Comparison of duration of immunity in chickens infected with a live infectious bronchitis vaccine by three different routes.

Three groups of chicks were vaccinated by aerosol, intra-ocular and drinking water routes with a live infectious bronchitis (IB) vaccine. At one, two, six, 15 and 32 weeks after vaccination five birds from each group were sampled for testing for IB haemagglutination-inhibiting (HI) antibodies and challenged. Assessment of susceptibility to infection was measured by recovery of virus from individual tracheas and from kidney and gonad pools four days after challenge. Virus was isolated from all kidney and gonad pools of birds challenged one week after vaccination, the kidney and gonad pools of the drinking water vaccinates at two weeks, the kidney pool of the intra-ocular group at 15 weeks and all organ pools except the gonads of the intra-ocular group at 32 weeks. Tracheal resistance was found in most of the birds challenged one week after vaccination and in all the birds tested at two weeks but had begun to wane by six weeks after vaccination. No correlation was found between low HI antibody titres of individual birds and their susceptibility to challenge measured by reisolation of virus from the traches, but birds with titres over log2 6 were always resistant.     

Establishment of persistent avian infectious bronchitis virus infection in antibody-free and antibody-positive chickens

Avian infectious bronchitis virus (IBV) causes a highly contagious and economically significant disease in chickens. Establishment of a carrier state in IBV infection and the potential for the persistent virus to undergo mutations and recombination in chicken tissues have important consequences for disease management. Nevertheless, whether chickens can maintain persistent IBV infection in the absence of reinfection from exogenous sources or the presence of antibody in the host can modulate virus persistence remains unclear. Indeed, whether or not IBV genome can undergo genetic changes during in vivo infection has not been demonstrated experimentally. In the present study, IBV shedding and tissue persistence were monitored in individual chickens maintained under strict isolation that precluded reinfection from exogenous sources. In the first of two experiments, intranasal exposure of 6-wk-old antibody-free chickens to IBV vaccine virus resulted in intermittent shedding of the virus from both trachea and cloaca of individual birds for up to 63 days. Also, the virus was recovered from the internal organs (spleen, gonad, kidney, lung, cecal tonsil, and cloacal bursa) of six of eight birds killed at various intervals between 27 and 163 days postinoculation (DPI). In the second experiment, IBV exposure of 1-day-old maternal antibody-positive chicks led to periodic virus shedding from the trachea and cloaca in all chickens until 77 days; however, internal organs (lungs and kidneys) of only one of seven birds (killed at 175 DPI) were virus positive, suggesting that presence of antibody at the time of infection protects internal organs from IBV infection. When the lung and kidney isolates of IBV from the latter experiment were compared with the parent-vaccine virus, no changes in their antigenicity, tissue tropism, or the nucleotide sequence of the S1 glycoprotein gene were observed. These findings indicate that, unlike the mammalian coronaviruses, propensity for frequent genetic change may not be inherent in the IBV genome.     

鸭瘟病毒强毒株在感染鸭实质器官内的增殖与分布

鸭瘟病毒(DPV)CHv强毒株经皮下注射、滴鼻和口服3种途径分别感染20日龄天府肉鸭,于攻毒后10、30、60、90min以及4、12、48、72h和9、15d每组分别剖杀2只鸭,采集心、肝、脾、肺、肾、脑、胸腺、法氏囊、哈德氏腺等实质器官,应用TaqMan-MGB探针实时荧光定量PCR对DPV在这些器官的分布和增殖进行检测。结果表明,DPV分布到具体器官的速度与感染的途径、鸭的解剖结构密切相关,其中皮下注射是DPV分布到各实质器官速度最快的途径。30min于皮下感染鸭的肝、脾、胸腺、法氏囊、哈德氏腺、肺、脑、肾,口服感染鸭的肺和法氏囊,滴鼻感染鸭的心脏和哈德氏腺均检测到DPV-DNA;90min所有受检样品中检测到DPV-DNA。鸭抗DPV感染的免疫器官的重要性依次是脾、胸腺、法氏囊和哈德氏腺,30min内DPV-DNA分布到脾、胸腺、法氏囊的速度和数量决定了DPV感染的潜伏期和疾病的严重程度。不同途经感染鸭的相同器官在同一时间内的DPV-DNA拷贝数大多以皮下感染鸭为最高。DPV致死鸭的法氏囊和肾是DPV-DNA含量最高的实质器官。     

