Chicken embryo propagation of type I avian adenoviruses.

Forty-two clone-purified, cell-culture-propagated type I avian adenoviruses (AAV) representing 11 serotypes and two intermediate strains were evaluated for virus replication (evidenced by embryo death and lesions) resulting from the inoculation of specific-pathogen-free chicken embryos via the chorioallantoic sac or yolk sac. Commonly observed embryonic changes were death, stunting and curling, hepatitis, splenomegaly, congestion and hemorrhage of body parts, and urate formation in the kidneys. Basophilic or eosinophilic intranuclear inclusion bodies characteristic of fowl adenoviruses were observed in hepatocytes. The magnitude and relative uniformity of intra- and interserotypic embryo mortality, gross lesions, and virus titers was greater in embryos inoculated via the yolk sac. This work identifies the yolk sac as a practical and sensitive chicken embryo inoculation route for poultry diagnosticians to employ. It is suggested that the yolk sac may be a reliable alternative to cell culture for the successful isolation of all type I avian adenoviruses.

A trivalent antigen for the detection of type I avian adenovirus precipitin.

A double immunodiffusion antigen prepared from cell-culture-propagated CELO virus was not capable of detecting precipitin directed against all of the type I avian adenovirus (fowl adenoviruses) isolates tested. However, an antigen pool containing CELO-4, B-3, and IBH-2 (Tipton) fowl adenovirus isolates detected precipitin directed against representative isolates of 10 type I serotypes. Additionally, this antigen pool markedly improved detection of adenovirus field exposure.

The propagation of avian viruses in a continuous cell line (QT35) of Japanese quail origin.

Seven of nine avian virus families tested (Birnaviridae, Coronaviridae, Herpesviridae, Paramyxoviridae, Poxviridae, Reoviridae, and Retroviridae) were found to replicate in a quail fibroblast cell line, designated QT35, resulting in a cytopathic effect (CPE) visible with the naked eye or by low-power microscopy. In comparison, only one (Paramyxoviridae) of seven mammalian virus families tested produced an observable CPE. Cytopathic changes induced by examined viruses were round cell, syncytial, and focus formation. Trypsin did not promote cytopathic changes by selected CPE-negative avian and mammalian viruses in QT35 cells. Several avian viruses (infectious bursal disease virus, Newcastle disease virus, Canary pox virus, and reovirus) formed plaques under agar. Avian reovirus and infectious bursal disease virus produced similar titers in chicken embryo fibroblast (CEF) and QT35 cell cultures. Chicken-egg-yolk neutralizing-antibody titers to IBDV were comparable in CEF and QT35 cell-culture systems.

Adenoviruses of chickens: serologic groups.

By literature review and experimental studies, 57 adenovirus isolants from chickens were grouped into 10 distinct serotypes. In plaque-reduction tests, 20 antibody units were reacted with 32-320 plaque-forming units of virus. Viruses that were neutralized (80% or greater reduction in titer) by a given serum were considered to belong to that serotype. Used to distinguish among serotypes were their antisera. One-way neutralization occurred in only two instances; in both cases it was traced to contamination of the virus inocula used to induce specific antiserum. Recloning corrected the problem. Of the 10 serotypes described, all are known to exist in the United States, 7 were found in Northern Ireland, and at least 6 have been shown to occur in Japan.

Characterization of a new infectious bronchitis virus isolate. III. Cell-culture adaptation of Clark 333.

The Clark 333 strain of infectious bronchitis virus (IBV) was substantially resistant to primary chicken cell-culture adaptation. More than 40 serial embryo passages were required before the virus would produce cytopathic alterations upon cell-culture inoculation. The cytopathic effect was characteristic of the effect of reported for IBV. Adaptation was not accomplished by alternating serial passages in embryo and cell-cultured systems. A careful monitoring of cell-culture fluid infectivity by embryo inoculation was found to be essential because absence of adaptation is accompanied by a loss in virus titer (3 to 5 serial passages). Helpful additional aids were immunofluorescence and cytopathology.

Serotyping of avian infectious bronchitis viruses by the virus-neutralization test.

