Microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptase-polymerase chain reaction.

A method is described for enabling safe transit of denatured virus samples for polymerase chain reaction (PCR) identification without the risk of unwanted viable viruses. Cotton swabs dipped in avian infectious bronchitis virus (IBV) or avian pneumovirus (APV) were allowed to dry. Newcastle disease virus and avian influenza viruses were used as controls. Autoclaving and microwave treatment for as little as 20 sec destroyed the infectivity of all four viruses. However, both IBV and APV could be detected by reverse transcriptase (RT)-PCR after autoclaving and as long as 5 min microwave treatment (Newcastle disease virus and avian influenza viruses were not tested). Double microwave treatment of IBV and APV with an interval of 2 to 7 days between was tested. After the second treatment, RT-PCR products were readily detected in all samples. Swabs from the tracheas and cloacas of chicks infected with IBV shown to contain infectious virus were microwaved. Swabs from both sources were positive by RT-PCR. Microwave treatment appears to be a satisfactory method of inactivating virus while preserving nucleic acid for PCR identification.

In vitro effects of some steroidal hormones on the replication of avian pneumovirus.

The effects of testosterone, oestradiol, progesterone and cortisone on the in vitro replication of avian pneumovirus in tracheal organ cultures (TOC) were investigated. Samples of cell-associated and cell-free virus from TOC, grown in medium containing these hormones, were taken at selected intervals. Progesterone and cortisone caused a slight increase in cell-associated virus. Testosterone and oestradiol caused a slight delay and decrease in virus replication when compared with the controls. All groups shared the same time interval to reach peak cell-free virus titre, 96 h post inoculation. In comparison with the controls, only a small drop (0.25-0.50 log10) in the peak of virus titre was observed in the hormone treated groups.

Effect of heterophil depletion by 5-fluorouracil on infectious bronchitis virus infection in chickens.

An anti-tumour drug, 5-fluorouracil (5 FU) was used to deplete heterophils in 11-day-old white leghorn chickens. The reduction in heterophil numbers was monitored by total and differential white cell counts in the peripheral blood. Three days after injection of 5 FU, when the heterophil numbers were significantly reduced, chickens were infected with the Massachusetts strain of infectious bronchitis virus (IBV). Following infection, although the numbers of birds exhibiting clinical signs (nasal exudate) were significantly higher in the 5 FU treated group, the consistency of the nasal exudate was characteristically thin and watery. No significant differences were seen in the virus titres in trachea, lung and kidney between normal and heteropaenic chickens infected with IBV. However, the epithelial cell damage in the tracheal sections was less in the heterophil-depleted chickens.

Effect of storage of chicken and turkey blood on the lymphocyte responses to concanavalin A and pokeweed mitogen.

The effect of storage of whole blood of chicken and turkeys on the mitogen responses of lymphocytes to concanavalin A (con A) and pokeweed mitogen was investigated. It was found that, despite slight differences in optimum storage conditions with respect to mitogens, species and age of birds, blood could be stored for 24 h at 4 degrees C without significant reduction in the lymphoproliferative responses. However, even during the initial 24 h storage of blood at 4 degrees C, the levels of reduction in stimulation index (SI) values following con A stimulation, ranged from 27% for 3-4 week-old turkeys to 60% for 3-4 week-old white leghorn chickens.

Experimental inoculation of mice with trypsin-resistant and trypsin-sensitive avian reoviruses.

Groups of sucking Swiss albino mice were inoculated by the intracerebral (i.c.), intraperitoneal (i.p.) or oral route with a trypsin-sensitive avian reovirus (TR1) or a trypsin-resistant (R2) reovirus. The viruses caused a number of effects, the most severe occurring after i.c. inoculation and the least after oral inoculation. They included incoordination and tremors, oiliness of the hair, and retarded growth. Patterns of viral persistence in tissues were similar for the two viruses, with high titres in the brain on days 3 and 6 after i.c. or i.p. injection. Both viruses were still present in the brain 21 days after i.c. injection. No virus was found in any tissue when TR1 was given orally. All groups "seroconverted" except the one infected orally with TR1, but neutralization titres were low. The effects resembled those described for mammalian reoviruses in mice. The results indicate that, for short periods, wild mice may be capable of transmitting avian reoviruses between poultry flocks. Furthermore, in the production of monoclonal antibodies to avian reoviruses in mice, it is possible that pathological changes will occur.

