On-line version ISSN 2224-9435
J. S. Afr. Vet. Assoc. vol.85 n.1 Cape Town Jan. 2014
Elizabeth M.d.S. SchmidtI; Ivan F.C. SantosII; Antonio C. PaulilloIII; Gislaine R.V. MartinsIII; Janine DenadaiIII; Ivan M. LapelaIII
IDepartment of Veterinary Clinic, São Paulo State University, Brazil
IIDepartment of Veterinary Surgery, Eduardo Mondlane University, Mozambique
IIIDepartment of Veterinary Pathology, Säo Paulo State University, Brazil
The effects of vaccination on avian blood parameters are poorly understood. The present study was designed to evaluate whether different strains (Ulster 2C, B1, live LaSota and inactivated LaSota) of Newcastle disease vaccines had an effect on the haematological profile of female turkeys. Seventy-five female turkeys were allocated to treatment groups according to vaccination strain. All the birds, except those in the control group, were vaccinated at 32 weeks of age and revaccinated at 40 and 48 weeks of age. Blood samples were obtained for haematological analyses and serum samples for the haemagglutination inhibition test. Haemoglobin concentration was significantly lower (p < 0.05) in vaccinated female turkeys than in the control birds 28 days after vaccination. Monocytes were significantly higher (p < 0.05) in 44-week-old female turkeys vaccinated with inactivated LaSota strain compared with the other groups. Turkeys vaccinated with the B1 strain showed significantly higher (p < 0.05) total white blood cell counts compared with the other groups vaccinated with various commercial strains of the Newcastle disease virus. In conclusion, female turkeys showed significant differences in haemoglobin concentrations, monocytes and white blood cell counts when vaccinated against Newcastle disease.
The bronze turkey (Meleagris gallopavo, Linnaeus, 1758) is the result of a cross between domestic turkeys brought from Europe and the wild turkey. They are selected to produce highly nutritive meat and feathers and for breeding flocks in United States, France, Italy, Chile, Brazil, Germany, United Kingdom, Portugal and Mexico (Brant 1998; Windhorst 2006).
Haematology is becoming a valuable routine diagnostic tool in the evaluation of avian patients and may also provide information that identifies diseases that affect the cells in the peripheral blood of birds (Campbell 2004). However, avian clinical pathology is poorly understood because there is a lack of information about reference values and haemogram responses after antigen exposure and immune stimulation. This study was conducted to determine whether different Newcastle disease (ND) vaccine strains (Ulster 2C, B1, live LaSota and inactivated LaSota) had an effect on the haematological profile of female turkeys vaccinated with various commercial strains of ND, as the effects of vaccination on avian haematology are poorly understood.
Material and methods
Seventy-five female bronze turkeys (M. gallopavo, Linnaeus, 1758) were used in the study. They were kept in a floor-pen house and received water and food ad libitum. The feed was formulated with corn, soybean, lysine, methionine, methionine + cystine and threonine according to specific National Research Council (NRC) recommendations for turkeys (NRC 1994). The hens were laying eggs for 20 weeks, from 32 to 52 weeks of age.
The 75 female turkeys were assigned in a completely randomised experimental design into five different treatment groups of 15 birds each. They were designated according to vaccination strain as G1 (Ulster 2C), G2 (B1), G3 (live LaSota), G4 (inactivated LaSota) and G5 (control group - not vaccinated). Commercial line ND vaccines (Ulster 2C, B1 and LaSota strains) were used (Paulillo et al. 1996). All the birds except those in the control group were vaccinated at 32 weeks of age and revaccinated at 40 and 48 weeks of age with the same vaccine strain that was applied in the first vaccination. Birds in G1 to G3 were vaccinated by eye drop using the Ulster 2C, B1 and live LaSota strains respectively and birds in G4 were vaccinated subcutaneously with inactivated LaSota strain.
Blood samples were collected from the superficial ulnar vein into a 2 mL glass tube containing ethylenediaminetetraacetic acid (EDTA) at 32, 36, 38, 44, 48 and 52 weeks for haematological analyses and serum samples were obtained and submitted for the inhibition of haemagglutination (HI) test (ed. Cunningham 1971).
Thin blood smears were made immediately after blood collection to avoid any interference with cell structure and the haematological analyses were performed. Total red blood cell (RBC) and white blood cell (WBC) counts were performed by a manual method using a haemocytometer with blood diluted with 0.01% of toluidine blue (Zinkl 1986).
The haemoglobin concentration was measured by the cyanmethaemoglobin method. The mixture of cyanmethaemoglobin reagent and blood was centrifuged prior to analysis to remove the free nuclei from lysed erythrocytes to avoid an overestimation of the haemoglobin. The haematocrit (HT) was determined by the microhaematocrit method in capillary tubes and centrifuged at 12 000 g for 5 min. Differential WBC counts, using an average of 200 cells, were made from blood films stained with Wright's stain (ed. Jain 1986).
