火鸡冠状病毒的分离和初步鉴定

2003年国内某火鸡场发生了一种以侵害15～25日龄雏火鸡为主的急性传染病，主要表现为腹泻，十二指肠、直肠充血和出血，盲肠肿大，肠道内充满黄绿色内容物，死亡率约为10％～20％。取病死火鸡肝、脾、肠匀浆，取上清液通过尿囊腔接种15日龄SPF鸡胚。连续传代至第5代，收集接种后72h内死亡或存活鸡胚的卵黄和肠道，用于病毒分离和提纯。试验中发现该病毒能凝集兔红细胞，不能凝集鸡红细胞。经电镜观察，在病毒提纯液中发现有圆形或椭圆形、带花冠状纤突的病毒粒子，初步诊断为火鸡冠状病毒感染。进而设计针对火鸡冠状病毒S2基因引物，进行RT-PCR扩增，结果扩增出预期大小的片段。运用所分离病毒进行动物回归试验，感染火鸡出现与自然病例一致的临床症状和病理变化，并能从发病火鸡分离出该病毒。以上结果表明所分离的病毒为火鸡冠状病毒。此病毒的分离在国内尚属首例。     

Pathogenicity of embryo-adapted serotype 2 OH strain of infectious bursal disease virus in chickens and turkeys

The pathogenicity of serotype 2 OH strain of infectious bursal disease virus (IBDV) to specific-pathogen-free (SPF) chicken embryos and 2-wk-old SPF chickens and turkey poults was investigated. The virus was pathogenic for chicken embryos after five passages as evidenced by pathologic changes in inoculated embryos. The embryo-adapted virus was not pathogenic for 2-wk-old SPF chickens and turkey poults as indicated by lack of clinical signs, gross or microscopic lesions in the bursa of Fabricius of inoculated birds. Bursa-to-body-weight ratios of the inoculated chickens and turkey poults were not significantly different from those of uninoculated controls. Virus-neutralizing antibodies to serotype 2 IBDV were detected in inoculated chickens and turkeys. Results of this study indicated that the embryo-adapted serotype 2 OH IBDV isolate that is pathogenic for chicken embryos is infectious but not pathogenic in chickens and turkeys.     

Isolation of avian paramyxovirus-2 from domestic and wild birds in Costa Rica

A survey for Newcastle disease virus (NDV) in wild birds of Costa Rica was conducted by swabbing wild-caught pet birds, backyard chickens, and wild birds captured in Japanese mist nets in tropical rain forests and agricultural areas. Cloacal swabs were collected from 876 birds of approximately 132 species representing 24 taxonomic families. Hemagglutinating agents were isolated from 18.7% of the birds. Paramyxovirus type 2(PMV-2) (Yucaipa-like), unreported in free-flying passerines in the Americas, was recovered from a finch, wren, and chicken, each from a different location. Pathogenicity trials with infected turkey poults and newly hatched chicks did not result in growth impairment or significant clinical signs of disease. Attempts to isolate NDV were negative.     

The effect of infectious bursal disease virus on the immune system of turkeys

In 1979 it was reported that an infectious bursal disease virus (IBDV) isolated from a case of respiratory disease of turkeys differed antigenically from the chicken isolates of this virus. We injected turkey poults with the turkey-originating TY89 and chicken-originating BD/6 isolates of IBDV and studied their effects on antibody production to the virus, serum immunoglobulin G (IgG), antibody response to sheep erythrocytes, in vitro response of peripheral blood lymphocytes to mitogens, and microscopic structure of the bursa of Fabricius. The chicken isolate BD/6 caused a significant decrease in the response to sheep erythrocytes, lower serum IgG, transient decrease in the response of lymphocytes to PHA, and mild microscopic lesions in the bursa of Fabricius. The turkey isolate TY89, however, caused no obvious damage to the immune system of the infected poults. We suggest that a partial and transient functional disorder of the immune system of poults can occur after infection with IBDV originating from chickens, even if the poults exhibited no clinical signs.     

