Assessing the infiltration of immune cells in the upper trachea mucosa after infectious laryngotracheitis virus (ILTV) vaccination and challenge.

The types of immune cells that populate the trachea after ILTV vaccination and infection have not been assessed. The objective of this study was to quantify CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells that infiltrate the trachea after vaccination with chicken embryo origin (CEO), tissue culture origin (TCO), and recombinant herpesvirus of turkey-laryngotracheitis (rHVT-LT) vaccines, and after challenge of vaccinated and non-vaccinated chickens with a virulent ILTV strain. Eye-drop vaccination with CEO, or TCO, or in ovo vaccination with rHVT-LT did not alter the number of CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells in the trachea. After challenge, the CEO vaccinated group of chickens showed swift clearance of the challenge virus, the mucosa epithelium of the trachea remained intact, and a limited number of CD4+, CD8α+, and CD8ß+ cells were detected in the upper trachea mucosa. The TCO and rHVT-LT vaccinated groups of chickens showed narrow viral clearance with moderate disruption of the trachea epithelial integrity, and a significant increase in CD4+, CD8α+, CD8ß+, and TCRγδ+ cells infiltrated the upper trachea mucosa. Non-vaccinated challenged chickens showed high levels of viral replication, the epithelial organization of the upper trachea mucosa was heavily disrupted, and the predominant infiltrates were CD4+, TCRγδ+, and MRC1LB+ cells. Hence, the very robust protection provided by CEO vaccination was characterized by minimal immune cell infiltration to the trachea mucosa. In contrast, partial protection induced by the TCO and rHVT-LT vaccines requires a prolonged period of T cell expansion to overcome the established infection in the trachea mucosa.

Isolation and Propagation of Coronaviruses in Embryonated Eggs.

The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, and yolk). The developing embryo and its membranes provide a diversity of cell types that allow for the successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). IBV replicates well in the embryonated chicken egg, regardless of inoculation route; however, the allantoic route is favored as the virus replicates well in epithelium lining the chorioallantoic membrane, with high virus titers found in these membranes and associated allantoic fluids. TCoV replicates only in epithelium lining the embryo intestines and bursa of Fabricius; thus, amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for detection, propagation, and characterization of other, novel coronaviruses.

A Retrospective Study of Transmissible Viral Proventriculitis in Broiler Chickens in California: 2000-18.

Transmissible viral proventriculitis (TVP) is a disease of chickens, mostly in broilers of 2-8 wk of age. Chicken proventricular necrosis virus (CPNV), a birnavirus, is the etiologic agent. Characteristic gross lesions are enlargement, atony, and pallor of the proventriculus. Cases diagnosed in California between 2000 and 2018 (n = 477), originating from 93 different farms representing all major companies in the region, were analyzed. Frequency of cases varied widely between years, with no recognizable seasonality. The flocks were between 6 and 61 days of age; the average age was 34.0 days, and the median age was 35 days. In 166 cases, between 6.3% and 100% of the submitted birds had gross lesions in the proventriculus. The most common findings were enlarged or dilated proventriculi, thickened walls, and pale or mottled serosal appearance. Histopathologically, inflammation of the glands was the most frequent finding. Other lesions included necrosis, hyperplasia, or both conditions of the glandular epithelium; dilated glands; and occasionally fibrin deposition, fibrosis, and hemorrhages. Twenty-three proventriculi from six cases were tested by immunohistochemistry for the presence of CPNV antigen; 21 stained positive. In 209 cases, birds also had lesions in the bursa fabricii attributed to infectious bursal disease, but with no significant difference in the mean percentage of birds with gross lesions in the proventriculus between cases with or without lesions in the bursa fabricii. The results show that TVP is a common disease of broiler flocks in California and confirms that CPNV is the likely causative agent.

Lack of influence by endosymbiont Wolbachia on virus titer in the common bed bug, Cimex lectularius.

