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Determination of in - Vivo Growth Kinetics of Virulent Newcastle Disease Virus in Layer Chicken

Md. Mostofa Kamal
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Mohammad Aynul Haque
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Shuvho Chakra Borty
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh.

AKM Khashruzzaman
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Mohammad Nizam Uddin Chowdhury
Bangladesh Forest Department, Bangladesh.

Md. Alimul Islam
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Abstract/ Executive Summary:

The research work was conducted on 105 layer chicks with a view to determine the rate of distribution of neurotropic virulent Newcastle disease virus (NVNDV) in various organs following infection through natural (intranasal, intraocular and oral) and parenteral (intravenous, intramuscular and subcutaneous) routes of inoculation at different ages (7, 15 and 28 days of old). Each bird received a dose of 0.2 ml contained 300 ELD₅₀ of reference NVNDV. The highest body temperature (≥108⁰F) was recorded in the birds of almost all the experimental groups within 48 to 72 hours of PI. Appearance of clinical signs was observed earlier (48 to 72 hours of PI) in parenterally infected birds than those of inoculated through natural routes. The shortest duration (24-48 hours of PI) and longest duration (74-138 hours of PI) of death time were recorded in birds those inoculated through IV and oral routes of infection respectively. Isolation of NDV was positive from day 2 of PI and onward in all the groups with some minor variations in some cases. The CEF system was found more sensitive for the isolation of viruses compare to that of avian embryo. The highest HA titre of NDV was found in the brain tissue followed by lungs and kidney. Significantly (p<0.01) higher HA titre of NDV isolate was recorded in the birds of all the experimental groups inoculated through IV route. Following infection, the MDA titre decreased day by day in the birds with the increase of HA titres of NDV.

Keywords: Newcastle disease virus, Growth kinetics, HA titer, MDA, Layer chickens.

Location: Department of Microbiology and Hygiene, BAU, Mymensingh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

To determine the rate of distribution of neurotropic virulent Newcastle disease virus (NVNDV) in various organs following infection through natural (intranasal, intraocular and oral) and parenteral (intravenous, intramuscular and subcutaneous) routes of inoculation at different ages (7, 15 and 28 days of old). Each bird received a dose of 0.2 ml contained 300 ELD50 of reference NVNDV.

Methodology:

