Comparative Haemato-biochemical and Histopathological Studies in Birds Inoculated with Vaccine and Field Strain of Infectious Bursal Disease Virus

Infectious bursal disease (IBD) in birds caused by a Birnavirus is an economically important due to the immunosuppression as well as direct losses due to mortality. The problem of immuno-suppression in birds is one of the leading cause of increase in incidences and intensity of already existing diseases and also of new emerging diseases. Infectious bursal disease chiefly affects immature chicks (Lasher and Shane, 1994) with highest susceptibility around 3-5 weeks of age (Panisup et al., 1984). Chicks younger than three weeks of age upon exposure to IBDV may not show clinical manifestation but the microscopic lesions would characteristically induce immunosuppression. The variability in pathogenicity may be attributed to the presence of B cells in birds above three weeks of age. The virus in the affected chicks causes lymphocytolysis and wrecksthe immune system (Verma et al., 1981) and ultimately causes the immunosuppression. The growth rate, liveability and productivity may be adversely affected by reduced resistance to a wide range of viral, bacterial and protozoa agents, poor vaccinal response, vaccine failures and enhanced mortality rates (McFerran, 1993). 
 
Vaccination is mostly used for prevention of such diseases. However vaccine are often pathogenic either by reverting to virulent form or by causing detrimental changes in biochemical pathways of biological processes (Jackwood and Sommer, 2002).There has been reports of prevalence of IBD due to highly virulent pathotypes across many parts of the country. Hence, hot vaccines i.e. less attenuated form of vaccines are becoming a necessity and are being used to prevent the outbreaks of IBD. IBD vaccines are known to cause bursal damage and tissue lesions in birds (Al-Mayah and Al-Mayah, 2013). Moreover, in endemic areas affected with IBD, some farmers also starts using hot stains of vaccines at an early age. Hence it is important to understand the damage caused by vaccine virus. The present communication aims at analysing the extent of pathogenic changes caused by vaccine virus and its comparative accessment with that of field virus.

Birds and viruses
 
Ninety specific pathogen free (SPF) day old broiler chicks were brought from Venkateshwar hatcheries, Pune, Maharashtra of either sex were randomly divided into appropriate groups as mentioned in Table 1. Birds of different experimental groups were reared in seperate isolation units. Feed and water were provided ad-libitum. A conventional serotype-1 Infectious Bursal Disease virus, isolated from Nammakal district of Tamilnadu, taken from repository of Department of Biotechnology, College of Veterinary Science and A.H., Pantnagar, was used as field strain. The virus was propogated in SPF chicken bursa and was titrated in SPF chiken embroyos as previously described  (Abdel-Alim and Saif, 2001). An intermediate invasive strain of IBD virus vaccine, IV-95 was used as vaccine strain. 


 
Experimental design
 
Experiment was conducted in two phases. In first experiment, forty five, 5-days-old birds were divided into 3 groups; viz. group A, B and C. In second experiment, forty five, 25-days-old birds were divided into 3 groups; viz. group D, E and F. As per group, the field virus @ 0.2 ml per bird having a virus concentration of 10⁶ EID₅₀/ml was inoculatedthrough intra-nasal and intra-ocular route (0.1 ml each). Vaccine virus was given at dose rate of ≥ 10³ EID₅₀ of virus through intra-nasal and intra-ocular route (0.1 ml each). Control group were given sterile normal saline by the same route. The flock was observed twice daily for assessing their health status and  any clinical signs or alteration of behaviour including mortality. Three birds were sacrificed daily upto 5 days post infection. Each bird was weighed, bled and PM was done to investigate the lesion and collect organs in 10% formaline for histopathology.Bursa was weighed seperately and bursal body weight index was determined as follows:  
Where,
Bursa: Body weight ratio =  
Hematology
 
Total Leucocyte Count (TLC) and Total Erythrocyte Count (TEC) were performed as per the method of Natt and Herrick (1952) using poultry diluting fluid and the results were expressed in thousand per cubic mm (10³ /cu mm) and millions per cubic mm (10⁶ /cu mm), respectively. Differential Leucocyte Count (DLC) was performed as method described by Lucas and Jamraz (1961) and recorded in percentage (%). Hemoglobin (Hb) Concentration was estimated by Sahli’shemoglobinometer (Schalm, 1965) and the results were expressed in grams per decilitre (g/dl). Packed Cell Volume (PCV) was estimated by microhematocrit method (Schalm, 1965) and the results were expressed in percentage (%).
 
