Mechanisms Of Occurrence And Transmission Of Newcastle Disease Virus Aerosol And Monitoring Of Newcastle Disease Virus Aerosol In Chicken Houses

Airborne and droplet transmission are one of the major routes for spreading animal viral diseases, such as Newcastle disease, Avian Influenza and foot-and-mouth disease. Recent emergence of respiratory viral pathogens such as SARS and avian influenza viruses have attracted public attention and are believed to be transmitted via the airborne route, raising various scientific and public health issues related to airborne transmission and control of infectious agents. Newcastle disease (ND) is included in the World Organization (OIE) for Animal Health list of notifiable diseases (former list A). Airborne transmission was also one of the important routes for spreading of Newcastle disease, which was considered a significant factor in the 1970-1971 ND outbreaks in England and the 1973 ND epidemic in Northern Ireland. Airborne transport over long distances was known to have occurred. Despite the recognition of airborne as a spread route for NDV, the mechanism of aerosol generation, transmission is still unclear. The notion of airborne spread needs experiment authentication. Therefore, we experimentally investigated airborne transmission of ND in a model system. Using this model system, we monitored occurrence and transmission of NDV aerosol. We have not only established the time of aerosol occurrence but also determined the aerosol concentrations, dynamic changes and their relationship with virus shedding. This experiment also confirmed the airborne transmission of the F48E9 strain. NDV aerosol originating from infected chickens could spread to their surrounding air (10m, 20m away) from the infected chickens and could infect the healthy chickens at that point. The infectious and lethal doses of NDV aerosol were compared by other infectious routs. The difference of intestinal flora of NDV aerosol infected group and healthy group by ERIC-PCR based fringerprint on molecular ecology. Airborne Newcastle disease (ND) viruses in the air of five chicken houses were detected and differentiated by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers. Airborne ND viruses were also isolated and virulence identified by in-vivo tests. Part of the fusion gene of isolate s were amplified by RT- PCR and sequenced.Not only is this result important in better understanding the mechanism of ND transmission, it also has implications in controlling avian influenza and other contagious diseases.The study consists of six parts:1. Occurrence and transmission of Newcastle Disease Virus aerosol originating from infected chickens under experimental conditions.One model was established to better study emission, transmission and infection mechanism of NDV (Newcastle Disease Virus, NDV) aerosol. Two trials (T1) and (T2) were performed. SPF chickens (G1) were housed in the isolator A and were inoculated with NDV strain F48E9 by means of dripping noses and eyes. After G1 was inoculated, the air in the isolator A was sampled by AGI-30 and airborne NDV was detected and quantified (Plaque per m3 air). SPF chickens were housed in the isolator B, which were used as aerosol challenged group (G2). Oropharyngeal and cloacal swabs were collected from each chicken, which used for NDV detection and determined virus shedding of chickens. The antibody response to NDV infection of Serum samples was assessed by the hemagglutination inhibition (HI) test. In T1 and T2, NDV aerosol could be detected at 2 dpi (days post inoculation) and 3 dpi respectively, at 13 dpi and 11 dpi the concentration of NDV aerosol respective was 1.69×104 PFU/m3 air and 9.14×103 PFU/m3 air which reached a peak, at 40 dpi the concentration still was 6.98×103 PFU/m3 air and 3.78×103 PFU/m3 air; at 2 dpi the shedding NDV in the swaps of chickens in T1G1 and T2G1 were confirmed; 7 days post infected by NDV aerosol, the presence of NDV in the swaps of 80% chickens in T1G2 and 60% chickens in T2G2 could be detected, The chickens of T1G2 and T2G2 had antibody titers for NDV reaching 4.07 and 7.40 at 7 dpi and 14 dpi respectively, positive results according to OIE standards, the antibody was consistently detected positive up to 40 dpi.The results of two trials showed that infected SPF chickens could emission NDV within 3 days post inoculation and come into NDV aerosol and then infected healthy chickens.2. Spread and infection of Newcastle disease virus aerosol under field conditions.In this study, two trials were conducted, I: Study on spread of NDV aerosol to their surrounding air; II: Infection of NDV aerosol. I: After SPF chickens were inoculated with NDV and were housed in one cage , the NDV aerosol originating from inoculated chickens (S0) and10m (S10),20m (S20) away were collected with All Glass Impinger-30 (AGI-30) to studied the occurrence and concentration of NDV aerosol.The spreading of NDV aerosol from the infected chickens to the ambient air was characterized. Th results showed that NDV aerosol was initially detectabl at day 3 post inoculation (dpi) at the S0 Aand at day5 post inoculation (dpi) at S10 and S20. The aerosol concentration peaked at 11dpi at S0 and was consistently detectable up to 30 dpi. , at 11-13 dpi at S10 and S20 and was consistently detectable up to 25 dpi. II: 3 groups (infected group, 10m aerosol infected group and 