A total of 105 day-old ISA Brown layer chicks were obtained with the courtesy of CP, Gazipur and very carefully transported to the experimental shed of the Department of Microbiology and Hygiene, BAU, Mymensingh for rearing through out the experiment. Neurotropic velogenic Newcastle disease virus (NVNDV) was obtained from the repository of the department of Microbiology and Hygiene, BAU, Mymensingh and it was used for experimental infection, as positive control during identification of the isolate of NDV from different tissue samples and in haemagglutination (HA) and haemagglutination inhibition (HI) tests. Fertile hen eggs with a good hatching rate (95-98%) from sero-negative Lohmann breed of chickens were collected from Higro Hatchery of FnF Pharmaceuticals Ltd., Jenaidah. The eggs were incubated at 37°C in an egg incubator of the department of Microbiology and Hygeine, BAU, Mymensingh maintaining relative humidity of 85% for 10 days. The well-developed healthy embryos were used for the propagation of reference NVNDV as well as various tissue samples of birds of different experimental groups for the isolation of NDV, to determine ELD50 of reference NVNDV and also preparation of chicken embryo fibroblast (CEF) cell culture. All the layer chicks (105) were divided into four groups named as A, B, C and D. Each group except group D consists of 30 chicks. Further, chicks of group A, B and C were equally subdivided into six sub groups based on the natural (intraocular, intranasal and oral) and the parenteral (intramuscular, intravenous and subcutaneous) routes of infection. All the chicks of group A were experimentally infected at day 7 of age, chicks of group B at day 15 of age and group C at day 28 of age. Each birds of all sub groups was received 0.2 ml of NVNDV (300 ELD50/bird) .The group D containing 15 served as non-infected control. Rectal temperature and clinical signs manifested by the birds of infected groups were recorded daily until death after experimental infection with NVNDV. One bird from each sub-group was subjected to post-mortem examination daily until day five of PI and various tissue samples (brain, lungs, kidney, spleen, bursa of Fabricious, colon and thymus) and feces were also collected for isolation of NDV. The inoculum was prepared from all the samples and inoculated into ten-day-old embryonated hen eggs. The embryos died within 24 hours of incubation were discarded and those died after 24 hours of inoculation were chilled at 4oC for 2 to 4 hours and then the clear allantoic fluid (AF) was carefully collected. All the collected AF was subjected to rapid slide haemagglutination and microplate HA tests following the procedure of Reed and Muench (1938). The 9-day-old healthy avian embryos were taken for cell culture. The bodies of embryos without head, viscera and appendages were chopped and washed 2-3 times with PBS.Two percent trypsin was added for 15 minutes to trypsinize (worm trypsinization) the tissues. Then the cells were washed five times with PBS. Then the cell were transferred into cell culture bottle containing growth promoting media and incubated at 370C for 48 hours to observe confluent growth of cells. The confluent monolayer was allowed to infect with 0.2 ml of inoculum prepared from NDV suspected tissue suspension and kept at 37⁰C for 45-50 minutes for the establishment of better attachment of the viruses to the cells. Two drops of infected AF collected from each of the freshly dead and chilled embryonated eggs or ICF were placed into five well of a 6- well HA slide and another two drops of sterile PBS were placed on another well as control. Two drops of 2% suspension of freshly prepared cRBC was dispensed with a unichannel micropipette upon the AF/ ICF and control PBS and mixed thoroughly with individual pipette tips and observed for clumping of the cRBC within 1 or 2 minutes. 25 μl of PBS was first dispensed into each well of a plastic U-bottomed microtitre plate. Then 25 μl of the virus suspension (i.e. infective AF/ ICF) was placed in the first well and mixed properly. Two fold dilutions of 25 μl volumes of the virus suspension were made across the plate. Again 25 μl of PBS was added into each well. 50 μl of 0.5% (v/v) cRBCs was added into each well. The cell-virus mixture was mixed well by stirring the plate gently. The cRBCs were allowed to settle for about 45 minutes at room temperature when control cRBCs were settled to make a distinct button. HA was determined by tilting the plate and observing the presence or absence of tear-shaped streaming of the cRBCs. The highest dilution of the virus at which the cRBC were found to form a clump and not regimented at the center of the well as button was considered as the end point of the HA activity of the virus sample. Finally, the titration of virus of each sample was determined and calculated by the Reed and Muench (1938) mathematical method. At first 25 μl of PBS was dispensed into each well of a U-bottomed 96-well plastic microtitre plate. Then 25 μl of serum sample was added into each of the first well of the 8 series of the wells and twofold dilutions of 25 μl volumes of the sera were prepared across the plate. 25 μl of 4 HAU virus suspensions of virus was added to each well and the plate was left for at least one hour at room temperature. Then 50 μl of 0.5% (v/v) cRBCs was added to each well and gently mixed and kept at room temperature for 45 minutes when the control cRBCs settled to a distinct button. The finding of hemagglutination inhibition was recorded by tilting the plates. Only those wells in which the cRBCs streamed at the same rate like tears as the control wells containing 50 μl of cRBCs and 50 μl PBS were considered to show inhibition.

Source of Information: Asian J. Med. Biol. Res. 2015, 1 (3), 526-536, (ISSN 2411-4472)

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Achievement/Findings:

This research work indicated that the CEF system is more sensitive for the isolation of viruses compare to that of avian embryo. The highest HA titre of NDV was found in the brain tissue followed by lungs and kidney. Significantly (p<0.01) higher HA titre of NDV isolate was recorded in the birds of all the experimental groups inoculated through IV route. Following infection, the MDA titre decreased day by day in the birds with the increase of HA titres of NDV. That findings will help to develop fathom about the rate of distribution of NDV in different organs of layer chickens in Bangladesh.

Knowledge Management: Journal