Serum biochemistry
 
Biochemical analysis of serum was carried out using auto analyzer (ACE/NexCT^TM clinical chemistry system). Total Protein (TP), Albumin (A) and Uric Acid (UA) were estimated by using Alfa Wassermann kit. Globulin concentration was determined by deducting albumin concentration from total protein concentration.
 
Gross and microscopic examination
 
Detailed necropsy of birds was conducted for gross examination. Tissue samples were collected in 10% buffered formalin for fixation. These tissue samples were processed further for routine processing and stained with haematoxylin and eosin (Lillie, 1965) and observed under microscope.
 
Statistical analysis
 
Statistics analysis one way ANOVA among the group was carried out as per standard Method as described by Snedecor and Cochran (1987).

Clinical signs
 
The birds in group B showed no apparent clinical signs except slight dullness and whitish diarrhea from 3^rd day onward. In group C also, no apparent clinical signs were seen except slight dullness and diarrhea, which was less as compared to group B. However, no mortality was seen in either group. In group E, severe clinical signs were seen in the birds with 13.33% mortality (2 out of 15 birds died during the experiment). Birds were dull and depressed and showed ruffled feathers. Severe whitish diarrhea was also seen along with vent pecking in extremely diarrheic birds. Blood tinged faeces was also seen in some cases. Feed consumption was lowered and the birds showing severe symptoms were recumbent. In group F, no other clinical signs except slight depression with whitish diarrhea were seen. The severity of these signs was slightly greater as compared to group C, however no mortality was observed in this group also during entire experiment. The present experiment indicated lack of frank clinical signs in birds below 3-weeks of age (group B and C), which might be attributed to smaller bursal size and thus lesser availability of bursal cells. The findings were in accordance with the data reported in other studies (Fadly and Nazarein, 1983), which suggests that the availability of a large number of susceptible bursal cells was a crucial point in development of clinical IBD. Hitchner (1971) showed that birds less than 3 weeks of age did not exhibit clinical signs. In other group of birds above 3-weeks of age and infected with field virus, the clinical signs were diagnostic of IBD and were in accordance with previous works (Panisup et al., 1988). The clinical signs such as depression, diarrhea, anorexia, recumbency and death within 5 days of study were suggestive of an acute viral infection. Morbidity was 100% and mortality was 11.2%. However, simple presence of virus could not account for clinical signs as bursectomized chickens did not develop clinical IBD despite presence of infection in thymus and spleen (Hiraga et al., 1994).
 
Bursal: body weight index
 
Bursal: body weight index was used as a measure of bursal enlargement or atrophy. The mean values of bursal: body weight index of all the six groups are presented in Table 2.  In 1^st experiment of birds below three weeks of age, bursal index trend showed enlargement immediately on 1^st day, followed by atrophy. In 2^nd experiment of birds above three weeks of age, bursal index trend showed enlargement from 1^st day onward, which continued up to 5^th day of experiment. Group B showed significant increase in bursal index on 1st day followed by significant decrease on 3^rd, 4^th and 5^th day. Group C also showed similar trend with increase on 1^st day followed by significant decrease on 2^nd, 4^th and 5^thday.In group E, significant increase was observed on 2^nd, 3^rd, 4^th and 5^th day, whereas group F showed significant increase on 2^nd, 3^rd and 5^th days post-infection. It was found that infection with field virus or vaccine virus of hot strain in birds below three weeks of age (5-days of age), revealed immediate atrophy of bursa of Fabricius without inflammatory signs in the bursa, while in birds of 24-days of age the infection was followed by an increase in size of bursa indicating an inflammatory response. This also shows the correlation between clinical signs and bursal enlargement. The birds below 3-weeks of age did not show any gross lesions except muscle hemorrhage and atrophy of bursa from 2/3 day in either group. This was in accordance with the appearance of clinical signs and findings of Abu Tabeekh and Al-Mayah (2009).