Egg transmission of avian reoviruses in chickens: comparison of a trypsin-sensitive and a trypsin-resistant strain.

Two groups of commercial Light Sussex hens with no cultural evidence of reovirus infection and very low titres of neutralising antibodies were mated with cockerels from 17 weeks of age. At 27 weeks of age the birds were separated into three groups, and were inoculated intranasally and intravenously with avian reovirus strain R2 which is resistant to trypsin, with strain TR1 which is sensitive to the enzyme or sham-inoculated. Of the eggs laid by the hens infected with strain R2, 13/29 infertile eggs and embryos which fails to hatch were positive for virus, as were 6/70 hatched chicks. Despite this, virus was never isolated from cloacal swabs from the hens. Virus-infected eggs were laid between days 5 to 17 post inoculation (p.i.). Virus was isolated from the liver of all six R2 virus-positive chicks, from the hock joint of four and from the intestine of three. In contrast, for the group infected with the trypsin-sensitive virus TR1, of 120 eggs laid in the 5-week period, virus was isolated once only, from a chick hatched from an egg laid 7 days p.i. This infected chick was one of 83 which hatched and virus was found only in the joint. In a further experiment, two groups of mature SPF hens were inoculated with the reoviruses as above. Cloacal swabs and tissue examination showed greater virus excretion and tissue distribution of R2 than TR1. These results helped to explain the much higher egg transmission rate of R2 than TR1. However, the rate of vertical transmission of chicken reoviruses in nature, where the infectious dose would normally be lower than given here, is likely to be low.

Re-excretion of infectious bronchitis virus in chickens induced by cyclosporin.

Following inoculation of day-old chicks with Moroccan strain 'G' of infectious bronchitis virus (IBV) which has a predilection for the gut, virus was recovered from the trachea up to day 7 and from the cloaca up to day 35. After this no virus could be detected, even following the natural stress of re-housing with unfamiliar birds at 9 weeks. When the birds were 12.5 weeks old, they were injected with cyclosporin, a selective T-cell suppressor. Four of the five birds re-excreted virus very erratically, as did two of five contacts. This was accompanied by the appearance of IBV-specific IgM in the sera of both groups. The results suggest that in long-term infections with IBV, virus persistence is controlled by T lymphocytes.

Early pathogenesis in chicks of infection with a trypsin-sensitive avian reovirus.

Experiments are described which show how the sensitivity to trypsin of avian reovirus strain TR1 restricts its replication in the intestine of the chicken in comparison with a trypsin-resistant strain R2. Following oral infection with a high dose (5.3 log10 TCID50), the trypsin-sensitive virus generally showed lower titres than the resistant one in all tissues examined. Infection of chicks with strain TR1 via the respiratory route enabled the virus to spread throughout the body and localize in the hock joint, an important target site for reoviruses. Trypsin-sensitive reoviruses might be transmitted via the respiratory route, even though TR1 caused little damage to the respiratory epithelium. Dose-response studies showed that TR1 injected via the footpad can localize in the hock joint after very low doses, but high oral doses (4-5 log10) are necessary for such localization. Intranasal infection was intermediate in effect.

A comparison of direct electron microscopy, virus isolation and a DNA amplification method for the detection of avian infectious laryngotracheitis virus in field material.

Seventy-two post-mortem samples of mainly tracheal tissue from commercial chickens from 25 commercial chicken flocks with suspected infectious laryngotracheitis (ILT) were examined for the presence of the virus using direct electron microscopy (EM), virus isolation (VI) in primary chick embryo liver cell culture and a DNA amplification method (polymerase chain reaction; PCR). ILT virus was identified in 22 outbreaks, and in 58 of the 72 specimens. PCR detected virus in 52 of the 72 specimens and VI was positive in 48. In five instances, VI was positive where the other methods were negative and in three, PCR was the only test positive. Direct EM examination detected virus in only 19 of the 58 positive samples and in no case was EM the only method positive. An advantage of PCR was that it could sometimes detect virus in samples that were too heavily contaminated with bacteria for virus to be isolated and on other occasions it was positive for ILT virus when the only virus that could be detected by growth in tissue culture was adenovirus.