Virulence and toxigenicity of capsular serogroup D Pasteurella multocida strains isolated from avian hosts

Five capsular serogroup D strains of Pasteurella multocida isolated from avian hosts were examined for virulence and toxigenicity. Virulence was based on development of lethal infections or lesions following intramuscular exposure of turkey poults. The four strains isolated from turkeys varied from slightly to moderately virulent; the strain isolated from a chicken was avirulent. Poults exposed by intra-airsac inoculation with relatively few organisms of the more virulent of the strains had a high mortality rate; however, intranasal exposure of poults with this strain did not cause clinical disease or establish infections. All strains from turkeys were toxigenic, producing heat-labile toxins that killed poults when administered intraperitoneally and caused focal dermal lesions when administered intradermally. Using these criteria, the strain from a chicken was not toxigenic. The demonstration of virulence, particularly the high mortality in poults exposed via air sacs, indicates avian capsular serogroup D strains are a potential cause of fowl cholera.     

The absence of cecal colonization of chicks by a mutant of Campylobacter jejuni not expressing bacterial fibronectin-binding protein.

Campylobacter jejuni is a common cause of human gastrointestinal illness throughout the world. Infections with C. jejuni and Campylobacter coli are frequently acquired by eating undercooked chicken. The ability of C. jejuni to become established in the gastrointestinal tract of chickens is believed to involve binding of the bacterium to the gastrointestinal surface. A 37-kD outer membrane protein, termed CadF, has been described that facilitates the binding of Campylobacter to fibronectin. This study was conducted to determine whether the CadF protein is required for C. jejuni to colonize the cecum of newly hatched chicks. Day-of-hatch chicks were orally challenged with C. jejuni F38011, a human clinical isolate, or challenged with a mutant in which the cadF gene was disrupted via homologous recombination with a suicide vector. This method of mutagenesis targets a predetermined DNA sequence and does not produce random mutations in unrelated genes. The parental C. jejuni F38011 readily colonized the cecum of newly hatched chicks. In contrast, the cadF mutant was not recovered from any of 60 chicks challenged, indicating that disruption of the cadF gene renders C. jejuni incapable of colonizing the cecum. CadF protein appears to be required for the colonization of newly hatched leghorn chickens.     

Pathogenicity for chickens of a reovirus isolated from turkeys

A viral agent that was isolated from livers of commercial turkey poults that died at approximately two weeks of age was characterized as a reovirus. Experimental infection of day-old chickens with this reovirus isolate resulted in the development of tenosynovitis, hepatitis, and myocarditis. In vitro neutralization of the turkey reovirus isolate by antiserum against chicken reovirus correlated with in vivo protection of maternally immune chickens from day-old oral challenge with the turkey reovirus isolate.     

Failure of two serotype II infectious bursal disease viruses to affect the humoral immune response of turkeys

The effect of serotype II infectious bursal disease virus (IBDV) isolates from turkeys on the homoral immune response of turkey poults was determined. Following exposure to the OH IBDV isolate, poults in two experiments were inoculated with sheep red blood cells, which is a T-dependent antigen, and poults in two other experiments were inoculated with Salmonella heidelberg O antigen, which is a T-independent antigen. Prior exposure to serotype II IBDV did not affect serum antibody titers to these antigens. IBDV infection also did not affect the concentrations of serum immunoglobulin M (IgM), IgG, or IgA in these poults. Bursa:body weight ratios of OH IBDV-infected poults were not significantly different from those of uninfected controls. In one experiment, the humoral immune response of poults to the LaSota Newcastle disease vaccine was not affected by infection with the MO IBDV isolate. Although no clinical infectious bursal disease was observed in any poult in these experiments, the serotype II IBDV isolates were infectious and transmissible in poults.     

