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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.

Backround Astrovirus, Norovirus and Sapovirus exhibit a wide distribution in swine pig herds worldwide. However, the association of porcine Astrovirus (PAstV), porcine Norovirus (PoNoV) and porcine Sapovirus (PoSaV) with disease in pigs remains uncertain. In this study, we investigated the prevalence of PAstV, PoNoV and PoSaV in Greek pig farms using both conventional RT-PCR and SYBR-Green Real-time RT-PCR in an effort to compare the sensitivity of the two methods. We examined 1400 stool samples of asymptomatic pigs originating from 28 swine farms throughout Greece in pools of five. Results PAstV was detected in all 28 swine farms examined, with an overall prevalence of 267/280 positive pools (95.4%). Porcine Caliciviruses prevalence was found at 36 and 57 out of the 280 examined samples, by the conventional and SYBR-Green Real time RT-PCR, respectively. Sequencing and phylogenetic analysis of the positive samples revealed that the detected PAstV sequences are clustered within PAstV1, 3 and 4 lineages, with PAstV3 being the predominant haplotype (91.2%). Interestingly, sequencing of the Calicivirus positive samples demonstrated the presence of non-target viruses, i.e. Sapovirus, Kobuvirus and Sapelovirus sequences and one sequence highly similar to bat Astrovirus, while no Norovirus sequence was detected. Conclusions The high prevalence of PAstV in Greek pig farms poses a necessity for further investigation of the pathogenicity of this virus and its inclusion in surveillance programs in case that it proves to be important. To our knowledge, this is the first epidemiological study of these viruses in pig farms in Greece.

Simple Summary Often tumors are eventually observed at poultry slaughter, leading to condemnation, not of zoonotic, but because of esthetic reasons. Most avian tumors are caused by infection with oncogenic viruses, where the most prominent is a herpesvirus, Marek’s disease virus (MDV). MDV-infections are economically important in the poultry industry as they cause immunosuppression, decreases in productivity and profitability and increased morbidity, mortality, and condemnation at slaughter. MDV was discovered about 50 years ago, and still its study is ongoing until now. This review is presenting the MDV biology, and protection by vaccination, but also topics that were less brought to wider knowledge, like the vaccine and virulent virus presence in feathers, and their use in assessing the efficacy of vaccines application in commercial settings. In addition, two relatively novel topics are reviewed, like the meq MDV oncogene and its recent implication in molecular epidemiology and MDV virulence determination, and second, the importance of native, three-dimensional conformational epitopes of the immunodominant glycoprotein B and the molecular recombination between DNA-DNA and DNA-RNA viruses. Our studies were particular, as they were originally describing how the native virion is activating the chicken immune response to create virus-neutralizing antibodies. The topic might shed light on items to be focused on in future searches for effective vaccines. Abstract Marek’s disease virus is an economically important avian herpesvirus that causes tumors and immunosuppression in chickens and turkeys. The virus, disease, and vaccines have been known for more than 50 years, but as knowledge gaps still exists, intensive research is still ongoing. The understanding of MDV complexity can provide scientific insight in topics that cannot be experimented in humans, providing a unique model that is dually useful for the benefit of the poultry industry and for studying general herpesvirology. The present review presents the following topics: the MDV biology, the vaccine’s and virulent virus’ peculiar presence in feathers, protection by vaccination. In addition, two relatively behind the scenes topics are reviewed; first, the meq MDV oncogene and its recent implication in molecular epidemiology and in the MDV virulence determination, and second, the functionality of conformational epitopes of the MDV immunodominant protein, glycoprotein B. Our studies were particular, as they were the only ones describing three-dimensional MDV gB oligomers. MDV gB (glycoprotein B) continuous and discontinuous epitopes were shown to possess distinctive neutralization activities. In contrast, the significance of oligomerization of the viral membrane proteins for the creation of discontinuous epitopes in other herpesviruses was explored extensively.

