Rapid passage of avian reovirus in one-day-old chicks: Clinical and virological findings

Two avian reoviruses, strain Reo-25 and isolate W3-492 were inoculated orally in 1-day-old chicks. Three to seven days post inoculation (dpi), the liver, spleen, pancreas, caecal tonsil and duodenum were collected, weighed and titrated in cell culture for their viral content. The different tissue homogenates collected were passaged several times in 1-day-old chicks. Reo-25 virus was passaged only at 3-day intervals and W3-492 virus was passaged at 3- and 7- or 14-day intervals. For both Reo-25 and W3-492 viruses, pathological effects and virus yields in tissues decreased with continued passages. In direct comparisons of reovirus W3-492 prior to chicken passage (PO) and after four passages at 7-day intervals (P4) using standardised amounts of virus for inoculation of chickens, no major differences in pathological effects were observed. P4 virus could be recovered from duodenal tissue at 28 dpi and from liver tissue at 14 dpi. In contrast, PO virus could be recovered from duodenal tissue at 14 dpi and from liver tissue at 10 dpi.     

The use of virus isolation, histopathology and immunoperoxidase techniques to study the dissemination of a chicken isolate of avian pneumovirus in chickens

A subgroup B isolate of turkey rhinotracheitis virus (TRTV) or avian pneumovirus (APV), obtained from a flock of commercial breeding chickens experiencing poor egg production, mortality and swollen head syndrome, was shown to cause substantial respiratory signs in both young SPF chickens and chicks with high levels of maternally derived TRT antibodies. This isolate replicated to high titre in the respiratory tract of experimentally inoculated SPF chickens for approximately 5 days after inoculation, but was recovered only occasionally after that time. It was never recovered from non-respiratory tract tissues. A detailed, sequential histological and immunoperoxidase study was performed. This revealed that, whilst TRT virus could be demonstrated consistently in the epithelium of upper respiratory tract tissue, although only for a short time after inoculation, the damage which it caused was minimal and recovery was rapid. This study, using a pathogenic TRT isolate obtained from diseased chickens, provides clear evidence that TRT virus can cause damage to the respiratory tract of chickens and that this damage is both localized and short lived.     

Intracloacal infection with avian infectious bronchitis virus

An infectious bronchitis virus (IBV) strain HS-91 isolated from kidneys of chicks which died of nephrosis was inoculated via the cloaca of specific pathogen-free (SPF) chicks. These chicks showed more severe clinical signs, grosser kidney lesions and higher mortality than chicks inoculated with the same IBV strain via the trachea.     

Pathogenicity of chicken anaemia virus (isolate 10343) for young and older chickens.

One-day-old chicks, inoculated intramuscularly (i.m.) with the chicken anaemia virus (CAV) isolate 10343, showed depression of body weight gain and anaemia, particularly between days 14 and 21 post-inoculation (p.i.)- The weights of thymus and bursa were substantially reduced compared to controls at days 14 and 21 p.i. The histological lesions detected in thymus, bursa, spleen and liver were similar in frequency at days 14 and 21 p.i. Eosinophilic intranuclear inclusion bodies, lymphocyte depletion, and focal necrosis were detected in the thymus, spleen, bursa and liver of more than 50% of the inoculated chicks at days 14 and 21 p.i. Focal necrosis and vacuolar degeneration in the liver, as well as apoptosis in different organs were more evident at days 14 and 21 p.i. Ten-week-old broiler breeders, inoculated i.m. with isolate 10343 showed pathological changes that were less severe than the changes shown by 1-day-old chicks. No anaemia could be detected in this group. However, severe thymus atrophy, and histological lesions in bursa, spleen, and liver, were also evident at days 14 and 21 p.i. in some of the inoculated birds. Viral detection by immunofluorescence using a monoclonal antibody revealed a wide distribution of the CAV isolate. CAV antigen was detected until day 21 p.i. in thymus, spleen, bursa and liver. According to the severity of the lesions shown by 1-day-old chicks, the length of the period in which CAV antigen could be detected in tissues, and the fact that CAV isolate 10343 was capable of inducing disease in 10-week-old chickens, it seems that this CAV isolate may be particularly virulent.     

