Immune complex vaccines for chicken infectious anemia virus.

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 10(5.4) 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 ICN 160 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 Marek's 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.

Selection for increased nitric oxide production does not increase resistance to Marek's disease in a primary broiler breeder line.

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-gamma. 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 Marek's 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.

Marek's disease virus phosphorylated polypeptide pp38 alters transcription rates of mitochondrial electron transport and oxidative phosphorylation genes.

Two splice variants of the Marek's disease virus phosphorylated polypeptide (pp)38 were previously identified in the quail cell line QTP32 expressing pp38 under the control of an inducible promoter. We developed QT35-derived cell lines expressing these splice variants or full length pp38 with the splice acceptor sites mutated to further elucidate the role of pp38. Only induction of full length pp38 resulted in an increase in mitochondrial succinate dehydrogenase activity compared to non-induced cells. Transcript copy numbers of cytochrome C oxidase subunit I and ATP synthase were reduced in induced cells. The ATP content of isolated mitochondria from induced cells was greatly reduced compared to those of non-induced cells. Mitochondrial and pp38 staining suggests that there is no direct interaction between pp38 and the mitochondria. Mitochondrial transcripts were also reduced in DF-1 cells expressing full length pp38 and in MDV-infected chick kidney cells indicating that this effect occurs independent of other viral genes and after in vitro infection with MDV.

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.

Pro-inflammatory responses in chicken spleen and brain tissues after infection with very virulent plus Marek's disease virus.

In chickens infected with virulent (v) or very virulent (vv) Marek's disease (MD) virus (MDV) strains, small to moderate increases in plasma nitric oxide (NO) levels are seen, respectively, whereas very virulent plus (vv+) strains induce very high levels in vivo. The data presented in this report show that chickens presenting with clinical neurological disease following infection with the vv+ RK-1 strain have significantly higher in vivo NO levels compared to RK-1-infected non-symptomatic chickens. Using quantitative real-time PCR (qPCR) assays, DNA was used to measure MDV copy numbers in the spleen and brain of P2a (MD-susceptible) and N2a (MD-resistant) chickens following infection with the JM-16 (v) or RK-1 (vv+) strains. RNA was used to measure inducible NO synthase (iNOS), interferon-gamma (IFN-gamma), interleukin (IL)-1beta, IL-6, and IL-8 mRNA levels, in addition to MDV-specific mRNA expression using quantitative RT-PCR (qRT-PCR) assays. Viral DNA loads were found to be considerably higher in RK-1-infected chickens than JM-16-infected chickens at most time points in both organs, with viral copy numbers being two to four logs lower in the brain. Large increases in iNOS, IFN-alpha, IL-1beta, IL-6, and IL-8 were seen in the brains of RK-1-infected chickens. These data strongly support the hypothesis that pro-inflammatory responses, including high levels of iNOS/NO, IFN-alpha, and pro-inflammatory cytokine expression in the chicken brain, may play a major role in the neurological diseases associated with vv+MDV strains.

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.

Marek's 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.