Marek's disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2), which primarily affects chickens. However, the virus is also able to induce tumours in turkeys, albeit less frequently than in chickens. This study reports the molecular characterization of a GaHV-2 strain detected in a flock of Italian meat-type turkeys exhibiting visceral lymphomas. Sequencing and phylogenetic analysis of the meq gene revealed that the turkey GaHV-2 has molecular features of high virulence and genetic similarity with GaHV-2 strains recently detected in Italian commercial and backyard chickens. GaHV-2 is ubiquitous among chickens despite vaccination, and chicken-to-turkey transmission is hypothesized due to the presence of broilers in neighbouring pens.RESEARCH HIGHLIGHTS A GaHV-2 strain from Italian turkeys was molecularly characterized.The turkey strain presented molecular characteristics of high virulence in its meq gene.The turkey strain was closely related to previously detected chicken strains.

The avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, drooping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA has now been upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the envelope and the non-structural NS5 gene, increasing the sensitivity by 10-100-fold compared to the previously existing assays. Based on the recent complete sequences of five TMEV isolates we have now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA assay was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009 and 2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV diagnosis in brain and feather-pulp RNA were similarly useful for diagnosis, at least in experimentally infected turkeys and in three cases of disease encountered in commercial flocks.

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.

The differentiation of Marek's disease virus (MDV)-infected and vaccinated animal (DIVA) test, based on the MDV pp38 gene was described by Baigent et al. [(2016). Real-time PCR for differential quantification of CVI988 vaccine and virulent MDV strains. Journal of Virological Methods, 233, 23-36], using similar primers and alternate probes for virulent MDV-1 and the vaccine CVI988 virus. We explored the assay's applicability for commercial vaccines and commercial chickens, as the above-mentioned study employed tissue-cultured MDV strains and tissues from experimental trials. DNA of visceral organs and feathers of vaccinated or naturally infected chickens was used. Further, the applicability of the DIVA assay was evaluated using single or duplexed probes for the two viruses in the same amplification tube. Due to the high viral content in the commercial vaccines and in the clinical cases of MDV-1 infected commercial chickens, their examination by the MDV-1 DIVA real-time PCR was performed in one step. However, for the feather DNAs of commercially vaccinated birds, a step of pre-amplification was required. The MDV-1 DIVA real-time PCR performed as single probe in separate tubes using the Vir3 probe was very sensitive for virulent MDV-1 strains, but not very specific, as it also gave a clear signal with CVI988 vaccine virus. In contrast, the CVI vaccine probe was specific for CVI988, and did not recognize the MDV-1 strains. When both probes were present in one tube, the CVI probe showed a greater sensitivity for CV1988, while the Vir3 probe showed a much better specificity for virulent MDV-1.

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.

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.

The aim of this study was to determine serological values of lysozyme, hemolytic complement levels (alternative pathway), and bactericidal activ- ity of serum in turkeys kept in different rearing sys- tems (industrial, backyard, and experimental). Results showed that the values for serum bactericidal activity and hemolytic complement levels increased with age, and their values were higher in experimental and in industrial turkeys than in turkeys reared in backyard. Lysozyme concentration showed a similar pattern; its value was higher in the industrial and experimental groups than in the backyard group. Data obtained sug- gest that rearing system can have an influence on the natural immune parameters considered; experimental and industrial groups showed a similar trend, differenti- ated from that observed in the backyard group. In the backyard group, the values observed may suggest that hybrid turkeys, selected for high production, have dif- ficulty with being reared outside where predators (foxes and weasels) and weather conditions could be respon- sible for a stress situation.

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.

