance of continued genomic characterization in European swine.

Field observations indicate that the impact of velogenic Newcastle disease virus (vNDV) is more severe in countries with concomitant circulation of low pathogenicity avian influenza virus, as is the case in the Middle East, in particular in Israel, where H9N2 and NDV are endemic. In our study, we evaluated how the exposure of chickens to an H9N2 challenge either favours or interferes with a subsequent vNDV infection and its transmission to sentinels. For this purpose, single vNDV and sequential H9/NDV challenges were performed with increasing doses of vNDV (101-106 EID50). The H9N2 challenge made birds more susceptible to the vNDV, lowering the minimum dose required to cause an infection, exacerbating the clinical outcome, while delaying the onset of the disease and time of death. Interestingly, the presence and degree of these seemingly contrasting effects were dose-dependent and not mutually exclusive.

Equine influenza and equine rhinopneumonitis are among the Office International des Epizooties or the World Organisation for Animal Health notifiable, contagious respiratory diseases. Although vaccination of horses in Israel against equine influenza virus (EIV) and against equine herpesvirus (EHV) is routinely performed, information regarding the occurrence and the epidemiology of the diseases is lacking. We hereby attempt to determine seroprevalence and rate of infection for EHV-1 and 4 and for EIV in horses distributed throughout Israel and describe demographic and environmental risk factors associated with seroprevalence. Despite the fact that last reported isolation of EIV in Israel occurred in 2007, we found a 26.4% (29/110) (95% confidence interval [CI]: 18.18–34.62) seroprevalence for H3N8, a 16.4% (18/110) (95% CI: 9.49–23.31) for H7N7, and a 6.4% (7/110) (95% CI: 1.83–10.97) rate of seroconversion for H3N8, suggesting current and active circulation of EIV in horses in Israel. Age, housing management type, and type of farm activity were significantly associated with seroprevalence, with activities allowing exposure to new horses positively associated with seroprevalence to EIV and an only pasture housing management negatively associated with seroprevalence. No association was detected between other demographic variables (gender, breed, and color) and environmental factors (climatic regions). Seroprevalence to EHV-1 and 4 were very low ( 99%), respectively, raising questions regarding the appropriate vaccination guidelines. Our findings of the occurrence of EIV in horses in Israel imply an underdiagnosis of this virus in this country and warrant further investigation as to the strains that circulate in this region and their accordance with the current vaccine strains.

Highlights • The review discusses the biotic factors and complexities of diagnosis. • The review exemplify the diagnosis complexities by avian viruses. • Important factors are the sample nature, its size and timing. • In multiple virus-infections diagnosis is complex., The great advance in the field of diagnosis of avian viruses is reflecting the highly sophisticated molecular assays of the human and general virology in providing highly sensitive and fast methods of diagnosis. The present review will discuss the biotic factors and the complexities that became evident with the evolution of the novel molecular diagnostic assays with emphasis on 4 avian viruses, chicken anemia, infectious laryngotracheitis, turkey meningoencephalitis, but mainly on Marek’s disease virus. To create a biologically meaningful diagnosis, attention should be dedicated to various biotic factors and not only of the diagnostic assay. Included among the important factors are, (a) the sample examined and the sampling strategy, (b) the outcomes of the pathogen amplification ex vivo, (c) the sampling time and its reflection on the disease diagnosis, (d) the impact of simultaneous multiple virus-infections regarding the ability to demonstrate all pathogens and inter- and intra-interactions between the pathogens. A concerted consideration of the relevant factors and the use of advanced molecular diagnostic assay would yield biologically significant diagnosis in real-time that would beneficiate the poultry industry.

H9N2 influenza viruses are isolated in Israel since 2000 and became endemic. From November 2006 to the beginning of 2012, many H9N2 viruses were identified, all belonged to the Asian G1-like lineage represented by A/qu/Hong Kong/G1/97 (H9N2). In the present study, 66 isolates were selected for their hemagglutinin gene characterization. Most H9N2 isolates were distributed between two main groups, identified as the 4th and 5th introductions. The 5th introduction, was represented by a compact cluster containing viruses isolated in 2011-2012; the 4th introduction was subdivided into two subgroups, A and B, each containing at least two clusters, which can be identified as A-1, A-2, B-1, and B2, respectively. Genetic analysis of the deduced HA proteins of viruses, belonging to the 4th and 5th introductions, revealed amino acid variations in 79 out of 542 positions. All isolates had typical low pathogenicity motifs at the hemagglutinin (HA) cleavage site. Most viruses had leucine at position 216 in a receptor binding pocket that enables the virus to bind successfully with the cellular receptors intrinsic to mammals, including humans. It was shown that the differences between the HA proteins of viruses used for vaccine production and local field isolates increased in parallel with the duration and intensity of vaccine use, illustrating the genetic diversity of the H9N2 viruses in Israel.

