Front. Vet. Sci.Frontiers in Veterinary ScienceFront. Vet. Sci.2297-1769Frontiers Media S.A.10.3389/fvets.2021.673398Veterinary ScienceOriginal ResearchPigeons (Columba livia domestica) Are Susceptible to Infectious Bursal Disease: A Comparative Study of Their Hematological and Serum Biochemical AlterationsOrakpoghenorOchuko1*OladeleSunday Blessing1AbduPaul Ayuba2MarkusTalatu Patience3EnamSamson James1AndaminAliyu Danlami2MuhammedMuhammed Shuaib1UsmanSuleman Geidam1EsievoKing Akpofure Nelson11Veterinary Pathology, Ahmadu Bello University, Zaria, Nigeria2Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria3Veterinary Microbiology, Ahmadu Bello University, Zaria, Nigeria
Edited by: Huanmin Zhang, Agricultural Research Service (USDA), United States
Reviewed by: John Ihedioha, University of Nigeria, Nsukka, Nigeria; Sébastien Mathieu Soubies, Agence Nationale de Sécurité Sanitaire de l'Alimentation, de l'Environnement et du Travail (ANSES), France
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The hematological and serum biochemical alterations following very virulent infectious bursal disease virus (IBDV) infection in pigeons and chickens were evaluated in this study. Sixty IBDV seronegative birds comprising 30 (3–6 weeks old) pigeons and 30 (3 weeks old) chickens were randomly divided as follows: 10 uninoculated pigeons only, 10 inoculated pigeons + 10 sentinel chickens, 10 inoculated chickens + 10 sentinel pigeons, and 10 uninoculated chickens. Inoculated birds were administered 0.20 ml of inoculum containing very virulent IBDV (vvIBDV). Blood was collected postinoculation/exposure (pi/pe) and processed for hematology and biochemistry. The results revealed significantly (P < 0.05) increased packed cell volume, decreased mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC) in inoculated and sentinel chickens. Total leukocyte count (TLC), heterophil, and heterophil/lymphocyte (H/L) ratio were increased from 3 dpi/dpe in inoculated pigeons and from 3 to 14 dpi/dpe in inoculated and sentinel chickens. At 10 and 14 dpi/dpe, there was significantly (P < 0.05) increased serum total protein and globulin concentrations and decreased albumin/globulin ratio in pigeons and chickens. Serum urea concentration showed significant (P < 0.05) increase in inoculated and sentinel chickens at 3 and 4 dpi/dpe. To the authors' knowledge, this is the first report on hematological and serum biochemical alterations due to vvIBDV infection in pigeons.
Infectious bursal disease (IBD) or Gumboro disease (GD) caused by infectious bursal disease virus (IBDV) is characterized by inflammation of the bursa, bursal atrophy, and immunosuppression in chickens between 3 weeks and 3 months of age (1–3). The virus responsible for IBD possesses selective tropism for lymphoid tissue with the bursa of Fabricius (BF) being the primary target organ where it affects immature B lymphocytes (4, 5). Other lymphoid cells in the bone marrow, thymus, spleen, Peyer's patches, cecal tonsils, and Harderian glands have been reported to be also affected by IBDV (6). Chickens and turkeys constitute the natural host of IBDV, and transmission is primarily via fecal–oral route. Two serotypes of IBDV (1 and 2) have been identified, with serotype 1 considered to be pathogenic to chickens and serotype 2 considered avirulent (7, 8). Based on antigenicity and virulence, serotype 1 has been further classified into classical virulent, attenuated, antigenic variant, and very virulent IBDV (5, 9). In free-living wild birds, IBDV has been reported to induce lymphoid depletion with subclinical infection (10). In addition, serological evidence of serotype 1 IBDV infection in free-living wild birds suggests their possible susceptibility and role in the epidemiology of IBDV (7, 11).
Studies have documented hematological alterations in chickens, turkeys, and ducks due to IBDV with no information on the hematological alterations in domestic pigeons (Columba livia domestica) (12, 13). Pigeons have close interactions with chickens at backyard and on commercial poultry farms as well as live bird markets. In addition, there is an increase in pigeon production for consumption as meat and for research needs. Information on their susceptibility to various poultry diseases such as IBD would provide the impetus for enhanced production. Hence, in this study, the hematological and serum biochemical alterations following very virulent infectious bursal disease virus (IBDV) infection in pigeons (C. livia domestica) and chickens were evaluated.
Materials and MethodsEthical Approval
The ethics governing the use and conduct of experiments on animals were strictly observed, and the experimental protocol was approved by the Ahmadu Bello University Committee on Animal Use and Care (ABUCAUC) with approval number ABUCAUC/2021/002.
Experimental Birds
Thirty-day-old Institut de Sélection Animale (ISA) white cockerels were acquired from a reputable hatchery. When the chickens were 3 weeks old, they were used for the experiment. In addition, thirty 3–8-week-old domestic pigeons were acquired from local breeders and used for the experiment.
Experimental Design
The pigeons were divided into three groups (A–C) comprising 10 birds each. In addition, the chickens were divided into three groups (I–III) comprising 10 birds each. Pigeons in group A (uninoculated pigeons) and chickens in group I (uninoculated chickens) were not inoculated with vvIBDV and kept separately; pigeons in group B (inoculated pigeons) and chickens in group II (inoculated chickens) were inoculated with Nigerian strain of vvIBDV orally (109.76 CID50/ml). Pigeons in group C (sentinel pigeons) and chickens in group III (sentinel chickens) were uninoculated and kept together with inoculated chickens and inoculated pigeons, respectively. Hence, the four isolated groups of birds used for the experiment comprised of the following: 10 uninoculated pigeons only, 10 uninoculated chickens only, 10 inoculated pigeons + 10 sentinel chickens, and 10 inoculated chickens + sentinel pigeons. Birds in each group were kept in four separate pens and provided feed and water ad libitum. In addition, the surroundings of each pen were continuously fumigated.
Blood Sample Collection
Prior to inoculation with vvIBDV, blood samples were collected from all pigeons and chickens via brachial veins, and the sera were harvested. The sera were confirmed seronegative for IBDV antibody using enzyme-linked immunosorbent assay (ELISA).
Blood was also collected from pigeons and chickens on days 0, 1, 2, 3, 4, 5, 6, 7, 10, and 14 postinoculation and/or exposure (dpi/dpe) and divided into two parts. One part was emptied into a labeled tube containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant for hematological analyses. The other part of blood was poured into a labeled plain tube to harvest serum and used for serum biochemical analyses and IBDV antibody detection using ELISA (ID Screen® IBD VP2, ID.vet, Innovative Diagnostics, Grabels, France).