Mareks disease virus (MDV), a herpesvirus, and avian leucosis virus subgroup J (ALV-J), a retrovirus, were used for experimental coinfection of chickens. Chimeric molecules having sequences of both viruses were detected by the hotspot-combined polymerase chain reaction (HS-cPCR) system. The detection of chimeric molecules provided evidence for avian retroviral inserts in the herpesvirus genome. The persistence of chimeric molecules on in vivo passage served to indicate the infectivity of the recombinant virus. The evaluation of formation and persistence of the chimeric molecules was performed in two trials involving three in vivo passages. The chimeric molecules were identified according to the primer sets, their product length, and pattern. The persistence of chimeric molecules on in vivo passages served as an indication of their ability to replicate in and infect chickens. In the first experimental passage, MDV and ALV-J prototype strains, MD11 and HC-1, were intraperitoneally (i.p.) injected into 1-day-old chicks. The second trial included two passages. Passage II chicks were injected i.p. and passage III chickens were in contact with the chickens of passage II. For passage II, enriched white blood cells from blood samples of chickens from the first trial that had chimeric molecules were injected i.p. into 1-day-old chicks. For passage III, uninfected chicks were included together with the infected chicks. Synthesis evidence for the various species of chimeric molecules was assessed in the tissues of birds of the second trial. DNA was extracted from blood and feathers and analyzed by the hotspot-combined PCR and by pulsed field gel electrophoresis. To overcome the limits of detection, three amplification assays followed by hybridization of the products to specific viral probes were conducted. A variety of chimeric molecules were detected in low concentrations. Five species of chimeric molecules were characterized in blood, tumors, and feathers. Chimeric molecules were detected in 18 of 36 dually infected birds from the first trial and in 14 of 21 dually infected birds from the second trial. The findings show that, in four out of seven groups of the second trial, the chimeric molecule species persisted on passage.

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.

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.

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.

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.

Marek's disease (MD) is a highly contagious lymphoproliferative disease of chickens caused by a herpesvirus, while reticuloendotheliosis (REV) virus is an avian C-type retrovirus that causes bursal and nonbursal lymphomas which often closely resemble MDV lymphomas. To provide a rapid and sensitive means of diagnosing and differentiating between these two neoplastic conditions, we have applied the PCR. The primers chosen to detect MDV sequences flank the 132 bp tandem repeat of the BamHI-H fragment, whose PCR product is specific for serotype 1 MDVs. The primers selected for REV are based on proviral spleen necrosis virus-long terminal repeat (SNV-LTR) sequences, and can identify both defective and non-defective REVs. The PCR for field isolates of both viruses was standardized with DNA extracted from chick embryo fibroblasts (CEF) infected with various Israeli isolates of MDV and REV. All chickens were examined for REV antibodies by ELISA, REV isolation was performed in CEF and the tumours were examined histopathologically. In some instances, MDV- and REV-PCRs were performed on whole blood DNA and tumour DNA from the same bird. Normal and tumour-bearing birds from a total of 16 chicken and turkey flocks were studied by PCR. These included three laying flocks with MD, two pullet flocks with neurolymphomatosis, four laying flocks that were the progeny of a REV antibody positive flock, four other REV antibody positive flocks, one turkey flock and two chicken flocks with an immunodeficiency syndrome. Some flocks were bled two or three times at 1- to 3-month intervals. We also analysed 30 normal grandparent light breeding birds. Compared to virus isolation, PCR was more sensitive in detecting MDV and REV sequences. It appears, therefore, that PCR is an efficacious and sensitive method for differential diagnosis of tumour-bearing and immunodeficient birds.

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.

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.

The presence of herpesvirus of turkeys (HVT) DNA in the feather tips of chickens vaccinated with HVT was assessed by dot blot hybridisation with a probe specific for HVT and lacking homology to MDV DNA. Only small amounts of HVT DNA were detected in the feather tips of chickens that were vaccinated or left in contact with HVT vaccinated chickens. However when chickens were challenged with virulent MDV, HVT DNA was detected in the feather tips of vaccinated chickens and the largest amount was detected 35 days after vaccination. HVT DNA was recovered in significantly higher quantities from some of the MDV-infected chickens than from those infected by contact. This suggests that MDV infection may provide helper functions for HVT. MDV DNA was identified in the feather tips of MDV-challenged chickens from 25 to 45 days after challenge. Thus, immunisation of chickens with HVT did not prevent the replication of MDV in the feather tips but only diminished it.

