Pathotyping of Newcastle disease virus isolates from pet birds.

Four Newcastle disease virus (NDV) isolates obtained from a pigeon, lory, parrot, and love bird were subjected to biological and molecular characterization. All the isolates were identified as velogenic with intracerebral pathogenicity indices (ICPI) of 1.9-2.0. All the isolates had a 112RRQKRF117 motif in the fusion protein cleavage site (FPCS), typical for pathogenic NDV. Phylogenetic analysis placed the isolates along with a velogenic Indian isolate of Cl group recovered during 1987.

Interaction between genomes of infectious bronchitis and Newcastle disease viruses studied by reverse transcription-polymerase chain reaction.

Reverse transcription-polymerase chain reaction (RT-PCR) with specific primers for the S1 gene of IBV and for the fusion protein cleavage site of NDV was used for detection of Infectious bronchitis virus (IBV, the family Coronaviridae) and Newcastle disease virus (NDV) genomes. The sensitivity of IBV and NDV RT-PCR was 10(3.7) and 10(3.0) EID50, respectively. Although a multiplex RT-PCR could detect and differentiate NDV and IBV genomes present in the same sample, there was a slight inhibition of the IBV PCR if a high amount of NDV genome was present in the sample. To overcome this problem a separate PCR for each virus was used to assess the interaction between vaccine IBV and NDV either inoculated singly or together into chickens. In the group vaccinated with the Newcastle disease (ND) vaccine alone, the viral genome was detected on days 2, 4 and 7 post vaccination (p.v.), while in the chickens given the infectious bronchitis (IB) vaccine alone, the viral genome was detected only on day 4 p.v. In the group inoculated with both vaccine viruses there was a 10(3)-fold reduction in the cDNA dilution factor on day 4 p.v. for both IBV and NDV genomes. This demonstrated clearly that when both these vaccines are administered there is a transient reduction in the replication of both viruses, probably due to their competition for the same target epithelial cells in the respiratory tract.

Egg:embryo weight ratio as an indicator of dwarfism induced by infectious bronchitis virus.

A simple objective method to quantify embryo dwarfism induced by infectious bronchitis virus in embryonated chicken eggs has been used to determine endpoints in virus titration and neutralization assays. The eggs and the respective embryos were weighed and embryo:egg weight (EE) ratios were calculated. The EE ratios were compared with the uninoculated control eggs and endpoints could be calculated objectively. EE indices were also calculated by dividing the EE ratios of inoculated embryonated chicken eggs by the mean EE ratio of uninoculated controls, or in the case of virus neutralization tests by the mean EE ratio of eggs inoculated with virus alone. Although this mean EE index did not reflect the dwarfing (or lack of it) in individual eggs, it served as a group indicator. This method would be useful to observe embryo lesions especially in field (non-egg adapted) infectious bronchitis virus isolates, which does not cause observable dwarfing until several embryo passages.

An in vitro and in vivo evaluation of the virulence of egg drop syndrome virus for the chicken reproductive tract.

The virulence of strains of egg drop syndrome (EDS) 1976 virus for the female reproductive tract of chickens was assessed in vitro using oviduct organ cultures (OOC) prepared from precociously induced oviducts in young chicks by oestrogen treatment. Ciliostasis, haemagglutination and virus titres in infected OOC supernatants, histology and immunoperoxidase test results indicated the pathogenic ability of the four viruses for the precocious oviducts. One of the isolates, EDS TN4, produced higher virus titres in the supernatants of infected OOC and more severe glandular atrophy and necrosis, but caused slightly delayed ciliostasis. When this isolate was used in vivo, virus could not be detected by haemagglutination, but was detected in a few birds using a polymerase chain reaction on the allantoic fluids of infected duck embryos. Ciliostasis of OOC and histological lesions were confined to early stages of infection. This technique could be a pointer to possible variations in virulence of EDS virus isolates, and warrants further investigation. The potential value of OOC from young chickens for EDS diagnosis is emphasized.

Comparison of virus isolation and polymerase chain reaction for diagnosis of peste des petits ruminants.

Oculonasal swabs and tissue samples collected from peste des petits ruminants (PPR) suspected sheep and goats were tested for presence of the virus of peste des petits ruminants (PPRV) or its RNA by reverse transcription-PCR (RT-PCR) and virus isolation (VI). Of 44 samples 31.8% and 40.9% were positive by VI and RT-PCR, respectively. The RT-PCR-positive samples were subjected to the nested PCR. Three of six samples positive by RT-PCR but negative by VI were negative by the nested PCR. The specificity and accuracy of the nested PCR were higher than those of the RT-PCR although the sensitivity of both tests were similar. Nucleotide sequencing of one nested PCR product revealed a 92% homology with the sequence available in the GenBank (Acc. No. Z37017).

Relative efficacy of conventional sperm parameters and sperm penetration bioassay to assess bull fertility in vitro.

Frozen semen samples from 10 bulls were thawed and actively motile sperm recovered using a swim-up technique. Calcium ionophore A23187 at 0.5 microM concentration was used for 1 min to induce the acrosome reaction in the sperm. Mature female golden hamsters were superovulated with 50 IU of equine chorionic gonadotrophin followed 56 h later with 75 IU of human chorionic gonadotrophin. The cumulus mass was recovered 17 h after hCG treatment by puncturing the oviducts in the infundibulum region. Subsequently, cumulus cell mass and zona pellucida were digested by 0.1% hyaluronidase and 0.1% trypsin, respectively, to yield zona-free hamster eggs (ZFE). A sperm penetration bioassay was performed by coincubating capacitated sperm at 5 X 10(6) concentration and ZFE for 3 h at 38 degrees C in an air incubator. The conception rate of the bulls was based of an average of 82.6 cows per bull with pregnancy status confirmed by rectal palpation. It was found to be strongly correlated (p < 0.01, r = 0.723) with fertilization percentage, whereas percent motile sperm, percent viable sperm and percent sperm with intact acrosomes were not significantly correlated with the conception rate (r = 0.210, -0.021 and -0.468, respectively). Results of the present study suggest that the sperm penetration bioassay can be reliably used to test the fertilizing potential of bull sperm in vitro.

High salt method: a simple and rapid procedure for isolation of genomic DNA from buffalo (Bubalus bubalis) white blood cells.

Three different methods, namely guanidine hydrochloride, phenol-chloroform extraction and high salt method, were compared in order to develop a simple method for the extraction of high yield of DNA from buffalo blood suitable for genome analysis. Both phenol-chloroform and high salt methods produced good yields of high molecular weight DNA as determined by agarose gel electrophoresis. The yield and quality of DNA extracted by high salt method was comparable to that of phenol-chloroform method. The mean yields of DNA from 10 ml of whole blood extracted by either the phenol-chloroform or the high salt methods were 446.16 micrograms (SE = 26.68) and 432.83 micrograms (SE = 19.34) respectively. The DNA obtained from both methods was suitable for conventional as well as polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) studies. Extraction using the guanidine hydrochloride method resulted in a gelatinous material that failed to resuspend in TE buffer. The high salt method is quick and reliable and can be routinely used for the extraction of DNA from buffalo samples instead of phenol-chloroform extraction which is hazardous and time-consuming.