Hematological Analyses
The packed cell volume (PCV) was determined using a standard technique (14). This involved filling up of a non-heparinized capillary tube with blood to about three-quarters of its length from one end and sealing the other end and centrifugating it at 4,383 × g for 5 min using a Saitexiangyi TG12MX® Microhematocrit centrifuge machine (Hunan Saite xiangyi centrifuge instrument Co., Ltd., Changsha, China). After that, the PCV value was measured using the Hawksley® Microhematocrit Reader (Hawksley, Lancing, UK) and recorded as percentage of blood volume.
The Natt–Herrick solution (1:200 dilution) and improved Neubauer hemocytometer (15) were used to determine total leukocyte (TLC) or white blood cell (TWBC) counts. The cells were viewed using a light microscope (Olympus XSZ-107BN, Wincom Company Limited, Changsha, China) at a low power magnification (×40) and counted by means of a tally counter. Both sides of the hemocytometer were counted, and the average was calculated.
Differential leukocyte count (DLC) was determined using the Giemsa technique (16, 17). The standard slide-to-slide technique was used to prepare a pair of smears for each blood sample using about 2 μl of blood on a clean microscope slide. The slide was then labeled, air dried, and fixed for 3 min in methanol followed by another air-drying brief period. The slides were then flooded with Wright–Giemsa stain for 3 min followed by addition of an equal amount of Sørensen's buffer (pH 6.8) and gently mixed until the formation of green metallic sheen is seen on the surface. This was followed by washing the stained slides with the Sørensen's buffer, using tissue paper to wipe off the excess stain from the back of the slides and then air dried. The blood smears were examined using a light microscope (Olympus XSZ-107BN) under high-power magnification with oil immersion (×1,000).
The mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentrations (MCHCs) were calculated as described by Coles (1986).
The total protein concentration in each serum sample was determined colorimetrically based on the principle of the Biuret reaction (copper salt in an alkaline medium) by following the manufacturer's instruction (AGAPPE—total protein; AGAPPE Diagnostics Switzerland, GmbH, Cham, Switzerland, 51013002). The absorbance of the standard and sample was measured against the reagent blank using a spectrophotometer (S23A, Techmel, New York, NY, USA) at 546 nm.
The albumin concentration in each serum sample was determined by bromocresol green methodology following the manufacturer's instruction (AGAPPE—albumin; AGAPPE Diagnostics Switzerland, GmbH, 51001002). The sample absorbance was measured against that of reagent blank using a spectrophotometer (S23A, Techmel, USA) at 630 nm.
The globulin concentration in each sample was calculated by subtracting the albumin concentration from the total protein concentration. The albumin–globulin (ALB/GLB) in each sample was calculated by dividing the albumin concentration with globulin concentration.
The urea concentration in each serum sample was determined using the modified Berthelot methodology by following the manufacturer's instruction (AGAPPE—urea B; AGAPPE Diagnostics Switzerland, GmbH, 51216001). The absorbance of the standard and sample against the reagent blank was measured using a spectrophotometer (S23A, Techmel, USA) at 600 nm.
The creatinine concentration in each serum sample was determined using the modified Jaffe's method following the manufacturer's instruction (AGAPPE—creatinine; AGAPPE Diagnostics Switzerland, GmbH, 51009001). The principle is based on the reaction of creatinine with picric acid to produce creatinine alkaline picrate (a colored product). The optical density of mixtures was first taken (T1) 60 s after addition of the standard/sample and then again was taken for the second time (T2) 60 s after the first reading using a spectrophotometer (S23A, Techmel, USA) at 600 nm.
Data Analyses
Data were presented using tables and charts. Data were expressed as mean ± SEM and subjected to a two-way analysis of variance (two-way ANOVA) followed by Bonferroni post-hoc test (not shown in the tables) using GraphPad Prism version 5.0 (GraphPad, San Diego, CA, USA). Values of P ≤ 0.05 were considered significant.
ResultsClinical Signs
The incubation period of IBD in pigeons and chickens was 2 days. Anorexia and reduced activity were the clinical signs observed in inoculated pigeons at 2 and 3 dpi, and these signs were absent by 4 dpi. There was no mortality in the pigeons. In inoculated and sentinel chickens, the clinical signs observed included ruffled feathers, anorexia, reduced activity, huddling, somnolence, prostration, and watery diarrhea. Mortalities were 60 and 50% in inoculated and sentinel chickens, respectively.
Antibody Response
Anti-IBDV antibodies were detected in inoculated and sentinel chickens from 3 dpi, in inoculated pigeons from 4 dpi, and in sentinel pigeons from 5 dpi (Table 1).
Mean IBDV ELISA titer (at 1:500) of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Mean ELISA titer (at 1:500)
0
26.00 ± 12.00
21.67 ± 10.81
24.00 ± 6.00
174.50 ± 14.50
167.00 ± 0.00
162.50 ± 30.50
1
22.00 ± 5.00
16.00 ± 2.00
21.00 ± 3.00
136.00 ± 29.50
213.50 ± 29.50
166.00 ± 4.36
2
13.00 ± 2.08
139.67 ± 7.97
28.00 ± 31.12
92.00 ± 0.00
404.33 ± 39.87
278.67 ± 85.87
3
13.00 ± 1.00
327.33 ± 5.24
223.33 ± 13.02
100.50 ± 8.50
512.33 ± 18.02
530.00 ± 25.00
4
8.00 ± 2.00
557.00 ± 2.00
258.67 ± 29.76
83.67 ± 22.26
544.33 ± 7.84
552.00 ± 11.02
5
11.00 ± 3.00
797.00 ± 40.00
715.00 ± 90.37
87.00 ± 51.05
1006.00 ± 16.00
928.50 ± 153.50
6
2.00 ± 2.00
1245.67 ± 110.64
1000.00 ± 68.00
71.67 ± 32.54
1274.00 ± 56.00
1028.50 ± 16.50
7
0.00 ± 0.00
1463.33 ± 66.16
1337.67 ± 67.77
80.67 ± 35.43
1468 ± 6.50
1545.00 ± 136.00
10
0.00 ± 0.00
1840.50 ± 26.50
1525.00 ± 30.00
60.00 ± 9.00
1932.00 ± 44.00
1822.50 ± 52.50
14
0.00 ± 0.00
2016.33 ± 21.84
1594.00 ± 25.89
43.67 ± 10.17
2143.50 ± 20.50
2175.50 ± 6.50
Mean ELISA titer >875 indicated positive IBDV antibody detection, while ELISA titer ≤ 875 indicated negative IBDV antibody detection according to the manufacturer's instructions.