SUMMARY. An avian influenza virus (AIV), A/Emu/Israel/552/2010/ (H5N1), was isolated from a dead emu that was found in the Ein Gedi oasis near the Dead Sea. The virus molecular characterization was performed by reverse transcriptase–polymerase chain reaction (RT-PCR) and real-time RT-PCR using AIV subtype-specific primers. The virus was of high pathogenicity, according to its intravenous pathogenicity index of 2.85 and the nucleotide sequencing at the cleavage site of the hemagglutinin gene, GERRRKKR, which is typical for highly pathogenic chicken influenza A viruses.

Our aim was to characterize the A/ck/Israeli/1055/2008 (H5N1) avian influenza virus that was isolated at the beginning of 2008, and to establish the phylogenetic relationship of this isolate to other H5N1 viruses that were recently isolated in adjacent countries. In light of a study of complete nucleotide sequences of all the genes we found that the isolate (year 2008) was closely related to the H5N1 viruses isolated in Egypt, Israel and Gaza in 2006. The Israeli isolate had the hemagglutinin-connecting peptide with a polybasic amino acid insertion. The most host-restriction sites of the 2008 isolate were typical of avian hosts, with one exception: K627 at the PB2 protein. As compared with previous local H5N1 isolates, a high mutation rate was found at the HA gene, which antigenic sites were under positive selection pressure.

Avian influenza viruses (AIVs) of the H9N2 subtype are a major economic problem in the poultry industry in Israel. Most field isolates from the last decade differ significantly from H9N2 isolates from Europe and the USA, rendering published detection methods inadequate. This study aimed to develop a real-time TaqMan((R)) RT-PCR assay, based on a conserved region in the HA9 gene. The assay was validated with viruses representing different genetic subtypes and other common avian pathogens, and was found specific to H9N2. The real-time RT-PCR assay was compared to RT-PCR, which is in routine diagnostic use. Real-time RT-PCR was found to be more sensitive than RT-PCR by 1.5-2.5 orders of magnitude when testing tracheal swabs directly and by 2-3 orders of magnitude allantoic fluid after AIV propagation in embryonated eggs. Sensitivity was quantified by using 10-fold dilutions of the H9-gene amplification fragment, and real-time RT-PCR was found to be 10(4)-fold more sensitive than RT-PCR. Clinical samples, which included tracheal and cloacal swabs, as well as allantoic fluid, were tested by both methods. By real-time RT-PCR 20% more positive H9N2 samples were detected than by RT-PCR. The real-time RT-PCR assay was found suitable for detection and epidemiological survey not only of Israeli H9N2 viruses, but also for isolates from other parts of the world.

The main aims of the present study were to characterize NS1 protein from H9N2 avian influenza viruses (AIVs) isolated in Israel and to investigate the possibility to use NS1-based indirect ELISA. To achieve these purposes, the non-structural gene (NS1) of 79 AIVs of the H9N2 subtype isolated in Israel in 2000-2009 was sequenced and genetically analyzed. The phylogenetic analysis demonstrated that four distinct introductions of H9N2 occurred in Israel during this period. Analysis of the inferred amino acid sequences of the NS1 proteins showed high, about 10%, differences between viruses of the 3rd and 4th introductions. Antibodies against NS1 protein in immune sera were tested by means of indirect ELISA using recombinant NS1 as antigen. Immune sera were obtained from experimentally H9N2-infected chicken after infection on 4, 7, 10, 14, and 21 days. All sera from chickens experimentally infected with 3rd- or 4th-introduction AIV contained anti-NS1 antibodies that were detected by enzyme-linked immunosorbent assay (NS1-ELISA) even though the recombinant NS1 used as antigen for NS1-ELISA differed significantly in its amino acid sequences from the NS1 protein of AIV that caused infection in experimental birds. These findings indicate that the sites of the NS1 protein by which viruses belonging to 3rd and 4th introduction are out of antigenic epitope positions were responsible for the results of NS1-based iELISA.