20 m aerosol infected group as sentinel chickens). Aerosol infection did occur, as shown by NDV shedding and seroconversion to NDV in sentinel chickens. The detailed methods were in accordance with the methods in Trial I. Th results showed that after 7 days of aerosol exposed infection, all the sentinel chickens were tested positive. Up to 28 dpi, some sentinel chickens were still positive.The results indicated that viruses shed from infected chickens readily aerosolized and aerosol could spread into the ambient air (20m away) and the chickens there were infected by the NDV aerosol.3. Studies to quantify the infectious and lethal dose of Newcastle disease virus for chickens through defferent routs.In this study, SPF chickens were ramdomly divided into 3 groups: aerosol infectedgroup, intramuscular injection, gastrointestinal tract infected group. Each chicken was challenged with the same volume of different dilution of NDV. Infection was determined by the specific antibody to NDV. Computation Method for ID50 and LD50 Based on Bliss. The results showed that aerosol routs: ID50 was 30.53 PFU, 95% confidence interval was 9.46 - 98.54 (PFU); LD50 was 117.05 PFU,95% confidence interval was 23.71 - 577.92 (PFU). Gastrointestinal tract infected rout: ID50 was 4419.78 PFU, 95% confidence interval was 1095.34 -17834.14 (PFU); LD50 was 44743.91 PFU,95% confidence interval was 10161.62 - 197017.62 (PFU). Intramuscular injection rout: ID50 was 16.63 PFU, 95% confidence interval was 14.85 - 57.00 (PFU); LD50 was 69.60 PFU,95% confidence interval was 14.24 - 340.27 (PFU).The results indicated that it was stronger with Intramuscular injection than aerosol infection. That with Gastrointestinal tract infection was weakest.4. Molecular detection and isolation of airborne chicken Newcastle disease virus in chicken houses Airborne Newcastle disease (ND) viruses in the air of five chicken houses were detected and differentiated by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers. Fifteen air samples were collected with All Glass Impinger-30 (AGI-30) air samplers in each house. Airborne ND viruses were also isolated and virulence identified by in-vivo tests. Only avirulent viruses were detected in two air samples and four swab samples in house A. Avirulent viruses were detected in seven air samples and five swab samples and virulent viruses were detected only in five air samples in house B. Avirulent viruses were detected in six air samples and five swab samples and virulent viruses were detected only in two air samples in house C. Avirulent viruses were detected in seven air samples and one swab samples in house E. Seven NDV strains were obtained. Three were virulent strains and four were avirulent strains. One strain avirulent (YTCX08) was isolated in house A; One virulent strain (DZ07) and one avirulent strain (DZ0702) were isolated in house B; Two virulent strains (MH07, MH0702) and one avirulent strain (MH0703) were isolated in house C; one avirulent strain (NY06) was isolated in house The results showed that it was feasible to detect and differentiate NDV in the air samples using degenerate primers based RT-PCR. This technique could decrease the time it required identify NDV infected flocks while distinguishing between virulent and avirulent viruses. It will help effectively to control Newcastle disease.5. Nucleotide Sequence Analysis of Main Function Domain of F Gene of airborne Newcastle disease virusOne pair of specific primers was designed and synthesized according to fusion ( F) protein gene sequences of Newcastle disease virus (NDV), Part of the fusion gene of seven isolates were amplified by RT- PCR and sequenced. A phylogenetic tree based on obtained sequences of reference NDV strains was constructed and the deduced amino acid sequences showed that the homologies of t he nucleotide are 79.3% - 100 % and the homologies of the deduced amino acid sequences are77.4% - 100%. NY06,MH0703,YTCX08 and DZ0702 belong to the genotype II and their amino acid sequences of the cleavage site region residues (111GG RQGRL117 ) matched to the characteristics of avirulent strains; MH07 belongs to the genotype IX and DZ07,MH0702 belong to genotype VII, and their amino acid sequences of the cleavage site region residues (111GRRQK RF117 ) matched to the characteristics of virulent strains.6. Analysis of the structure features of Intestinal communities of chickens infected with NDV aerosolThe article compare the difference of intestinal E. coli floras between healthy chickens and the chickens infected with NDV aerosol. The results showed that Significant difference in E. coli floras in the feces of the group infected with NDV aerosol increased (P<0.01) in Early infection and also was detected in the later phase (P<0.01). The difference of intestinal flora of NDV aerosol infected group and healthy group by ERIC-PCR based fringerprint on molecular ecology. Some difference was detected in the ERIC-PCR fingerprints between infected group and healthy group. The intestinal microbial population was changed when the chickens exhibited clinical symptoms (2-10 dpi), and was rebuilt in the recovery stage (10-15 dpi), and then was stable after fully recovery.The results showed that the intestinal flora of NDV aerosol infected chickens was changed and ERIC–PCR based fingerprints can analyze the dynamic monitoring of animal intestinal flora.