Changes in HematologyErythrocytic Parameters
In pigeons, the packed cell volume (PCV) showed no statistical (P > 0.05) difference preinoculation, postinoculation, and/or exposure to 14 dpi/dpe. At 1 dpi, the PCV was significantly (P < 0.05) higher in the inoculated chickens than both the uninoculated and sentinel chickens, but the latter two groups were not different from each other (P > 0.5). The PCVs of both the inoculated and sentinel groups at 2, 3, 4, and 5 dpi/dpe were significantly higher (P < 0.05) than the uninoculated group. From 6 to 14 dpi/dpe, the PCV showed no significant difference (P > 0.05) between the treatment groups (Figure 1).
Mean packed cell volume of chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus. Values with different alphabets in the same day differ significantly at P < 0.05.
The MCH showed no significant difference (P > 0.05) between the treatment groups of both pigeons and chickens except one time point in the chickens. At 3 dpi/dpe, the average MCH of the inoculated group of chickens was significantly lower (P < 0.05) than both the uninoculated and sentinel groups (Table 2).
Mean corpuscular hemoglobin of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Mean corpuscular hemoglobin (pg)
0
37.24 ± 3.98a
38.59 ± 3.87a
38.53 ± 2.02a
29.48 ± 0.73a
29.52 ± 0.38a
29.90 ± 0.01a
1
36.39 ± 2.92a
38.60 ± 1.95a
38.35 ± 2.14a
29.72 ± 0.48a
28.38 ± 1.53a
29.89 ± 0.01a
2
37.42 ± 3.33a
37.67 ± 2.03a
37.93 ± 0.61a
29.54 ± 0.23a
27.78 ± 1.41a
28.46 ± 1.47a
3
37.61 ± 2.93a
37.47 ± 1.03a
38.18 ± 1.42a
29.53 ± 0.90a
27.05 ± 1.64b
27.79 ± 1.39a
4
37.29 ± 1.14a
37.51 ± 1.72a
37.52 ± 1.91a
29.20 ± 2.17a
26.31 ± 1.86a
27.69 ± 1.69a
5
37.94 ± 1.43a
37.89 ± 1.27a
38.54 ± 1.02a
29.78 ± 0.13a
27.36 ± 2.37a
28.77 ± 0.90b
6
37.65 ± 1.98a
38.24 ± 1.50a
37.98 ± 1.56a
29.55 ± 1.29a
29.06 ± 0.71a
29.19 ± 0.37a
7
37.70 ± 0.82a
37.99 ± 3.28a
37.70 ± 0.82a
29.70 ± 0.05a
29.74 ± 0.02a
29.90 ± 0.02a
10
37.79 ± 2.24a
38.86 ± 4.25a
38.64 ± 3.23a
29.73 ± 0.02a
29.77 ± 0.01a
29.93 ± 0.01a
14
36.62 ± 1.16a
37.25 ± 2.00a
36.29 ± 1.18a
29.11 ± 0.69a
29.20 ± 1.06a
29.15 ± 0.81a
Values with different superscript alphabets in the same row for the same species of bird differ significantly at P < 0.05.
No significant difference was observed in MCHC between the treatment groups of pigeons from 0 to 14 dpi/dpe. The MCHC of the inoculated group of chickens was significantly lower (P < 0.05) than that of both the sentinel and uninoculated groups of chickens at 2 dpi/dpe, and the MCHCs of both the inoculated and sentinel groups were significantly lower (P < 0.05) than their uninoculated groups at 3 and 4 dpi/dpe (Table 3).
Mean corpuscular hemoglobin concentration of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days post-inoculation and/or exposure
Mean corpuscular hemoglobin concentration (g/dl)
0
28.80 ± 2.55a
29.59 ± 1.71a
29.67 ± 1.00a
27.25 ± 0.00a
27.24 ± 0.01a
27.24 ± 0.01a
1
28.18 ± 2.17a
29.33 ± 1.57a
29.53 ± 1.65a
27.25 ± 0.01a
26.03 ± 1.21a
27.25 ± 0.00a
2
29.12 ± 1.44a
29.03 ± 2.48a
29.40 ± 1.27a
27.10 ± 0.00a
25.34 ± 1.28b
25.93 ± 1.34a
3
29.63 ± 1.69a
29.42 ± 1.43a
29.21 ± 1.46a
27.11 ± 0.00a
24.67 ± 1.49b
25.32 ± 1.26b
4
29.07 ± 1.60a
29.14 ± 1.53a
28.89 ± 1.46a
27.10 ± 0.01a
23.99 ± 1.71b
25.25 ± 1.52b
5
29.67 ± 1.00a
29.26 ± 1.09a
29.67 ± 1.00a
27.10 ± 0.01a
24.95 ± 2.15a
26.22 ± 0.83a
6
29.08 ± 1.36a
29.67 ± 1.00a
29.39 ± 1.20a
27.11 ± 0.01a
26.48 ± 0.63a
26.62 ± 0.32a
7
29.14 ± 1.44a
28.68 ± 1.78a
29.03 ± 0.62a
27.11 ± 0.00a
27.10 ± 0.01a
27.26 ± 0.00a
10
28.30 ± 1.59a
28.21 ± 1.46a
28.65 ± 0.63a
27.10 ± 0.00a
27.11 ± 0.01a
27.24 ± 0.01a
14
28.35 ± 1.27a
29.12 ± 1.24a
28.34 ± 1.05a
27.14 ± 0.80a
27.17 ± 4.14a
27.19 ± 0.64a
Values with different superscript alphabets in the same row for the same species of bird differ significantly at P < 0.05.
Leukocytic Parameters
At 3 dpi/dpe, total leukocyte count (TLC) was significantly (P < 0.05) increased in the inoculated group of pigeons compared to the uninoculated and sentinel groups of pigeons. The TLC was observed significantly lower (P < 0.05) in the inoculated group of chickens than its counterparts of the sentinel and uninoculated groups at 1 dpi/dpe, and both the inoculated and sentinel groups had significantly lower (P < 0.05) TLC than the uninoculated group of chickens at 2 dpi/dpe. From 3 to 14 dpi/dpe, however, the observed TLC of the inoculated and sentinel groups of chickens were significantly higher (P < 0.05) than their counterpart of the uninoculated groups. The TLC of both the inoculated and the sentinel groups of chickens reached a peak at 5 dpi/dpe, followed with gradual decreases (Table 4).