 
Gross lesions
 
In group B, slight hemorrhages in thigh muscles were seen. No significant gross changes and enlargement in the bursa could be seen. Slight atrophy of bursa could be observed from 3^rd day onwards. The spleen was slightly enlarged. In group C, slight hemorrhages in skeletal muscles as seen in group B were seen, however, atrophy of bursa of Fabricius could be seen earlier, i.e., from 2^nd day onward (Fig 1). In group E, extensive skeletal muscle hemorrhages were observed. The bursa was found enlarged from 2^nd day up to 4^th day. No hemorrhages could be seen within lumen of bursa, although slight caseous exudate was observed. The spleen and gall bladder showed increase in size. Kidneys were congested in some birds up to 2^nd or 3^rd day and were enlarged and pale with urate deposits in some cases on 4^th and 5^th day. The liver was enlarged and hyperemic up to 2-3 days in most cases and showed mottled appearance on 4^th and 5^th day. In group F, extensive hemorrhages in thigh muscles were seen. Bursa was less enlarged than group E. The enlargement of the spleen and gall bladder were comparable to group E. Gross lesions in birds above 3-weeks of age infected with field isolate showed extensive hemorrhages on thigh muscles which were in accordance with findings of Verma et al., (1981) who also noticed hemorrhages on breast and thigh muscles and at junction of proventriculus and gizzard. In the present study, hemorrhages at junction of proventriculus and gizzard were not observed, although hemorrhages on intestinal mucosa were seen in some cases. The enlargement of bursa from 1^st day onward was seen. Similar changes were noticed earlier also (Camilotti, 2016). Hemorrhages inside bursal lumen were not observed. This might be due to variation in virulence of virus. Spleen was at times enlarged, but was not quantified and very often had small grey foci uniformly dispersed on the surface (Sivanandan and Maheshwaran, 1980). This might be due to virus induced inflammatory stress.Changes in kidney were in compliance with previous work (Lukert and Saif, 1991). Liver was hypertrophied and hyperemic up to 2-3 days in most cases. Several systemic infections are associated with various degrees of swelling of liver (Neilson et al., 1998). In the present study, the hypertrophy of liver may be explained by cellular hyperplasia due to an infection induced rise in metabolic turn-over and increased blood flow through the organ.