The antigenic relationships of 12 strains of infectious bronchitis virus (IBV) were evaluated by a virus-neutralization procedure similar to that used in typing human rhinoviruses. Such a procedure consists of reciprocal neutralization tests performed by reacting 32-320 EID50 or plaque-forming units of virus with 20 antibody units of antiserum. Eight serologic groups were identified by chicken embryo assay, and 4 by plaque-reduction (90%). In general, serologic groupings were not distinct but reflected numerous intergroup relationships. The contrasting results exhibited by indicator systems are viewed as differences in the accuracy of the methods employed. It is suggested that before an IBV classification scheme can be proposed, agreement must be reached on the most suitable indicator system, techniques must be standardized, and reference viruses and antisera distributed to several institutions for comparison testing. In addition, cross-protection studies are needed to determine the importance of IBV serotypes and/or variants on vaccine efficacy.

Some observations on the response of ring-necked pheasants to inoculation with various strains of cell-culture-propagated type II avian adenovirus.

The response of ring-necked pheasants to inoculation with three strains of cell-culture-propagated type II avian adenovirus was examined. Marble spleen disease (MSD) virus of pheasants and both avirulent and virulent strains of hemorrhagic enteritis virus (HEV) of turkeys all induced typical gross and microscopic splenic lesions of MSD; neither MSD-associated lung lesions nor mortality were noted in inoculated pheasants, regardless of strain of virus used. Pheasants inoculated with a cell-culture-propagated avirulent strain of HEV were properly immunized against challenge with virulent HEV, as indicated by seroconversion and by protection against virus-induced splenic lesions. We conclude that these strains of type II avian adenovirus are comparable in pathogenicity for pheasants and cannot be distinguished. Further, absence of MSD-associated lung lesions and mortality in pheasants maintained under controlled laboratory conditions suggest that other environmental factors are probably involved in induction of such lesions and mortality in field cases of MSD.

An outbreak of avian urolithiasis on a large commercial egg farm.

A significant outbreak of avian urolithiasis was observed on a large commercial egg farm. From the initial outbreak site (a single laying house), the incidence of urolithiasis slowly spread in the ensuing months to numerous other laying houses. Increasing mortality associated with urolithiasis commenced during late growout to early lay and then leveled off when egg production peaked. At the height of the outbreak, mortality was typically 0.5% per week; 75% of this mortality was due to urolithiasis. The clinical and pathologic features of this condition are described. Both infectious bronchitis virus (IBV) and fowl adenoviruses were isolated from organ homogenates of sampled birds. A clone of the IBV strain was found to induce nephritis in specific-pathogen-free white leghorns.

Experimental egg-transmission of avian reovirus.

Two experiments with an avian reovirus (FDO isolant), serologically identical to the Fahey-Crawley and WVU 2937 isolants, provided conclusive proof that egg-transmission can occur. In the first experiment, 4-day-old chicken embryos were inoculated into the yolk sac with serial dilutions of virus and subsequently hatched. High virus doses killed all embryos, whereas low doses (14 or less plaque-forming units) allowed some embryos to hatch normally although infected. In the second experiment, virus was administered by the nasal, esophageal, and tracheal routes to twenty 15-month-old breeders. Eggs from -6 to 21 days and 58 to 64 days postinoculation were incubated. Three newly hatched chicks from eggs laid on successive days (17, 18, and 19 days postinoculation) and one infertile egg (61 days postinoculation) were found infected. Active infection, as judged by virus isolations from cloacal swabs, was over by the 17th day postinoculation.

Localization of avian reovirus (FDO isolant) in tissues of mature chickens.

Mature hens were exposed to a reovirus (FDO isolant) serologically related to the viral arthritis agent. Examination of 23 tissues from the respiratory, alimentary, and reproductive tracts and tendons from the hock joint revealed widespread infection 4 days after inoculation via the respiratory and alimentary tracts. By 14-15 days, infection had largely subsided but was still detectable in the tendons, oviduct, and alimentary tract of some birds. No infection was found at 30 days. Virus isolation attempts in chick kidney cultures were more reliable than fluorescent-antibody tests for detecting infection. None of 156 chicks hatched from eggs laid between 12 and 33 days postinoculation were infected.

A standardized enzyme-linked immunosorbent assay for infectious bronchitis virus: comparison with hemagglutination-inhibition and virus-neutralization assays for measuring protective antibody levels in chickens.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure antibodies to infectious bronchitis virus (IBV) in chickens. The results are reported in IBV standard ELISA values calculated by comparing antibody levels in test sera with antibody levels in a series of standard reference sera. The IBV standard ELISA values were good indicators of responses to vaccination and the immune status of experimentally challenged birds. Although the assay was not serotype-specific, the sensitivity makes it ideally suited for determining the immune status of poultry flocks. The assay results compared favorably with other laboratory results, including virus-neutralization titers, hemagglutination-inhibition levels in sera, virus isolation from vaccinated/challenged birds, and the tracheal ring test results.