Immunopathogenesis of infection in SPF chicks and commercial broiler chickens of a variant infectious bronchitis virus of economic importance.

The immunopathogenesis of an economically important variant of infectious bronchitis virus (IBV) has been studied in day-old specific pathogen-free and 6-week-old broiler chickens. Experimental infection caused very mild respiratory disease and no mortalities. Diarrhoea was seen in the broilers. Virus distribution and titre in respiratory, urinary and enteric tissues were assessed. In SPF chicks, maximum virus isolations were from oesophagus, followed by trachea, kidney, lung and rectum. In contrast, in the broilers, maximum isolations were from ileum, followed by caecal tonsils, rectum, bursa of Fabricius and harderian gland, but virus was recovered less frequently from trachea, lung and kidney. In both groups virus replication was demonstrated in the epithelium of the trachea, kidneys and tissues of the lower gut by immunofluorescence. Immunostaining for CD4 and CD8 T-cell subsets in trachea, lung and kidney showed earlier and increased recruitment of CD8 cells over CD4 cells. From the kidneys of SPF chicks, no virus was isolated on days 10 or 14 p.i., but was recovered to high titre on day 21. Immunostaining showed the presence of IgM deposits in the glomeruli on those days when virus could not be recovered. In the broilers, gross muscle changes were produced which were less severe than reported in the field and without histopathological changes or increased creatine kinase levels in serum. There was no evidence of virus-induced immunosuppression. The spread of infection and rate of seroconversion to this IBV were similar to other IBV strains. The possible pathogenesis of the muscle changes is discussed.     

Influenza A virus (H5N1) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts

The ongoing outbreak of avian influenza A virus (subtype H5N1) infection in Asia is of great concern because of the high human case fatality rate and the threat of a new influenza pandemic. Case reports in humans and felids suggest that this virus may have a different tissue tropism from other influenza viruses, which are normally restricted to the respiratory tract in mammals. To study its pathogenesis in a mammalian host, domestic cats were inoculated with H5N1 virus intratracheally (n = 3), by feeding on virus-infected chicks (n = 3), or by horizontal transmission (n = 2) and examined by virological and pathological assays. In all cats, virus replicated not only in the respiratory tract but also in multiple extra-respiratory tissues. Virus antigen expression in these tissues was associated with severe necrosis and inflammation 7 days after inoculation. In cats fed on virus-infected chicks only, virus-associated ganglioneuritis also occurred in the submucosal and myenteric plexi of the small intestine, suggesting direct infection from the intestinal lumen. All cats excreted virus not only via the respiratory tract but also via the digestive tract. This study in cats demonstrates that H5N1 virus infection causes systemic disease and spreads by potentially novel routes within and between mammalian hosts.     

Attempts to reproduce swollen head syndrome in specific pathogen-free chickens by inoculating with Escherichia coli and/or turkey rhinotracheitis virus.

Attempts to reproduce swollen head syndrome (SHS) lesions were carried out in specific pathogen-free (SPF) chickens. In Experiment 1, chickens inoculated into the submucosal tissue of the nasal membrane or subcutaneous tissue of eyelids with four different strains of Escherichia coli, developed typical SHS lesions; purulent and necrotic lesions of facial subcutis (especially around their eyelids), periocular connective tissue, infraorbital sinus, air spaces, middle ears and eyeballs. The lesions elsewhere included splenic necrosis with fibrinous exudation, fibrin thrombi in hepatic sinusoids, and fibrinopurulent epicardi-tis and perihepatitis which occasionally accompanied lesions in SHS cases. In Experiment 2, SPF chickens inoculated intranasally with turkey rhinotracheitis (TRT) virus and/or E. coli showed no significant lesions in the facial skin, upper respiratory tract or other organs. However, the presence of TRT antibodies demonstrated that the virus infected the chickens. This study suggests that E. coli may play a significant role in the pathogenesis of SHS, but that the significance of TRT virus in the pathogenesis is still to be clarified.     