BACKGROUND: The common bed bug, Cimex lectularius, is an obligatory blood-feeding ectoparasite that requires a blood meal to molt and produce eggs. Their frequent biting to obtain blood meals and intimate association with humans increase the potential for disease transmission. However, despite more than 100 years of inquiry into bed bugs as potential disease vectors, they still have not been conclusively linked to any pathogen or disease. This ecological niche is extraordinarily rare, given that nearly every other blood-feeding arthropod is associated with some type of human or zoonotic disease. Bed bugs rely on the bacteria Wolbachia as an obligate endosymbiont to biosynthesize B vitamins, since they acquire a nutritionally deficient diet, but it is unknown if Wolbachia confers additional benefits to its bed bug host. In some insects, Wolbachia induces resistance to viruses such as Dengue, Chikungunya, West Nile, Drosophila C and Zika, and primes the insect immune system in other blood-feeding insects. Wolbachia might have evolved a similar role in its mutualistic association with the bed bug. In this study, we evaluated the influence of Wolbachia on virus replication within C. lectularius. METHODS: We used feline calicivirus as a model pathogen. We fed 40 bed bugs from an established line of Wolbachia-cured and a line of Wolbachia-positive C. lectularius a virus-laden blood meal, and quantified the amount of virus over five time intervals post-feeding. The antibiotic rifampicin was used to cure bed bugs of Wolbachia. RESULTS: There was a significant effect of time post-feeding, as the amount of virus declined by ~90% over 10 days in both groups, but no significant difference in virus titer was observed between the Wolbachia-positive and Wolbachia-cured groups. CONCLUSIONS: These findings suggest that other mechanisms are involved in virus suppression within bed bugs, independent of the influence of Wolbachia, and our conclusions underscore the need for future research.

Differential attenuation of Marek's disease virus-induced tumours and late-Marek's disease virus-induced immunosuppression.

Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and a variety of non-neoplastic syndromes in chickens. Furthermore, very virulent plus (vv+) MDVs induce a form of immunosuppression (late-MDV-IS) that might involve both neoplastic and non-neoplastic mechanisms. The objective of this study was to evaluate whether the attenuation of MDV-induced tumours and late-MDV-IS occurs simultaneously or can be dissociated. The immunosuppressive ability of three viruses derived from vv+ MDV strain 686 (wild-type 686, the somewhat attenuated molecular clone 686-BAC, and the nononcogenic molecular clone lacking the two copies of the oncogene meq 686-BACΔMEQ) was evaluated. Late-MDV-IS was evaluated indirectly by assessing the negative effect of MDV strains on the protection conferred by infectious laryngotracheitis (ILT) vaccines. Our results showed that the ability to induce late-MDV-IS was attenuated before the ability to induce tumours. Strain 686 induced both tumours and late-MDV-IS, 686-BAC induced tumours but did not induce late-MDV-IS and 686-BACΔMEQ did not induce either tumours or late-MDV-IS. Further comparison of strains 686 and 686-BAC revealed that strain 686 reduced the humoral immune responses to ILTV (1132 vs 2167) more severely, showed higher levels of meq transcripts (2.1E+09 vs 4.98E+8) and higher expression of MDV microRNAs (mdv1-miR-M4-5p and mdv1-miR-M2-3p) in the spleen, and further reduced the percentage of CD45+-MHC-I+splenocytes (13 vs32 %) compared to molecular clone 686-BAC. This study suggests that the immunosuppressive ability of MDV might follow a continuous spectrum and only the most virulent MDVs can overcome a certain threshold level and induce clinical MDV-IS in the ILT model.

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

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

Evaluation of factors influencing the development of late Marek's disease virus-induced immunosuppression: virus pathotype and host sex.

Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is complex and can be divided into two phases: early-MDV-IS associated with cytolytic infection in the lymphoid organs in chickens lacking maternal antibodies against MDV (MAbs) and late-MDV-IS that appears later in the pathogenesis and occurs even in chickens bearing MAbs. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model evaluates late-MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccines against challenge with ILT virus. In the present study, we have used this model to investigate the role of two factors (MDV pathotype and host sex) on the development of late-MDV-IS. Five MDV strains representing three different pathotypes: virulent (vMDV; 617A, GA), very virulent (vvMDV; Md5), and very virulent plus (vv+MDV; 648A, 686), were evaluated. Only vv+ strains were able to induce late-MDV-IS. An immunosuppression rank (IS-rank) was established based on the ability of MDV to reduce the efficacy of chicken embryo origin vaccine (values go from 0 to 100, with 100 being the highest immunosuppressive ability). The IS-rank of the evaluated MDV strains ranged from 5.97 (GA) to 20.8 (617A) in the vMDV strains, 5.97 to 16.24 in the vvMDV strain Md5, and 39.08 to 68.2 in the vv+ strains 648A and 686. In this study both male and female chickens were equally susceptible to MDV-IS by vv+MDV 686. Our findings suggest that late-MDV-IS is a unique feature of vv+ strains.

