Pathogenicity and antigenicity of eleven isolates of chicken anaemia agent (CAA)

Pathogenicity for chicks of 11 isolates of chicken anaemia agent (CAA) isolated in Japan was examined. Each strain produced aplastic anaemia in 100% of chicks when inoculated at 1 day of age with mortalities ranging from 20 to 90%. Incidence of anaemia in chicks inoculated at 7 days of age varied from 0 to 87.5% according to the isolates, but no chick died. The susceptibility of chicks at various ages against the G1 (Gifu-1) and A2 strains was compared. Both strains produced anaemia in 100% of the chicks when inoculated at 1, 2, 3 or 4 days of age. Half of the chicks inoculated with the A2 strain at 8 days of age showed anaemia, but none of those inoculated with the G1 strain did. No chick showed anaemia from either strain when inoculated at 14 days of age. The antigenicities of the isolates were compared by cross neutralisation and indirect immunofluorescent antibody methods. No serological distinction was recognised among the isolates.     

Interaction of avian reovirus with chicken lymphoblastoid cell lines

Four chicken lymphoblastoid cell lines were inoculated with avian reovirus strain S1133 and two local isolates, 965 and 615. Of the inoculated cell lines, TLT, a B-cell line, was productively infected with the three viruses as demonstrated by immunofluorescence assay (IFA) and radioimmunoprecipitation assay. A comparative growth curve analysis of the three avian reoviruses was done at 37 degrees and 41 degrees C. Isolate 965 replicated to a higher titre at both temperatures while the replication of S1133 and 615 was found to be inhibited at 41 degrees C. IFA revealed that among the transformed T lymphoblastoid cells used in this study, only MDCC-RP1 was permissive to virus infection with isolate 965, and at 41 degrees C, but not 37 degrees C.     

Pathogenicity of avian reoviruses: examination of six isolates and a vaccine strain.

Six avian reovirus isolates and a vaccine reovirus strain were compared for invasiveness, virulence, and pathological characteristics upon infection of day-old specific-pathogen-free chicks by the footpad, subcutaneous, and oral routes of inoculation. No significant differences were noted regarding the ability of individual isolates to infect target tissues. However, virulence (measured as the 50% lethal dose) among the isolates varied markedly from 2 x 10(5) to less than 10 PFU per chick for the most virulent isolate; between the parental wild-type virus and the derivative vaccine virus strain, a million-fold (10(6)) difference in virulence was demonstrated. All strains revealed, with considerable variation, arthrogenic potential.     

Biological and molecular characterization of chicken anaemia virus isolates of Indian origin

In the present study, four chicken anaemia virus (CAV) isolates (CAV-A, -B, -E and -P) recovered from different geographical regions of India were characterized. CAV genome of 1,766 bp nucleotide region containing the complete coding region of VP2 and VP3 proteins, and partial coding region of VP1 protein were sequenced. The nucleotide and deduced amino acid sequence of the Indian CAV isolates were aligned and compared with CAV isolates of European, Asian, American and Australian origin. Phylogenetic analysis of the Indian CAV isolates were also carried out based on the nucleotide and deduced amino acid sequences. The results indicated that Indian isolates were genetically evolved from different parts of the world. Indian isolate, CAV-A was found closely related to European Cux-1 strain, CAV-B and -P were closely related to Bangladesh BD-3 strain and CAV-E was closely related to Australian 704 strain. The pathogenicity of the four CAV isolates was studied in day-old specific pathogen free (SPF) chicks. Day-old SPF chicks (n=50) were divided into five groups comprised of 10 chicks in each group. Group 1 was kept as control and groups 2-5 were infected with each CAV isolate separately. The chicks were infected at a dose rate of 1 ml cell culture fluid (10(4.5)TCID(50)/0.1 ml) per bird intramuscularly. The clinical signs, mortality and packed cell volume (PCV) and body weight gain were recorded on 5, 10 and 15 days post-infection. At 15th day, all the birds were sacrificed and various organs, viz., thymus, bone marrow, spleen, liver and bursa were examined for gross and microscopic changes. The pathogenicity study indicated that all the CAVs except CAV-B were able to produce clinical disease and immunosuppression in young chicks whereas the isolate CAV-B produced no clinical disease but only induced immunosuppression, which was revealed by microscopic examination of the lymphoid organs. The study showed valuable information on molecular epidemiological status of CAV isolates prevalent in India for the first time.     