Isolation of infectious laryngotracheitis virus from proximal femora of lame broiler chickens.

Two herpesviruses previously isolated from seven of 19 affected joint/bone samples in an earlier survey of lameness in broilers were identified. They were characterised as infectious laryngotracheitis (ILT) virus using serum neutralisation, immunofluorescence, restriction enzyme analysis and polymerase chain reaction techniques. In experimentally infected chicks, one of the isolates caused mild ILT and intranuclear inclusion bodies were present in the tracheal epithelium after four days. It is considered unlikely that these viruses were involved in the pathological changes in the affected legs. The possibility that ILT pathogenesis and epidemiology are more complex than currently understood is discussed.

Further studies on the development of a live attenuated vaccine against turkey rhinotracheitis.

Turkey rhinotracheitis (TRT) virus attenuated by passaging in Vero cells was tested at two different passage levels (15 or 25 passages) and two dose levels [10(3) or 10(4) TCID50 (50% tissue culture infectious doses) per bird] to determine the efficacy in protecting turkey poults against experimental challenge with virulent TRT virus. Following administration by the eyedrop route at 10 days of age, all four preparations proved successful in providing protection against clinical disease and establishment of challenge virus in the trachea when challenged with virulent virus 3 weeks later. Twelve-day-old poults given the 25th Vero passage TRT virus at a dose of 10(3.5) TCID50 per bird were protected against experimental challenge with virulent virus for at least 22 weeks post-primary inoculation. The 25th passage virus was tested for safety by administering ten times the dose (10(4.5) TCID50 per bird) used in the previous trial to a group of 10-day-old turkey poults. None of the birds showed any clinical signs during 21 days post-inoculation. Attempts to back-passage the virus from bird to bird were unsuccessful.

Development of a live attenuated vaccine against turkey rhinotracheitis.

Three preparations of a strain of turkey rhinotracheitis (TRT) virus were tested for their ability to protect turkey poults against challenge with virulent virus given 3 weeks later. The preparations were as follows: one had been passaged in turkey embryo tracheal organ culture (TOC) 98 times, another had been passaged in primary chick embryo fibroblast (CEF) monolayers 28 times and the third had undergone 17 passages in Vero cell monolayers. Each was administered by the eyedrop route to groups of 21-day-old TRT-seronegative turkey poults. The TOC preparation caused clinical signs consistent with TRT infection, indicating the virus had not been attenuated. The CEF and Vero preparations produced no clinical effects. Following challenge with virulent TRT virus at 21 days post-inoculation, the CEF group developed clinical signs consistent with TRT but the TOC and Vero virus groups showed none. All other parameters correlated with these findings. All groups showed an increase in specific SN and ELISA antibodies following challenge. These results indicated that after relatively few passages in Vero cells, this strain of TRT virus became satisfactorily attenuated and was able to offer protection against clinical disease following experimental challenge. Two of the three virus preparations (TOC and Vero) were also shown to spread from the inoculated birds to uninoculated contact birds, introduced into the groups at 5 days post-inoculation, and they induced protection in these contacts against virulent virus challenge.

Experimental infection of laying turkeys with rhinotracheitis virus: distribution of virus in the tissues and serological response.