Efficacy of various Marek's disease vaccines protocols for prevention of Marek's disease virus-induced immunosuppression.

Marek's disease virus (MDV) induces tumors and severe immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is very complex and difficult to study. In particular, the late MDV-IS (late-MDV-IS) is of great concern since it can occur in the absence of lymphoid organ atrophy or gross tumors. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model measures MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccination; hence the name late-MDV-IS ILT model. In this study, we have used the late-MDV-IS ILT model to evaluate if MD vaccination can protect against late-MDV-IS. One experiment was conducted to determine whether serotype 1 MD vaccines (CVI988 and Md5ΔMEQ) could induce late-MDV-IS by themselves. Three additional experiments were conducted to evaluate efficacy of different MD vaccines (HVT, HVT+SB-1, CVI988, and Md5ΔMEQ) and different vaccine protocols (day-old vaccination, in ovo vaccination, and double vaccination) against late-MDV-IS. Our results show that none of the currently used vaccine protocols (HVT, HVT+SB-1, or CVI988 administered at day of age, in ovo, or in double vaccination protocols) protected against late-MDV-IS induced by vv+MDV strains 648A and 686. Experimental vaccine Md5ΔMEQ administered subcutaneously at one day of age was the only vaccine protocol that significantly reduced late-MDV-IS induced by vv+MDV strain 686. This study demonstrates that currently used vaccine protocols confer high levels of protection against MDV-induced tumors (protection index=100), but do not protect against late-MDV-IS; thus, commercial poultry flocks could suffer late-MDV-IS even in complete absence of tumors. Our results suggest that MDV-IS might not be related to the development of tumors and novel control methods are needed. Further evaluation of the experimental vaccine Md5ΔMEQ might shed light on protective mechanisms against late-MDV-IS.

Early infection with Marek's disease virus can jeopardize protection conferred by laryngotracheitis vaccines: a method to study MDV-induced immunosuppression.

Marek's disease virus (MDV) is a herpesvirus that induces lymphomas and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is divided into two phases: early-MDV-IS occurring mainly in chickens lacking maternal antibodies (MAb) against MDV and associated with lymphoid organ atrophy; and late-MDV-IS occurring once MDV enters latency and during tumour development. Our objectives were to document the impact of late-MDV-IS on commercial poultry (meat-type chickens bearing MAb against MDV and that were vaccinated or unvaccinated against MD) and to optimize a model to study late-MDV-IS under laboratory conditions. The impact of late-MDV-IS was evaluated by assessing the effect of early infection (day of age) with a very virulent plus MDV (vv+MDV) on the efficacy of chicken-embryo-origin (CEO) infectious laryngotracheitis (ILT) virus vaccine against ILT challenge. The CEO ILT vaccine was administered in water at 14 days of age and ILT virus (ILTV) challenge was done intratracheally at 30 days of age. Development of ILT was monitored by daily evaluation of clinical signs, development of gross and histological lesions in trachea, and quantification of ILTV transcripts in trachea. Infection with vv+MDV strain 648A resulted in total abrogation of protection conferred by the CEO vaccine against ILTV challenge even in chickens vaccinated at 1 day of age with either HVT, HVT+SB-1, or CVI988. Chickens exposed to vv+MDV prior to vaccination with CEO ILTV vaccine had similar (P < 0.05) clinical scores, gross lesions, histopathologic lesion scores, and load of ILTV transcripts in trachea after ILTV challenge, as chickens that were not vaccinated with CEO ILTV vaccine.

Evaluation of the Protection Efficacy of a Serotype 1 Marek's Disease Virus-Vectored Bivalent Vaccine Against Infectious Laryngotracheitis and Marek's Disease.