 
Microscopic lesions
 
Histopathological examination was carried out on tissue samples of the bursa of Fabricius, spleen, thymus, skeletal-muscle, liver and kidney. Almost every group was affected by the virus especially the changes were pronounced in the bursa of Fabricius. In group B, bursa revealed only slight heterophilic infiltration only on 1^st day post-inoculation. From 2^nd day onward, necrosis in peripheral follicles was mostly seen followed by changes in other follicles from 3^rd day onward, along with extensive depletion of lymphocytes from 2^nd day onward. The thymus revealed lymphocytic necrosis and depletion from 3^rd day onward. The changes in spleen comprised of slight necrosis of cortical areas from 2^nd day. Paucity of lymphocytes was observed from 3^rd day with necrosis around germinal centres in white pulp. The lesions in muscles comprised of oedema and rare occurrence of RBC infiltration between bundles of muscle fibers. The liver did not reveal any specific changes except some areas of coagulative necrosis on 4^th and 5^th day. Kidney showed certain casts in the lumen of distal convoluted tubules on 5^th day only. In group C, changes in the bursa were of greater intensity than group B, with necrosis appearing on 1^st day followed by extensive depletion of lymphocytes from 2^nd day only. The spleen showed paucity of lymphocytes in the white pulp. The thymus, muscles, liver and kidney did not show any change. In group E, microscopic lesions in the bursa of Fabricius were evident as early as on 1^st day post-inoculation. There was oedema, congestion and depletion of lymphocytes in some follicles. By 3^rd day post-inoculation almost all follicles showed 50-80% depletion of lymphocytes and was accompanied with inter and intra follicular epithelial cells proliferation and folding of mucosal epithelium and infiltration of heterophils. Lymphocytic depletion also started in the medulla on 3^rd day post-inoculation. Accumulation of degenerative mass in medulla was observed. By 4^th day extensive depletion of lymphocytes was observed in all parts of bursa (Fig 2). In some cases there were haemorrhages on the inter follicular connective tissue. The thymus revealed lymphocytic necrosis from 4^th day onward. The spleen revealed microscopic changes from 2^nd day onward with congestion, haemorrhages and perivascularreticulo-endothelial hyperplasia on 2^nd day followed with lymphoid necrosis in the germinal centres on 3^rd day post-inoculation. The muscles showed extensive haemorrhages and oedema along with heterophilic infiltration from 2^nd day onward which subsided on 5^th day post-inoculation. The liver showed extensive congestion as early as 1^st day post-inoculation (Fig 3). This was followed by focal areas of necrosis, haemorrhages and fatty degeneration up to 5^th day. The kidney revealed changes from 3-4^th day post-inoculation with some glomeruli showing hypertrophy filling the Bowman’s capsule. Proximal segments of the tubules were dilated and extensive casts were observed within lumen of distal tubules. In group F, the changes in the bursa, thymus and spleen were almost same as that of group E but of less intensity. The liver and kidney were affected to a less extent with major changes being confined to congestion in liver and appearance of casts in kidney on 5^th day post-inoculation. The lesions in muscles were comparable to group E. The birds in control groups did not show any microscopic lesions. Histo-pathological lesions showed severity in intensity of changes in group E, F, B and C in decreasing order. This is in compliance with other pathological changes showing that, the birds of age above 3-weeks were most susceptible to infection and also suffers from damage to internal organs from vaccine strains. The bursa showed damage in birds of both age groups. The vaccine used in this experiment caused more severe depletion of lymphocytes in birds below 3-weeks of age and almost equal damage in birds above 3-weeks of age as compared to field isolate indicating that hot vaccine was harmful to the birds and caused severe depletion of lymphocytes and in turn immunosuppression. The histopathological lesions observed in infected groups were in accordance with previous works (Hiragaet al., 1994). The damaging effects of hot vaccine in birds were as per observations recorded by other workers (Moraes et al., 2004, Sahar et al., 2004).




 
Haematological studies
 
Haemoglobin
 
The haemoglobin concentration showed decreasing trend as compared to normal values. The mean values of all the six groups are presented in Table 3. Group B showed a significant decrease on 3^rd and 5^th day as compared to control. Group C showed relatively more decrease in haemoglobin concentration than group B. There was significant decrease in haemoglobin values on 1^st, 3^rd, 4^th and 5^th day as compared to control group A. Group E showed marked decrease in haemoglobin concentration which was evident from 1^st to 5^th day, while group F showed significant decrease on 1^st and 2^nd day only  as compared to control on 1^st to 5^th day. There was a decrease in haemoglobin concentration in all the groups. The greatest decrease was in group E, which was in susceptible age range and infected with field isolate. This gives an idea that more the pathogenicity of virus more is its effect on haemoglobin concentration. Thus the depression of haemoglobin concentration might be a factor in causation of clinical signs. In birds below 3-weeks of age, the decrease in haemoglobin concentration was comparable in both groups, i.e., infected and vaccinated, while in birds above 3-weeks of age infected group was suffering from a greater decrease in haemoglobin concentration compared to vaccinated group. Hence vaccination with hot strain was more detrimental in neonatal birds. PCV and TEC were decreased in all the groups with greater effect on infected birds of age above 3-weeks.  


