K. Nachimuthu

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.

Postvaccinal immune response to regimens of Newcastle disease vaccination by filter paper sampling technique.

SummarySeven hundred and ten blood samples were collected at random from commercial layers in Tamil Nadu on Whatman filter paper No. 1 instead of the conventional method of serum collection. The birds were subjected to different Newcastle disease (ND) vaccination schedules and samples were collected to study the vaccinal response to ND at field level. Eluates were obtained from sample areas of filter paper using Brij-35 solution [detergent] and subjected to the micro haemagglutination inhibition (HI) test for ND antibodies. The HI titre ranged from less than 24 to 29. The possible causes of poor immune response to ND vaccinations are discussed.Résumé710 échantillons de sang furent collectés au hasard dans des élevages industriels dans le Tamil Nadu utilisant des filtres Whatman No. 1 au lieu de la méthode conventionnelle d’échantillonnage des serums. Les oiseaux furent soumis à différents traitements de vaccination contre la maladie de Newcastle et les échantillons furent recueillis pour étudier la réponse vaccinale contre la maladie de Newcastle au miveau environnemental. Les éluats furent obtenus à partir de la surface des filtres utilisant une solution de Brij-35 (détergent) et soumis à un test d’inhibition par micro-hémagglutination des anticorps contre la maladie Newcastle. Les titres d’inhibition par hémagglutination furent compris entre 24 et 29, les causes probables de cette faible réponse immunitaire face à la vaccination contre la maladie de Newcastle sont discutées.ResumenSe obtuvieron 710 muestras de sangre de ponedoras seleccionadas al azar en Tamil Nadu. En lugar de utilizar el método tradicional de obtención de suero, las muestras de sangre se recognieron sobre papel de filtro Waltham no. 1. Los animales habían recibido diferentes protocolos de vacunación frente a la enfermedad de Newcastle y las muestras se recogieron para estudiar la respuesta vacunal a nivel de campo. El papel de filtro se lavó con solución Brij-35 y después se realizó el test de inhibición de la microhemaglutinación para detectar anticuerpos frente a Newcastle. Los títulos de anticuerpos variaron entre 24 y 29. Se discuten las posibles causas de la baja respuesta observada.

Demonstration of Alternative and Classical Complement Pathway Activity in Colostrum from Buffalo (Bubalus bubalis)

Buffalo colostrum caused lysis of unsensitized red blood cells (RBC) from sheep, goats, rabbits and chickens. RBC from cattle and buffalo were resistant to lysis. That lysis was due to the presence of natural antibodies to these RBC was ruled out since there was no reduction in haemolytic titres even after adsorption with the respective RBC. The addition of EGTA to the diluent had no effect on the haemolytic activity. These findings indicate the presence of alternative complement pathway (ACP) activity in buffalo colostrum. The haemolytic activity of buffalo complement for unsensitized rabbit RBC was reduced to very low levels by heating at 50°C for 45 min. Treatment with zymosan also inhibited the haemolytic activity, while inulin had no effect. The maximum activity of ACP occurred in the presence of 4 mmol/L Mg2+ in the diluent. The range of ACP activities in colostrum from buffaloes varied from 4.06 to 8.48 CH50 units/ml. Using a standard system for titrating the classical complement pathway and rabbit red blood cells sensitized with goat haemolysin, the range of complement activity in buffalo colostrum was 4.81–6.77 CH50/ml.

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.

Comparison of Various Diagnostic Methods in Characterizing Newcastle Disease Virus Isolates from Desi Chickens

Eleven Newcastle disease viruses (NDV), isolated from apparently healthy and ailing Desi chickens were subjected to both conventional and modern characterization techniques. The virulence and strain differentiating experiments placed 10 isolates in the velogenic group and one in the mesogenic group. In MDBK cells, 9 isolates produced characteristic cytopathogenic effects up to 5 and 2 up to 3 passages. Molecular characterization with a 21-mer oligonucleotide probe placed all the isolates in the velogenic/mesogenic group. The results of this study clearly indicated that the isolates obtained are either velogenic or mesogenic but not lentogenic.

