village indigenous birds are constantly exposed to immunosuppressive conditions such as aflatoxicosis and infectious bursal disease virus .
in addition , management and ecological factors such as confinement , climatic and seasonal fluctuations , poor feeding , and worm infestations have been associated with stress and reduced immune response .
stressful factors have been reported to cause functional and morphological changes in chickens .   in tanzania , it was observed that newcastle disease ( nd ) was a greater problem in villages with ducks .
earlier reports indicated that newcastle disease virus ( ndv ) persisted for a long time in a flock of ducks in a village situation in indonesia .
however , the factors leading to shedding of the virus by the carrier ducks are not well documented .  
it was hypothesized that immunosuppression of immunised carrier ducks does not influence persistence of ndv in these birds . in this experiment ,
thus , the aim of the present study was to determine the effect of immunosuppression on the viral persistence and immune status of ducks .
it was designed to simulate field situation where ducks that have varying levels of ndv antibodies undergo immunosuppression in the presence of high ndv challenge .
one - day - old indigenous ducklings were hatched from the duck flock maintained at the university of nairobi premises .
all the birds were reared in isolation and transferred to experimental units at one year of age .
they were wing tagged , tested , and confirmed to be free of ndv and respective antibodies .
water and food were provided ad libitum .   a kenyan virulent newcastle disease virus isolate ( vndv )
was obtained from the repository maintained at the university of nairobi and characterized by standard methods .
the inactivated vaccine was prepared by mixing 40% formalin and allantoic fluid with a titer of 2 of vndv in a ratio of 1  :  40 , that is , formalin to virus .
the reparation was kept at room temperature ( 24c to 26c ) for 24 hours before use .
all the ducks were vaccinated via an initial dose of 1  ml of the vaccine intramuscularly on the thighs and a booster of 0.5  ml of the same vaccine 16 days later .
the live virulent kenyan newcastle diseases virus isolate , previously characterized by standard methods , was later used to challenge vaccinated and naive ducks .
dexamethasone ( dexamethasone sodium phosphate and sodium methyl hydroxybenzoate , coophavet , france ) was used to stress ducks in this study .
the respective groups of ducks were injected intramuscularly with the dexamethasone , following the protocol of corrier et al .  
modified as follows : the dosage was given at the rate of 2  mg per kilogram of body weight per day for 4 days continuously , then the ducks were rested for 2 days and the injections resumed at the same dosage for 2 more days .
sixty - four ducks were vaccinated with 1  ml of inactivated nd vaccine intramuscularly and 14 days later , they were bled from the brachial vein and sera prepared .
they were later boosted with a single dose of 0.5  ml of the nd inactivated vaccine and bled 7 days later .
seven days after the booster dose , the ducks were divided into two groups , each with 32 birds , namely , low antibody level group ( 1  :  32 ) and medium antibody level group ( 1  :  64 ) .
each group of 32 ducks was further subdivided into 4 minigroups , as follows : ( i ) immunosuppressed and challenged ( 1a , 2a ) , ( ii ) immunosuppressed only ( 1b , 2b ) , ( iii ) challenged only ( 1c , 2c ) , and ( iv ) not challenged nor immunosuppressed ( 1d , 2d ) . another group ( group 3 ) of 30 nonimmunized ducks were subdivided into 4 groups .
groups 3a and 3c had 12 ducks each while 3b and 3d had 3 birds each .
immunosuppression was done before respective groups were inoculated intranasally with 0.2  ml of undiluted amnioallantoic fluids of vndv having a titer of 1  :  1024 .
five birds from each of the challenge groups and all the 3 ducks from each control group were sampled throughout the experimental period ( 28 days ) .
the samples were taken on days 0 , 1 , 4 , 8 , 14 , and 28-after inoculation ( p.i . ) .
blood for serum was sampled each time from the five ducks in each challenge group , and the three ducks from each of the controls .
further , two ducks from each of the ndv challenged groups were killed serially and brain , kidney , lung , cecal tonsils , liver , and spleen collected separately from each bird .
the tissues were processed for nd viral recovery using chicken embryo fibroblasts , while serum samples were tested for ndv - specific antibodies by hemagglutination inhibition ( hi ) test .  
virus recovery from the tissues was carried out in primary chicken embryo fibroblasts as described by oie .
analysis of variance was performed using sas software ( sas institute inc . , cary , nc , usa , 2002 - 2003 ) to determine the treatments ' main effects and the interaction between time ( days ) and treatment , on various responses .
immunosuppressed virus - challenged ducks ( group 1a ) had low mean antibody levels ( 5.0 ) up to day 4 after - inoculation ( p.i . ) compared with day 0 ( 4.5 ) .
thereafter , there was marked increase ( from 4.5 to 7.0 ) in antibody titers up to 14 days p.i .
after day 14  p.i . , there was a slight decrease ( 6.9 ) in antibody levels up to 28 days p.i . although the levels were still higher than any period between day 0 and 8  p.i .
the nonimmunosuppressed virus - challenged group ( 1c ) had a moderate increase ( 5.0 to 6.0 ) in antibody levels from day 1 up to day 14  p.i .