Systemic and mucosal isotype-specific antibody responses in pigs to experimental influenza virus infection

The immunoglobulin isotype-specific responses in serum and at the respiratory mucosa of pigs after a primary infection with influenza virus were studied. To do this, we developed an aerosol challenge model for influenza in specified pathogen-free (SPF) pigs and isotype-specific enzyme-linked immunosorbent assays (ELISAs). Ten-week-old pigs were inoculated without anesthesia in the nostrils with an aerosol of the field isolate influenza A/swine/Neth/St. Oedenrode/96 (H3N2). The infection caused acute respiratory disease that closely resembled the disease observed in some outbreaks of influenza among finishing pigs, which were not complicated by bacterial infections. Pigs showed clinical signs characterized by fever, dyspnea, and anorexia. At necropsy on postinfection days 1 and 2, an exudative endobronchitis was observed throughout the lung. Viral antigen was present in the epithelial cells of the bronchi and bronchioli and virus was isolated from bronchioalveolar and nasal lavage fluids and from pharyngeal swabs until 5 days after infection. With the isotype-specific ELISAs, viral nucleoprotein specific immunoglobulin (Ig) M, IgG1, and IgA antibody responses were measured in serum and bronchioalveolar and nasal lavage fluids. To determine whether the antibodies were produced and secreted at the respiratory mucosa or were serum-derived, the specific activity (ie, the ratio of antibody titer to Ig concentration) was calculated for each isotype. The IgA and interestingly also a substantial part of the IgG1 antibody response in pigs upon infection with influenza virus was shown to be a mucosal response. Local production of specific IgA in the nasal mucosa, and of specific IgA and IgG1 in the lung was demonstrated. These results indicate that protective efficacy of vaccination can be improved by an immunization procedure that preferentially stimulates a mucosal immune response. The aerosol challenge model in SPF pigs and the isotype-specific ELISAs that we developed can be useful for evaluating various strategies to improve efficacy of porcine influenza vaccines and to study the immune mechanisms underlying the observed protection.     

Reovirus-induced tenosynovitis in chickens: the effect of breed

The effect of breed of chicken on infection with an arthrotropic avian reovirus strain R2 was studied by oral or footpad inoculation of 1-day-old chicks of the following breeds: (1) specific pathogen-free (SPF) light-hybrid, (2) commercial White Leghorn egg-layer, and (3) commercial Ross I broiler, and observed to 12 weeks of age. Although most inoculated birds of all three breeds developed swelling of one or both legs below the hock joint at 3 to 4 weeks of age, gross lesions of tenosynovitis became progressively more severe and extended above the joints only in broilers, whereas in most orally-infected SPF and commercial light chickens gross lesions were intermittently severe and regressed with time. Cloacal virus shedding continued up to 2 weeks in the lighter breeds and 3 weeks after infection in broilers. From a small proportion of infected chickens, reovirus was also reisolated from heart, pancreas and caecal tonsils. In all breeds, the tissue in which virus persisted longest was the hock joint/tendon. There was a poor correlation between isolation of virus and the presence of gross lesions in chickens of 12 weeks of age, especially in broilers. Virus-neutralisation tests demonstrated that seroconversion in the lighter breeds occurred predominantly at 3 weeks and in broilers at 4 weeks after infection. In all three breeds the footpad infection gave significantly lower growth rates than were found in the control and oral-infection groups. Oral infection had no apparent effect on growth rates. The greater susceptibility of broilers to reovirus infection is discussed.     

Effect of multiple cell culture passages on the biological behaviour of chicken anaemia virus.