Molecular Epidemiology of Peste Des Petits Ruminants Viruses from Southern India

PESTE des petits ruminants (PPR) is a highly contagious disease affecting sheep and goats which is widely distributed in sub-Saharan Africa and the Arabian Peninsula (Taylor 1984). The virus is antigenically related to rinderpest virus, which infects cattle and other large ruminants. It is a member of the morbillivirus genus, which also includes measles, canine distemper virus and viruses of marine mammals, in the family Paramyxoviridae. In India, PPR virus was first identified in an outbreak in 1987 (Shaila and others 1989) and since then it has been reported in all parts of the country (Nanda and others 1996). It is now assumed that PPR was probably present in India before 1987 but was confused with rinderpest (Taylor and others 2002). In order to obtain epidemiological information on the nature of the viruses involved in PPR outbreaks, the genetic relationships between the viruses from different geographical regions of the world have been determined by sequence comparisons of a segment of the virus fusion (F) protein gene, by Shaila and others (1996). In that study, a phylogenetic tree was constructed using several different PPR viruses and it was found that there were four different lineages. PPR viruses from Africa were found to group into three distinct lineages. Viruses isolated from Africa in the early 1970s formed one lineage (group 1), those isolated from the Ivory Coast and Guinea in the late 1980s formed a second lineage (group 2) and the third group included viruses isolated from Sudan, Oman and southern India over a 20-year period. The more recent outbreaks, which occurred across Asia (including northern India) in the 1990s, fell into a fourth lineage (group 4). The existence of distinct lineages of viruses in northern and southern India calls for a more detailed molecular analysis of PPR viruses involved in later outbreaks. The present study was undertaken to confirm the present situation with respect to the molecular epidemiology of the PPR viruses circulating in southern India since 1996. Seven PPR virus isolates were used in this study, six from Tamil Nadu and one from Andhra Pradesh, two states of southern India. The place and year of isolation and the nature of the sample used for RNA isolation are given in Table 1. Viruses were grown on Vero cells maintained in minimum essential medium supplemented with antibiotics and fetal calf serum. Infected cells were harvested when extensive cytopathic effects were seen. RNA was isolated from infected Vero cell monolayers, from tissues obtained from dead animals at postmortem examination or from oculonasal swabs from live animals using the method described by Chomczynski and Sacchi (1987). The total RNA extracted was reverse transcribed into CDNA using the Thermoscript reverse-transcriptase PCR kit (Life Technologies), following the method of Forsyth and Barrett (1995). For the PCR, two sets of F gene-specific primers were used. Primers F 1 and F2 amplified a 372 base-pair product, while primers FIA and F2A amplified a 308 base-pair product, in a nested reaction. The sequences of the primers are described by Forsyth and Barrett (1995). The PCR was performed in a thermal cycler (MJ Research) using the Taq PCR core kit (Qiagen) and the following programme: step 1, one cycle, 94°C for five minutes; step 2, 35 cycles, 94°C for one minute, 55°C for one minute, 72°C for one minute; step 3, one cycle, 720C for seven minutes. The amplified PCR products of the first reaction were confirmed in the nested reaction. After confirmation of the product as part of the PPR virus genome, the outer PCR product (produced by primers Fl and F2) was gel purified using a DNA extraction kit (Fermentas). The purified PCR products were sequenced using the automated ABI prism module at Bangalore Genei, Bangalore, India, using cycle sequencing methods. The nucleotide sequences were aligned with each other using the ClustalV programme and the phylogenetic analysis was carried out using molecular evolutionary genetic analysis (MEGA) software, version 1.02 (Sudhir and others 1993). The parameters used were Universal genetic code, Jukes Cantor distance and UPGMA. These partial F gene sequences were compared with the already available PPR F sequences and an updated phylogenetic tree was generated. When the nucleotide sequences of the seven isolates from southern India were aligned along the sequence available for one other isolate from southern India (Ind TN 92/1) (Shaila and others 1996) it was seen that all of the isolates collected from 1996 to 2001 belonged to the same group, and differed from the Ind TN 92/1 isolate (Fig 1). The first isolate of PPR virus from India, from an outbreak in 1987 and named as Ind TN 88/1 (Shaila and others 1989) also grouped with the six more recent virus isolates. Another candidate vaccine virus Ind TN 97, subgrouped with the Ind TN 88/1 isolate, but was slightly different from the others. However, when the amino acid sequences coded by the fragment of the F gene amplified by PCR were compared among these isolates, no differences were seen. Thus, it appears that only the Ind TN 92/1 isolate had different nucleotide sequences, aligning itselfwith Middle Eastern virus isolates, while the other viruses from southern India did not. In a previous study (Shaila and others 1996), it was also reported that a unique epidemiological situation was present

Detection of Antibodies to Egg Drop Syndrome Virus in Chicken Serum Using a Field-Based Immunofiltration (Flow-Through) Test

Abstract A simple, user-friendly, and rapid method to detect the presence of antibodies to egg drop syndrome 76 (EDS) virus in chicken sera based on an immunofiltration (flow-through) test was developed. Purified EDS virus antigen was coated onto nitrocellulose membranes housed in a plastic module with layers of absorbent filter pads underneath. Following addition of serum to be tested and washing, monoclonal antibodies or polyclonal serum to chicken immunoglobulin G (IgG) was used as a bridge antibody to mediate binding between EDS virus-specific IgG and protein A gold conjugate. The appearance of a pink dot indicated the presence of antibodies to EDS virus in the sample tested. The results could be obtained within 5–10 min. The developed immunofiltration test could detect antibodies in the sera of experimentally vaccinated chickens from 2 wk postvaccination. With field sera samples, this test was positive in samples having hemagglutination inhibition titers of 8 and above. This test has the potential to be used as a field-based kit to assess seroconversion in EDS-vaccinated flocks.