Avian tumor viruses are important, economically significant pathogens in the poultry industry, and can provide scientific insight in topics that cannot be experimented in humans. The present review will present viral-induced tumors in poultry and will emphasize the specific findings that can be taken from their study, regarding (a) multiple virus infections in vivo, (b) molecular interactions in vitro and in vivo between avian oncogenic viruses, (c) creation of new viable viruses with altered biological properties by the integration of two retroviruses in vivo, (d) the diversity of clinical signs that appear in viral-infected poultry prior to tumor formation. The inter-viral molecular interaction were studied in vitro and in vivo, in natural infections and in experimentally-infected birds. About 25% of flocks were double-virus-infected, and molecular integrations occurred spontaneously in about 5% of the samples, as by the chimeric molecules detection. In experimentally-infected birds the chimeric molecules rate was higher, while in herpes- and retroviruses co-infected tissue cultures, recombinant virus creation was efficient, leading to a new virus with altered biology. Spontaneous inter-viral recombination occurred between retroviruses, lead to the emergence of a delirious new virus, avian leukosis virus, subgroup J, which actually caused great economic losses to the poultry industry.

Our aim was to establish the phylogenetic and genetic relationships among avian influenza viruses (AIV) recently isolated from poultry in Israel. During this study we analyzed complete nucleotide sequences of two envelope (hemagglutinin and neuraminidase) and six internal genes (polymerase B1, polymerase B2, polymerase A, nucleoprotein, nonstructural, and matrix) of 29 selected H9N2 and six internal genes of five H5N1 viruses isolated in Israel during 2000-2006. Comparative genetic and phylogenetic analyses of these sequences revealed that the local H5N1 viruses are closely related to H5N1 viruses isolated in European, Asian, and Middle Eastern countries in 2005-2006. The H9N2 Israeli isolates, together with viruses isolated in Jordan and Saudi Arabia formed a single group. Our data support the claim that during recent years a new endemic focus of H9N2 has been formed in the Middle East. The introduction of H5N1 and co-circulation of these two subtypes of AIV in this region may augment the risk of potentially pandemic strains emergence.

Blood samples from nine broiler breeder flocks comprising five flocks clinically affected with myeloid leukosis tumours (ML+) and four tumour-free flocks from the same commercial background (ML-) were compared for avian leukosis virus subgroup J (ALV-J) serum antibodies by enzyme-linked immunosorbent assay (ELISA), for antigenemia (group-specific antigen) by antigen-trapping ELISA and for viremia. Group-specific antigen was detected in the sera of 58.1% of ML+ birds and 46.4% of the ML- birds (P=not significant), while 45.5% of ML+ birds and 24.1% of the ML- birds had ALV-J antibodies (P=0.065). In inoculated cell culture, 64.1% of the ML+ sera were viremic compared with 16.7% of the ML- sera (P=0.001). Similar significant differences were found between the two groups of flocks when ALV-J viremia was detected by immunofluorescence using a monoclonal env antibody (P=0.004), and for proviral DNA by polymerase chain reaction using two different sets of env-gene primers, H5-H7 (P=0.001) and R5-F5 (P=0.001). Using the primer pair R5-F5 the product size was approximately 1 kbp, while some heterogeneity in size among isolates was discernable. Our results indicate that a combination of diagnostic tests should be adopted in routine examination of tumour material in order to rule out false-negative findings.

Marek's disease virus (MDV) productive replication occurs in the feather follicle epithelium and the feather tips are valuable both for research and disease diagnosis. Three novel applications of feather tip extracts are described now: (A). As a source of DNA for amplifying either MDV and/or ALV-J. In two clinical situations a marked advantage was obtained compared to blood and organs; in broiler breeder flocks with a mixed MDV and ALV-J infection, and in young broilers with neurological Marek's disease (MD). (B). Separation of the large ( approximately 200 kbp) MDV genome directly from the infected chickens. Using pulsed field gel electrophoresis, the DNA extracted from tumors or feather tips was separated and hybridized to a 132 bp tandem repeat MDV probe. Compared to 2/55 polymerase chain reaction (PCR) positive tumor samples, 15/61 feather tip extracts contained whole MDV genomes. (C). Experimental MDV infection was induced by the mucosal route by dripping feather tip extract to the eye and mouth of the bird. That attempted to reproduce the native infection process, however the use of extracts, instead of dry feather dust was a compromise, aimed to synchronize the infection. In one trial, tumors were induced 6 weeks after dripping day-old broilers, while in another, feather tips were PCR positive 16 days after dripping of 2-month-old layers.

The major histocompatibility complex (MHC) is a part of the immune system which presents epitopes of intracellular antigens on the cell surface. MHC molecules have receptor–ligand binding affinities with T lymphocytes, permitting the latter to detect foreign intracellular infectious agents. Some pathogens, such as herpesviruses, have developed strategies of evading the host response by MHC. This pressure on the immune system brought, in turn, improvements in the antigen-presenting pathway, for example through the effect of interferon (IFN), which can upregulate MHC expression. The main objective of this work was on the one hand, to determine the abilities of three strains of Marek's disease virus (MDV), a chicken herpesvirus, in interfering with the expression of MHC class I molecules in chicken embryo fibroblasts. On the other hand, we analyzed the ability of IFN to reinstate this important immune capability to the infected cells. Our results show that only an oncogenic serotype 1 strain of MDV (RB1B) was able to markedly decrease MHC class I expression, and that addition of IFN reversed this MDV effect.