 
Packed cell volume
 
The mean values of PCV showed a decreasing trend as compared to normal values. The mean values of PCV of all the six groups are presented in Table 3. In group B, significant decrease was seen on 3^rd, 4^th and 5^th day as compared to control. In group C, significant decrease was seen only on 4^th day. In group E, significant decrease in PCV was observed from 2^nd day onwards as compared to control. In group F, significant decrease was seen from 1^st to 3^rd day as compared to control. PCV and TEC were decreased in all the groups with greater effect on infected birds of age above 3-weeks. A significant decrease in haemoglobin concentration, PCV and TEC was found in similar study (Nunoya et al., 1992).
 
Total erythrocyte count
 
The TEC showed decreasing trend from that of normal values but were mostly insignificant due to greater variability (SE) in values of same group. The mean values of TEC of all the six groups are presented in Table 3. Group B showed significant decrease in TEC at 2^nd, 3^rd and 5^th day as compared to control. In group C, significant decrease was seen on 1^st day as compared to control followed by non-significant decrease. In group E, TEC was significantly low only on 5^th day as compared to control. In group F, significant decrease was seen on 1^st, 2^nd, 4^th and 5^th as compared to control. A reduction in RBC count was reported from 1 to 7 days post-infection (Cho and Edgar, 1969).
 
Total leucocyte count
 
The TLC showed increasing trend except in 1^st experiment where, birds were below three weeks of age. The mean values of all the six groups are presented in Table 4. In group B, insignificant increase in TLC was seen from 1^st day followed by significant increase on 3^rd day as compared to normal which was again followed by a significant decrease on 4^th day as compared to control. In group C, insignificant increase was seen up to 3^rd day followed by significant decrease on 4^th day as compared to control.  In group E, significant increase was observed on 3^rd and 4^th day as compared to control whereas, group F showed non-significant increase from 1^st day followed by significant increase on 3^rd day as compared to control. In the present study, significant increase in TLC was found in birds above 3-weeks of age. This might be due to a virus induced inflammatory response where there is increase in TLC to combat the infection. In birds below 3-weeks of age, there was significant decrease in TLC. This may be due to the fact that the virus was capable of causing destruction of lymphocytes to a greater extent in young birds, which is also confirmed with greater apoptotic indices in birds below 3-weeks of age.


 
Lymphocyte %
 
There was initial lymphocytopenia followed by lymphocytophilia with some exceptions. The mean values of all the six groups are presented in Table 4. Group B showed significant decrease in lymphocyte values from 1^st to 4^th as compared to control followed by significant increase on 5^th day as compared to control. Group C showed significant decrease on 2^ndand 3^rd day as compared to control with insignificant decrease on other days. Group E showed significant decrease of lymphocyte values on 1^st and 2^nd day as compared to control followed by non-significant increase on 4^th and 5^th day. In group F, there was significant decrease on 2^nd and 3^rd day as compared to control followed by significant increase on 5^th day as compared to control. The initial lymphocytopenia followed by lymphocytophilia is in accordance with previous work (Juranova et al., 2001). The lymphocytopenia was more severe in birds below 3-weeks of age. In a work (Sivanandan and Maheswaran, 1980) absolute peripheral blood lymphocytes in chickens infected with IBD virus at one day and three weeks of age was studied. The severity of depression of B lymphocytes was found to be age dependent. The birds infected at one day of age showed a more severe depletion than those infected at three weeks of age. The T-lymphocyte numbers were less markedly affected in either group of birds. This might be due to the fact that IBDV is extremely lymphocidal especially to young dividing lymphocytes which are in greater number in young birds below 3-weeks of age.
 
Heterophils %
 
The heterophil % showed a trend of initial heterophilia which returned to near normal values. The mean values of heterophil % of all the six groups are presented in Table 4.  In group B, significant increase was observed on 1^st, 2^nd and 4^th day as compared to control. In group C, no significant changes were observed. In group E, significant increase was seen on 1^st, 2^nd and 3^rd day as compared to control followed by non-significant decrease. In group F, significant increase was seen only on 4^th day as compared to control. In the present study, heterophilia was seen in infected birds but not in vaccinated birds. This finding was in both age groups, which is in accordance with the histopathological findings. It may be because of an initial inflammatory response against virus infection. Similar findings, i.e., initial heterophilia up to 2-3 days followed by heteropenia) were also recorded previously (Juranova et al., 2001).
 