Isolation and characterization of avian rotaviruses

Rotaviruses were isolated from intestinal contents obtained from flocks of turkey poults and pheasant poults with diarrhea and from different age groups of chickens showing various signs of intestinal disorders. The incorporation of 5 micrograms trypsin/ml in the inoculum and medium was essential for virus isolation in chicken kidney cells. All isolates were identified as rotaviruses by fluorescent-antibody technique using a National Institutes of Health reference rotavirus antiserum against human rotavirus strain "D," Type 2. Negative-staining and transmission electron microscopy showed the presence of rotavirus particles. Furthermore, polyacrylamide gel electrophoresis pattern of viral RNA segments of our isolates confirmed that they are rotaviruses. Seven-day-old turkey poults could be infected with turkey and chicken rotavirus isolates; in contrast, chicks of the same age were refractory.     

Enhancement of Campylobacter jejuni virulence by serial passage in chicks

Four isolates of Campylobacter jejuni were studied to determine changes in virulence following six serial passages in chicks. Chicks that received invasive isolates exhibited diarrhea and depressed weight gain. Immature mice were used to assess virulence of the passaged isolates of C. jejuni. Nine-day-old mice infected with passaged isolates showed lethargy, dehydration, depression, decreased weight gain, and occult blood in feces. Mouse pups inoculated with the third and sixth chick passage levels of an invasive isolate showed significant depression in mean daily weight gain and elevated mortality compared with controls and subjects inoculated with unpassaged isolates. This study demonstrated enhancement of virulence in a C. jejuni isolate following chick passage. In contrast, three other passaged isolates failed to show any consistent increase in virulence.     

Field studies with convalescent serum and infectious bursal disease vaccine to control turkey coryza

Convalescent serum given to 1-day-old poults delayed clinical signs of turkey coryza by several days and reduced mortality on infected farms. Turkey breeders immunized with cell-culture-adapted infectious bursal disease virus (IBDV) or turkey infectious bursal disease virus (TIBDV) had a marked increase in virus-neutralization (VN) antibody titers. The VN antibody titer was significantly higher in progeny poults than in poults from unimmunized breeders. Clinical turkey coryza and mortality was considerably less in poults from IBD- or TIBD-vaccinated breeders than in control poults. They also responded more favorably to hemorrhagic enteritis and fowl cholera vaccination.     

Viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus

Intestinal samples from turkey poults affected with poult enteritis and mortality syndrome (PEMS) were examined for viruses by immune electron microscopy and double-stranded RNA virus genome electropherotyping. Turkey coronavirus (TCV), avian rotaviruses, reovirus, and a yet undefined small round virus (SRV) were detected. The SRV and TCV were isolated and propagated in turkey embryos. Challenge of specific-pathogen-free turkey poults with SRV, TCV, or both resulted in mortality and clinical responses similar to those of natural PEMS. Our experiments indicate that SRV and TCV are possibly important agents in the etiology of PEMS and the combination of these infections might result in outbreaks with high mortality. The severity of clinical signs and mortality of PEMS are postulated to be partly related to the virus agents involved in individual outbreaks.     

Comparison of post‐hatch holding time and subsequent early performance of broiler chicks and Turkey poults

1. The effect of a holding period, lasting up to 48 h post‐hatch, on early growth and mortality rate was studied in broiler chicks and turkey poults.

2. After 48 h of food and water deprivation, birds of both species lost about a tenth of their initial post‐hatch weight. Weight loss was more marked in poults subjected to heat stress. Thus, duration of the holding period adversely affected early growth.

3. During the 48‐h holding period, the relative weight of residual yolk decreased in both species by approximately half of its weight in the just hatched chicks and poults. Relative yolk size was much higher in poults than in broilers.

4. On day 1 weight loss was attributed to degradation of the yolk sac, whereas on day 2 only about two‐thirds of the loss could be attributed to residual yolk. Body composition of newly‐hatched broiler chicks did not differ from those held for 24 h despite shortages of energy. However, after 48 h body fat content was markedly reduced compared to newly‐hatched or 24‐h‐old birds.

5. Irrespective of access to food, the relative size of the gastrointestinal tract increased with holding time in both species, reflecting its importance.