The Cux-1 isolate of chicken anaemia virus (CAV) was passaged over 320 times in Marek's disease virus transformed chicken lymphoblastoid (MDCC-MSB1) cells. Comparison of the infectivity titres of virus pools derived from viruses that had received 0 (P0), 49 (P49), 170 (P170) and 320 (P320) passages in our laboratory indicated that the yields of infectious virus increased over 100-fold with passage number from P0 to P170. P320 exhibited unusual cell culture growth characteristics in that, unlike its lower passage counterparts, virus-specific immunofluorescence (IF) and cytopathic effect were detected at very low levels at 2 days post infection, with an additional passage of infected cells into fresh medium being required to produce high levels of infectious virus. Experimental infection of 1-day-old SPF chicks showed that P170 and P320 were substantially attenuated compared to P49 and a pathogenic, low-passage isolate used as control. When assessed by the indirect IF test, infection of 1-day-old chicks with the P49 and P170 isolates elicited similar levels of CAV-specific antibody to those elicited by infection with the pathogenic, low-passage virus and higher than those elicited by infection with the P320 isolate. Experimental infection of 5-week-old chicks indicated that the attenuated P170 and P320 isolates invoked similar CAV-specific antibody levels to those invoked by the pathogenic P49 and control isolates.     

Astrovirus-induced “white chicks” condition – field observation,virus detection and preliminary characterization

Chicken astrovirus (CAstV) was recently indicated as the factor of the “white chicks” condition associated not only with increased embryo/chick mortality but also with weakness and white plumage of hatched chicks. In February 2014, organ samples (livers and kidneys) from dead-in-shell embryos, as well as 1-day-old whitish and normal chicks, were delivered from one hatchery in Poland for disease diagnosis. The samples originated from the same 30-week-old breeder flock in which the only observed abnormal signs were 4–5% decrease in the number of hatched chickens and the presence (about 1%) of weaker chicks with characteristic whitish plumage among normal ones. CAstV was detected in submitted samples and was then isolated in 10-day-old embryonated specific pathogen free (SPF) chicken eggs. We also reproduced an infection model for the “white chicks” condition in SPF layer chickens using the isolated PL/G059/2014 strain as the infectious agent. Results of experimental reproduction of the “white chicks” condition were somewhat more serious than field observation. The administration of the CAstV material into the yolk sac of 8-day-old SPF chicken eggs caused delay and prolongation of hatching, as well as death of embryos/chicks, and also a change of plumage pigmentation. Only two chicks of a total of 10 inoculated SPF eggs survived and were observed for 2 months. A gradual elimination of the CAstV genome was noted in this period. Moreover, a few contact-naive SPF chicks, which had been placed in the same cage, were infected with CAstV. Molecular characterization of detected CAstV was performed by nucleotide sequencing of the full ORF2 region encoding the capsid precursor protein gene. Phylogenetic studies showed that the PL/G059/2014 isolate clustered in the subgroup Aiii of CAstV. In the light of the new classification rules, the Polish PL/G059/2014 CAstV isolate could be assigned to a new species of the Avastrovirus genus.     

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.     

Reisolation of avian arthrotropic reovirus r2 from chicks infected as embryos

Four groups of specific-pathogen-free (SPF) chicken eggs of 7 days' incubation were inoculated via the yolk sac with a range of 10-fold dilutions of avian reovirus R2. Deaths between days 8 and 18 of incubation increased in number with increased virus dose inoculated, and virus was recovered with high frequency from all groups except those given the lowest dose. For those chicks which hatched, cloacal swabs at day-old failed to demonstrate virus in the chicks given the lowest dose, although those infected with the two higher doses were positive. At 7 days post-hatch, however, virus could be isolated from target tissues e.g. liver, hock joints and rectal contents, of most chicks in all groups, but many samples were not positive until after three cell culture passages. It is considered that this method of experimental infection could conveniently simulate natural egg transmission of avian reoviruses.     