, after which there was a decrease to day 0 level titers by day 28  p.i .
the immunosuppressed group ( 1b ) had marked decrease in antibody titers from day 1 to 4 and gradual decrease ( 5.0 to 3.8 ) up to day 28  p.i .
the immunosuppressed virus - challenged group for the medium antibody level ducks ( 2a ) had a gradual decline ( 6.0 to 5.7 ) in antibody titers up to day 4 followed by an increase in antibody titers ( 6.9 ) up to day 14  p.i .
this was followed by a marked decrease ( 6.1 ) and by day 28  p.i .
the nonimmunosuppressed virus - challenged group ( 2c ) showed a slight decrease ( from 6.0 to 5.9 ) in the antibody titer followed by a gradual decrease and then an increase up to the end of the experiment . from day 1 up to day 4 after - inoculation , the immunosuppressed , immunised noninfected ( 2b ) group showed a more rapid decrease ( 6.0 , 5.2 , 4.8 , 3.7 , 2.3 , and finally 2.0 ) in antibody levels as compared ( 6.0 , 4.7 , 4.5 , 3.8 , 2.7 , and finally 2.2 ) to the nonimmunosuppressed controls ( 2d ) .
in general , all the nonchallenged , but immunized control ducks showed decrease in antibody titers with time ( figure 1(b ) ) .  
the immunosuppressed virus - challenged group ( 3a ) had a gradual antibody response ( from 0.0 to 6.5 ) up to the end of the experimental period .
the nonimmunosuppressed virus - challenged group ( 3c ) showed a massive increase ( 0.0 to 6.6 ) in antibody levels similar to immunosuppressed virus - challenged group 3a .
the group 3c also had a marked decrease ( from 6.6 to 4.6 ) in antibody titres after day 14  p.i . and by 28 days p.i . , the titers were quite low .
negative control ducks ( 3b and d ) , sampled at the same time , were negative for ndv antibodies ( figure 1(c ) ) .   for days 4 , 8 , 14 , and 28  p.i .
antibody titres of the following groups were found to be significantly different ( p < 0.05 ) . when considering the antibody levels elicited in the different duck - groups , with respect to their initial antibody levels , both sets ( low - antibody group and medium - antibody group ) , and both the immunosuppressed and non - immunosuppressed ducks showed higher responses in ducks that were challenged with virulent virus than in those that were not challenged .
all the control naive ducks ( groups 3b and 3d ) did not sero - convert .
immunosuppressed medium - antibody - level , challenged with ndv ducks ( group 2a ) versus immunosuppressed medium - antibody - level , nonchallenged ( group 2b ) , was lowest in the latter group .
nonimmunosuppressed low antibody level , challenged with ndv ducks ( group 2c ) versus 2d , the latter group had lower levels of antibodies .
in addition , antibody titres of group 1a versus 1d were significantly different ( p < 0.05 ) on day 14 , being lower in the latter .
all the control nave ( groups 3b and 3d ) birds did not seroconvert . on day 1 after inoculation ,
ndv titers were recorded in liver tissues of group 1a ( low - antibody - level group , immunosuppressed and challenged with vndv ) ducks only . on day 4 
, high titres of the ndv were recorded in the kidneys ( figure 2(a ) ) . on day 8  p.i .
, ndv was isolated in the liver , kidneys , caecal tonsils , and lungs of all treatment groups ( figure 2(b ) ) .
the highest ndv titers were recorded in the liver and kidney tissues of immunosuppressed medium ( 2a ) and nonimmune ( 3a ) challenged ducks and nonimmunosuppressed , low - antibody - level challenged ducks ( 1c ) .
no ndv was isolated by day 14  p.i . in the brain and spleen from any of the groups .  
high ndv titres were recorded from the liver tissues of ducks in all treatment groups .
however , titres were recorded in the cecal tonsils only in group 2a on day 14  p.i . and in groups 1a and 1c at day 28  p.i .
in addition , the immunosuppressed ducks of groups 1a ( low - antibody - level group , immunosuppressed and challenged with vndv ) and 2a ( medium - antibody - level group , immunosuppressed and challenged with vndv ) yielded the highest ndv titres as compared to other treatment groups .
newcastle disease virus was recovered from the brain and lung on day 28 p.i from immunosuppressed ducks only ( figure 2(d ) ) .
