In vitro infection studies with infectious laryngotracheitis virus

Infectious laryngotracheitis (ILT) virus strains were studied for their ability to infect chicken macrophages, lymphocytes, and kidney cells in vitro. Although macrophages were as susceptible as chicken kidney cells to infection, replication of most virus strains in macrophages was markedly restricted. Only a few isolates induced progressive infections in macrophages, and even with these the donor of the macrophages influenced replication. Thus, it appears that both cell genotype and virus genotype may help determine the extent of restriction of virus replication. Macrophages were more susceptible to an attenuated vaccine strain of ILT virus than to virulent virus strains. Spleen lymphocytes, peripheral blood lymphocytes, thymocytes, bursal lymphocytes, buffy coat leukocytes, and activated T-cells were nearly or totally refractory to infection by ILT virus.     

鸭感染鸡传染性法氏囊病病毒的调查和人工感染试验

鸡传染性法氏囊病 ( IBD)是危害幼鸡的一种急性、传染性和高度接触性病毒性感染 ,发病率和死亡率均很高 ,给养鸡业造成严重的经济损失。该病呈世界性流行。IBD病毒有两个血清型 ,即血清 1型病毒和血清 2型病毒。血清 1型病毒能使鸡产生明显的临床症状和病理变化。我国流行的多属血清 1型病毒 ,血清 1型病毒又分不同的变异株或血清亚型。近年来国内有关 IBDV对雏鸭的感染 ,并引起临床症状、病理变化和暴发疾病的报道 ,我们对此很感兴趣 ,因为我们在 IBD诊断试剂盒的研制过程中 ,通过雏鸡法氏囊繁殖病毒 ,制备兔抗 IBDV高免血清 ,这样…     

Labeled avidin-biotin enzyme-linked immunosorbent assay for detecting antibody to infectious laryngotracheitis virus in chickens

A labeled avidin-biotin enzyme-linked immunosorbent assay (LAB-ELISA) for detecting antibody to infectious laryngotracheitis (ILT) virus in chicken sera was developed and compared with ordinary ELISA. Purified ILT virus, biotin-labeled anti-chicken IgG rabbit IgG conjugate, and horseradish-peroxidase-labeled avidin were used in the LAB-ELISA. When sera from farm chickens were tested by serum neutralization (SN) and two kinds of ELISA, the correlation rate between SN and LAB-ELISA was 50/50 (100%), and that between SN and ordinary ELISA was 39/50 (78%). In LAB-ELISA, all of the sera that were antibody-negative by SN had low absorbance (A) values (below 0.05), and the A values were closely correlated with the SN indexes. In ordinary ELISA, however, the sera antibody-negative by SN had various A values ranging from 0.06 to 0.32. LAB-ELISA had much lower nonspecific reactions than ordinary ELISA against sera from ILT-negative chickens, even when chickens were 30 weeks old. ILT antibody production after ILT vaccination could be detected by LAB-ELISA. A values peaked 5 weeks postinoculation and were maintained for 17 weeks.     

Development and evaluation of an ELISA for the detection of antibody to infectious laryngotracheitis virus in chickens

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to infectious laryngotracheitis (ILT) virus in chickens was developed and compared with the serum-neutralization assay. The ELISA routinely yielded 16-to-32-fold higher titers than the serum-neutralization test. To overcome the requirement for large amounts of purified viral antigen, the microtiter trays were initially coated with an antibody prepared against purified ILT virus. A relatively crude viral preparation could then be used to coat the trays. Sera from specific-pathogen-free chickens less than 12 weeks of age did not show nonspecific binding, although 2.7% of all sera from chickens between 13 and 64 weeks of age had nonspecific activity. The majority of nonspecific reactors came from one highly inbred flock of specific-pathogen-free chickens. A number of modifications of ELISA procedures reported to reduce the nonspecific binding of chicken sera were investigated. Treatment of the serum or the plate and changes in the composition of the diluent did not increase the relative sensitivity of the anti-ILT assay.     

Pathological changes of tracheal mucosa in chickens infected with infectious laryngotracheitis virus

Six-week-old chickens were inoculated via the posterior thoracic air sac with infectious laryngotracheitis virus. Chickens were sacrificed on various days through day 16 postinoculation (PI), and the trachea was examined by scanning electron microscopy (SEM) and light microscopy (LM). The pathological changes observed on day 1 PI were hypertrophy and hyperplasia of goblet cells. From day 3 PI, the epithelial cells protruded collectively and fused to form syncytia, which contained many intranuclear inclusion bodies. Subsequently, epithelial syncytia desquamated, one after another, and connective tissues were exposed in places. Serofibrinous exudate and detritus were abundant on the surface of the exposed connective tissues and seemed to form a pseudomembrane. On day 5 PI, the remaining epithelial cells began to repair the devastated mucosa just under the pseudomembrane. On day 6 PI, microvillus-rich regenerating epithelial cells were arranged like paving stones. On day 8 PI, the epithelial cells proliferated extensively and formed folds with cyst-like structures. By day 16 PI, the tracheal epithelium was covered with cilia and regained its normal histologic appearance.     