Pathogenesis of Influenza A/H5N1 virus infection in ferrets differs between intranasal and intratracheal routes of inoculation

Most patients infected with highly pathogenic avian influenza A/H5N1 virus develop severe pneumonia resulting in acute respiratory distress syndrome, with extrarespiratory disease as an uncommon complication. Intranasal inoculation of ferrets with influenza A/H5N1 virus causes lesions in both the respiratory tract and extrarespiratory organs (primarily brain). However, the route of spread to extrarespiratory organs and the relative contribution of extrarespiratory disease to pathogenicity are largely unknown. In the present study, we characterized lesions in the respiratory tract and central nervous system (CNS) of ferrets (n = 8) inoculated intranasally with influenza virus A/Indonesia/5/2005 (H5N1). By 7 days after inoculation, only 3 of 8 ferrets had a mild or moderate bronchointerstitial pneumonia. In contrast, all 8 ferrets had moderate or severe CNS lesions, characterized by meningoencephalitis, choroiditis, and ependymitis, and centered on tissues adjoining the cerebrospinal fluid. These findings indicate that influenza A/H5N1 virus spread directly from nasal cavity to brain, and that CNS lesions contributed more than pulmonary lesions to the pathogenicity of influenza A/H5N1 virus infection in ferrets. In comparison, intratracheal inoculation of ferrets with the same virus reproducibly caused severe bronchointerstitial pneumonia. The method of virus inoculation requires careful consideration in the design of ferret experiments as a model for influenza A/H5N1 in humans.     

Type A influenza: Postmortem virus isolations from different organs in human lethal cases

Summary Trachea, lung, liver, spleen, pancreas and brain of 77 human patients who had died in the course of clinically diagnosed influenza were subjected to virological and histopathological examination. Type A influenza viruses closely related to the virus variants contemporarily in circulation were isolated from 12 of the lethal cases. In 10 of them, virus was demonstrated in organs other than respiratory, most often the brain. Influenza antigen was also demonstrated in brain tissue by immunofluorescence.With 2 Figures     

In vivo antiviral activity of D-glucosamine

Summary Intraperitoneal treatments with D-glucosamine, an inhibitor of the glycosylation of the viral envelope, decreased the growth rate of tumors induced in quails or in chicks by Rous sarcoma virus and increased the survival of mice inoculated with human influenza virus.With 2 Figures     

Tissue tropism of a Thailand strain of high-pathogenicity avian influenza virus (H5N1) in tissues of naturally infected native chickens (Gallus gallus), Japanese quail (Coturnix coturnix japonica) and ducks (Anas spp.).

The tropism of a Thailand strain of highly pathogenic avian influenza H5N1 virus was demonstrated on tissues (lung, trachea, heart, liver, spleen, pancreas, rectum, kidney, brain, skeletal muscle, duodenum, and oviduct) from naturally infected native chickens (Gallus gallus), Japanese quail (Coturnix coturnix japonica) and ducks (Anas spp.) by indirect immunofluorescence assay. In chickens and quail, the distribution and localization of nucleoprotein viral antigen was similar and detected at the highest level in cardiac myocytes, at 88% (chickens) and 89% (quail), and respiratory, digestive and urinary systems all showed high levels of antigen. Antigen in duck tissues were detected at significantly lower levels (P < 0.05) with the exception of brain and skeletal muscle samples. In most cases, antigen in duck tissue was absent in the digestive organs but present in respiratory organs, which supports the hypothesis that aerosol and oral-oral transmission are the main method of highly pathogenic avian influenza virus transmission from this species.     

Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens

We have evaluated the ability of two carbohydrate biopolymers, chitosan and gellan, to enhance antibody responses to subunit influenza virus vaccines delivered to the respiratory tracts of mice. Groups of mice were vaccinated three times intranasally (i.n.) with 10 microg of purified influenza B/Panama virus surface antigens (PSAs), which consist of hemagglutinin (HA) and neuraminidase (NA), either alone or admixed with chitosan or gellan solutions. Separate groups were vaccinated subcutaneously (s.c.) with PSAs adsorbed to Alhydrogel or chitosan or gellan alone i.n. Serum antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) for influenza virus-specific immunoglobulin G (IgG) and by HA inhibition (HAI) and NA inhibition (NAI) assays. The local respiratory immune response was measured by assaying for influenza virus-specific IgA antibody in nasal secretions and by enumerating nasal and pulmonary lymphocytes secreting IgA, IgG, and IgM anti-influenza virus-specific antibodies by enzyme-linked immunospotting (ELISPOT). When administered alone i.n., B/Panama PSA was poorly immunogenic. Parenteral immunization with B/Panama PSA with Alhydrogel elicited high titers of anti-B/Panama antibodies in serum but a very poor respiratory anti-B/Panama IgA response. In contrast, i.n. immunization with PSA plus chitosan stimulated very strong local and systemic anti-B/Panama responses. Gellan also enhanced the local and serum antibody responses to i.n. PSA but not to the same extent as chitosan. The ability of chitosan to augment the immunogenicity of influenza vaccines given i.n. was confirmed using PSA prepared from an influenza A virus (A/Texas H1N1).     

An entero-like virus associated with the runting syndrome in broiler chickens

A small round unenveloped virus, 31 nm in diameter, and with no obvious surface structure, was identified during the first week of life in the gut contents of broiler chickens which later developed runting. This virus grew in the cytoplasm of the villous epithelial cells of the small intestine, with a predilection for the mid small intestine. Broilers orally infected at 1 day old with a crude inoculum containing the small round virus and reovirus passed abnormal faeces, gained weight and feathered more slowly than controls, which were either uninoculated or inoculated with faeces from SPF chicks. The small round virus was passaged four times through broilers using gut contents collected from experimentally infected birds between 2 and 3 days after inoculation. Clinical signs as described above were obtained at each passage. The small round virus could not be grown serially in cell cultures. However, immunofluorescence showed that viral antigen was synthesised in the cytoplasm of infected chicken embryo liver and chick kidney cell cultures. The small round virus was resistant to pH3. It is suggested that the small round virus is an enterovirus but no evidence for an antigenic relationship with avian encephalomyelitis virus was obtained.     

Pathogenicity of Salmonella enteritidis phage types 4, 8 and 23 in specific pathogen free chicks

The pathogenicity of two isolates of Salmonella enterica serovar Enteritidis (SE) phage type (PT) 4, three of PT8 and one of PT23 was investigated in groups of 1-day-old specific pathogen free White Leghorn chicks. Two groups were crop gavaged with each culture but at two different doses. Two additional groups were given Salmonella enterica serovar Pullorum (SP) at similar doses and one further group served as uninoculated controls. Body weights were recorded at 14, 21, and 28 days postinoculation (d.p.i), and mortality was monitored throughout. In most treatment groups, the average body weights were significantly lower than the controls. Birds inoculated with SP had the highest mortality followed by those given SE PT4 of human or chicken origin. At 14 and 21 d.p.i., four chicks from each group were killed and examined for gross lesions. Selected tissues were collected for histopathology and cultured for bacteria. Dead birds had fibrinous exudate in the pericardium and also, in a few, on the liver capsule. They had enlarged livers, sometimes with congestion and white foci. At 7 d.p.i., several birds, especially those inoculated with SE PT4, had retained yolk sacs containing coagulated material. Microscopic lesions of pericarditis, myocarditis, hepatitis, splenitis, peritonitis and enteritis were present at 7 d.p.i. in most birds inoculated with SE, but was greatly variable at 14 d.p.i.. This study shows that 1-day-old SPF chicks are susceptible to various phage types of SE, with yolk-sac infection as the most prominent feature.