Priscilla H. O'Connell

Influence of Genetic Resistance of the Chicken and Virulence of Marek's Disease Virus (MDV) on Nitric Oxide Responses After MDV Infection

SUMMARY. Nitric oxide (NO), a free radical produced by the enzyme NO synthase (NOS), is a potent antiviral agent in addition to having immune regulating functions. Recently, it was reported that chickens resistant (N2a, MHC: B21B21) to the development of Mareks disease (MD) had a greater potential to produce NO than MD-susceptible chickens (P2a, MHC: B19B19). This difference was shown by measuring NO levels in chick embryo fibroblast cultures obtained from these chickens after treatment with lipopolysaccharide and recombinant chicken interferon-gamma (IFN-γ). To extend these results, the levels of NO in blood plasma from N2a and P2a chickens inoculated with the nonattenuated JM-16 strain of MD virus (MDV) were examined. In four out of five experiments, N2a chickens had increased NO levels at 7 days postinoculation (DPI). In contrast, P2a chickens challenged with JM-16 had a significant increase in NO in only one of four experiments, and in that experiment the increase was delayed (10 DPI) compared with N2a chickens. Attenuation abrogated MDV-induced NO in chickens. Inoculation with MDV strains ranging from mild to very virulent plus showed that the more virulent strains induced the highest level of NO in blood plasma, suggesting a role of NO in the pathogenesis of MD with more virulent strains. On the basis of quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) assays for analysis of mRNA expression, IFN-γ does not appear to be the primary inducer of inducible (i)NOS gene expression during MDV infection. iNOS gene expression and NO production are mediated during the cytolytic phase of MDV infection on the basis of real-time RT-PCR assays with primers specific for glycoprotein B, a late gene expressed only during the cytolytic phase of MDV infection. These findings implicate NO as a factor potentially involved in increasing virulence of MDV, possibly through immune suppression.

Detection and quantification of Mycoplasma gallisepticum genome load in conjunctival samples of experimentally infected house finches (Carpodacus mexicanus) using real-time polymerase chain reaction.

A TaqMan®-based real-time, quantitative polymerase chain reaction (qPCR) assay utilizing the mgc2 gene was developed to detect Mycoplasma gallisepticum in conjunctival swabs of experimentally infected house finches. The assay was demonstrated to be quantitative by the standard curve method with reproducible results within runs and between runs. The detection limit of the mgc2 assay was examined using two standards. The test had a detection limit of less than 14 copies per reaction when tested with a plasmid standard and less than 10 copies per reaction when tested with M. gallisepticum genomic DNA. All M. gallisepticum-negative birds (10 specific pathogen free chickens and 10 house finches) were negative by mgc2 qPCR assay. Existing evidence suggests that an important part of M. gallisepticum pathogenesis includes both its attachment to and invasion of host cells. Thus, our test also made use of rag-1 as an internal control gene. The rag-1 qPCR results showed that host cell quantity varied greatly between conjunctival samples. After inoculation, M. gallisepticum levels in the house finch conjunctiva increased over the 7-day period post infection. The bird with the most pronounced clinical conjunctivitis harboured the highest level of M. gallisepticum and the bird that did not develop conjunctivitis had very low numbers of M. gallisepticum. Thus, it appears that development of conjunctivitis may correlate with M. gallisepticum load.

Humoral Immune Responses to Chicken Infectious Anemia Virus in Three Strains of Chickens in a Closed Flock

This is a comparative study on seroconversion to chicken infectious anemia virus (CIAV) in a closed flock of specific-pathogen-free chickens undergoing a natural outbreak and after vaccination of some of these flocks with a commercial, live vaccine. The N2a strain (B21B21 haplotype) had the highest seroconversion after natural infection (94%) or vaccination (100%), followed by the P2a strain (B19B19) at 75%-82% seroconversion after natural infection and 85% seroconversion after vaccination. The S13 (B13B13) chickens were 26% seropositive after natural infection and 75% seropositive after vaccination. N2a chickens with polymerase chain reaction (PCR)-positive tissues were 97% seropositive compared to 80%-83% PCR-positive and seropositive for the P2a chickens and only 8% seropositive and PCR-positive for the S13 chickens. Seroconversion occurred at or near sexual maturity after natural infection in seven flocks studied.

