Detection of pigeon paramyxovirus antibodies using haemagglutinaton inhibition test

Newcastle Disease (ND) is a highly contagious avian disease which causes severe economic losses in domestic poultry especially in chickens due to mortality and morbidity in high percentage of infected chickens, which depends on virulence of the infecting strain and host susceptibility (1, 2). Newcastle disease is a virus disease of birds characterised by variable combinations of gastroenteritis, respiratory distress and nervous signs (3). All birds are susceptible but the disease is more severe in chickens and turkeys and less severe in ducks and pigeons(4). Over 200 species of birds have been reported to be susceptible to natural and/or experimental infection with ND virus and it seems probable that more are fully susceptible (5). Ducks and geese tend to show few signs even when infected with the most virulent strains of ND virus for chickens (5). Although many species of bird are susceptible to ND it occurs mainly in galliformes such as pheasants, partridges and quails and also in birds of prey, including pigeons and psittaciformes. In most species of bird the young are more susceptible than the adult (6). ND virus (NDV), the only member of the avian paramyxovirus type 1, belongs to the Avulavirus genus of the family Paramyxoviridae (7). The disease normally affects the respiratory, gastrointestinal and nervous systems. The clinical signs reported in birds infected with NDV vary widely but mainly depend on strain of the virus. Other factors determine the outcome of the disease such as the host species, age, immune status, infection with other organisms, and environmental stress (2). In some circumstances, with extremely virulent viruses the disease may result in sudden death (8). The antibody response to NDV occurs rapidly with detectable neutralizing antibodies, usually detected in the serum of birds within 4-6 days after vaccination with live attenuated vaccines (9).

Materials and Methods

Serum samples. Eighty Blood samples were collected in test tubes to get serum from local domestic pigeon in Ninavah governorate in Iraq via the slaughter from May- August 2009.

Chicken red blood cells (RBC). A 1% suspension of washed RBC was prepared for use in haemagglutination (HA) and haemagglutination inhibition (HI) tests (10).

Antigen. Pigeon paramyxovirus obtained from department of microbiology/ college of veterinary medicine/ university of mosul/ Mosul/ Iraq was used as the antigen for HI test. The HA titres of the Pigeon paramyxovirus antigen were determined as described by (10) and (11) and diluted to contain 4-

HA units. This concentration was used for the HI test. The HI titre for each bird was determined and expressed in log2, and the mean for each species was calculated.

Haemagglutination inhibition test (HI): Two- fold serial dilutions of serum samples were made with normal saline in macro titer plates. 0.25 ml of PBS was dispensed into each well of a macrotitre plate, 0.25 ml of each serum sample placed into the first wells of the plate.Two fold dilutions were made by transferring 0.25 ml of each serum sample from first row of wells to second and so on thus diluting each serum sample as 1:2, 1:4,1:8,1:16: 1:32...1:2048, etc. 0.25 ml of 4 HA units of virus were dispensed in each well with the help of 8-channel micropipette and plates were left for of 30 minutes at room temperature. 0.25 ml of 1% chicken RBCs added to each well and after gentle mixing, the RBCs allowed to settle for about 30 minutes at room temperature (12). The base two logarithmic titer was calculated.

Results

90% of 80 local domestic pigeon suffered from different clinical signs, mainly nervous, digestive and respiratory signs, and the nervous signs revealed the highest percentage representing 48.75% (figure 1).

Figure 1: percentages of clinical signs of sampled pigeon

All of the serum samples were collected from local domestic pigeon revealed positive reaction for HI test with different titers of antibodies. Pigeon suffered from nervous and digestive showed highest titer of antibodies 9log 2 , but 6log 2 screened a highest number of 80 samples.

Figure 2: Log of HI paramyxovirus antibody titers of Pigeon

Discussion

There was serological evidence of paramyxovirus antibodies in local pigeons. This might be due to occasional visits of these birds to the poultry premises where they could have contracted the virus from materials from poultry houses. So, there is the possibility of transmission of paramyxovirus among these specie of birds.

Pigeons are among a group of domesticated birds that were primarily affected by the paramyxovirus (13). The disease spread to all parts of the world, largely as a result of contact between birds at races and shows and the large international trade in such birds (14).

There is serological evidence of paramyxovirus antibodies in pigeons in this work, the spread of paramyxovirus to chickens from pigeons has occurred in several countries including Great Britain where 20 outbreaks in unvaccinated chickens occurred in 1984 as a result of feed that had been contaminated by infected pigeons (15).

The presence of specific antibodies in the serum of a bird gives indication of presence an infection, as well as an immunological response (16) .

In this study, all of the serum samples were collected from local domestic pigeon revealed positive reaction for HI test with different titers of antibodies, and the highest HI paramyxovirus specific antibodies titer "9log2" was recorded in the pigeon suffered from nervous and digestive signs, and that indicate these birds maybe infected with the virulent strain of virus (17).