6. It was concluded that post‐hatch birds deprived of food and water for 48 h face a severe shortage of energy, causing alterations in body composition. This in turn adversely affects early growth and increases mortality rate under heat stress.     

Isolation of a reovirus from poult enteritis and mortality syndrome and its pathogenicity in turkey poults

Poult enteritis and mortality syndrome (PEMS) is an acute, infectious intestinal disease of turkey poults, characterized by high mortality and 100% morbidity, that decimated the turkey industry in the mid-1990s. The etiology of PEMS is not completely understood. This report describes the testing of various filtrates of fecal material from control and PEMS-affected poults by oral inoculation into poults under experimental conditions, the subsequent isolation of a reovirus, ARV-CU98, from one of the PEMS fecal filtrates, and in vivo and in vitro studies conducted to determine the pathogenicity of ARV-CU98 in turkey poults. In order to identify a filtrate fraction of fecal material containing a putative etiologic agent, poults were challenged in two independent experiments with 220- and 100-nm filtrates of fecal material from PEMS-negative and PEMS-positive poults. The 100-nm filtrate was chosen for further evaluation because poults inoculated with this filtrate exhibited mortality and significantly lower (P < or = 0.05) body weight and relative bursa weight, three clinical signs associated with PEMS. These results were confirmed in a third experiment with 100-nm fecal filtrates from a separate batch of PEMS fecal material. In Experiment 3, body weight and relative bursa and thymus weights were significantly lower (P < or = 0.05) in poults inoculated with 100-nm filtrate of PEMS fecal material as compared with poults inoculated with 100-nm filtrate of control fecal material. Subsequently, a virus was isolated from the 100-nm PEMS fecal filtrate and propagated in liver cells. This virus was identified as a reovirus on the basis of cross-reaction with antisera against avian reovirus (FDO strain) as well as by electrophoretic analysis and was designated ARV-CU98. When inoculated orally into poults reared under controlled environmental conditions in isolators, ARV-CU98 was associated with a higher incidence of thymic hemorrhaging and gaseous intestines. In addition, relative bursa and liver weights were significantly lower (P < or = 0.05) in virus-inoculated poults as compared with controls. Virus was successfully reisolated from virus-challenged poults but not from control birds. Furthermore, viral antigen was detected by immunofluorescence in liver sections from virus-challenged poults at 3 and 6 days postinfection and virus was isolated from liver at 6 days postinfection, suggesting that ARV-CU98 replicates in the liver. In addition to a decrease in liver weight, there was a functional degeneration as indicated by altered plasma alanine aminotransferase and aspartate aminotransferase activities in virus poults as compared with controls. Although this reovirus does not induce fulminating PEMS, our results demonstrated that ARV-CU98 does cause some of the clinical signs in PEMS, including intestinal alterations and significantly lower relative bursa and liver weights. ARV-CU98 may contribute directly to PEMS by affecting the intestine, bursa, and liver and may contribute indirectly by increasing susceptibility to opportunistic pathogens that facilitate development of clinical PEMS.     

Transmissible enteritis of turkeys: experimental inoculation studies with tissue-culture-adapted turkey and bovine coronaviruses.

Four Quebec isolates of turkey enteric coronaviruses (TCVs) and three isolates of bovine enteric coronaviruses (BCVs) were serially propagated in HRT-18 and compared for their pathogenicity in turkey embryos and turkey poults. By immunoelectron microscopy, hemagglutination-inhibition, and Western immunoblotting assays, tissue-culture-adapted Quebec TCV isolates were found to be closely related to the reference Minnesota strain of TCV and the Mebus strain of BCV. Genomic relationships between TCV isolates and the reference BCV strain were confirmed by hybridization assays with BCV-specific radiolabeled recombinant plasmids containing sequences of the N and M genes. Only TCV isolates could be propagated by inoculation in the amniotic cavity of chicken and turkey embryonating eggs, and induced clinical disease in turkey poults. Nevertheless, coronavirus particles or antigens were detected by electron microscopy or indirect enzyme-linked immunosorbent assay in the clarified intestinal contents of BCV-infected poults up to day 14 PI, and genomic viral RNA was detected by slot-blot hybridization using BCV cDNA probes.     