鸭源鸡杆菌对SPF雏鸡的感染特性研究

为研究鸭源鸡杆菌的流行病学特性,本研究采用从自然病例分离得到的鸭源鸡杆菌人工感染4日龄SPF雏鸡,通过形态学、PCR、荧光定量PCR和组织学等方法分别对感染后SPF雏鸡的组织脏器进行了细菌分离、检测和鉴定,用ELISA对其血清抗体水平进行检测.结果表明,人工感染鸡无临床症状,组织学检查感染鸡的肝脏、气管和肺脏组织均有损伤.人工感染后第3d和同居感染第2d鸡体内可分离出鸭源鸡杆菌,人工感染后第96d仍能够分离到鸡杆菌.ELISA方法证实人工感染后47 d和同居后32 d出现抗体高峰,持续2～3周,人工感染组和同居组抗体水平均高于对照组(p＜0.05);荧光定量PCR检测病料结果显示人工感染组和同居组的气管组织中细菌含量最高.本研究表明雏鸡在4日龄即可感染鸭源鸡杆菌,并能通过同居传播,长期带菌、排菌;感染后抗体产生缓慢、持续期短.本研究为鸭源鸡杆菌的流行病学、致病机理研究及防治措施的制定等提供了参考依据.     

Duration of excretion of virulent Newcastle disease virus following challenge of chickens with different titres of serum antibody to the virus

Virulent Newcastle disease virus (NDV) was isolated from susceptible and immune chickens following intra-ocular challenge with the Essex '70 strain. Challenge virus was isolated from the trachea and cloaca of susceptible birds until they died 7 to 9 days after challenge. This virus was isolated from immunised chickens for up to 14 days after challenge. The duration of excretion was influenced by the prechallenge serum antibody titre to NDV. It persisted longest in chickens with titres of 2(3) to 2(7) and decreased in length and frequency from chickens with titres in the range 2(8) to 2(12). Chickens with pre-challenge titres of 2(3) to 2(5) developed 2- to 3- fold increases in post-challenge titres, whereas those with higher pre-challenge titres had smaller proportional increases in titre. Excretion of virulent virus from immunised birds should be considered in the development of Newcastle disease control programs.     

Effect of intratesticular inoculation with Aujeszky's disease virus on genital organs of boars

Ten 8-10-month-old Belgian Landrace boars were intratesticularly inoculated with 500 TCID50 of a virulent Belgian Aujeszky's disease virus (ADV) isolate (75V19) in 0.1 ml volume. One control boar was similarly inoculated with phosphate-buffered saline solution. The genital organs of six inoculated boars were examined by virus isolation and immunofluorescence. In spite of high virus titers, the fluorescence in the testicles remained limited to a few small foci in the interstitial connective tissue and tunica albuginea at or close to the inoculation site. Neither virus replication, necrosis nor inflammatory lesions could be demonstrated in the epithelium of the seminiferous tubules. However, virus replication was regularly demonstrated in the serosa covering testicles, plexus pampiniformis, ductus deferens and tunica vaginalis. Virus was also isolated from the scrotal fluid. It is suggested that the serosa is the primary target tissue for ADV. The other four boars were inoculated to study the effect of ADV on semen. Severe morphologic alteration and lowered sperm cell concentrations were observed during several weeks after inoculation or until slaughter at 47, 53 and 58 days post inoculation. Virus was isolated from semen of only two out of four boars examined at 9 and 10 days post inoculation.     

Viral and bacterial agents associated with experimental transmission of infectious proventriculitis of broiler chickens.