Mean total leukocyte count of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Total leukocytes count (×109/l)
0
22.20 ± 1.18a
21.90 ± 1.13a
21.66 ± 1.19a
25.68 ± 0.66a
25.96 ± 1.53a
25.98 ± 0.89a
1
21.88 ± 0.52a
21.68 ± 0.78a
21.62 ± 1.10a
25.62 ± 1.01a
22.42 ± 1.05b
25.72 ± 1.02a
2
22.12 ± 0.91a
20.92 ± 1.29a
21.10 ± 1.47a
25.96 ± 1.03a
23.02 ± 0.76b
23.48 ± 0.80b
3
21.90 ± 0.95a
23.86 ± 1.71b
22.02 ± 1.51a
25.52 ± 1.04a
28.64 ± 1.06b
29.22 ± 1.45b
4
21.80 ± 0.99a
21.42 ± 1.20a
21.58 ± 1.29a
25.50 ± 0.58a
36.07 ± 1.67b
37.63 ± 1.50b
5
21.56 ± 1.01a
21.90 ± 1.43a
20.68 ± 1.50a
26.18 ± 1.02a
48.43 ± 2.48b
46.30 ± 3.04b
6
21.76 ± 0.90a
21.70 ± 1.11a
20.72 ± 1.01a
25.96 ± 1.56a
40.80 ± 1.23b
38.83 ± 3.64b
7
22.06 ± 1.26a
21.26 ± 1.04a
20.72 ± 1.03a
26.38 ± 1.75a
32.77 ± 1.24b
31.13 ± 1.96b
10
21.84 ± 0.90a
21.36 ± 1.23a
21.58 ± 0.99a
26.60 ± 1.16a
29.35 ± 1.65b
29.00 ± 0.80b
14
21.88 ± 0.61a
21.52 ± 0.78a
21.80 ± 0.64a
26.28 ± 1.76a
28.20 ± 1.70b
28.35 ± 1.65b
Values with different superscript alphabets in the same row for the same species of bird differ significantly at P < 0.05.
The heterophil counts of pigeons and chickens pre- and postinoculation/exposure to IBDV are presented in Table 5. There was a significant (P < 0.05) increase in heterophil count of inoculated pigeons at 3 dpi compared to the uninoculated and sentinel pigeons. In chickens, from 3 to 14 dpi/dpe, heterophil counts of both the inoculated and sentinel groups of chickens were significantly higher (P < 0.05) than the uninoculated control groups. The heterophil count reached the highest at 5 dpi/dpe in the inoculated and sentinel chickens and then gradually declined all the way to the 14 dpi/dpe.
Mean heterophil count of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Heterophil count (×109/l)
0
7.70 ± 0.29a
7.42 ± 0.66a
7.84 ± 0.84a
9.99 ± 0.23a
9.38 ± 0.78a
9.37 ± 0.89a
1
8.01 ± 0.67a
7.94 ± 0.19a
8.10 ± 0.46a
9.99 ± 0.50a
9.11 ± 0.60a
9.80 ± 1.02a
2
7.94 ± 0.29a
8.06 ± 0.60a
8.02 ± 0.43a
8.98 ± 0.32a
9.30 ± 0.34a
9.47 ± 0.80a
3
7.68 ± 0.52a
8.40 ± 0.73b
7.96 ± 0.69a
8.59 ± 0.47a
12.43 ± 0.71b
11.20 ± 1.45b
4
7.60 ± 0.37a
7.58 ± 0.38a
7.44 ± 0.73a
9.22 ± 0.40a
17.00 ± 1.57b
17.88 ± 1.50b
5
7.62 ± 0.71a
7.72 ± 0.60a
7.52 ± 0.72a
8.58 ± 0.72a
28.02 ± 0.70b
25.98 ± 3.04b
6
7.42 ± 0.84a
7.58 ± 0.60a
7.42 ± 0.54a
8.70 ± 0.85a
23.02 ± 1.61b
23.44 ± 3.64b
7
7.54 ± 0.35a
7.66 ± 0.60a
7.56 ± 0.46a
9.23 ± 0.72a
14.24 ± 1.11b
15.97 ± 1.96b
10
7.66 ± 0.57a
7.58 ± 0.62a
7.68 ± 0.32a
8.57 ± 0.45a
11.69 ± 0.40b
11.96 ± 1.30b
14
7.32 ± 0.54a
7.64 ± 0.41a
7.52 ± 0.47a
8.95 ± 1.29a
11.42 ± 0.35b
11.48 ± 2.15b
Values with different superscript alphabets in the same row for the same species of bird differ significantly at P < 0.05.
The lymphocyte counts of pigeons and chickens following inoculation and/or exposure to IBDV are presented in Table 6. No difference in lymphocyte count was observed between the treatment groups of pigeons (P > 0.05). The observed lymphocyte counts were significantly lower in the inoculated groups of chickens at 1–4 dpi and in the sentinel groups at 2–4 dpe (P < 0.05). No significant difference in lymphocyte count was observed between the treatment groups of chickens from 5 to 14 dpi/dpe.
Mean lymphocyte count of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Lymphocyte count (×109/l)
13.38 ± 0.78a
13.24 ± 0.50a
13.36 ± 0.77a
15.82 ± 0.60a
16.38 ± 0.97a
16.35 ± 0.51a
1
12.96 ± 0.74a
13.04 ± 0.90a
13.34 ± 0.73a
15.98 ± 0.90a
13.03 ± 0.73b
15.99 ± 0.73a
2
13.22 ± 0.86a
13.18 ± 1.50a
13.14 ± 1.04a
16.78 ± 1.14a
13.20 ± 0.42b
13.48 ± 0.53b
3
13.30 ± 0.65a
12.98 ± 0.65a
13.04 ± 1.40a
16.63 ± 0.65a
14.34 ± 0.45b
14.89 ± 0.69b
4
13.30 ± 0.90a
12.92 ± 0.75a
13.26 ± 0.69a
16.43 ± 0.66a
14.53 ± 0.68b
15.35 ± 0.82b
5
13.24 ± 0.50a
13.14 ± 1.08a
13.18 ± 0.65a
17.15 ± 1.16a
15.84 ± 0.76a
16.19 ± 0.69a
6
13.16 ± 0.68a
12.94 ± 0.57a
12.98 ± 0.57a
17.01 ± 1.35a
17.23 ± 1.00a
16.86 ± 0.61a
7
13.38 ± 0.92a
13.16 ± 0.43a
13.30 ± 0.58a
16.89 ± 1.39a
17.13 ± 0.26a
17.21 ± 0.71a
10
13.28 ± 0.62a
12.98 ± 0.49a
12.98 ± 0.97a
17.04 ± 1.04a
17.22 ± 0.31a
17.13 ± 0.23a
14
13.30 ± 0.42a
13.00 ± 0.49a
13.10 ± 0.48a
17.19 ± 1.04a
17.16 ± 1.08a
16.79 ± 1.00a
Values with different superscript alphabets in the same row for the same species of bird differ significantly at P < 0.05.