Marek's disease virus (MDV), a herpesvirus, and avian leukosis virus, subgroup J (ALV-J), a retrovirus, are oncogenic viruses of poultry. The present report describes a case-report study aimed at examining the efficacy of amplifying MDV and/or ALV-J from feather-tip DNA as compared with DNA purified from liver and spleen. We show that the polymerase chain reaction for MDV and ALV-J env using DNA from feather tips was more effective for diagnosis of naturally infected commercial chickens than using the liver and spleen.

SUMMARY. Marek's disease virus (MDV) causes immunosuppression and tumors in chickens, but the turkey is an unusual host for the virus, and tumors caused by MDV in turkeys are unique. We describe the prevalence of turkey tumors in Israel between 1993 and 2000, their molecular diagnosis by polymerase chain reaction (PCR), and the natural distribution of herpesvirus of turkeys (HVT). Most clinical cases with tumors in commercial turkeys were diagnosed as MDV. The reproduction of Marek's disease (MD) in turkeys by two turkey MDV strains, Ar and La, was analyzed, and it was shown that these strains can induce tumors in experimental trials. The severity of experimental disease differed from those features of the original outbreak, since a less severe disease was recorded.

SUMMARY. Marek's disease virus (MDV) causes immunosuppression and tumors in chickens. As sporadic cases of Marek's disease (MD) were recorded in turkeys, the antigenic and genomic characteristics of the MDV glycoprotein B (gB) gene and antigen of turkeys were compared to the chicken MDV gB. The whole chicken and turkey gB genes were sequenced and found identical. By immunoblotting of infected-cell culture lysates using chicken convalescent and gB monoclonal antibodies, the antigenic epitopes of the chicken and turkey viruses were found to differ. The turkey MDV had a unique epitope, compared to the chicken MDV and compared with our previous findings. While the chicken MDV had two epitope types, heat-labile but dithiothreitol (DTT)-stable and heat-stable but DTT-labile, the turkey MDV gB epitope is both heat and DTT-labile.

Marek's disease virus is an oncogenic herpes virus of poultry that is highly cell associated. In the infected tissues and tumors the virus replicates in a low copy number. The propagation and dissemination of the virus takes place at the feather follicle epithelium, where the viral genome is produced in high copy number. As the viral genome is a large circular DNA molecule (200 kbp), pulsed field gel electrophoresis was used for separation of the viral genome directly from the infected chicken. DNA was extracted from tumors or feather tips by the phenol:chloroform technique or by low melting agar technique. It was found that feathers, being the site of virus productive replication, are useful for separation of free Marek's disease virus DNA from in vivo infections.

Infectious laryngotracheitis virus (ILTV) and fowlpox virus (FPV) cause diphtheritic lesions in chicken tracheas and can simultaneously infect the same bird. A differential molecular diagnostic test, the duplex real-time polymerase chain reaction, is now reported using ILTV and FPV vaccine viruses and clinical samples from chickens, either uninfected or naturally infected with ILTV or FPV, or with both viruses. The dual virus amplification by real-time polymerase chain reaction was demonstrated to behave similarly to monoplex amplification, in spite of the fact that the real-time exponential amplification plots of the vaccine viruses were more illustrative than those of the clinical samples.

A two-step PCR, the Hot Spot-combined PCR assay, was developed for the identification and characterization of recombinant viruses in Marek's disease (herpes) and retrovirus co-infections. In the first PCR the herpesvirus genomic fragment, that was recognized in previous studies as a hot spot site for retroviral integration was amplified [reviewed in Bronovskis, P., Kung, H.-J., 1996. Retrotransposition and herpesvirus evolution. Virus Genes 11, 259-270]. The products served for a second amplification step, performed in six PCR sets, using the six possible combinations of the two herpes and the retrovirus primer sets. Development of the assay employed DNA of the recombinant virus, RM1, which was created by in vitro co-cultivation of Marek's disease and reticuloendotheliosis viruses [Isfort et al., 1992. Proc. Natl. Acad. Sci. 89, 991-995; Witter et al., 1997. Avian Dis. 41, 407-421]. As the retroviral insertion site and junction sequences were determined previously [Jones et al., 1996. J. Virol. 70, 2460-2467], RMI served in the present study as a test virus for the development of the new assay. It is shown now that the Hot Spot-combined PCR can detect the retroviral insert in RM1. the MDV integration site and the insert orientation. For confirmation the herpes and retrovirus chimeric PCR products were sequenced and the results were similar to those published previously [Jones et al., 1996. J. Virol. 70, 2460-2467]. This assay might be adopted in additional systems to detect foreign inserts at suspected genomic sites.