Proventriculitis of broilers can be reproduced by oral inoculation of day-old chicks with a proventricular homogenate from affected 3-wk-old broilers. The objective of the following studies was to isolate from this homogenate viral and bacterial isolates that could produce proventriculitis. A monoclonal antibody to infectious bursal disease virus (IBDV) was used to precipitate virus from the homogenate. A primary chicken digestive tract cell culture system was also used to isolate virus from a 0.2-microm filtrate of the homogenate, and a bacterium was also isolated from the homogenate. In trial 1, day-old birds were orally inoculated with either proventriculus homogenate or monoclonal antibody immunoprecipitated IBDV (MAB-IBDV). At 4, 7, 14, and 21 days postinfection (PI), 12 birds from each treatment group were subjected to necropsy. In trial 2, day-old birds were orally inoculated with either infectious proventriculus homogenate, suspect virus isolated in cell culture and propagated in embryo livers and spleens, or a bacterial isolate. Twelve birds from each treatment were subjected to necropsy at days 7, 14, 21, and 28 PI. In trial 3, treatments were maintained in negative pressure isolation chambers, and an additional treatment included virus plus bacterial isolate. Twenty-four birds from each treatment were subjected to necropsy at day 21 PI. In trial 1, infectious homogenate decreased body weight and relative gizzard weights at 4, 7, 14, and 21 days PI. Proventriculus relative weight was increased at days 7, 14, and 21 PI, and proventriculus lesion scores were increased at days 14 and 21 PI. Bursa/spleen weight ratios were decreased at day 14, and feed conversion was increased at days 4 and 21. The MAB-IBDV treatment decreased proventriculus and gizzard relative weights at day 4 PI, increased proventriculus lesion scores and bursa/spleen weight ratios at day 14, and decreased heterophil/lymphocyte ratios at day 21. In trial 2, all infected birds had significantly higher mean relative proventriculus weights at 21 days PI and had higher 4-wk mean proventriculus scores as compared with both control groups. In trial 3, birds treated with homogenate and birds treated with both suspect virus and the bacterial isolate had significantly higher proventriculus lesion scores; higher relative weights of proventriculus, gizzard, liver, and heart; lower body weights; and lower relative bursa weights compared with the saline control group. These studies suggest that infectious proventriculitis has a complex etiology involving both viral and bacterial infection.     

Pathogenicity and transmission of H5N1 avian influenza viruses in different birds

In this study, we selected three H5N1 highly pathogenic avian influenza viruses (HPAIVs), A/Goose/Guangdong/1/1996 (clades 0), A/Duck/Guangdong/E35/2012 (clade 2.3.2.1) and A/Chicken/Henan/B30/2012 (clade 7.2) isolated from different birds in China, to investigate the pathogenicity and transmission of the viruses in terrestrial birds and waterfowl. To observe the replication and shedding of the H5N1 HPAIVs in birds, the chickens were inoculated intranasally with 10⁶ EID₅₀ of GSGD/1/96, 10³ EID₅₀ of DkE35 and CkB30, and the ducks and geese were inoculated intranasally with 10⁶ EID₅₀ of each virus. Meanwhile, the naive contact groups were set up to detect the transmission of the viruses in tested birds. Our results showed that DkE35 was highly pathogenic to chickens and geese, but not fatal to ducks. It could be detected from all the tested organs, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. GSGD/1/96 could infect chickens, ducks and geese, but only caused death in chickens. It could transmit to the chickens and ducks, but was not transmittable to geese. CkB30 was highly pathogenic to chickens, low pathogenic to ducks and not pathogenic to geese. It could be transmitted to the naive contact chickens, but not to the ducks or geese. Our findings suggested that H5N1 HPAIVs from different birds show different host ranges and tissue tropisms. Therefore, we should enhance serological and virological surveillance of H5N1 HPAIVs, and pay more attention to the pathogenic and antigenic evolution of these viruses.     

Post-epizootic surveys of waterfowl for duck plague (duck virus enteritis)

Surviving birds from nine duck plague outbreaks in urban and confined waterfowl were sampled for duck plague (DP) virus and DP antibody during 1979-86. Duck plague virus was found in combined oral and cloacal swabs of birds from three outbreaks, and DP-neutralizing antibody was demonstrated in some birds from all nine outbreaks. Greater prevalence of DP antibody and higher titers were found in survivors from confined populations than from free-flying urban populations. Free-flying waterfowl from within 52 km of four DP outbreak sites were also sampled; virus was not found in any birds, but DP antibody was found in urban waterfowl in the vicinity of an outbreak in Potterville, Michigan. No evidence of exposure to or shedding of DP virus in migratory waterfowl was found in two regions where DP appears enzootic in urban and confined waterfowl (Eastern Shore of Maryland and the vicinity of Sacramento, California).     