Evaluation of the egg transmission and pathogenicity of Mycoplasma gallisepticum isolates genotyped as ts-11

Live Mycoplasma gallisepticum vaccines are used for the control of respiratory disease, egg production losses and egg transmission associated with M. gallisepticum infection in long-lived poultry. The first field case of apparent increased virulence and vertical transmission of ts-11, a live M. gallisepticum vaccine, has been reported. In that study a M. gallisepticum isolate from the broiler progeny of ts-11-vaccinated breeders was genotyped as ts-11 by sequence analysis of four different genetic targets and Random Amplified Polymorphic DNA and found to be significantly more virulent than ts-11 vaccine. The objective of the current study was to evaluate the rate of egg transmission and pathogenicity of ts-11 vaccine and isolates recovered from ts-11-vaccinated breeders (K6222B) and their broiler progeny (K6216D) which had been genotyped as ts-11. Groups of 28-week-old specific pathogen-free chickens at 87% average weekly egg production were inoculated with sterile broth media (negative controls), ts-11 vaccine, K6222B, K6216D or R strain (positive controls) by eye-drop and aerosol. K6216D transmitted via the egg at an average rate of 4.0% in the third and fourth weeks post-infection, while egg transmission of K6222B and ts-11 vaccine was not detected. M. gallisepticum was isolated from the air sacs, ovaries and oviducts of hens infected with K6216D and K6222B, but not from those infected with ts-11 vaccine. K6216D and K6222B both induced respiratory signs and significantly more tracheal colonization and more severe tracheal and air sac lesions than ts-11 vaccine (P ≤ 0.05). There were no substantial differences in the egg production of ts-11, K6216D and K6222B infected groups. These results provide the first conclusive evidence of transovarian transmission of an isolate genotyped as ts-11 and indicate that isolates genotyed as ts-11 vary in their virulence and ability to transmit via the egg.     

Newcastle disease virus pathotypes

The clinical signs, mortality and postmortem findings following infection of six-week-old chicks with nine strains of Newcastle disease virus were studied. Although strains could be divided into four pathotypes the divisions were not clear-cut. The most prominent feature of disease following infection by two isolates from the post-1970 USA epidemic, a 1962 UK isolate and a 1972 UK isolate were haemorrhagic gut lesions. A virus isolate from the post-1970 UK epidemic, Lamb-Essex '70, rarely caused gut lesions in infected birds although this was the only strain to consistently induce oedema of the eye in infected birds.     

Some strains of serotype 4 fowl adenoviruses cause inclusion body hepatitis and hydropericardium syndrome in chickens.

Fowl adenoviruses were isolated and characterized from severe cases of hydropericardium syndrome in Ecuador and Pakistan. All were neutralized by antibodies against serotypes 4 and 11. Cross-neutralization tests and restriction enzyme analysis strengthen the classification as serotype 4 strains. The restriction endonucleases used allowed the differentiation among field isolates and reference strains. All field isolates tested induced high embryo mortality. One-day-old specific pathogen free (SPF) chicks were infected with 10(5) plaque-forming units by natural routes to reproduce the disease with plaque purified virus. Whereas no mortality was seen with the reference strain, the mortality with the field isolates was 100%. A reduced mortality occurred with a lower infectious dose. Field isolate K1013 from Ecuador was also highly pathogenic for 1- to 3-week-old SPF chicks after intramuscular inoculation. The main pathological signs were swollen livers, severe hydropericardium and nephritis, reflecting the field situation and underlining the role of FAV4 strains in the aetiology of infectious hydropericardium.     