Congenital transmission of reticuloendotheliosis virus (REV) is known to occur when one of the parents in either tolerantly infected or viraemic; however little attention has been paid to replication of the virus within the embryo itself. In this study five non-defective and one replication-defective strain of REV were inoculated into the yolk sac of 6-day-old embryonated eggs. With the exception of one non-defective strain, the others induced embryonic deformation or lesions that were apparent on the day of hatching. Replicative integration of the provirus was determined for several tissues of each embryo by the polymerase chain reaction employing a set of primers specific for the long terminal repeat. Indirect immunofluorescent detection of whole virus was performed on the same tissues in parallel. A variation in replication was found among the strains and between embryos inoculated with the same strain of virus, therefore, the results for each virus strain were expressed as cumulative values. The liver and spleen were the most commonly affected organs, while the bursa of Fabricius and thymus supported viral replication to a lesser degree. Detection of proviral DNA in blood was evidence that the chicks had been congenitally infected. Such an infection would likely lead to tolerance. Isolates REV-S and REV-D were found to replicate to the highest extent, and they also caused lesions in the commercial type of embryonated eggs, while REV-S and mainly the REV-T prototype were pathogenic for the SPF embryonated eggs.

While the previous phylogenetic analyses of AIV H9N2 in Israel had mainly focused on phylogenetics and on describing different virus introductions into the country, for the first time, the H9N2-HA gene evolutionary history has been examined taking into account its origin, evolution and phylodynamics. The present study reveals the Israeli H9N2 molecular evolution rate, the virus molecular clock and skyline plot. The molecular skyline plot showed two major increments in population diversity sizes, the first which had occurred in 2003, the second between the end of 2007 and the first half of 2008. Between 2004 and 2007 the population size had proved to be constant. The two peaks correspond to the appearance of the 3rd and 4th major genetic groups, as well as to the introduction of two H9N2 vaccines. The mean evolution rate was 6.123 E-3 substitutions/site/year, typical of avian influenza viruses. The time interval from the most recent common ancestor was 12.3 years, corresponding to the year 2000, when H9N2 was first isolated in Israel.

The worldwide distribution of chicken anemia virus (CAV) and Marek's disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated.

The antigenic profiles of three REV prototype strains, CSV, SNV and REV-T and eight Israeli isolates were analysed by SDS-PAGE and immunoblotting with convalescent chicken serum, three mAbs, 11A25, 11C237 and 11C100, a rabbit antiserum to REV-T whole virus (Cui et al., 1986) and a rabbit antiserum to REV-A p30 gag protein (Tsai et al., 1985). Under both reducing (+DTT) and non-reducing conditions of SDS-PAGE, a major immunodominant 75-100 kDa band was shared by all strains examined. In contrast to the chicken serum that recognized both continuous and discontinuous epitopes on the 75-100 kDa band of all the isolates, the mAbs and the two rabbit sera behaved otherwise. Only the DTT-resistant epitopes on the 75-100 kDa band of REV-T were recognized by the rabbit antisera and the mAb 11C237, and only the DTT-labile epitopes of REV-T 75-100 kDa antigen were detected by mAb 11C100. The two mAbs 11A25 and 11C237 detected discontinuous epitopes of all the strains except SNV, while the rabbit antisera recognized the discontinuous epitopes on the 75-100 kDa band of all the 11 strains. The rabbit antisera and mAb 11C237 detected additional lower molecular weight proteins and the mAb 11C237 also detected three proteins of high molecular weight under non-reducing conditions only. The p30 antiserum detected the low molecular weight proteins demonstrating their gag gene-encoded identity. From these results we conclude that the major immunogen of REV is the 75-100 kDa protein that contains both continuous and discontinuous epitopes. With this panel of antibodies the eight new isolates appeared to belong antigenically to REV subtype 3 (Chen et al., 1987).