The data was analysed by analysis of variance (ANOVA) and those with statistical differences were submitted to Tukey's test at 0.05% using Statview® (version 5.0).
The study was conducted according to the Ethical Principles of Animal Experimentation adopted by the Brazilian College of Animal Experimentation (COBEA) and was approved by the Ethics Committee of Animal Welfare (CEBEA) with protocol number 005624-08.
There were no physical or psychological risks involved in the experimental trials.
The birds were observed daily for mortality; none was recorded. Egg production and weekly weights were not recorded because they were not the focus of the study.
Mean antibody titres against ND from female turkeys are presented in Table 1. As the control group (G5) was not vaccinated, its antibodies titres were nil from 32 to 52 weeks of age.
The RBC, HT, haemoglobin (HB), WBC, heterophils (Het.), lymphocytes (Lymp.), eosinophils (Eosin.), monocytes (Mon.) and basophils (Bas.) results are presented in Tables 2-7. There were no significant differences for RBC and HT amongst the different groups of female turkeys vaccinated against ND or not (Tables 2-7). At 36 weeks of age, the haemoglobin concentration was significantly lower (p < 0.05) in female turkeys vaccinated with Ulster 2C, B1, live LaSota and inactivated LaSota strains compared with the control birds (G5) 28 days after vaccination (Table 3). At this age, these turkeys showed high mean antibody titres (Table 1).
No significant differences were found in this study for heterophils, lymphocytes, eosinophils and basophils of female turkeys vaccinated with various commercial strains of ND. Monocytes were significantly higher (p < 0.05) in 44-week-old female turkeys vaccinated with inactivated LaSota strain in relation to other groups, showing the highest antibody mean levels.
At 52 weeks of age, the WBC count was significantly higher (p < 0.05) for B1-vaccinated female turkeys compared with the other groups vaccinated with various commercial strains of ND, although no significant differences were observed for lymphocytes.
The results from Tables 2 to 7 suggest that vaccinated birds may have developed anaemia. According to Dein (1986), viral infections in birds (chicken anaemia virus) have resulted in non-regenerative anaemia. In these cases, the bone marrow was hypoplastic, suggesting a severe inhibition of haematopoiesis. Campbell (2004) and Morrisey (2000) also observed anaemia caused by decreased production of RBC in inflammatory diseases of infectious aetiology. Also, 38-week-old and 44-week-old female turkeys vaccinated against ND with Ulster 2C strain showed significant decreases (p < 0.05) in haemoglobin concentrations, suggesting a prolonged anaemia in turkeys, probably caused by the vaccine antigens. On the other hand, the stress of vaccination can decrease food consumption and therefore cause anaemia (ed. Jain 1986).
Juvenile ring-necked pheasants also showed decreased haemoglobin concentrations when vaccinated against ND with Ulster 2C strain (Schmidt et al. 2008).
Lymphocytes were the major circulating leukocyte in these female turkeys vaccinated with various commercial strains of ND, irrespective of the age at which they were evaluated. Haematological studies in chickens, bronze and wild turkeys showed similar results, although heterophils are the most abundant leukocyte in peripheral blood of most species of birds (Bounous & Stedman 2000; Bounous et al. 2000; Dein 1986; Latimer & Bienzle 2000; Maxwell & Robertson 1998; Schmidt et al. 2009). However, the predominance of lymphocytes was also found by Schmidt et al. (2007) and Schmidt et al. (2008) in ring-necked pheasants vaccinated with various commercial strains of ND.
The monocytes were significantly higher (p < 0.05) in female turkeys aged 44 weeks vaccinated with inactivated LaSota strain - this group of turkeys also had the highest antibody mean levels in relation to other groups. It seems reasonable to suggest that vaccination with inactivated LaSota strain elevated the number of monocytes, demonstrating the participation of these cells in the immune response, as they are one of the major effector cells of non-specific immunity in birds (Klansing 1991).
It is possible to speculate that the leukocytosis may be present during one phase of the immune response (Dein 1986) because no significant differences were observed for lymphocytes, but the WBC count was significantly higher (p < 0.05) at 52 weeks of age for B1-vaccinated female turkeys when compared with the other groups vaccinated with various commercial strains of ND.
Female bronze turkeys showed significant differences in haemoglobin concentrations, monocytes and WBC when vaccinated against ND, especially with Ulster 2C, inactivated LaSota and B1 strains.
The authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article.
E.M.d.S.S. (São Paulo State University) was responsible for the experimental and project design and was the project leader. A.C.P. (Säo Paulo State University) was also responsible for the project design. I.F.C.S. (Eduardo Mondlane University), G.R.V.M (Säo Paulo State University), J.D. (Säo Paulo State University) and I.M.L. (Säo Paulo State University) made conceptual contributions.
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Botucatu, São Paulo
Received: 20 Feb. 2013
Accepted: 07 Mar. 2014
Published: 25 Aug. 2014