Pathogenesis of pancreatic atrophy by avian influenza a virus infection

Specific-pathogen-free (SPF), 2-day-old chicks were inoculated with type A influenza virus (A/whistling swan/Shimane/499/83/(H5N3)) into their caudal thoracic air sac. The original isolate of the virus was of low virulence (ICPI 0. 20 to 0.40), and was passaged 10 times through the respiratory organs of SPF chicks. Most of the chicks inoculated with the passaged virus (strain 499) showed respiratory and alimentary signs. Three of 30 chicks died on days 2, 6 and 7 post-inoculation (p.i.). Almost half of the infected chicks showed poor growth, and the variation of body size in the flock became prominent from day 10 p.i. Infected chicks consistently had pathological changes in the pancreas, liver, kidneys and respiratory tracts, and occasionally in the brain, duodenum and bone marrow. Positive immunoreaction to avian influenza virus (AIV) antigen and recovery of the virus persisted for longer period in the pancreas than in other organs. The pancreatic lesions were caused by a direct, lytic virus infection of the acinar cells and contributed to poor growth of the chicks.     

Evaluation of the protective efficacy of the anticoccidial vaccine Coccivac-B in broilers, when challenged with Egyptian field isolates of E. tenella

The present study was performed to explore the efficacy of the commercial anticoccidial vaccine (Coccivac B®) in broiler chickens using five field strains of Eimeria tenella that were isolated from five provinces in Egypt. This study also analyzed the ITS-1-rDNA sequence of these five strains and its corresponding sequence in the vaccine. In a floor pen experiment, 216 one-day-old commercial broiler chicks were classified into vaccinated and non-vaccinated groups. Each main group was classified into six subgroups. The chicks were challenged on the 28th day of age with 10⁴ sporulated oocysts of one of the five field strains of E. tenella. Our results indicated that Coccivac B® produced variable degrees of protection in the birds infected with the five different strains of E. tenella. Aligning the ITS-1 sequences from the five strains with the ITS-1 sequence of E. tenella from the vaccine revealed 96 % sequence similarity with the Kafer El-Sheikh strain, 94 % with the Gharbia strain, 90 % with the Alexandria strain, and 78 % with the Matrouh and Behera strains. While interesting, these similarity values were not useful for predicting the protection conferred by the vaccine against the five field isolates. However, based on the data reported here, we can conclude that Coccivac B® produced variable degrees of protection in the birds infected with the five different strains of E. tenella     

Infectivity of Blastocystis isolates from chickens, quails and geese in chickens

The infectivity of six Blastocystis isolates obtained from two domestic chickens, two Japanese quails and two domestic geese, were examined in 1-week-old male chicks. All six isolates were able to infect the chicks via the intracecal inoculation of 1×10⁶ cells of cultured organisms. Since the infected chicks discharged many cysts in their feces, the infectivity of the concentrated cysts in chicks was compared among three isolates from different bird species. The CK86-1 and QQ93-3 isolates, which were obtained from a chicken and a quail, respectively, were successfully infected in chicks by orally inoculating with 1×10²–1×10⁶ cysts. On the other hand, the AC03-1 isolate from a goose required more cysts to infect the chicks, from 1×10³ cysts to 1×10⁶ cysts. In addition, when an uninfected normal chick was housed with five experimentally inoculated chicks with cysts of the QQ93-3 isolate, the normal chick became infected, indicating the fecal–oral transmission of the cyst form among the birds. These results show that the transmission of Blastocystis infection occurs easily between the same or different bird species. Therefore, the proposal of new Blastocystis species on the basis of different avian host species is problematic.     

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.     