Biochemical studies
 
Total protein
 
The mean values of total protein concentration followed a decreasing order as compared to control. The mean values of all the six groups are presented in Table 5. In group B, non-significant decrease of total protein was from 1^st to 3^rd day, followed by significant decrease on 4^th and 5^th day as compared to control. In group C also, same trend was noticed with significant decrease on 4^th and 5^th day. In group E, significant decrease was seen from 3^rd day onward as compared to control. In group F, non-significant changes were noticed up to 4^th day. Significant decrease was seen on 5^th day as compared to control. Total protein was decreased in all the groups but to a greater extent in infected group of birds above 3-weeks of age. Significant decrease was seen from 3-4 day onwards corresponding with subsiding of hyperaemic stage of liver when it became pale with mottled appearance and microscopic lesion of vacuolar degeneration. This may be attributed to hepatic dysfunction and loss of its synthetic ability. Similar finding was reported earlier also (Zeryehun et al., 2012).


 
Albumin
 
The albumin concentration showed a decreasing trend as compared to normal values. The mean values of albumin concentration of all the six groups are presented in Table 5.  Group B birds showed significant decrease on 3^rd day as compared to control with non-significant decrease on other days. Group C showed significant decrease on 5^th day as compared to control with decrease on other days being non-significant. Group E showed significant decrease on 4^th and 5^th day. Group F showed same trend as group E with significant decrease on 4^th and 5^th day as compared to same control. Albumin was also showing quite similar trend as total protein. Panigrahy et al. (1986) and Al-Afaleq (1998) also reported significant decrease in total protein and albumin concentration after IBDV infection.
 
Globulin
 
No specific trend could be traced out in case of globulin concentration. The mean values of globulin concentration of all the six groups are presented in Table 5. Group B showed significant increase only on 5^th day as compared to control with other changes being non-significant. Group C also showed significant increase only on 5^th day. Group E showed significant increase on 1^st day as compared to control, whereas Group F showed significant increase on 1^st, 3^rd, 4^th and 5^th day. In birds below 3-weeks of age, significant increase in globulin concentration was noticed on 5^th day. In birds above 3-weeks of age, significant increase was noticed from 1^st day but only in vaccinated group. This might be due to an increase in antibody component of globulin which is especially raised in case of vaccinated strain. Similar findings of significant increase in globulin concentration after IBDV infection also reported earlier (Ley et al., 1983).
 
Uric acid
 
The uric acid concentration showed an increasing trend as compared to normal values. The mean values of uric acid concentration of all the six groups are presented in table 5. Group B showed increase in uric acid concentration from 1^st day, which was significant on 3^rd, 4^th and 5^th day as compared to control. In group C, significant increase in uric acid concentration was seen on 3^rd and 4^th day as compared to control. In group E, significant increase was seen from 1^st day onward. In group F also, same trend was seen and significant increase was seen from 1^st day onward. Uric acid concentration was significantly increased in all the groups which were more severe in infected birds of both age groups. Birds below 3-weeks of age showed increase from 3^rd day onwards while birds with age above 3-weeks showed increase from 1^st day onward only. This shows a severe effect of IBDV on kidneys in birds within susceptible age range. The increase in uric-acid may be attributed to dehydration caused by viral infection which hampers the urine flow and thus accumulation of uric-acid is seen. Previous workers also noticed rise in uric-acid concentration (Ley et al., 1983).

From the current work, it can be concluded that pathogenesis of IBD is dependent on the age of birds with greater visible damage three-weeks onwards. The clinical signs were dependent and could be correlated to aspects such as haemato-biochemical alterations, gross and histopatho--logical lesions in organs other than bursa of Fabricius. Bursal changes could not be correlated to clinical signs as the birds lacking frank clinical signs were equally suffering from bursal damage and depletion of lymphocytes. Hence bursal body weight index is not a good indicator for IBD pathogenicity. This study further indicates the requirement of better vaccines which do not cause bursal damage and pathogenic changes while giving good protection against emerging variant and very virulent strains of IBD.