Heterophil/lymphocyte ratios (H/L) were calculated. IBDV infection significantly upregulated the H/L in pigeons only at 3 dpi (P < 0.05). Significantly higher H/L (P < 0.05) were observed in the inoculated groups of chickens from 1 to 14 dpi and in the sentinel groups of chickens from 2 to 14 dpe than the uninoculated control group of chickens (Table 7).
Mean heterophil/lymphocyte ratio of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Heterophil/lymphocyte ratio
0
0.55 ± 0.01a
0.54 ± 0.04a
0.52 ± 0.04a
0.63 ± 0.03a
0.57 ± 0.04a
0.57 ± 0.03a
1
0.53 ± 0.04a
0.57 ± 0.03a
0.55 ± 0.04a
0.63 ± 0.05a
0.70 ± 0.03b
0.62 ± 0.03a
2
0.55 ± 0.03a
0.57 ± 0.04a
0.56 ± 0.06a
0.55 ± 0.04a
0.70 ± 0.01b
0.70 ± 0.01b
3
0.51 ± 0.05a
0.60 ± 0.06b
0.53 ± 0.07ab
0.52 ± 0.01a
0.87 ± 0.04b
0.76 ± 0.06b
4
0.53 ± 0.04a
0.57 ± 0.04a
0.56 ± 0.05a
0.58 ± 0.04a
1.17 ± 0.05b
1.14 ± 0.07b
5
0.54 ± 0.03a
0.56 ± 0.08a
0.52 ± 0.05a
0.51 ± 0.06a
1.67 ± 0.06b
1.59 ± 0.14b
6
0.56 ± 0.07a
0.58 ± 0.05a
0.57 ± 0.03a
0.53 ± 0.07a
1.35 ± 0.17b
1.34 ± 0.06b
7
0.56 ± 0.04a
0.58 ± 0.05a
0.57 ± 0.04a
0.56 ± 0.07a
0.82 ± 0.07b
0.81 ± 0.10b
10
0.55 ± 0.04a
0.57 ± 0.04a
0.56 ± 0.02a
0.51 ± 0.02a
0.68 ± 0.04b
0.66 ± 0.06b
14
0.50 ± 0.04a
0.53 ± 0.04a
0.52 ± 0.03a
0.52 ± 0.08a
0.67 ± 0.06b
0.65 ± 0.05b
Values with different superscript alphabets in the same row for the same species of bird differ significantly at P < 0.05.
Changes in Serum BiochemistryTotal Protein, Globulin Concentrations, and Albumin/Globulin Ratio
The average serum total protein concentration was significantly upregulated in the treatment groups of pigeons in response to IBDV inoculation or exposure at 10 and 14 dpi/dpe in contrast to the uninoculated control groups (P < 0.05). The significantly upregulated total protein concentration was also observed (P < 0.05) in inoculated and sentinel groups of chickens at 3, 4, and 7–14 dpi/dpe in comparison with the counterpart of uninoculated control groups (Table 8).
Mean serum total protein concentration of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Serum total protein concentration (g/dl)
0
4.05 ± 0.25a
4.05 ± 0.47a
4.08 ± 0.37a
3.28 ± 0.09a
3.30 ± 0.12a
3.28 ± 0.11a
1
4.25 ± 0.23a
4.10 ± 0.15a
4.13 ± 0.11a
3.35 ± 0.30a
3.40 ± 0.16a
3.28 ± 0.11a
2
4.03 ± 0.40a
4.03 ± 0.20a
4.10 ± 0.41a
3.38 ± 0.06a
3.43 ± 0.14a
3.43 ± 0.17a
3
4.20 ± 0.27a
4.05 ± 0.06a
4.08 ± 0.32a
3.35 ± 0.09a
3.60 ± 0.15b
3.57 ± 0.20b
4
4.10 ± 0.45a
4.10 ± 0.27a
4.13 ± 0.38a
3.40 ± 0.15a
3.65 ± 0.05b
3.63 ± 0.12b
5
4.28 ± 0.40a
4.13 ± 0.18a
4.13 ± 0.41a
3.50 ± 0.20a
3.55 ± 0.25a
3.47 ± 0.35a
6
4.10 ± 0.34a
4.35 ± 0.30a
4.33 ± 0.39a
3.50 ± 0.20a
3.55 ± 0.35a
3.53 ± 0.27a
7
4.30 ± 0.31a
4.50 ± 0.51a
4.48 ± 0.23a
3.35 ± 0.09a
3.50 ± 0.10b
3.53 ± 0.15b
10
4.18 ± 0.29a
4.75 ± 0.16b
4.70 ± 0.29b
3.38 ± 0.09a
3.60 ± 0.10b
3.65 ± 0.15b
14
4.33 ± 0.30a
4.85 ± 0.23b
4.78 ± 0.24b
3.48 ± 0.20a
3.75 ± 0.15b
3.80 ± 0.20b
Values with different superscript alphabets in the same row for the same species of bird differ significantly at P < 0.05.
The average serum globulin concentration of the IBDV inoculated and sentinel groups of both pigeons and chickens was significantly higher (P < 0.05) at 10 and 14 dpi/dpe than the uninoculated control groups (Table 9). The average albumin–globulin ratios (A/G) of both the inoculated and sentinel groups of pigeons and chickens were significantly lower (P < 0.05) than their uninoculated control groups at 10 and 14 dpi/dpe (Table 10).
Mean serum globulin concentration of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Serum globulin concentration (g/dl)
0
2.01 ± 0.30a
2.03 ± 0.50a
2.04 ± 0.30a
1.83 ± 0.08a
1.87 ± 0.08a
1.84 ± 0.18a
1
2.18 ± 0.15a
2.10 ± 0.22a
2.17 ± 0.25a
1.86 ± 0.32a
1.82 ± 0.24a
1.76 ± 0.16a
2
2.05 ± 0.22a
1.94 ± 0.11a
2.14 ± 0.40a
1.81 ± 0.08a
1.79 ± 0.22a
1.78 ± 0.25a
3
2.12 ± 0.28a
2.18 ± 0.19a
2.19 ± 0.50a
1.84 ± 0.15a
2.05 ± 0.19a
2.01 ± 0.08a
4
2.06 ± 0.35a
2.16 ± 0.23a
2.09 ± 0.60a
1.90 ± 0.16a
2.08 ± 0.03a
2.06 ± 0.25a
5
2.00 ± 0.29a
2.06 ± 0.31a
2.10 ± 0.45a
1.90 ± 0.18a
1.89 ± 0.46a
1.77 ± 0.33a
6
2.10 ± 0.34a
2.29 ± 0.35a
2.35 ± 0.47a
1.87 ± 0.33a
1.97 ± 0.00a
1.94 ± 0.40a
7
2.11 ± 0.32a
2.54 ± 0.24a
2.56 ± 0.36a
1.82 ± 0.12a
2.01 ± 0.08a
2.02 ± 0.26a
10
2.18 ± 0.35a
2.68 ± 0.04b
2.73 ± 0.14b
1.77 ± 0.14a
2.17 ± 0.04b
2.21 ± 0.23b
14
2.21 ± 0.16a
2.74 ± 0.24b
2.65 ± 0.26b
1.84 ± 0.26a
2.25 ± 0.12b
2.24 ± 0.09b
Values with different superscript alphabets in the same row for the same species of bird differ significantly at P < 0.05.