Laryngotracheitis (LT) is a highly contagious respiratory disease of chickens that produces significant economic losses to the poultry industry. Traditionally, LT has been controlled by administration of modified live vaccines. In recent years, the use of recombinant DNA-derived vaccines using turkey herpesvirus (HVT) and fowlpox virus has expanded, as they protect not only against the vector used but also against LT. However, HVT-based vaccines confer limited protection against challenge, with emergent very virulent plus Marek's disease virus (vv+MDV). Serotype 1 vaccines have been proven to be the most efficient against vv+MDV. In particular, deletion of oncogene MEQ from the oncogenic vvMDV strain Md5 (BACδMEQ) resulted in a very efficient vaccine against vv+MDV. In this work, we have developed two recombinant vaccines against MD and LT by using BACδMEQ as a vector that carries either the LT virus (LTV) gene glycoprotein B (gB; BACΔMEQ-gB) or LTV gene glycoprotein J (gJ; BACδMEQ-gJ). We have evaluated the protection that these recombinant vaccines confer against MD and LT challenge when administered alone or in combination. Our results demonstrated that both bivalent vaccines (BACΔMEQ-gB and BACδMEQ-gJ) replicated in chickens and were safe to use in commercial meat-type chickens bearing maternal antibodies against MDV. BACΔMEQ-gB protected as well as a commercial recombinant (r)HVT-LT vaccine against challenge with LTV. However, BACδMEQ-gJ did not protect adequately against LT challenge or increase protection conferred by BACΔMEQ-gB when administered in combination. On the other hand, both BACΔMEQ-gB and BACδMEQ-gJ, administered alone or in combination, protected better against an early challenge with vv+MDV strain 648A than commercial strains of rHVT-LT or CVI988. Our results open a new avenue in the development of recombinant vaccines by using serotype 1 MDV as vectors.

Isolation and propagation of coronaviruses in embryonated eggs.

The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, yolk). The developing embryo and its membranes provide the diversity of cell types that are needed for successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). The embryonated egg also has been shown to be suitable for isolation and propagation of pheasant coronavirus, a proposed member of the Gammacoronavirus genus. IBV and pheasant coronavirus replicate well in the embryonated chicken egg, regardless of inoculation route; however, the allantoic route is favored as these viruses replicate well in epithelium lining the chorioallantoic membrane, with high virus titers found in these membranes and associated allantoic fluids. TCoV replicates only in epithelium lining the embryo intestines and bursa of Fabricius, thus amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for detection and characterization of other, novel coronaviruses.

A chicken embryo lethality assay for pathogenic Enterococcus cecorum.

Pathogenic strains of Enterococcus cecorum cause outbreaks of arthritis and osteomyelitis in chickens worldwide. Enterococcal spondylitis (ES) is a specific manifestation of E. cecorum-associated disease of broilers and broiler breeders characterized by increased flock mortality, resulting from unresolved infection of the free thoracic vertebra by pathogenic E. cecorum. A study of 22 ES outbreaks in the southeast United States revealed that pathogenic E. cecorum strains isolated from spinal lesions were genetically clonal. Here, we compare the virulence of previously genotyped pathogenic strains (n = 8) isolated from spinal lesions and nonpathogenic strains (n = 9) isolated from ceca of unaffected birds in a chicken embryo lethality model. Strains were inoculated into the allantoic cavity of 12-day-old broiler and specific-pathogen-free (SPF) layer embryos; embryo survival was determined by candling eggs daily for 4 days. Significantly decreased survival occurred in both broiler and SPF embryos inoculated with pathogenic genotype strains compared with embryos inoculated with nonpathogenic genotype strains (broiler embryos, 23% vs. 60%; SPF embryos, 9% vs. 61%). Embryos infected with pathogenic strains were unable to control infection and consistently showed gross changes typical of sepsis, including hemorrhage and edema. After 48 hr, similar changes were not observed in embryos infected with nonpathogenic strains. This embryo lethality assay provides a useful tool for understanding the genetic basis of E. cecorum virulence.

Novel avian coronavirus and fulminating disease in guinea fowl, France.

For decades, French guinea fowl have been affected by fulminating enteritis of unclear origin. By using metagenomics, we identified a novel avian gammacoronavirus associated with this disease that is distantly related to turkey coronaviruses. Fatal respiratory diseases in humans have recently been caused by coronaviruses of animal origin.