Twenty-four laying turkey hens shown to be free of antibodies to turkey rhinotracheitis virus were inoculated intranasally with an isolate of the virus. A mild respiratory disease developed between 5 and 9 days post infection (pi). Two birds were selected at random at intervals between days 1 and 20 pi, killed and tissues examined for the presence of virus. At autopsy between days 2 and 12 abnormalities were found in the oviducts including the deposition of inspissated albumen. Yolk material was occasionally found in the abdominal cavity and there was one instance of egg peritonitis. Eggs with abnormal shells were found in the uterus on days 3 and 9. By direct immunofluorescence (IF) staining, virus was detected in the trachea between days 1 and 7 pi and in the turbinates between days 2 and 5 pi. Virus could also be isolated from these sites using turkey embryo tracheal organ cultures but this method was slightly less sensitive than IF for these tissues. No virus was demonstrated in the lungs or air sacs. Viral antigens were detected by IF in the epithelium of the uterus on day 7 pi and in this and all other regions of the oviduct on day 9 pi. Virus was isolated only from middle magnum and vagina on day 9 pi. On other occasions up to 20 days pi the above tissues and spleen, ovary, liver, kidney and hypothalamus were all negative for virus. Antibodies detected by ELISA and serum neutralisation both reached, high titre by 12 days pi and were maintained at a high level (Iog2 12-15) throughout the period of observation (89 days).

Effects of experimental immunosuppression on reovirus-induced tenosynovitis in light-hybrid chickens.

Groups of specific pathogen-free light-hybrid chickens which had been immunosuppressed either by surgical thymectomy (Tx) or surgical bursectomy (Bx) or cyclophosphamide (Cy) treatment or Tx plus Cy treatment (Tx + Cy), as well as intact (untreated) birds, were inoculated with graded doses of an arthrotropic avian reovirus at 1 day of age and observed up to 5 weeks post-inoculation (p.i.). Cy-treatment with or without Tx considerably increased the mortality, incidence of gross leg lesions and severity of microscopic lesions due to reovirus infection. The Bx group showed only a significant increase in mortality, and the Tx group response was generally similar to the untreated group. Dead birds showed hepatic necrosis, which in Cy-treated groups (Cy, Tx + Cy) was associated with calcification. Surviving Cy-treated birds had acute tenosynovitis characterised grossly by large amounts of serous exudate in leg tendon sheaths, and microscopically by a massive heterophilic but only mild lymphocytic infiltration of tendon sheaths. Tenosynovitis lesions in Bx birds were generally similar to those of the untreated chickens, i.e. grossly small amounts of yellowish brown gelatinous exudate and microscopically moderate chronic inflammatory changes in leg tendon sheaths. In Tx birds gross lesions were rarely seen and the microscopic lesions were often very mild. Reovirus could be recovered from cloacal swabs from untreated and Tx birds for 2 weeks, Bx birds for 3 to 4 weeks, and Cy and Tx + Cy chickens continuously throughout. Reovirus was isolated from tendon tissue of all Cy and Tx + Cy infected birds examined at 5 weeks p.i. and gross tenosynovitis lesions were seen in all birds. The virus was recovered from the tendons of only a proportion of the infected untreated, Tx and Bx groups, and overall more frequently from apparently normal birds. This was especially marked in the infected Tx group. Antibody responses as shown by gel precipitation and virus neutralisation were positive and similar in untreated and Tx birds, were delayed in the Bx group with the precipitation test only, and absent from most of the Cy and Tx + Cy birds. The results of these experiments indicate that recovery from reovirus infection probably involves both B- and T-cell systems but that the B-cell system is predominantly protective.

Experimental reovirus infection in chickens: observations on early viraemia and virus distribution in bone marrow, liver and enteric tissues.

The nature of viraemia and tissue distribution of reovirus were studied in the early phase after oral infection of 1-day-old specific-pathogen-free (SPF) White Leghorn chicks with the R2 strain of avian reovirus. A range of tissues collected up to 3 weeks after infection was titrated for their viral content. Virus was present in the plasma, erythrocyte and mononuclear fractions of the blood within 30 hours post-inoculation (p.i.) and was widely distributed in tissues, including the bone marrow by 3 to 5 days p.i. A greater part of the viraemia was associated with plasma, virus in the blood mononuclear fraction being detected only occasionally. There was more infectious virus in the duodenum than the liver and the highest virus titres were found in cloacal swabs taken 1 to 5 days p.i. It was also evident that virus reached the liver within a very short time after infection (<6 hours p.i.) although the source of this early hepatic virus was considered to be residual inoculum absorbed directly into the portal blood. Viraemic virus titres could not be correlated either with duodenal or hepatic virus titre alone.