The antigenic cross-reactivity between the Marek's disease virus glycoprotein B (MDV gB) and glycoprotein B (gB) of herpes simplex virus type 1 and 2 (HSV1 and HSV2) was analysed by the immunoblotting method. We studied cell lysates in both denatured and in undenatured form (i.e., unheated) and reacted them with convalescent sera from chickens infected with the RBIB MDV strain and with human anti-HSV1 gB. Both sera detected the heat-labile MDV gB and the HSV gB oligomers. In addition, monospecific antibodies to the MDV gB 230 kDa oligomer (strain CVI988) were immunoaffinity purified from both the chicken and the human sera. The chicken and human monospecific antibodies detected the homologous and the heterologous gB oligomers in native MDV- and HSV1-infected cell lysates. 15 human sera were tested by immunoblotting and by immunofluorescence on HSV1-, CVI988-and herpes virus of turkeys (HVT)-infected cells. By both assays about half of the human sera reacted with MDV-infected cells. This study demonstrates that the MDV gB heat-labile oligomers possess conformational epitopes shared with the human alpha-herpes virus HSV1 and HSV2 gB heat-labile oligomers.

An avian influenza virus (AIV), A/turkey/Israel/09 subtype H6N1, was isolated from turkey poults exhibiting typical pathology associated with AIV infection. The virus was characterized by RT-PCR using AIV subtype-specific primers and by the haemagglutination inhibition test using AIV subtype-specific antisera. The virus has an intravenous pathogenicity index of 0 and possessed a nucleotide sequence at the cleavage site of the hemagglutinin gene, PQIETR*GLF, associated with avian influenza viruses of low pathogenicity. Unlike the two previous H6N2 isolates originating from domestic ducks and mallard, the A/turkey/Israel/09 (H6N1) was isolated from turkeys. The gene sequences of the A/turkey/Israel/09 (H6N1) virus show divergence from the former Israeli H6 isolates.

The H9N2 avian influenza virus (AIV) subtype has become endemic in Israel since its introduction in 2000. The disease has been economically damaging to the commercial poultry industry, in part because of the synergistic pathology of coinfection with other viral and/or bacterial pathogens. Avian influenza virus viability in the environment depends on the cumulative effects of chemical and physical factors, such as humidity, temperature, pH, salinity, and organic compounds, as well as differences in the virus itself. We sought to analyze the viability of AIV H9N2 strains at three temperatures (37, 20, and 4 C) and at 2 pHs (5.0 and 7.0). Our findings indicated that at 37 C AIV H9N2 isolate 1525 (subgroup IV) survived for a period of time 18 times shorter at 20 C, and 70 times shorter period at 4 C, as measured by a decrease in titer. In addition, the virus was sensitive to a lower pH (pH 5.0) with no detectable virus after 1 wk incubation at 20 C as compared to virus at pH 7.0, which was viable for at least 3 wk at that temperature. The temperature sensitivity of the virus corresponds to the occurrence of H9N2 outbreaks during the winter, and lower pH can greatly affect the viability of the virus.

The aim of this study was to evaluate the presence of Helicobacter (H.) spp. in swine affected by gastric ulceration. Stomachs from 400 regularly slaughtered swine were subjected to gross pathological examination to evaluate the presence of gastric ulcers. Sixty-five samples collected from ulcerated pars esophagea and 15 samples from non-ulcerated pyloric portions were submitted to histopathological and molecular analyses, to detect Helicobacter spp., H. suis and H. pylori by PCR. Feces and saliva swabs were also collected from 25 animals in order to detect in vivo the presence of Helicobacter spp.. Gastric ulcers were detected in 373 cases (93%). The presence of ulcers in association with inflammatory processes was further confirmed by histological examination. Forty-nine percent (32/65) of the ulcerated esophageal portions as well as 53% (8/15) of the non-ulcerated pyloric portions were positive for Helicobacter spp. by PCR. The Helicobacter spp. positive samples were also positive for H. suis, while H. pylori was not detected. These results were confirmed by restriction enzyme analysis. With regard to feces and saliva samples, 15/25 (60%) and 16/25 (64%) were positive for Helicobacter spp. PCR, respectively but all were negative in H. suis and H. pylori specific PCR.

Turkey meningoencephalitis (TME) is a paralytic epornitic disease of turkeys caused by turkey meningoencephalitis virus (TMEV), an arthopod-borne flavivirus belonging to the Ntaya serogroup VI. A TMEV specific RT-PCR was compared with classical techniques for TMEV diagnosis, which include virus isolation in 8-day-old chicken embryonated eggs and suckling mice, on 17 TME flocks with neurological signs that occurred during the fall of 1997. In 11/17 flocks both the RT-PCR and the virus isolation methods detected virus, in 4/17 flocks a negative diagnosis was obtained by both methods, and two flocks were positive by RT-PCR only. In four flocks RT-PCR only detected virus after inoculation into embryonated eggs or suckling mice. There was a dose response effect in the yield of the RT-PCR product. Direct examination of turkey brains yielded bands of low to medium intensity. Use of RT-PCR after embryo and/or mouse inoculation resulted in products of far greater intensity. Thus, RT-PCR can be successfully used to amplify TMEV RNA in the brains of diseased turkeys but a negative result would require egg and mouse inoculation for enrichment of virus prior to RT-PCR.