Impact of deletions within the Bam HI-L fragment of attenuated Marek's disease virus on vIL-8 expression and the newly identified transcript of open reading frame LORF4

Mareks disease (MD) in chickens is caused by MD herpesvirus (MDV), which induces T cell lymphomas. The early pathogenesis of MDV infection is characterized by a primary infection in B lymphocytes followed by infection of activated T lymphocytes. It has been speculated that a MDV-encoded homologue of interleukin-8 (vIL-8) may be important to attract activated T lymphocytes to infected B lymphocytes. Recently, more virulent strains of MDV have emerged, named very virulent plus (vv+)MDV, that cause earlier and more prolonged cytolytic infections compared to less virulent strains. In this report, it was found that vIL-8 mRNA expression in vivo was increased in very virulent (vv) and vv+MDV strains compared to mild (m) and virulent (v) strains, and could not be detected in two attenuated MDV strains examined using very sensitive real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assays. In order to identify potential mechanisms for the increased vIL-8 mRNA expression in more virulent strains, and lack thereof in attenuated strains, the vIL-8 gene and putative promoter sequences upstream of the vIL-8 gene were compared from 10 different MDV strains, including attenuated derivatives. Only the JM-16 strain (both non-attenuated and attenuated) and attenuated 584A (584Ap80C) encoded a predicted vIL-8 gene sequence different from all other strains examined. Within the putative vIL-8 gene promoter sequence, there was little difference among the non-attenuated strains; however significant deletions were identified in the attenuated JM-16/p71, Md11 (R2/23), and 584Ap80C strains. Additionally, these deletions were located within a previously hypothetical open reading frame (ORF) named LORF4. Rapid amplification of cDNA ends identified a full-length transcript of LORF4 in the MDV-transformed lymphoblastoid cell line MSB-1, and deletions within this ORF caused truncated predicted proteins in 4 out of 6 attenuated MDV strains examined.

Characterization of Proteins of Chicken Infectious Anemia Virus with Monoclonal Antibodies

Eight monoclonal antibodies (MAbs) against chicken infectious anemia virus (CIAV) were developed. These MAbs identified three isolates adapted to grow in the Mareks disease chicken cell line MSB1 (Cux-1, GA-1, and Conn-B) and the chicken-propagated CIA-1 isolate. All MAbs stained MSB1 in the same way with mostly perinuclear staining, although larger nuclear inclusions and cytoplasmic staining were also detected. None of the MAbs neutralized Cux-1. All MAbs reacted in a direct enzyme-linked immunosorbent assay with Cux-1 antigen treated with 0.5% sodium dodecyl sulfate followed by extraction with chloroform, but not with MSB1 cells infected with Cux-1 or chloroform-extracts of these cells. Three viral proteins--VP1, VP2, and VP3--with estimated sizes of 45, 30, and 16 kilodaltons (kd), respectively, were immunoprecipitated using the MAbs and Cux-1-infected cell lysates. The 16-kd protein was the major VP. In addition, a 79-kd protein was detected in infected cell lysates by immunoprecipitation with CIAV-antibody-positive and -negative chicken serum, and CIAV-specific and non-specific MAbs.

Detection of retrovirus sequences in budgerigars with tumours.

Renal tumours are a common neoplastic disease of budgerigars. Although a retro-virus has been implicated as the aetiological agent, there is no definitive proof for this hypothesis. Sixteen birds suspected to have renal tumours were examined in an attempt to elucidate the possible role of retroviruses. Thirteen birds had renal tumours and the majority of these birds showed abdominal enlargement and paresis. Renal masses were detected by radiography in nine birds. Post-mortem examination confirmed the presence of abdominal tumours which were mostly confined to the kidneys. All of the renal tumours were carcinomas. ELISA tests to detect the presence of p27 of avian leukosis virus and virus isolation attempts were negative. DNA from eight tumours was examined by dot-blot hybridization for the presence of sequences hybridizing with a full length clone of the RAV-2 strain of the avian leukosis virus. A positive reaction was detected with DNA from 6/8 tumours. Southern blot hybridization demonstrated the presence of a 7.2 kb fragment following restriction with BamHI and a 4.6 kb fragment in an additional tumour following digestion with EcoRI that were recognized by the RAV-2 probe. These results suggest the presence of a retrovirus in tumours of budgerigars.