there have been comparatively few sequential virological studies on the pathogenesis of nd in ducks and the reported studies involved only fully susceptible chickens [ 8 , 9 ] .
reports in other studies have documented frequent isolation of virulent ndv from captive caged birds , healthy wild birds , and village chickens [ 1012 ] . in some cases , the ducks expressed clinical nd as a result of confinement that was understood to have induced stress .
our findings indicate that ndv carrier status in nonnatural hosts such as ducks would possibly , under stress , recrudescence virulent virus from sequestered sites in the kidney , liver , and caecal tonsils , leading to virus release in faecal and respiratory exudates .
the excreted virus would set up an infectious index case in chickens , thus maintaining ndv endemicity .
immunosuppression induced by injection of dexamethasone in the three treatments influenced the pattern of antibody response and the ndv recovery rate .
the immunosuppressed ducks that had low and medium antibody level showed a decrease in antibody titers up to day 4 after challenge with ndv .
the nonimmunosuppressed virus - challenged ducks of low - to - medium antibody level had an increase in antibody titres up to day 14  p.i .
the nonimmunized ducks manifested increased antibody titres after day 4  p.i . and had a massive increase in antibody levels as compared to immunosuppressed challenged group . in the present study ,
the number of immunosuppressed ducks that yielded the nd virus was higher compared to the nonimmunosuppressed .
the prechallenge antibody titers may therefore play a significant role in shedding off the virus as well as clinical manifestation of the disease .
.   noted that a few hours of treatment with low concentrations of synthetic glucocorticoid ( analogue dexamethasone ) are sufficient to inhibit the synthesis of interferon , a virus inhibitor .
this may , in part , explain the observation that treatment with glucocorticoids increased virus yield and lethality in mice infected with coxsackie virus .
our present study using ducks concurs with those of asdell and hanson   who showed that prior treatment of chickens with dexamethasone lead to massive nd virus multiplication .  
there was significant difference in geometric mean antibody titers between the immunosuppressed ducks of group 1a and nonimmunosuppressed counterparts ( group 1d ) and also between immunosuppressed ducks of group 3a and nonimmunosuppressed group 3d .
this means that whereas dexamethasone seems to have an effect on immune system of ndv - infected ducks , the prechallenge titres also play a major role in the immune response of immunosuppressed birds in that immunosuppression of ducks with high viral titers allows virus multiplication making ducks better carriers .
the effect of immunosuppression with dexamethasone in ducks appears to be the same as that induced by aflatoxin in chicks with ndv infections .
chickens fed on aflatoxin produced lower antibody levels when compared to the non - aflatoxin - treated ones .  
the nonchallenged preimmunized ducks had a progressive decrease in antibody levels suggesting that if they were to be exposed to the virus , they could come down with the nd or if the antibody titers were within the protective levels ( 2 to 2 ) , they might not develop clinical disease but instead may remain as virus carriers .
the fact that the ducks in these experiments had high levels of antibodies may not necessarily prevent subclinical infection and excretion of virulent virus as supported by other studies elsewhere .   based on these results ,
it is possible to assume that immunosuppressed ducks carrying ndv are more likely to shed virus under stress than nonimmunosuppressed ducks .
furthermore , the ducks showed progressively declining antibodies titres to very low levels making them more susceptible to infection by challenge nd virus , thus leading to clinical disease and virus excretion .
the excreted virus would contaminate the birds ' environment and be transferred to susceptible chickens and other birds .
thus , immunosuppression seems to have epidemiologically significant effects on ndv carrier ducks having varying antibody titer levels .
 this study was carried out to verify the possibility that ducks are sources of newcastle disease ( nd ) virus infection for chickens in mixed flocks . 
 immunosuppressed ( is ) and non immunosuppressed ( nis ) birds , at three different antibody levels ( medium , low and absent ) were used ; the titres having been induced through vaccination , and immunosuppression done using dexamethazone . 
 each of the 3 respective groups was further divided into 2 groups of about 12 ducks each : one challenged with velogenic nd virus ; the other not challenged . selected ducks from all groups 
 had their antibody titres monitored serially using hemagglutination inhibition test , while two birds from each of the challenged groups were killed and respective tissues processed for nd viral recovery , using chicken embryo fibroblasts . in general , antibody titres of is and nis challenged ducks were significantly higher than their unchallenged counterparts ( p < 0.05 ) . 
 non - challenged pre - immunised ducks had a progressive decrease in antibody levels ; non - immunised ducks did not seroconvert . 
 newcastle disease virus was isolated from livers and kidneys of the challenged ducks throughout the experimental period ; indicating a possibility of viral excretion , especially when the birds are stressed . 
 it , therefore , provides another possible model of viral circulation within mixed flocks . 