Mean albumin/globulin ratio of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Albumin/globulin ratio
0
1.12 ± 0.25a
1.05 ± 0.24a
1.10 ± 0.30a
0.79 ± 0.03a
0.77 ± 0.02a
0.79 ± 0.12a
1
1.08 ± 0.14a
1.02 ± 0.19a
0.98 ± 0.24a
0.82 ± 0.13a
0.86 ± 0.12a
0.85 ± 0.10a
2
1.09 ± 0.17a
1.08 ± 0.06a
1.12 ± 0.35a
0.87 ± 0.10a
0.88 ± 0.12a
0.87 ± 0.11a
3
1.14 ± 0.21a
1.14 ± 0.14a
1.14 ± 0.43a
0.85 ± 0.12a
0.78 ± 0.13a
0.77 ± 0.05a
4
1.05 ± 0.13a
0.93 ± 0.11a
0.97 ± 0.12a
0.81 ± 0.07a
0.76 ± 0.05a
0.79 ± 0.12a
5
1.05 ± 0.23a
1.11 ± 0.23a
1.01 ± 0.31a
0.86 ± 0.07a
0.86 ± 0.25a
0.85 ± 0.10a
6
1.04 ± 0.21a
0.97 ± 0.16a
1.09 ± 0.43a
0.86 ± 0.11a
0.81 ± 0.18a
0.82 ± 0.20a
7
0.95 ± 0.27a
0.87 ± 0.23a
0.93 ± 0.13a
0.85 ± 0.08a
0.74 ± 0.11a
0.75 ± 0.15a
10
1.13 ± 0.32a
0.77 ± 0.04b
0.78 ± 0.14b
0.94 ± 0.12a
0.66 ± 0.08b
0.64 ± 0.11b
14
1.06 ± 0.17a
0.82 ± 0.13b
0.83 ± 0.12b
0.92 ± 0.15a
0.60 ± 0.08b
0.65 ± 0.12b
Values with different superscript alphabets in the same row for the same species of bird differ significantly at P < 0.05.
Urea and Creatinine Concentrations
The average serum urea concentration was significantly higher (P < 0.05) in the IBDV inoculated (1.01 ± 0.05 mg/dl, 1.02 ± 0.01 mg/dl, 0.92 ± 0.04 mg/dl) and sentinel (0.97 ± 0.05 mg/dl, 0.99 ± 0.07 mg/dl, 0.85 ± 0.08 mg/dl) groups of chickens than the corresponding uninoculated control groups at 3, 4, and 5 dpi/dpe (respectively). No significant difference was observed between the treatment groups of chickens at the rest of dpi/dpe, and no difference was observed between the treatment groups of pigeons at any dpi/dpe (Table 11). No significant difference was observed in average serum creatinine concentration between the treatment groups of either pigeons or chickens in response to the IBDV inoculation or exposure during the experimental period (Table 12).
Mean serum urea concentration of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Serum urea concentration (mg/dl)
0
0.30 ± 0.06a
0.30 ± 0.05a
0.29 ± 0.06a
0.86 ± 0.06a
0.89 ± 0.11a
0.90 ± 0.04a
1
0.31 ± 0.03a
0.32 ± 0.06a
0.30 ± 0.04a
0.88 ± 0.05a
0.89 ± 0.10a
0.85 ± 0.06a
2
0.32 ± 0.09a
0.34 ± 0.07a
0.34 ± 0.09a
0.85 ± 0.03a
0.92 ± 0.07a
0.88 ± 0.01a
3
0.34 ± 0.08a
0.36 ± 0.08a
0.33 ± 0.08a
0.86 ± 0.06a
1.01 ± 0.05b
0.97 ± 0.05b
4
0.31 ± 0.05a
0.33 ± 0.04a
0.32 ± 0.12a
0.81 ± 0.05a
1.02 ± 0.01b
0.99 ± 0.07b
5
0.32 ± 0.06a
0.29 ± 0.06a
0.34 ± 0.05a
0.76 ± 0.05a
0.92 ± 0.04b
0.85 ± 0.08ab
6
0.32 ± 0.07a
0.30 ± 0.04a
0.31 ± 0.03a
0.81 ± 0.11a
0.82 ± 0.03a
0.81 ± 0.09a
7
0.34 ± 0.07a
0.34 ± 0.09a
0.34 ± 0.05a
0.83 ± 0.05a
0.85 ± 0.07a
0.84 ± 0.04a
10
0.33 ± 0.06a
0.33 ± 0.08a
0.33 ± 0.10a
0.82 ± 0.08a
0.85 ± 0.10a
0.86 ± 0.03a
14
0.33 ± 0.04a
0.32 ± 0.12a
0.32 ± 0.06a
0.81 ± 0.11a
0.84 ± 0.03a
0.83 ± 0.02a
Values with different alphabets in the same row for the same species differ significantly at P < 0.05.
Mean serum creatinine concentration of pigeons and chickens following inoculation with and/or exposure to a very virulent infectious bursal disease virus.