Fowlpox virus (FWPV) is found worldwide in poultry and wild birds. FWPV is a natural example of recombination between viruses, as reticuloendotheliosis virus (REV) fragments have been found in all poultry FWPVs and these are implicated in virulence alteration. We aimed to determine the commonality of this phenomenon and analysed FWPVs collected from 128 poultry flocks and birds over the last 10 years. Various fragments of both viruses were amplified and sequenced at the FWPV integration site, located between FWPV open reading frames 201 and 203. Seven isolates were found to contain no REV insertions, including fragments of the REV env, gag and 5′ REV-long terminal repeat (LTR). We demonstrate here for the first time, the existence of poultry FWPVs without REV inserts (two from chickens, one from turkey FWPV and four from wild birds). The REV inserts were heterogeneous in size. In addition to poultry and wild bird isolates, three FWPV vaccine virus strains were examined and found to contain only remnant REV-LTR and no REV envelope gene fragments.

Four novel phylogenetic clades of canine rabies virus (RABV) variants have been identified in the Middle East and North Africa. The three novel Middle Eastern clades comprise RABV isolates from the borders between Israel and neighbouring countries. The North African clade (Africa 4) comprises four RABV isolates from Egypt and one from Israel. We characterized various RABV lineages antigenically by using a panel of monoclonal antibodies to the nucleoprotein (N) and phylogenetically by analysis of entire N gene sequences. The estimated mean substitution rate for the N gene alignment (2.7×10−4substitutions per site per year) is comparable with previous estimates for RABV. The application of a molecular clock indicates the emergence of current canine RABV diversity to have occurred at about the same time (approx. 1870) in the Middle East and Europe, following divergence from established lineages in Africa and Asia.

Viruses encounter the innate immune system immediately after infection of the host; specifically, soluble molecules that are both directly lethal and that initiate acquired immunity. Using the oncogenic Marek's disease alpha-herpesvirus (MDV) model, we quantified the effect of a interferon-containing supernatants (ICS), on MDV replication, gene transcription and antigen expression kinetics. We used an established cell culture system and a well-defined virulent MDV (RB-1B). RB-1B was cultured without ICS, or pretreated and then continuously treated with ICS. We compared (i) RB-1B infectivity; (ii) RB-1B growth by microscopy; (iii) numbers of cells expressing RB-1B antigens by flow cytometry; (iv) RB-1B-DNA load per cell by duplex real-time PCR, and (v) gene transcription kinetics for key MDV-life stages by duplex real-time reverse-transcriptase PCR (RT-PCR). ICS inhibited RB-1B infection, completion of productive life cycle and cell-to-cell infection. The numbers of cells expressing glycoprotein B (a kinetically late antigen) greatly decreased, but the numbers of cells expressing pp38 (a kinetically early antigen) decreased only slightly. The two greatest effects were increases in both RB-1B-DNA per infected cell and pp38 mRNA. We propose MDV has evolved to increase specific gene transcription and genome copies per cell to compensate for ICS. We speculate that the bi-directional shared pp38/origin of replication promoter, is central to this mechanism.

Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus (ILTV). To evaluate differential detection of ILTVs belonging to the two types, wild-type or vaccine-type, both causing clinical signs, five PCRs were evaluated to detect wild-type and vaccine-type ILTV in clinical samples. By directly sampling the organs, we aimed to avoid changes in the virus genome and to facilitate a fast diagnosis. The samples were tracheal and spleen homogenates and feather shafts. The latter are easy to collect, nonlethal for the bird, and advantageous for monitoring purposes. We investigated the time interval for vaccine virus detection following commercial vaccination by the vent application, which is successfully practiced in Israel. The study indicated that ILTV amplification from feather shafts was possible in clinical cases for about a one-month period after vaccination. Vaccine strains were identified by nested PCR for the ILTV-gE gene and differed from wild-type ILTV strains by two criteria: (1) While avirulent vaccines could be detected for about a month after the vent application, wild-type virus could be detected, in conjunction with clinical signs, for an unlimited time period; and (2) The ILTV vaccine was present in the bird in minute quantities compared to the wild-type virus. We assessed the virus type that appeared in conjunction with the clinical signs and determined that the clinical signs appeared in conjunction with both molecular forms of ILTV. The vaccine virus-type and the wild-type ILTV differed by their distinct restriction pattern when using the HaeIII restriction enzyme digestion of the nested amplification product.