Comparative susceptibility of chickens, turkeys and ducks to infectious bursal disease virus using immunohistochemistry

Twenty chicks, 12 turkey poults and 10 ducklings, all 5 weeks old were infected with 2 × 10^3.5 chick LD₅₀ IBD virus to determine the course of the virus in the 3 poultry species. Uninfected control birds were kept separately. Two infected and 2 control birds/species were euthanized at time intervals between 3 and 168 hours post infection (pi). Sections of thymus, bursa of Fabricius, spleen, liver, kidney, proventriculus and ceacal tonsil were stained for the detection of IBD virus antigen using immunoperoxidase technique. IBD virus antigen positive cells stained reddish-brown and the amount of such cells in tissue sections were noted and scored. Stained cells were present in all organs examined for up to 168 hours pi in the 3 poultry species except ceacal tonsils of ducks at 72 and 120 hours pi. Antigen score was highest in chickens and least in ducks as reflected by average of total scores/sampling time of 12, 10.8 and 8 in chickens, turkeys and ducks respectively. Total antigen score/sampling time in infected chickens peaked twice; 24/48 and 144 hours pi, whereas such bi-phasic peaks were absent in turkeys and ducks. Range of total antigen score at different sampling times was 7–17.5 in chickens, 10–13 in turkeys and 7–10 in ducks indicative of marked viral replication in chickens. Presence of IBD viral antigen in organs of all 3 poultry species is indicative of infections. The innate ability of turkeys and ducks to prevent appreciable replication of IBD virus after infection requires further investigation.     

Outbreak of duck plague (duck herpesvirus enteritis) in numerous species of captive ducks and geese in temporal conjunction with enforced biosecurity (in-house keeping) due to the threat of avian influenza A virus of the subtype Asia H5N1

The continuing westward spread of avian influenza A virus of the subtype H5N1 in free-living and domestic birds forced the European Union and the German federal government to enhance all biosecurity measures including in-house keeping of all captive birds from October 20 to December 15, 2005. Movement of captive ducks and geese of many different species from a free-range system to tight enclosures and maintenance for prolonged times in such overcrowded sheds resulted in pronounced disturbance of natural behaviour, interruption of mating and breeding activities and possibly additional stress. Under these conditions the birds developed signs of severe disease and enhanced mortality twentyfour days later. A total of 17 out of 124 (14%) adult birds and 149 out of 184 year-old birds (81 %) died during the outbreak. A herpesvirus was isolated from many organs of succumbed ducks and geese that was identified as a duck plague herpesvirus by cross neutralization test using known antisera against duck plague virus. The published host range of duck plague comprises 34 species within the order Anseriformes. We report here on additional 14 species of this order that were found to be susceptible to duck plague virus. The exact source of the herpesvirus could not identified. However, low antibody titres in some ducks at day of vaccination indicate that at least some of the birds were latently infected with a duck plague herpesvirus. The remaining healthy appearing birds were subcutaneously vaccinated with a modified live duck plague vaccine (Intervet, Boxmeer, NL) that stopped losses and resulted in seroconversion in most of the vaccinated birds.     

Efficacy of avian pneumovirus vaccines against an avian pneumovirus/Escherichia coli O2:K1 dual infection in turkeys

The clinical, pathological and microbiological outcome of a challenge with avian pneumovirus (APV) and Escherichia coli O2:K1 was evaluated in turkeys vaccinated with an attenuated APV vaccine and with or without maternally derived antibodies. Two groups of two-week-old poults, one with and one without maternally derived antibodies against APV, were vaccinated oculonasally with attenuated APV subtype A or B. A third group remained unvaccinated. Eleven weeks later, the turkeys were inoculated intranasally with either virulent APV subtype A, or E. coli O2:K1, or with both agents three days apart. After the dual infection, birds vaccinated with attenuated subtype A or B, and with or without maternally derived antibodies, had lower mean clinical scores than the unvaccinated birds. In the vaccinated birds, virus replication was significantly reduced and no bacteria were isolated, except from the birds vaccinated with attenuated subtype B. In the unvaccinated turkeys, large numbers of E. coli O2:K1 were isolated from the turbinates of the dually infected birds between one-and-a-half and seven days after they were inoculated.     