Selection for Increased Nitric Oxide Production Does Not Increase Resistance to Marek's Disease in a Primary Broiler Breeder Line

Abstract Two primary broiler breeder lines, A and B, were examined for their potential to produce nitric oxide (NO) after stimulating splenocytes from 20-day-old embryos with lipopolysaccharide and interferon-γ. Significant differences were found between lines A and B. Overall, line A had a higher response than line B, but line A also had a large degree of variation between individual sire families. Selection for high and low responders within line A resulted in the segregation of high- and low-responder sire families. Offspring from sire families selected for high and low NO responses and from a nonselected control group from line A were challenged with RB-1B Mareks disease (MD) virus to determine whether these differences could be used to select for improved resistance to MD. Virus isolation rates at 6 and 10 days postinfection were not significantly different, but unexpectedly, the MD incidence in the high-responder group was significantly higher than in the other two groups.

Immune Complex Vaccines for Chicken Infectious Anemia Virus

SUMMARY. Infection of maternal, antibody-negative chickens with chicken infectious anemia virus (CIAV) can cause clinical disease, while infection after maternal antibodies wane often results in subclinical infection and immunosuppression. Currently, vaccines are not available for vaccination in ovo or in newly hatched chickens. Development of CIAV vaccines for in ovo use depends on the ability to generate vaccines that do not cause lesions in newly hatched chicks and that can induce an immune response regardless of maternal immunity. Immune complex (IC) vaccines have been successfully used for control of infectious bursal disease, and we used a similar approach to determine if an IC vaccine is feasible for CIAV. Immune complexes were prepared that consisted of 0.1 ml containing 105.4 tissue culture infective dose 50% of CIA-1 and 0.1 ml containing 10 to 160 neutralizing units (IC Positive [ICP]10 to ICP160), in which one neutralizing unit is the reciprocal of the serum dilution required to protect 50% of CU147 cells from the cytopathic effects caused by CIA-1. Virus replication was delayed comparing ICP80 and ICP160 with combinations using negative serum (IC Negative [ICN]80 or ICN160). In addition, the number of birds with hematocrit values <28% were decreased with ICP80 or ICP160 compared to ICN80 or ICN160. Seroconversion was delayed in ICP80 and ICP160 groups. To determine if ICP80 or ICN160 protected against challenge, we vaccinated maternal, antibody-free birds at 1 day of age and challenged at 2 wk or 3 wk of age with the 01-4201 strain. Both ICP80 and ICP160 protected against replication of the challenge virus, which was measured using differential quantitative PCR with primers distinguishing between the two isolates. Thus, in principle, immune complex vaccines may offer a method to protect newly hatched chicks against challenge with field virus. However, additional studies using maternal, antibody-positive chicks in combination with in ovo vaccination will be needed to determine if immune complex vaccines will be useful to protect commercial chickens.

Role of Marek's disease herpesvirus in the induction of tumours in Japanese quail (Coturnix coturnix japonica) by methylcholanthrene.

The QT35 cell line, established from 20-methylcholanthrene (MCA)-induced tumours in Japanese quail, is positive for Mareks disease virus (MDV), and therefore we examined whether MDV is important for the development of MCA-induced tumours. Japanese quail were inoculated with the JM16 strain of MDV at 1 or 3 days of age or left uninoculated. At 3 weeks of age, quail were injected in the breast muscle with 4 mg MCA in corn oil or corn oil alone. Quail were observed for tumours three times/week and at post mortem at 11 to 12 weeks of age. MDV DNA was detected by polymerase chain reaction (PCR) in spleens of 14/20 birds inoculated with JM16+corn oil and of 53/71 birds inoculated with JM16+MCA. Interestingly, 1/74 quail was positive in the MCA group alone for MDV DNA. Tumours were collected for histopathology, cell line development, and PCR and reverse transcriptase-PCR for the presence of MDV. Tumours developed in 38/83 MCA-treated and 32/85 JM16+MCA-treated quail. Fibrosarcomas without metastasis were the only tumours observed in the MCA-treated quail, while quail treated with JM16 and MCA developed undifferentiated tumours, fibrosarcomas, lymphosarcomas or combinations with or without metastasis. One out of 20 quail receiving JM16 alone developed a lymphosarcoma. Cell line development was not influenced by JM16. Tumours from MCA-treated quail were negative for MDV, while 19/29 were positive in the JM16+MCA group. MDV transcripts were present in 13/18 tumours examined in the JM16+MCA group. In conclusion, MDV did not affect tumour development but did influence tumour aggression and histological type.