An in vitro and in vivo evaluation of the virulence of Newcastle disease virus and vaccines for the chicken reproductive tract

The virulence of two vaccine strains and two field strains of Newcastle disease virus (NDV) for the female reproductive tract of chickens was assessed using oviduct organ cultures (OOC) prepared from precociously-induced oviducts in young chicks by oestrogen treatment. Ciliostasis, haemagglutination and virus isolation from infected OOC supernatants, histopathology and immunoperoxidase test results indicated the pathogenic nature of both vaccine and virulent NDVs for the precocious oviducts. The virulent viruses, mesogenic and lentogenic vaccines caused damage in that order of magnitude and the uterus had a higher susceptibility than oviducts. One virulent and the mesogenic strain of NDV were used for in vivo trials. The pathogenicity was assessed in oestrogen-treated infected chickens using histopathology and immunoperoxidase test. The vaccine virus produced transient damage up to 6 days post-infection, while the damage with the virulent isolate persisted for at least 9 days post-infection. This technique could be a pointer to possible variations in virulence of NDV vaccine and field strains, and warrants further investigation. The potential value of OOC from young chickens for testing the possibility of NDV vaccines causing damage by themselves and offering protection against damage of the reproductive tract caused by virulent isolates is emphasized.     

Identification and characterization of an avian adenovirus isolated from a 'spiking mortality syndrome' field outbreak in broilers on the Delmarva Peninsula, USA

A fowl adenovirus isolated from a 'spiking mortality syndrome' field outbreak in broilers on the Delmarva Peninsula, USA, was identified by cross-neutralization tests and by restriction enzyme analysis using eight restriction enzymes as a strain of the fowl adenovirus serotype FAV12. The isolate is nearly identical to strain UF71 though it shows substantially higher virulence in vivo than UF71. Contrary to strains of other fowl adenovirus serotypes (except serotype FAV1), the isolate caused high embryo mortality in specific pathogen free (SPF) chicken embryos inoculated via the allantoic cavity. Infection of 83 one-day-old SPF chicks with 105 plaque forming units of the isolate via a natural route led to clinical signs and growth inhibition. Gross and histological lesions in the liver, bursa of Fabricius and thymus, and in five cases hydropericarditis were observed. An inclusion body hepatitis was found in two chicks.     

Kinetics of the appearance of Marek's disease virus DNA and antigens in the feathers of chickens

A comparison was made of the temporal appearance of six isolates of serotype 1 Marek's disease virus (MDV) in the feathers of specific pathogen-free (SPF) infected birds using three assays: agar gel precipitation (AGP), enzyme-linked immunosorbent assay (ELISA) and dot-blot DNA hybridisation. Isolate GA-5 served to standardise the in vivo pathogenicity assay, while the remaining five were recent isolates from Israel. Each isolate was inoculated into susceptible 4-day-old birds housed with an equal number of uninoculated birds. All six caused high mortality (80 to 100%) in the inoculated birds and a wide range of mortality (15 to 80%) in the contact groups. The transmission of infection from the inoculated group to the contact group was demonstrated for all six isolates by AGP and ELISA and for four isolates by dot-blot hybridisation. The other two isolates either showed a concurrent rise in MDV-DNA levels (isolate B) or failed to produce detectable levels of DNA in the inoculated and contact infected groups (isolate Ab). This could be due to the nature of the hybridisation reaction between the probe and the homologous sequence in the genome of isolate Ab. Antigenic activity was detected 11 days post-injection by ELISA, 14 days by AGP in some of the inoculated groups. In the contact infected birds the ELISA and dot-blot assays detected virus about 14 days earlier than did AGP. The time interval between the first detection of virus in the inoculated as compared with the contact infected groups differed for each assay and each isolate, viz; 10 to 14 days by ELISA, 14 to 24 days by AGP and 11 to 18 days by DNA-hybridisation.     

Involvement of birds in the epidemiology of the Lyme disease agent Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, was isolated from the liver of a passerine bird, Catharus fuscescens (veery), and from larval Ixodes dammini (tick) feeding on Pheucticus ludovicianus (rose-breasted grosbeak) and Geothlypis trichas (common yellowthroat). In indirect immunofluorescence antibody tests, isolates reacted with polyclonal and monoclonal (H5332) antibodies. Studies on the DNA composition of the veery liver isolate and the strain cultured from an I. dammini larva indicated that both were B. burgdorferi and not Borrelia anserina or Borrelia hermsii. The veery liver isolate infected hamsters and a chick. In contrast, B. anserina infected chicks but not hamsters. B. burgdorferi is unique among Borrelia spp. in being infectious to both mammals and birds. We suggest that the cosmopolitan distribution of B. burgdorferi may be caused by long-distance dispersal of infected birds that serve as hosts for ticks.     

Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51

The Brucella AMOS PCR assay was previously developed to identify and differentiate specific Brucella species. In this study, an abbreviated Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella abortus into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and Brucella AMOS PCR. This included 120 isolates identified as B. abortus S19, 9 identified as B. abortus strain RB51, 57 identified as B. abortus biovar 1, 15 identified as B. abortus bv. 2, 1 identified as B. abortus bv. 2 (M antigen dominant), 7 identified as B. abortus bv. 4, and 22 identified as B. abortus S2308 and isolated from experimentally infected cattle. The Brucella AMOS PCR correctly identified each isolate as RB51/S2308, S19, or a field strain of Brucella.     

Full-length infectious clone of an Iranian isolate of chicken anemia virus

An Iranian field strain of chicken anemia virus (CAV), designated IR CAV, was isolated in the Marek’s disease virus-transformed lymphoblastoid cell line MDCC-MSB1 (MSB1) culture for the first time. The full-length CAV DNA of this strain was cloned in the bacterial plasmid pTZ57R/T to create the molecular clone pTZ-CAV. The nucleotide and deduced amino acid sequences of viral proteins of IR CAV were compared with those of representative CAV sequences including reference and commercial vaccine strains. IR CAV was not related to vaccine strains and also found to have glutamine at positions 139 and 144 confirming previous studies in which such mutations were associated with a slow rate of virus spread in cell culture. pTZ-CAV was digested with PstI to release IR CAV DNA and then transfected into MSB1 cell by electroporation. The transfected cells showed cytopathic effect similar to virion-initiated infection. One-day old specific pathogen-free chicks were inoculated with the regenerated virus, which had been obtained from transfected MSB1 cells, and compared with the chicks inoculated with IR CAV. Gross lesions in the birds inoculated with the regenerated virus illustrated the infectious nature of the regenerated virus from the cloned IR CAV DNA.     

Histopathology and immunohistochemistry of renal lesions due to avian infectious bronchitis virus in chicks uninoculated and previously inoculated with highly virulent infectious bursal disease virus.

A nephropathogenic strain of infectious bronchitis virus (IBV) was inoculated intra-tracheally into 14-day-old specific-pathogen-free chicks or ones previously inoculated with highly virulent infectious bursal disease virus (IBDV) at 7 days of age. The renal lesions were examined histopathologically and immunohistochemi-cally at intervals up to 30 days post-inoculation. The mortality was 20% in the IBDV + IBV-inoculated group, but not in the IBV-inoculated one. Swollen and pale kidneys due to IBV infection were more severe and of longer duration in dually infected chicks. At the early stage of infection, the histopathological changes in the kidneys were similar in both groups, but the ducto-tubular damage was more severe in the dually infected chicks. At the late stage of infection, the renal lesions were characterized by chronic interstitial nephritis with formation of lymphoplasmacytic nodules in IBV-inoculated chicks and by chronic active nephritis which consisted of tubular degeneration, lymphoid cell reaction and interstitial fibrosis in IBDV + IBV-inoculated ones. More IBV antigen-positive cells persisted longer in the kidneys of dually infected chicks than in those of IBV-inoculated ones.     

Preliminary characterisation of isolates of chicken anaemia agent from the United Kingdom

Two isolates of chicken anaemia agent (CAA) were made from livers obtained from broiler flocks with runting stunting syndrome. Following primary isolation in SPF chicks, both isolates were propagated in MDCC-MSB1 cells. Both were resistant to heat and chloroform-treatment, and were antigenically related to the Cux-1 isolate of CAA by cross-immunofluorescence and cross-neutralisation tests. The isolates produced anaemia and characteristic lesions in 100% of experimentally inoculated 1-day-old susceptible chicks, but anaemia was observed in only 15 to 20% of inoculated 7-day-old chicks.