The effect of infectious bursal disease virus infection on local and systemic antibody responses following infection of 3-week-old broiler chickens

Broiler chickens infected at 3 weeks of age with infectious bursal disease virus (IBDV) were given Brucella abortus (BA) or sheep red blood cell (SRBC) antigens before, during, and after the acute phase of the infection. Gland of Harder (GH) extracts and serum samples were used to assay local and systemic antibody titer to each antigen 7 days after antigen was administered. Antibody titers to both BA and SRBC antigens were lower (P less than 0.05) in GH extracts and serum of IBDV-infected broilers than uninfected controls. The responses to BA, a thymus-independent antigen, took longer to become depressed than the responses to SRBC, a thymus-dependent antigen. The depression of antibody titers following IBDV inoculation suggests compromise of both local and systemic immune function, a finding of importance to the broiler industry.     

Cryptosporidiosis of the bursa of Fabricius of chickens.

Light-microscope and electron-microscope studies of a coccidial organism found in the bursa of Fabricius from 3 chickens clearly established the parasite as belonging to the family Cryptosporiidae. Hyperplasia and heterophil infiltration were associated with the presence of organisms attached to the microvillus border of epithelial cells lining the plicae of the bursa of Fabricius. Although there were no clinical signs or gross lesions common to the 3 cases described, all had similar histologic lesions in the epithelium lining the bursa of Fabricius.     

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.     

Concurrent infection in chickens with fowlpox virus and infectious laryngotracheitis virus as detected by immunohistochemistry and a multiplex polymerase chain reaction technique

A concurrent infection of chickens with infectious laryngotracheitis virus (ILTV), a herpesvirus, and fowlpox virus (FWPV), an avipoxvirus, is described. Two techniques, an immunohistochemistry (IHC) technique and a multiplex polymerase chain reaction (PCR), were used to examine 11 tissue samples from chickens clinically diagnosed as FWPV-infected, but only IHC was used to examine six tissue-paraffin blocks prepared from turkeys suspected of having FWPV infection. By multiplex PCR, both FWPV and ILTV were detected from three chicken samples (FI-90, FI-93, and FI-94); both FWPV and ILTV were detected from only two samples (FI-93 and FI-94) by IHC. All chicken samples were positive for FWPV by both PCR and IHC. Viral DNA from these samples was further confirmed by restriction enzyme analysis. When turkey samples were analyzed by the double-stain IHC, all six samples showed the presence of FWPV antigens, but no ILTV antigens. The double IHC technique, using monoclonal antibodies against FWPV and ILTV, was successful in simultaneous demonstration of specific FWPV and ILTV antigens colocalized in infected tissue samples as well as within individual cells. This paper emphasizes the importance of reliable tests that detect specifically the presence of ILTV and FWPV in infected tissue samples. The multiplex PCR assay holds potential to be versatile, rapid, and more sensitive (100%) than IHC (67%) for the simultaneous detection of two different avian viruses. Furthermore, the presence of mixed infection should always be kept in mind in the virologic analysis of respiratory sickness of poultry.     

Immunohistological identification of both infectious bursal and Marek virus antigens in the bursa of Fabricius

Indirect Immunoperoxidase (IIP) and Avidin Biotin-Peroxidase Complex (ABC) techniques were used for the detection of Infectious Bursal Virus (IBV) and Marek Disease Virus (MDV) antigens in alcohol and formalin-fixed, paraffin-embedded lymphoid tissues from broilers. Both techniques appeared potentially useful for the diagnosis of both viral antigens in alcohol-fixed tissues, and allowed the observation of dual infection in the bursa of Fabricius of the studied animals in a natural infection.     

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

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

Protection of chickens from Newcastle disease and infectious laryngotracheitis with a recombinant fowlpox virus co-expressing the F, HN genes of Newcastle disease virus and gB gene of infectious laryngotracheitis virus

A recombinant fowlpox virus (rFPV) coexpressing the Newcastle disease virus (NDV) fusion and hemagglutinin-neuraminidase genes and infectious laryngothracheitis virus (ILTV) glycoprotein B gene was constructed. This virus was then evaluated for its ability to protect specific-pathogen-free (SPF) chickens against clinical symptoms and death after challenge by virulent NDV and ILTV. SPF chickens were grouped and vaccinated with the rFPV and commercial NDV (La Sota) and ILTV attenuated live vaccine (Nobilis ILT), respectively. After challenge with NDV 10 days postvaccination, 70% of chickens vaccinated with rFPV were protected from death, whereas 100% of the commercial NDV-vaccinated chickens were protected from death. In contrast, 100% of the unvaccinated chickens died after challenge. After challenge with ILTV, both the rFPV and commercial ILTV-vaccinated chickens were completely protected from death and 70% of chickens were protected from respiratory signs. In comparison, 100% of the unvaccinated chickens developed severe respiratory disease and 10% of chickens died. The protective efficacy was also measured by the antibody responses and isolation of challenge viruses. Results showed that this rFPV could be a potential vaccine for preventing NDV and ILTV by a single immunization.