Transmissible viral proventriculitis identified in broiler breeder and layer hens.

Transmissible viral proventriculitis (TVP) is a recognized cause of production losses in broiler chickens, but previously it has not been reported in broiler breeder and commercial layer hens. In this study, TVP was identified in broiler breeder and commercial layer hens, 9-20 wk of age, based on histopathologic detection of characteristic microscopic lesions. Microscopic lesions in proventriculi of affected hens consisted of glandular epithelial necrosis, ductal epithelial hyperplasia, replacement of glandular epithelium with ductal epithelium, and diffuse interstitial lymphoid infiltration. Additionally, chicken proventricular necrosis virus (CPNV), a virus previously identified as the etiology of TVP in broiler chickens, was detected in proventriculi of TVP-affected hens using a reverse transcriptase-polymerase chain reaction procedure. The findings identify TVP as a potential cause of production losses in broiler breeder and commercial layer hens and provide additional evidence for etiologic involvement in TVP by CPNV.

Replication of recombinant herpesvirus of turkey expressing genes of infectious laryngotracheitis virus in specific pathogen free and broiler chickens following in ovo and subcutaneous vaccination.

Replication of a recombinant herpesvirus of turkey vaccine expressing infectious laryngotracheitis virus genes (rHVT-LT) was evaluated in specific pathogen free (SPF) and commercial broiler chickens after various vaccination protocols (amniotic route at embryonation day [ED] 18; intra-embryonic route at ED 19; and subcutaneous at 1 day of age [s.c.]). Three experiments were conducted: in the first experiment, replication of rHVT-LT vaccine was chronologically evaluated and compared with the replication of herpesvirus of turkey (HVT) in SPF chickens; in the second experiment, the effect of different in ovo vaccination procedures on rHVT-LT vaccine replication was evaluated in SPF chickens; and in the third experiment, the effect of different in ovo vaccination procedures on rHVT-LT vaccine replication was evaluated in commercial broiler chickens with maternal antibodies against HVT and infectious laryngotracheitis virus (LTV). rHVT-LT vaccine replicated in chickens after in ovo (ED 18 and ED 19) or s.c. administration at a similar level. In vivo replication of rHVT-LT vaccine was slower than HVT vaccine. However, in vivo both rHVT-LT and HVT vaccines replicated at similar levels. Both vaccines were consistently detected in the spleen and feather pulp and at lower frequency in the lung. The frequency of samples with detectable levels of rHVT-LT DNA was lower in broiler chickens than in SPF chickens, probably due to interactions with maternal antibodies. Differences between SPF chickens and broiler chickens were found also in the transcription of the LTV glycoprotein I gene (gI). In SPF chickens, in ovo inoculation resulted in a higher number of spleen samples with detectable gI transcripts than s.c. inoculation. In broiler chickens, however, no differences in the level of gI transcripts in spleen samples were found between chickens vaccinated in ovo and those vaccinated by the s.c. route. Transcription of LTV gI gene in lung samples was very low in both SPF and broiler chickens. Further studies to evaluate the mucosal immune response elicited by rHVT-LT in chicken with and without maternal antibodies are warranted.

Physical and genomic characteristics identify chicken proventricular necrosis virus (R11/3 virus) as a novel birnavirus.

Chicken proventricular necrosis virus (CPNV), isolate R11/3, previously was isolated from transmissible viral proventriculitis-affected chickens and was determined to be the likely etiology of this disease. CPNV was identified as a birnavirus on the basis of virion size and morphology (icosahedral, approximately 75 nm in diameter, nonenveloped); buoyant density in cesium chloride (1.32 g/ml); a genome comprising bisegmented, double-stranded RNA (approximately 3.8 and 3.4 kilobase pairs); and nucleotide sequence analyses. Nucleotide sequencing of CPNV RNA, segment B, identified a single large open reading frame that encodes a 903-amino acid protein. The 903-amino acid protein was identified as the putative VP1, the viral RNA-dependent RNA polymerase (RdRp), on the basis of sequence homologies with other birnavirus VP1 proteins. The CPNV VP1 possessed the unique permuted RdRp sequence motif arrangement characteristic of birnaviruses; however, phylogenetic analyses based on VP1 demonstrated that CPNV is deeply divergent from other birnaviruses.