Two paralytic conditions affecting flocks of young commercial chickens that were indistinguishable histopathologically from lesions caused by Marek's disease virus (MDV) were investigated. The first, transient paralysis, was observed only in broilers while the second condition, a spastic paralysis, was seen in replacement White Leghorn pullets aged between 6 and 16 weeks. PCR examination of DNA extracted from the blood, brain and spleens of affected birds using primers specific for MDV serotype 1 confirmed the diagnosis of MD for transient paralysis only. None of the Leghorn DNA samples was positive by PCR. Some evidence for a genetic predisposition is offered for the neuritis affecting the pullets based on epidemiological considerations.

SUMMARY. The present study demonstrated, for the first time, that not only in vitro, but also in vivo, coinfections with Marek's disease virus (MDV) and each of the three avian retroviruses (reticuloendotheliosis virus [REV], avian lymphoid leukosis virus [ALV], and ALV-J) lead to retroviral long terminal repeat (LTR) integration into MDV A total of 306 chicken and 59 turkey commercial flocks, submitted for differential avian oncogenic virus diagnosis, served to evaluate the flock mixed virus infection rate, the rate of birds with a multiple virus infection, and the issue of retroviral LTR integration into MDV in vivo. About a quarter of the tumor-bearing commercial flocks carried a mixed MDV and retrovirus infection. A total of 2926 DNA samples were analyzed, including 2428 chicken and 498 turkey DNA samples. Of these, 991 DNAs originated from flocks with a multiple virus infection. In 103 DNA preparations from that group (103/991, 10.4%), including 38 and 56 from chicken blood and tumor tissues, respectively, and nine samples from turkey blood, multiple virus sequences were detected by polymerase chain reaction (PCR). Fifty-six of the 103 samples were further analyzed by the previously developed hot spot-combined (HScPCR assay, of which 48% (27/56) contained chimeric MDV and retroviral LTR molecules. When extrapolated to the total samples derived from the flocks with multiple virus infection, that rate implies that about 5% of the DNA samples would carry MDV-retrovirus integration events. Several birds held a variety of chimeric molecules, indicating that several recombination events occurred simultaneously. The validation of the MDV and retroviral LTR chimeric constitution of these molecules was derived by the MDV and retroviral heterologous primers used for their creation by the HS-cPCR assay, Southern blotting and their detection by retroviral LTR probes, and LTR amplification from the gel-purified chimeric molecules. From several molecules, the LTR was sequenced, and a 161-bp retroviral LTR sequence was demonstrated. Our biochemical data imply that a recent integration occurred in the birds. The viability of recombinant viruses represented by the chimeric molecules will be further approached.

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.

A comparison was made of the temporal appearance of six isolates of serotype 1 Marek's disease virus (MDV) in the feathers of specific pathogen-free (SPF) infected birds using three assays: agar gel precipitation (AGP), enzyme-linked immunosorbent assay (ELISA) and dot-blot DNA hybridisation. Isolate GA-5 served to standardise the in vivo pathogenicity assay, while the remaining five were recent isolates from Israel. Each isolate was inoculated into susceptible 4-day-old birds housed with an equal number of uninoculated birds. All six caused high mortality (80 to 100%) in the inoculated birds and a wide range of mortality (15 to 80%) in the contact groups. The transmission of infection from the inoculated group to the contact group was demonstrated for all six isolates by AGP and ELISA and for four isolates by dot-blot hybridisation. The other two isolates either showed a concurrent rise in MDV-DNA levels (isolate B) or failed to produce detectable levels of DNA in the inoculated and contact infected groups (isolate Ab). This could be due to the nature of the hybridisation reaction between the probe and the homologous sequence in the genome of isolate Ab. Antigenic activity was detected 11 days post-injection by ELISA, 14 days by AGP in some of the inoculated groups. In the contact infected birds the ELISA and dot-blot assays detected virus about 14 days earlier than did AGP. The time interval between the first detection of virus in the inoculated as compared with the contact infected groups differed for each assay and each isolate, viz; 10 to 14 days by ELISA, 14 to 24 days by AGP and 11 to 18 days by DNA-hybridisation.

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