A case of acute pulmonary edema, splenomegaly, and ascites in guinea fowl.

Acute pulmonary edema, splenomegaly, and ascites were observed in a disease outbreak in adult white and pearl guinea fowl. The clinical history and gross and microscopic lesions resembled those described for marble spleen disease of pheasants and avian adenovirus group II splenomegaly of chickens. A small number of intranuclear inclusion bodies were found in liver, spleen, and lung sections of affected guinea fowl. Attempts to isolate virus and serological tests to detect the presence of viral antigens were unsuccessful. Adult female pearl guinea fowl experimentally exposed to pheasant and turkey isolates of type II avian adenoviruses developed gross and microscopic lesions similar to those seen in the field outbreak. The pheasant isolate was the more virulent. Intranuclear inclusion bodies were observed in liver, spleen, and lung sections of pearl guinea fowl inoculated with either of the virus isolates, and direct immunofluorescent examination revealed viral antigen in the spleen and lung.

Use of a hypobaric chamber as a model system for investigating ascites in broilers.

In a series of experiments, broilers were reared at simulated altitudes of 3000, 3500, and 5000 meters utilizing a hypobaric chamber. The purpose was to evaluate the chamber as a model for ascites syndrome and to further study the pathophysiology of this disease. Results show that this model can effectively reproduce the disease as it is seen in the field. Birds reared at the simulated altitude of 5000 meters had a statistically significant increase in EKG amplitude, packed cell volume, and heart-to-body-weight and lung-to-body-weight ratios when compared with controls reared at 366 meters. Additionally, there was a trend for birds dying of ascites to have larger-amplitude EKGs than survivors when reared at 5000 meters.

An infectious bronchitis virus isolated from chickens experiencing a urolithiasis outbreak. I. In vitro characterization studies.

An infectious bronchitis virus (IBV) strain isolated from commercial layers experiencing urolithiasis was 50-100 nm in size and possessed widely spaced, club-shaped surface projections. It was sensitive to lipid solvents and exhibited responses characteristic of IBV when exposed to heat, divalent cations, and trypsin. Reciprocal virus-neutralization tests demonstrated it to be closely related to Gray, JMK, Delaware 2868, and Delaware 2897 strains of IBV.

Experimentally induced necrotic enteritis in chickens.

A 1.3 to 10% incidence of necrotic enteritis was experimentally produced in broiler-type chickens in three of five trials. The incidence range observed was considerably narrower and lower than the 5.6-37.3% range reported in the literature. Clostridium perfringens was inconsistently isolated from the liver and intestine of dosed chickens.

Incidence and distribution of "avian adenovirus group II splenomegaly of chickens".

Forty-six percent of 3,585 serum samples from 174 broiler-breeder chicken flocks in 9 states were positive for the precipitating antibody of hemorrhagic enteritis (HE) of turkeys/marble spleen disease (MSD) of pheasants. Seroconversion was detected only in flocks 19 weeks of age and older. The percent of individual serum samples positive within a flock increased with the age of the flock.

Physiologic and electrocardiographic changes occurring in broilers reared at simulated high altitude.

An experiment was conducted to determine whether differences in the electrocardiograms (EKGs) of broilers reared at simulated high altitude from the day of hatch can be used to predict which birds are developing ascites. In three replicate experiments, conducted with 100 broilers per replicate, birds were reared at a simulated altitude of 3000 meters or at ambient atmospheric pressure. Lead I, II, and III EKGs were obtained from all birds on days 0, 14, 28, and 42. No consistent significant differences were seen on day 0 in the amplitude of the R or S wave or total amplitude of the QRS complex when broilers that developed ascites while being reared at simulated high altitude were compared with unaffected birds reared at simulated high altitude and with birds reared at ambient atmospheric pressure. On days 14 and 28, the average amplitude of the S wave and the total amplitude of the QRS complex were significantly higher in the ascites group than in the two other groups. Packed cell volumes were significantly higher in birds reared at simulated high altitude at all sampling days (days 14, 28, and 42) than in those reared at ambient atmospheric pressure, and they were significantly higher in the ascites group on day 28 than in the two other groups. Birds in the ascites group weighed significantly less than the two other groups by day 14, and this trend persisted.