Detection of chicken proventricular necrosis virus (R11/3 virus) in experimental and naturally occurring cases of transmissible viral proventriculitis with the use of a reverse transcriptase-PCR procedure.

A reverse-transcriptase-polymerase-chain-reaction (RT-PCR) procedure was evaluated for detection of chicken proventricular necrosis virus (CPNV) in transmissible viral proventriculitis (TVP) -affected chickens. The RT-PCR procedure was compared with indirect immunofluorescence (IFA) and virus isolation for detection of CPNV in experimentally infected chickens. Microscopic lesions characteristic of TVP were detected on days 5-35 postexposure (PE) in CPNV-infected chickens; CPNV was detected by RT-PCR on days 3-14 PE in freshly collected proventriculi, and on days 1-14 PE in formalin-fixed paraffin-embedded (FFPE) proventriculi. CPNV was detected in proventriculi of experimentally infected chickens by IFA on days 3-10 PE, and by virus isolation on days 1-14 PE. With IFA used as a reference, sensitivity of the RT-PCR procedure with freshly collected and FFPE proventriculi was 88% and 100%, respectively; specificity was 83% and 86%, respectively. Proventriculi (FFPE) obtained from suspect TVP cases (n=19) were evaluated for presence of CPNV by RT-PCR and microscopic lesions consistentwith TVP. CPNV was detected by RT-PCR in proventriculi from 8/11 TVP (+) cases (24/36 tissue sections). TVP (+) cases were defined by microscopic lesions characteristic of TVP; CPNV was not detected in proventriculi (0/8 cases, 0/32 tissue sections) in the absence of these lesions. The association between presence of TVP-characteristic microscopic lesions and presence of CPNV was highly significant (P = 0.0014). These findings indicate the utility of the RT-PCR procedure for detection of CPNV and provide additional evidence for an etiologic role for this virus in TVP.

Isolation and propagation of coronaviruses in embryonated eggs.

The embryonated egg is a complex structure comprising an embryo and its supporting membranes (chorioallantoic, amniotic, yolk). The developing embryo and its membranes provide the diversity of cell types that are needed for successful replication of a wide variety of different viruses. Within the family Coronaviridae, the embryonated egg has been used as a host system primarily for two group 3 coronaviruses, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV), but it also has been shown to be suitable for pheasant coronavirus. IBV replicates well in the embryonated chicken egg, regardless of the inoculation route; however, the allantoic route is favored as the virus replicates extensively in chorioallantoic membrane and high titers are found in allantoic fluid. TCoV replicates only in embryo tissues, within epithelium of the intestines and bursa of Fabricius; thus amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for studies aimed at identifying other, novel coronavirus species.

Experimental evaluation of Musca domestica (Diptera: Muscidae) as a vector of Newcastle disease virus.

House flies, Musca domestica L. (Diptera: Muscidae), were examined for their ability to harbor and transmit Newcastle disease virus (family Paramyxoviridae, genus Avulavirus, NDV) by using a mesogenic NDV strain. Laboratory-reared flies were experimentally exposed to NDV (Roakin strain) by allowing flies to imbibe an inoculum consisting of chicken embryo-propagated virus. NDV was detected in dissected crops and intestinal tissues from exposed flies for up to 96 and 24 h postexposure, respectively; no virus was detected in crops and intestines of sham-exposed flies. The potential of the house fly to directly transmit NDV to live chickens was examined by placing 14-d-old chickens in contact with NDV-exposed house flies 2 h after flies consumed NDV inoculum. NDV-exposed house flies contained approximately 10(4) 50% infectious doses (ID50) per fly, but no transmission of NDV was observed in chickens placed in contact with exposed flies at densities as high as 25 flies per bird. Subsequent dose-response studies demonstrated that oral exposure, the most likely route for fly-to-chicken transmission, required an NDV (Roakin) dose > or =10(6) ID50. These results indicate that house flies are capable of harboring NDV (Roakin) but that they are poor vectors of the virus because they carry an insufficient virus titer to cause infection.