Effects of chicken anaemia virus on experimental leukosis, induced by avian myelocytomatosis virus Mc29

The effects of concomitant infection with chicken anaemia virus (CAV) on the incidence, clinical manifestation and mortality from leukosis, induced by the avian myelocytomatosis virus strain Mc29 were studied. Experimental one-day-old 15 I line White Leghorn chickens were inoculated simultaneously with Mc29 and CAV or with Mc29 alone and observed daily for clinical signs and mortality. Both groups of chickens inoculated with Mc29 virus strain alone or in combination with CAV deve­loped tumours and died within 57 days. Necropsy has been performed on all dead birds following the standard protocol. Organ samples from thymuses, spleens, bone marrow, and livers were collected and histopathologically investigated. Neoplasms detected included myelocytomas, nephroblastomas and hepatocellular carcinomas. In addition, 50% of the CAV/Mc29-inoculated chickens developed epithelial type thymomas. However, no such lesions were found in chickens infected with Mc29 alone. No significant differences in the clinical course of leukosis between the two experimental groups of chickens were observed. The results indicated that CAV infection did not affect substantially the incidence and mortality from avian leukosis, induced by myelocytomatosis virus strain Mc29, but contributed to greater variety of the induced tumours.

CHICKEN ANAEMIA VIRUS IMPAIRS NITRIC OXIDE PRODUCTION IN HD11 CHICKEN MACROPHAGES

Immunosuppressive viruses cause substantial economic losses to the poultry industry. Chicken anaemia virus (CAV) causes severe disease in young chickens, whereas subclinical infection in older birds causes immunosuppression. In this study, we addressed the ability of CAV to interfere with production of antimicrobial molecule nitric oxide (NO) by macrophages. NO production in chicken macrophage cell line HD11 was induced using both Toll-like receptor 4 agonist, bacterial lipopolysaccharide, and an immune modulator, interferon-γ. In addition, we treated macrophages with CAV propagated in chicken lymphoblastoid cells. The levels of NO were measured by the Griess reaction. Addition of CAV decreased both the interferon-γ and the lipopolysaccharide associated induction of NO. Observed effect was not caused by CAV-related cytotoxicity, as no decrease in number of viable cells was observed. Although CAV could not completely abrogate NO production, attenuation of NO induction was clearly present. We have previously shown that CAV interferes with the expression of interferons in chickens during subclinical infection. Since the signalling pathways of expression of interferons and type 2 nitric oxide synthase, enzyme involved in NO formation, overlap, we conclude that measured decrease in NO levels is a consequence of CAV interference with interferon and NO synthase signalling. Regardless of the fact whether the attenuation of NO serves as a viral primary defence, or is only a secondary effect, it could impair the immune response to other pathogens and contribute to the global immunosuppression in chicken houses.Key words: chicken; immunosuppression; chicken anaemia virus (CAV); macrophage; nitric oxide (NO) VIRUS PIŠČANČJE ANEMIJE VPLIVA NA PROIZVODNJO DUŠIKOVIH OKSIDOV V MAKROFAGIH PIŠČANEV HD11 Povzetek: Imunosupresivni virusi povzročajo velike gospodarske izgube v perutninski industriji. Virus piščančje anemije (CAV) pri mladih piščancih povzroča hudo bolezen, medtem ko subklinična okužba pri starejših pticah povzroča oslabljen imunski odziv. V tej raziskavi je bil spremljan vpliv CAV na proizvodnjo dušikovih oksidov (NO) v makrofagih. Proizvodnja NO v piščančjih makrofagih v celični liniji HD11 je bila sprožena z uporabo agonista Toll-u podobnega receptorja 4, bakterijskega lipopolisaharida in imunskega modulatorja interferona-γ, makrofagi pa so bili okuženi s CAV, razmnoženim v piščančjih limfoblastoidnih celicah. Ravni NO so izmerili po Griessovi reakciji. Prisotnost CAV je zmanjšala proizvodnjo NO, spodbujeno tako z interferonom-γ, kot z lipopolisaharidom. Opaženega učinka ni povzročila citotoksičnost, povezana s CAV, saj ni bilo opaziti zmanjšanja števila živih celic. Čeprav CAV ni popolnoma zavrla nastajanja NO, je bilo očitno prisotno zmanjšanje nastajanja NO. Pred tem so pokazali, da CAV moti izražanje interferonov pri piščancih med subklinično okužbo. Ker se poti znotrajceličnega prenosa urejanja izražanja interferonov in sintaze dušikovih oksidov tipa 2, encima, ki sodeluje pri tvorbi NO, prekrivajo, predvidevamo, da je izmerjeno znižanje ravni NO posledica motenj CAV pri znotrajceličnem prenosu sporočila interferona do sintaze dušikovih oksidov. Ne glede na to, ali zaviranje nastajanja NO služi kot primarna virusna obramba ali je le sekundarni učinek, lahko poslabša imunski odziv na druge patogene in prispeva k splošnemu zmanjšanju imunskega odziva v kurnikih ali na kokošjih farmah.Ključne besede: piščanci; zmanjšanje imunskega odziva; virus piščančje anemije (CAV); makrofagi; dušikov oksid (NO)

Use of degenerate oligonucleotide primed polymerase chain reaction for detection of chicken anaemia virus contamination in avian viral vaccines

For quality control of biologicals of veterinary use, the absence of extraneous agents needs to be certified. One of the requirements for quality control of avian viral vaccines is to demonstrate freedom from extraneous and adventitious pathogenic agents, like chicken anaemia virus (CAV). In this study, a degenerate oligonucleotide primed PCR (DOP-PCR) for the detection of CAV was developed. Degenerate oligonucleotide primers were selected based on sequences corresponding to conserved regions of VP1 gene. After spiking of CAV genomic DNA to an infectious laryngotracheitis virus (ILTV) vaccine, detection limit for the test was 3.056×10-9 ng/µl. To evaluate the performance of the test, 11 avian viral vaccines including infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and ILTV vaccines from 5 manufacturers were screened for CAV and no contamination was detected. The test described here may provide a rapid, sensitive and specific method for contamination detection of avian viral vaccines with CAV, and may be applied for quality control of live and killed commercial vaccines.