Uninoculated pigeons
Inoculated pigeons
Sentinel pigeons
Uninoculated chickens
Inoculated chickens
Sentinel chickens
Days postinoculation and/or exposure
Serum creatinine concentration (mg/dl)
0
0.43 ± 0.08
0.42 ± 0.07
0.43 ± 0.08
0.43 ± 0.02
0.44 ± 0.02
0.43 ± 0.03
1
0.46 ± 0.05
0.47 ± 0.07
0.46 ± 0.04
0.44 ± 0.02
0.44 ± 0.04
0.43 ± 0.02
2
0.46 ± 0.11
0.49 ± 0.10
0.49 ± 0.13
0.46 ± 0.02
0.46 ± 0.02
0.45 ± 0.02
3
0.49 ± 0.11
0.52 ± 0.11
0.49 ± 0.10
0.45 ± 0.02
0.43 ± 0.03
0.44 ± 0.03
4
0.44 ± 0.07
0.47 ± 0.05
0.46 ± 0.17
0.47 ± 0.02
0.44 ± 0.05
0.45 ± 0.02
5
0.46 ± 0.09
0.47 ± 0.06
0.48 ± 0.06
0.46 ± 0.02
0.48 ± 0.00
0.46 ± 0.03
6
0.45 ± 0.09
0.45 ± 0.04
0.45 ± 0.03
0.45 ± 0.02
0.44 ± 0.00
0.45 ± 0.03
7
0.49 ± 0.10
0.49 ± 0.13
0.49 ± 0.05
0.48 ± 0.01
0.47 ± 0.02
0.48 ± 0.01
10
0.47 ± 0.09
0.49 ± 0.11
0.48 ± 0.13
0.47 ± 0.03
0.50 ± 0.02
0.49 ± 0.04
14
0.47 ± 0.06
0.46 ± 0.17
0.47 ± 0.08
0.44 ± 0.01
0.46 ± 0.05
0.44 ± 0.01
Discussion
The detection of anti-IBDV antibodies in pigeons and chickens in this study indicated serological evidence of IBDV infection. There was no significant alterations in PCV, MCH, and MCHC of inoculated and sentinel pigeons. In inoculated and sentinel chickens, there was significant polycythemia and decreased MCH and MCHC. However, the polycythemia was relative due to insignificant alterations in the red blood cell (RBC). Polycythemia following IBDV infection in chickens has also been reported by Oladele et al. (12), Bello et al. (13), and Chineme and Cho (18). In contrast, anemia due to IBDV infection has been documented in turkeys and guinea fowls (12). The relative polycythemia observed in inoculated and sentinel chickens might be associated with hemoconcentration due to dehydration resulting from anorexia and diarrhea (12, 13). The decrease in MCH and MCHC in this study suggested normocytic hypochromic anemia, and this might have resulted from hemorrhage with subsequent iron deficiency. Thus, the type of anemia encountered in vvIBDV infection in chickens in this study could be considered as normocytic hypochromic anemia.
Leukocytosis due to heterophilia in vvIBDV-inoculated pigeons was short lived and absent in sentinel pigeons. In the chickens, leukocytic response was characterized by initial leukopenia due to lymphopenia followed by leukocytosis due to heterophilia. Since heterophils have been known to play critical role in phagocytosis of tissue debris (16), the absence of heterophilic response in sentinel pigeons suggested no tissue destruction. The short-lived heterophilia in inoculated pigeons was suggestive of mild tissue destruction by the vvIBDV. In contrast, the persistence of heterophilia in chickens was indicative of massive tissue destruction (12). Lymphopenia, as observed in this study, has been reported to occur following IBDV infection in chickens, and this was attributed to excess glucocorticoid release (19, 20) and/or specific destruction of lymphocytes by IBDV within the BF (21).
Although heterophil/lymphocyte ratio has been documented to be an indicator of stress in birds, it does not indicate susceptibility or response to disease challenge (20, 22, 23). The transient high heterophil/lymphocyte ratio observed in the inoculated pigeons of this study might be due to the short-lived heterophilic response to vvIBDV infection. Similarly, the persistently high heterophil/lymphocyte ratio observed in the inoculated and sentinel chickens was due to a prolongedly heterophilic response to the IBDV infection. In addition, decreased relative space per bird resulting from competition with the pigeons might have induced further increase in corticosterone levels of the chickens with consequent lymphopenia and heterophilia (20, 24). Species differences and/or differences in susceptibility to vvIBDV might be the possible reason for the variation in erythocytic, heterophilic, and lymphocytic responses of pigeons and chickens observed in this study.
The significant hyperproteinemia, consequent hyperglobulinemia, and decreased albumin/globulin ratio observed in pigeons following infection and/or exposure at 10 and 14 dpi/dpe was possibly due to increase in immunoglobulins in response to vvIBDV infection. In chickens, the initial hyperproteinemia at 3 and 4 dpi/dpe might be due to dehydration (25). This suggestion is justified by the concomitant polycythemia with unaltered erythrocytes count. The later hyperproteinemia and subsequent decreased albumin/globulin ratio might probably have resulted from hyperglobulinemia due to increased immunoglobulins in response to vvIBDV infection and decreased albumin level. However, the absence of significant changes in albumin/globulin ratio at 3 and 4 dpi/dpe in chickens, despite hyperproteinemia, might be due to decreased albumin resulting from hemorrhages and anorexia caused by vvIBDV infection. This is consistent with the report by Lumeij (1987) who suggested possible hyperproteinemia with normal albumin/globulin ratio in dehydrated birds (26). The albumin/globulin ratio has been documented to be a good indicator of health and response to diseases (27, 28); hence, the decreased albumin/globulin ratio in pigeons and chickens postinfection/exposure in this study is indicative of immune response to the vvIBDV infection.
Serum/plasma urea and creatinine concentrations have been suggested to be indicators of the extent of renal damage and muscle destruction, respectively (29). In this study, there were no significant changes in serum urea and creatinine concentrations of inoculated and sentinel pigeons up to 14 dpi/dpe, and this is suggestive of minimal or absence of renal damage and muscle destruction as well as dehydration. The increased serum urea concentrations of inoculated and sentinel chickens might be due to dehydration resulting from anorexia and watery diarrhea caused by the vvIBDV infection (30). Since urea is excreted mainly by glomerular filtration, the rate of tubular reabsorption is dependent on hydration condition such that during dehydration all filtered urea is reabsorbed (31). This constitutes the possible reason for elevated urea concentrations in chickens in this study. However, there were no significant changes in creatinine concentrations of chickens following infection/exposure despite dehydration, and this suggests minimal muscle destruction. Another possible reason is that creatinine excretion occurs mainly by tubular secretion, which is not dependent on rate of urine flow (32) and so was not affected by dehydration.
Conclusion
The leukocytosis, heterophilia, hyperproteinemia, and hyperglobulinemia demonstrated in pigeons post-vvIBDV infection/exposure in this study suggest that they are susceptible to IBD. To the authors' knowledge, this is the first report on hematological and serum biochemical alterations in pigeons due to vvIBDV infection.
Data Availability Statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.
Ethics Statement
The animal study was reviewed and approved by Ahmadu Bello University Committee on Animal Use and Care.
Author Contributions
SO, PA, and KE conceptualized and design the experiment, edited, and review the manuscript. OO, TM, SE, AA, MM, and SU performed the experiment and analyzed the data. OO and TM drafted the manuscript. All authors have read and agreed to the published version of the manuscript.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors are grateful to the Clinical Pathology Laboratory, Department of Veterinary Pathology and Mr. David Leo, Department of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria.