Efficacy of maduramicin ammonium against coccidiosis in turkeys under laboratory and floor-pen conditions

Experimental infections with field isolates of Eimeria meleagrimitis, E. adenoeides, E. gallopavonis, and E. dispersa in turkey poults were used to test the efficacy of maduramicin ammonium at 2.5-10 ppm in laboratory experiments. Infection with single or mixed species of coccidia reduced the weight gain of unmedicated infected controls and caused 18.1-65% mortality in two experiments. Maduramicin ammonium given at 5-7 ppm prevented mortality, significantly reduced droppings scores and oocyst passage, and improved weight gain to near that of the unmedicated uninfected controls. Maduramicin ammonium was tested at 4-7 ppm in a floor-pen trial lasting 10 weeks. Mortality from coccidiosis averaged 11.9% in unmedicated controls, compared with 0.6% with 4 ppm of maduramicin or no mortality with 5-7 ppm. Average weight gain and feed conversion at 10 weeks were significantly improved over unmedicated infected controls when maduramicin ammonium was given at 5-7 ppm. These results suggest that maduramicin ammonium is highly efficacious against field isolates of Eimeria in turkeys, especially within the range of 5-7 ppm in the feed.     

Virulence of avian capsular serogroup B Pasteurella multocida for turkey poults

Two strains of capsular serogroup B Pasteurella multocida isolated from avian hosts (swan and turkey) were evaluated for virulence based on lethality for turkey poults. Groups of poults were exposed intramuscularly to various concentrations of organisms of each strain. Both strains were virulent. The strain isolated from a turkey was highly virulent: all exposed poults died in less than 24 hours, including those exposed to only 79 organisms. This highly virulent strain was neither highly invasive nor highly infective: intrapharyngeal exposure with 7.9 x 10(6) organisms resulted in death of only one of five poults, and attempts to isolate the organism from pharyngeal mucosae and livers of surviving poults were unsuccessful. The high degree of virulence of a B capsular group strain isolated from a turkey indicates a disease-producing potential for members of this uncommon serogroup of P. multocida.     

Efficacy and safety of a cell-culture live virus vaccine for hemorrhagic enteritis of turkeys: laboratory studies

Poults free from hemorrhagic enteritis (HE) antibody were vaccinated by gavage at 1 day or 2 weeks of age with a live HE vaccine virus that had been propagated in a Marek's disease (MD)-induced B-lymphoblastoid cell line of turkey origin. Vaccinated and unvaccinated poults were challenged with a virulent HE virus at various times postvaccination. One hundred tissue-culture-infectious doses of the vaccine virus per poult were sufficient to induce a serological response as well as to protect poults against HE lesions and mortality. Vaccinated poults were protected against the disease as early as 1 week and as late as 8 weeks PV. The vaccine was efficacious by several routes of application. The vaccine virus spread horizontally from vaccinated to contact-exposed poults, as indicated by seroconversion and resistance of contact-exposed poults to challenge. The vaccine had no detectable harmful effects on the humoral immune response to particulate antigens or on weight gain of vaccinated poults. The vaccine proved to be free from MD virus, as indicated by the absence of MD lesions and antibody in 8-week-old chickens inoculated intra-abdominally with the vaccine at hatching. These findings indicate that the cell-culture-propagated HE vaccine is efficacious and safe.     

Colonization of broilers with Campylobacter in conventional broiler-chicken flocks

Eight of 16 conventional broiler-chicken flocks examined contained Campylobacter. All isolates were identified as C. jejuni except from 1 flock were C. coli was isolated. One herd consisting of 6 different houses where Campylobacter regularly has been isolated was continuously examined. It was not possible to isolate Campylobacter from newly hatched chickens or from environmental samples and cloacal swabs during the 2 first weeks of growth.