Investigation into avian pneumovirus persistence in poults and chicks using cyclosporin A immunosuppression

One-day-old poults or two-week old chicks were infected oculonasally with avian pneumovirus. Cloacal swabs were collected for virus isolation as were selected tissues (Harderian gland, turbinates, trachea, lungs and kidneys) from birds killed at regular intervals up to 33 days post infection (p.i.) for poults, and up to 40 days p. i. for chicks. In an attempt to induce virus re-excretion, the T-cell-suppressor cyclosporin A (CSA) was given for 12 days starting from three weeks p.i. in poults and from four weeks p.i. in chicks. Birds were sampled for virus isolations up to day 12 post CSA treatment. Virus was recovered only up to day nine p.i. in poults, and day five p.i. in chicks during the acute phase of the infection. Despite T-cell suppression, there was no evidence of re-excretion of the virus, and hence no evidence for the persistence of virus in the tissues examined.     

The pathogenicity of three Australian fowl plague viruses for chickens,turkeys and ducks

The pathogenicity of three Australian fowl plague viruses, FPV-1, FPV-2, FPV-3, isolated during a natural outbreak of the disease varied for chickens, turkeys and ducks. FPV-1 and FPV-2 were pathogenic for chickens and turkeys, but not for ducks. However, these viruses were not highly pathogenic as they failed to cause illness or death in all birds that became infected. FPV-3 was non-pathogenic for the three species tested.The viruses spread from infected to in-contact birds, and more readily to ducks than to chickens or turkeys. All chickens and turkeys infected with the fowl plague viruses developed specific serum haemagglutination-inhibiting antibody which persisted for up to 85 days after infection. The titre of this antibody wan ed in six of 16 ducks over an 85-day period and two ducks failed to produce detectable specific HaI antibody despite being infected with the virus.     

REPLICATION OF INFECTIOUS LARYNGOTRACHEITIS VIRUS IN CHICKENS FOLLOWING VACCINATION

SUMMARY The site of replication of infectious laryngotracheitis vaccine virus (ILTV) was studied in chickens vaccinated by drinking water (DW), intraconjunctival (IC) and cloacal (CL) routes. The anatomical sites exposed to vaccine were determined by simulating vaccination with rhodamine red dye. Virus replication was determined by recovering virus from various organs at necropsy.
The dye simulation studies clearly demonstrated that DW vaccination did not usually expose susceptible target organs to virus while the CL and IC routes flooded susceptible organs with vaccine.
Virus replication was confined to the cloaca in CL vaccinated birds while in IC and DW vaccinated birds most replication took place in the nasal cavity. Vaccine virus did not always become established in DW vaccinated birds and its establishment did not appear to be related to the amount of vaccine these birds were exposed.
It was concluded that DW vaccination depends for its success upon the accidental contamination of the nasal cavity with vaccine virus during the act of drinking.     

Host range of A/Chicken/Pennsylvania/83 (H5N2) influenza virus

The highly pathogenic A/Chicken/Penn./1370/83 (H5N2) avian influenza virus, which caused 80% mortality in chickens in Pennsylvania, produced only mild transient illness in experimentally infected pheasants, little or no clinical signs in ring-billed gulls and pigs, and no clinical signs in pekin ducks. Virus could be recovered from only the upper respiratory tract of gulls and pigs for 1-2 days. Infection in ducks resulted in intestinal replication of virus in only 1 out of 12 ducks. By contrast, pheasants shed virus in feces (10(4.7) EID50) for at least 15 days. These studies reinforce wildlife surveillance findings indicating that gulls and ducks are unlikely to have transmitted virus between chicken farms during the 1983 outbreak. Although experimental data suggest that wild gallinaceous birds such as pheasants are potentially capable of virus transmission, there has been no evidence of this from wildlife surveillance in Pennsylvania. Experimental infection of chickens with H5N2 virus isolated from wild ducks one year before the Pennsylvania outbreak or a gull virus (H5N1) isolated in the quarantine area in 1983 resulted in asymptomatic infections and virus replication occurring only in the upper respiratory tract. These studies suggest that if the first H5N2 virus infecting chickens in Pennsylvania originated from waterbirds, changes in host specificity and pathogenicity for chickens and other gallinaceous birds probably occurred during emergence of the Chicken/Penn./83 virus. It is recommended that attention be given in the future to the isolation of domestic poultry from contact with wild aquatic birds.     