Filter paper technique for seromonitoring against infectious bursal disease.

Infectious bursal disease (IBD) causes immune suppression and various degrees of disease in chickens (Pejkovski et al., 1979). IBD is controlled by vaccinating chickens with live vaccines or hens with live and/or inactivated vaccines. Seroconversion after vaccination or natural outbreaks can be measured by the agar gel precipitation test (AGPT) but collection, storage and transport of serum samples are time consuming. Hence, an attempt was made to collect samples on Whatman No. 1 filter paper and assess the usefulness of filter paper eluate in AGPT. Blood samples were collected from birds on Whatman No. 1 filter paper strips and in test tubes for serum following the method of Brugh and Beard (I 980). Filter papers were then air dried, sealed in potypropylene bags and stored at room temperature. Serum samples removed after dotting were stored at -20°C. Samples were collected from birds 7 to 23 weeks old which l~ad been vaccinated with live vaccines against IBD at 3 and 6 weeks of age. Paired filter paper and serum samples were collected simultaneously from 52 birds. Another 44 blood samples were collected on filter paper from birds of different age groups from natural outbreaks of IBD. Filter paper samples were eluted as described by Rivetz et al. (1985). Two discs of 5mm diameter were cut from the sample area and treated with 75#1 of 0-1% v/v polyoxyethylene 23 lauryl ether (Brij-35, Loba, India; a non ionic detergent) in normal saline solution in a flat bottom microtitre plate for 2 hours. Complete elution was indicated by uniform light colouration of both sides of the filter paper discs. Antigen for AGPT was prepared and the AGPT carried out following the procedures of Hirai et al. (1972) modified by Karunakaran (1991). Three-week old birds were inoculated intraocularly with 10% w/v bursal homogenate containing IBD virus of serotype 1. After 72 hours the bursae were collected and added to an equal volume of phosphate buffer saline with 1% t-octylphenoxy-polyethoxyethanol solution (Triton X-100 Sigma, USA) and homogenised. Homogenate was centrifuged at 2,000 g for 30 minutes and the supernatant further clarified by centrifugation at 4,000 g for 30 minutes. For the AGPT, the central wells were charged with antigen and the peripheral wells with serum along with positive and negative controls. Filter paper eluates were tested simultaneously in the same way as the serum. The significance of the difference between the proportions of positive results was analysed by McNemars Change Test (McNemar, 1969). Specificity and sensitivity indices were calculated using the equations described by Ilstrup (1990). In the first trial, IBD antibodies were detected in 60% of the serum samples and 40% of the filter-paper eluates; the difference was very significant (McNemars chisquare = 6-75, P < 0.01). The specificity of the results with filter paper eluates was 95% but the sensitivity was only 64 per cent. A similar proportion of positives

Latex immunoassay for rapid detection of Newcastle disease virus.

SummaryA rapid test has been developed based on the technique of latex immunoassay for the detection of Newcastle disease virus from suspected tissue suspensions. The latex particles were sensitised with globulins and were used for antigen detection. Of the 258 samples tested, 165 samples were positive by this kit which was compared for its efficacy with the standard OIE approved haemagglutination (HA) and haemagglutination inhibition (HI) tests. No significant difference (P > 0·05) was observed between the tests. The sensitivity and specificity of the developed test was 94·19% and 87·63% respectively.RésuméUn test rapide a été développé grâce à une technique immunologique au latex pour la détection du virus de la maladie de Newcastle chez des suspensions de tissus pouvant être infectés. Les particules de latex, sensibilisées avec des globulines furent utilisées pour la détection de l’antigène. Sur les 258 échantillons testés, 165 furent positifs avec ce type de kit dont l’efficacité fut comparée avec les méthodes standards de l’OIE: l’hémaglutination et le test d’inhibition de l’hémaglutination. Aucune différence ne fut significative (P > 0,05) entre les différents tests. La sensibilité et la spécificité furent respectivement pour ce test de 94,19% et 87,63%.ResumenSe desarrolló un test rápido basado en la técnica del immunoensayo en látex para la detección del virus de la enfermedad de Newcastle en suspensiones tisulares. Las partíiculas de latex se sensibilizaron con globulinas y se utilizaron para la detección de antígenos. De un total de 258 muestras analizadas, 165 fueron positivas de acuerdo con este test. La eficacia del test se comparó con los tests estándar de hemoaglutinación (HA) e inhibición de la hemoaglutinación (HI). No se encontraron diferencias entre tests. La sensibilidad y especificidad del test desarrollado fue del 94·19% y 87·63% respectivamente.