Chick embryos infected at Day 13 of embryonic development (ED) with the oncogenic serotype 1 Marek's Disease Virus, isolate B (MDV-B) and three MDV vaccines (CVI988, SB1 and HVT, serotypes 1, 2 and 3, respectively) and uninfected chick embryos were studied for the distribution of Ia antigen positive dendritic cells (DC), B cells and MDV antigen positive (Ag+) cells in the lymphoid organs and chorioallantoic membrane (CAM). The immunofluorescence study was conducted on acetone-fixed organ touch impressions using monoclonal antibodies to Ia antigen, and MDV serotypes 1, 2 and 3 and polyclonal antibodies to bursal Ig-bearing (Ig+) B cells. DC were found mainly in the thymus and spleen and Ig+ cells in the bursa, thymus and spleen of normal embryos. All virus-infected embryos had MDV Ag+ cells in the spleen. MDV-B and SB1 infected embryos also had MDV Ag+ cells in the bursa, MDV-B Ag+ cells in the CAM and SB1-Ag+ cells in the thymus. Infection with MDV altered the distribution pattern of DC in a serotype-specific manner: to a lesser extent, infection with MDV-B and SB1 induced their appearance in the CAM, while HVT and CVI988 depleted the DC population from all organs except the bursa and the thymus, respectively. Infection with MDV-B depleted the Ig+ cells from all organs. These results suggest that virus-specific patterns of change in the distribution of DC and B cells occur in various tissues and organs of the chick embryo as a result of infection with oncogenic and apathogenic strains of MDV.

The antigenic profile of cell cultures infected with Marek's disease virus (MDV) was determined by the immunoblotting method using convalescent immune serum obtained from chickens that survived infection with MDV strain GA5. The MDV antigen profile in infected cell lysates could be accurately determined since this method has advantages over the immunoprecipitation method used in other studies. We studied six very virulent MDV isolates and the prototype of serotype 1 MDV, the GA5 strain. Immunoblots of NaDodSO4-polyacrylamide gel electrophoresis (PAGE) performed under reducing conditions revealed a main viral antigen (B) of 120-130 kD, which was present in all cell lysates infected with MDV isolates. Analysis of infected cell proteins by NaDodSO4-PAGE performed under nonreducing conditions, revealed a 205 kD major MDV antigen, which, under reducing conditions, was identical to the 130 kD major antigen. The unglycosylated precursors of the major MDV antigens were elucidated. Two polypeptides of 43 and 45 kD were found to be the unglycosylated precursors of MDV antigen A (the glycosylated form of which appears in 4 distinct bands). The unglycosylated precursors of the MDV major antigen B were found to be three polypeptides of 80, 110, and 125 kD.

A highly specific enzyme-linked immunosorbent assay (ELISA) is described for determination of bovine IgM and use in screening IgM-secreting hybridoma cell lines. The advantages of this assay over the method of radioactive labelling of cells followed by immune precipitation, SDS-PAGE, fluorography and autofluorography are demonstrated.

Two novel tests, enzyme-linked immunosorbent assay (ELISA) and dot-blot hybridization, were developed to detect and quantify the antigens and DNA of Marek's disease virus (MDV) in feather tips from infected chickens. In both methods, buffered extracts of the feathers served as the same test material. The ELISA technique was compared to the conventional agar-gel precipitation (AGP) test, using the same convalescent serum from a MDV-infected bird. Of 86 feather samples tested, 34 were negative by both methods, while 6 out of 52 were ELISA positive but AGP negative. Viral antigen detection by the AGP and ELISA methods was compared with the detection of MDV DNA by the dot-blot DNA hybridization technique. At an ELISA reading (OD 405) of 0.3 and above, only 5 out of 48 DNA extracts failed to hybridize with the MDV-DNA probe. The use of the radioactively labelled MDV-DNA probe for hybridization with DNA extracts from feather tips of MDV-infected chickens was both sensitive and specific, and there was good correlation among the different tests.

SUMMARY An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies in turkey serum to hemorrhagic enteritis virus. The ELISA antigen was extracted from turkey spleens and partially purified with fluorocarbon. Antibodies were demonstrated in serum samples of breeding and meat flocks that had been naturally exposed to infection. These samples were also examined in parallel by agar-gel precipitin (AGP); most of the sera were AGP-positive. ELISA, however, was more sensitive in detecting antibodies in day-old sera that were AGP-negative. The passively acquired antibodies were no longer detected by 4 weeks of age. A brisk but short-lived secondary response was detected by ELISA in the sera of turkeys immunized with ,-propiolactoneinactivated extract of infected spleens. RESUMEN