Detection of chicken anemia virus DNA in embryonal tissues and eggshell membranes

Chicken infectious anemia virus (CIAV) is a ubiquitous and highly resistant virus of chickens that causes anemia and death in chicks less than 3 wk of age and immunosuppression in chickens older than 3 wk of age. The production of specific-pathogen-free eggs free of CIAV is essential for research and vaccine production. Currently, flocks are screened for CIAV by antibody tests to ensure freedom from CIAV infection. Recent evidence, however, indicates that chickens may carry and vertically transmit CIAV DNA independently of their antibody status. In this study, we tested embryos and eggshell membrane residues by nested polymerase chain reaction (PCR) as a sensitive method of detecting CIAV DNA. CIAV DNA could be detected in the blastodisks and semen obtained from antibody-positive and -negative chickens. Examination of different tissues between 18 and 20 days of incubation indicated that many but not all organs of individual embryos were positive. The lymphoid organs and gonads had the highest incidence of CIAV DNA, which was significantly different (P < 0.05) from the incidence in the liver. Eggshell membrane samples from embryos or newly hatched chicks were an excellent noninvasive source for the detection of CIAV DNA, identifying significantly more positive embryos than did pooled lymphoid organs. The use of dexamethasone injections as a method to improve the detection of carrier birds did not result in an increase of vertical transmission or cause seroconversion in the treated hens. A combination of testing eggshell membrane residues at hatch and periodic testing of blood DNA by nested PCR can be used to identify chickens carrying CIAV DNA and may be used to eradicate carrier birds.     

Effect of dietary supplementation of probiotic on the performance of F1 crossbred (Rhode Island red male × Fayoumi female) cockerels

A study was conducted to evaluate four different probiotic levels, using diets supplemented with 0 (control), 0.1%, 0.2%, 0.3% or 0.5% of organic-green culture-zs (probiotic). The cockerels of 6-weeks old were randomly divided into 15 separate floor pens each comprising 25 birds and three pens (replicates) per treatment group following completely randomized design. At 12 weeks of age BW and feed to gain ratio (FCR) were determined. At the end of 42 days of experiment, nine birds per treatment were sacrificed to evaluate carcass characteristics, abdominal fat contents and the internal organs. Blood haemato-biochemical parameters were also determined. Haemagglutination inhibition antibody titres against Newcastle disease virus and lymphoid organs weight/body weight ratio were also determined. The BW of birds fed 0.2-0.5% of probiotic was significantly greater than birds fed without probiotic diet. Similarly, better FCR was observed in birds those fed diets of high level of probiotic. There was no mortality recorded at higher levels of probiotics. Differences in carcass characteristics, organs weight, meat composition, haematological values and HDL and LDL concentrations among all the diets were non-significant. However, abdominal fat contents reduced significantly in supplemented groups in relation to control and cholesterol contents were reduced significantly in 0.3% or 0.5% supplemented groups in relation to control. Feeding levels of 0.2-0.5% of probiotic, did positively affect the immune system within the parameters measured. It may be concluded that performance, blood chemistry and immunity against disease in cockerels could be maintained when supplementing 0.3% or 0.5% level of probiotic incorporated in crossbred cockerel's diets.     

Serum antibody responses of chickens following sequential inoculations with different infectious bronchitis virus serotypes

Sequential inoculations of chickens with different live infectious bronchitis virus (IBV) antigenic types had major effects on virus-neutralization (VN) and hemagglutination-inhibition (HI) serum antibody responses. Antibody production in IBV-inoculated chickens that were reinoculated 8 weeks later with heterologous virus was largely directed against the virus used for initial inoculation rather than the virus used for reinoculation. In addition, chickens inoculated sequentially with IBV produced a broadened spectrum of serum antibodies that reacted with IBV types to which the birds had never been exposed (JMK and Florida). Chickens inoculated sequentially with heterologous IBV tended to produce higher levels of cross-reacting antibody than birds given homologous virus inoculations. Levels of cross-reacting antibodies were lower than levels of specific antibodies directed against viruses that the birds had received. Limited studies indicated that birds with cross-reacting antibodies were not protected against challenge with the virus that the cross-reacting antibody was directed against. Implications of the research for interpreting serological data from commercial chicken flocks are discussed.