Influenza A viruses isolated from waterfowl in two wildlife management areas of Pennsylvania.

A survey was conducted at two wildlife management areas of Pennsylvania (USA) to evaluate an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) for the detection of avian influenza viruses (AIV) in cloacal swabs from waterfowl and to determine the influenza A virus subtypes and the distribution of these viruses among waterfowl. We collected 330 cloacal swabs from hunter-killed waterfowl in the fall of 1990 and from cage-captured waterfowl in the summer of 1991. Thirty-one hemagglutinating agents were isolated by chicken embryo inoculation (CEI) of which 27 were influenza A viruses and four Newcastle disease viruses (NDV). The prevalence of AIV infection was 8.2%. Compared to CEI, AC-ELISA was only 15% sensitive and 61% specific. Based on the distribution of AIV by species of waterfowl, mallards (Anas platyrhynchos) and American wigeons (Anas americana) were at equal risk of AIV infection even though most of the AIV isolates came from mallards. Although significant crude effects of sampling site and season on AIV recovery could be established, juvenile age was identified as the primary risk factor of AIV recovery. Twelve AIV subtypes were identified by hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests. The most prevalent subytpes were H4N8 and H6N8. We concluded that AC-ELISA was not useful for the detection of AIV in cloacal swabs from waterfowl and that CEI, HI, and NI tests remain as the method of choice for AIV screening in waterfowl. Based on the results AIV infected preferentially the young which represent the high risk group in waterfowl populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary acidification on kidney damage induced in immature chickens by excess calcium and infectious bronchitis virus.

Experiments were designed to evaluate the effect of dietary acidification on the development of kidney lesions induced by excess dietary calcium (Ca) and Gray strain infectious bronchitis virus (IBV). Specific pathogen-free (SPF) chicks and SPF chicks inoculated with Gray strain IBV were fed one of three diets: a commercial pullet grower ration (1% Ca); a commercial layer ration (3.25% Ca); or layer ration plus .5% ammonium chloride (acidified layer ration). Gray strain IBV significantly reduced total kidney weights in males, reduced total kidney weight as a percentage of body weight in males, increased the number of gross kidney lesions, and decreased the number of filtering nephrons when compared with uninoculated birds when both groups were fed the grower ration. The layer ration induced a 60% incidence of kidney lesions, caused a significant increase in kidney weight asymmetry ratios, and caused a 25% reduction in the number of filtering nephrons. Acidifying the layer ration significantly reduced the incidence of gross kidney lesions and reduced kidney weight asymmetry ratios, but did not prevent Ca-induced reductions in filtering nephrons.

Effect of dietary acidification and alkalinization on urolith formation and renal function in Single Comb White Leghorn laying hens.

One of the major causes of mortality in poultry is urolithiasis. Although two of the major causes of urolithiasis have been defined, active and preventative treatments for this disease have not been extensively field tested. Previous research demonstrated that a diet acidified with NH4Cl was effective in preventing the formation of uroliths in 16-wk-old pullets. In the present study, experiments were designed to evaluate the effects of dietary acidification and alkalinization on preformed uroliths in mature laying hens. Urolithiasis was induced by feeding excess dietary Ca (in commercial layer ration) during pullet growout (6 to 18 wk of age) and during the early laying period (18 to 32 wk of age). At 32 wk of age birds were randomly divided into three diet treatment groups: a normal layer ration group, an acidified layer ration group (1% NH4Cl added); and an alkalinized layer ration group (1% NaHCO3 added). Each group received the respective diets until 52 wk of age, when the experiment was terminated. Blood gas analysis at 44 wk of age demonstrated that hens fed the acidified diet had significantly higher blood H+ concentrations, lower blood bicarbonate concentrations, and lower total blood carbon dioxide levels than hens fed the alkalinized layer ration. At 52 wk of age, hens fed the acidified layer ration had higher urinary H+ concentrations, higher blood H+ concentrations, and lower blood HCO3 and total CO2 concentrations than hens fed the normal and alkalinized layer rations. The acidified layer ration group had no uroliths present in their ureters (0% urolithiasis), whereas the normal and alkalinized layer ration groups had 8.3 and 13.1% incidences of urolithiasis.(ABSTRACT TRUNCATED AT 250 WORDS)