ReferencesFatimaTBenaissaADrissB. Comparison of cell culture, integrated cell culture-PCR, and direct PCR methods for detection of infectious bursal disease virus. Int J Med Health Sci. (2015) 4:12–9.WahomeMWNjagiLWNyagaPNMbuthiaPGBeboraLCBwanaMO. Occurrence of antibodies to infectious bursal disease virus in non-vaccinated indigenous chickens, ducks and turkeys in Kenya. Int J Vet Sci. (2017) 6:159–62.OrakpoghenorOOladeleSBAbduPA. Infectious bursal disease: transmission, pathogenesis, pathology and prevention - An overview. Worlds Poult Sci J. (2020) 76:292–303. 10.1080/00439339.2020.1716652LukertPDSaifYM. Infectious Bursal Disease. Diseases of Poultry. 11th ed.SaifYMBarnesHJGlissonJRMcDougaldLRSwayneDE, editors. Ames, IA: Iowa State University Press (2003). p. 161–79. 18484000MagwodSAArafaASHusseinHA. Molecular genotyping of the infectious bursal disease virus (IBDV) isolated from broiler flocks in Egypt. Int J Vet Sci Med. (2014) 2:46–52. 10.1016/j.ijvsm.2014.02.004EterradossiNSaifYM. Infectious Bursal Disease. Diseases of Poultry. 12th ed.SaifYMFadlyAMGlissonJRMcDougaldLRNolanLKSwayneDE, editors. Ames, IA: Iowa State University Press (2008). p. 211–35. 18484000MotohikoOTakashiWTsuyoshiY. Seroprevalence of infectious bursal disease in free-living wild birds in Japan. J Vet Med Sci. (1998) 60:1277–9. 10.1292/jvms.60.12779853315SwaiESKessyMJSankaPNMtuiPF. A serological survey of village chickens in Northern Tanzania. J South Afr Vet Assoc. (2011) 82:32–5. 10.4102/jsava.v82i1.3021826835WangYSWangZCTangYDShiZLHeKWLiY. Comparison of four infectious bursal disease viruses isolated from different bird species. Arch Virol. (2007) 152:1787–97. 10.1007/s00705-007-1022-117619114Animal Health Australia (AHA). Disease Strategy: Infectious Bursal Disease Caused by Very Virulent IBD Virus or Exotic Antigenic VARIANT strains of IBD Virus (version 3.0). Canberra, ACT: Australian Veterinary Emergency Plan (AUSVETPLAN), Edition 3, Primary Industries Ministerial Council (2009). Available online at: http://www.animalhealthaustralia.com.au/fms/Animal%20Health%20Australia/AUSVETPLAN/IBD22PROOFGilchristP. Involvement of free-flying wild birds in the spread of the viruses of avian influenza, newcastle disease and infectious bursal disease from poultry products to commercial poultry. Worlds Poult Sci J. (2005) 61:198–210. 10.1079/WPS200451OladeleOAAdeneDFObiTUNottidgeHOAiyedunAI. Sequential hematological study of experimental infectious bursal disease virus infection in chickens, turkeys and ducks. Rev Med Vet Tropicaux. (2005) 58:211–5. 10.19182/remvt.9914BelloIIOladeleOABakreAAAkpanI. Haematology of infectious bursal disease virus infected chickens on garlic supplemented diet. Trop Vet. (2015) 33:72–9.RehmanHAbbasSLohahetN. Laboratory Manual of Physiology. Vol. 1. Lahore: Society of Veterinary Physiology (2003). p. 57–64.CampbellTWEllisCK. Haematology of birds. In: CampbellTWEllisCK, editors. Avian and Exotic Animal Haematology and Cytology.3rd ed.Ames, IA: Blackwell Publishing Professional (2007). p. 3–50.ColesEH. Veterinary Clinical Pathology. 4th ed.Philadelphia, PA: Harcourt Brace Jovanovidi; W.B. Sunders Company (1986). p. 17–39. 10.1136/jcp.39.12.1365WeissDJWardropKJ. Schalm's Veterinary Haematology. 6th ed. Iowa, IA: Wiley-Blackwell (2010). p. 67.ChinemeCNChoY. Clinicopathological and morphological changes in chickens experimentally infected with infectious bursal (Gumboro) disease virus. Trop Vet. (1984) 2:218–24.CampbellTWColesEH. Avian Clinical Pathology. Veterinary Clinical Pathology. ColesEH, editor. Philadelphia, PA: W.B. Saunders Company (1986). p. 289–90.CotterPF. An examination of the utility of heterophil/lymphocyte ratios in assessing stress of caged hens. Poult Sci. (2015) 94:512–7. 10.3382/ps/peu00925480738WeissEKaufer-WeissI. Pathology and pathogenesis of infectious bursal disease. In: Proceedings of International Symposium on Infectious Bursal Disease and Chicken Infectious Anaemia. Rauischholzhausen (1994).GrossWBSiegelHS. Evaluation of the heterophil/lymphocyte ratio as a measure of stress in chickens. Avian Dis. (1983) 27:972–9. 10.2307/15901986360120SiegelHS. Stress, strains and resistance. Br Poult Sci. (1995) 36:3–22. 10.1080/00071669508417748DavisAKManeyDLMaerzJC. The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct Ecol. (2008) 22:760–72. 10.1111/j.1365-2435.2008.01467.xHorowitzMBorutA. Blood volume regulation in dehydrated rodents: plasma protein turnover and sedimentation coefficients. Comp Biochem Physiol Part A Physiol. (1975) 51:827–31. 10.1016/0300-9629(75)90062-6237706LumeijJT. The diagnostic value of plasma proteins and non-protein nitrogen substances in birds. Vet Q. (1987) 9:262–8. 10.1080/01652176.1987.96941113314105HarrKE. Diagnostic Value of Biochemistry. Clinical Avian Medicine. HarrisonGJLightfootTL, editors. Ithaca, NY: International Veterinary Information Service (2009).IgweAOEzeDC. Leucogram, serum protein parameters and histopathology of broiler chickens (Gallus domesticus) treated with cyclophosphamide and infected with velogenic newcastle disease virus. Sokoto J Vet Sci. (2018) 16:63–74. 10.4314/sokjvs.v16i2.9WimsattJCanonNPearceRDVapLMGetzyDM. Assessment of novel avian renal disease markers for the detection of experimental nephrotoxicosis in pigeons (Columba livia). J Zoo Wildlife Med. (2009) 40:487–94. 10.1638/2008-0169.119746864LumeijJT. Plasma urea, creatinine and uric acid concentrations in response to dehydration in racing pigeons (Columba livia domestica). Avian Pathol. (1987) 16:377–82. 10.1080/0307945870843638818766627SkadhaugeE. Osmoregulation in Birds. Berlin: Springer Verlag (1981). p. 84–91. 10.1007/978-3-642-81585-0SturkiePD. Kidneys, Extrarenal Salt Excretion and Urine. Avian Physiology. Sturkie PD, editor. New York, NY: Springer Verlag (1976). p. 263–85. 10.1007/978-3-642-96274-5_14