Sheep polyclonal antibody to map <i>Haemonchus contortus</i> mimotopes using phage display library

Buzatti, Andréia; Fernandez, Arnielis Diaz; Arenal, Amilcar; Pereira, Erlán; Monteiro, Alda Lucia Gomes; Molento, Marcelo Beltrão

doi:10.1590/S1984-296120180023

Andréia Buzatti ^** Corresponding author: Andréia Buzatti. Laboratório de Doenças Parasitárias, Departamento de Medicina Veterinária, Universidade Federal do Paraná – UFPR, Rua dos Funcionários, 1540, CEP 80035-050, Curitiba, PR, Brasil. e-mail: deiabuzatti@gmail.com

Laboratório de Doenças Parasitárias, Departamento de Medicina Veterinária, Universidade Federal do Paraná – UFPR, Curitiba, PR, Brasil

Arnielis Diaz Fernandez

Facultad de Ciencias Agropecuarias, Universidad de Camagüey Ignacio Agramonte Loynaz, Camagüey, Cuba

Amilcar Arenal

Facultad de Ciencias Agropecuarias, Universidad de Camagüey Ignacio Agramonte Loynaz, Camagüey, Cuba

Erlán Pereira

Facultad de Ciencias Agropecuarias, Universidad de Camagüey Ignacio Agramonte Loynaz, Camagüey, Cuba

Alda Lucia Gomes Monteiro

Laboratório de Produção e Pesquisa em Ovinos e Caprinos, Departamento de Zootecnia, Universidade Federal do Paraná – UFPR, Curitiba, PR, Brasil

Marcelo Beltrão Molento

Laboratório de Doenças Parasitárias, Departamento de Medicina Veterinária, Universidade Federal do Paraná – UFPR, Curitiba, PR, Brasil

* Corresponding author: Andréia Buzatti. Laboratório de Doenças Parasitárias, Departamento de Medicina Veterinária, Universidade Federal do Paraná – UFPR, Rua dos Funcionários, 1540, CEP 80035-050, Curitiba, PR, Brasil. e-mail: deiabuzatti@gmail.com

Figures (3)
Tables (1)

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Figure 1
Phage display technology was employed to select mimotopes of Haemonchus contortus peptides. Analyses of the phage cloned sequences were performed with MEGA software Version 6.06. Consensus sequence (*) was observed among amino acids of some clones (a, b) and also to two H. contortus proteins, Glyceraldehyde-3-phosphate dehydrogenase (c) and a Disorganized muscle family member – Dim 1 (d).

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Figure 2
Mean levels of plasma IgG of 25 adult sheep naturally infected with Haemonchus contortus. The serum samples were analyzed against two chemically-synthesized peptides: (a) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (b) Disorganized muscle family member (Dim 1). These synthetic peptides have the same sequences (7 amino acids) displayed on two clones (6 and 17, respectively) selected by biopanning.

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Figure 3
Mean levels of plasma IgG of sheep against M13 phage (a), Clone 6 (b), Clone17 (c), Chemically-synthesized GAPDH mimotope (d) and Chemically-synthesized Dim 1 mimotope (e). Twelve 8-10 month-old Suffolk sheep were allocated into 3 groups of 4 animals as follows: Control: infected but not immunized; GAPDH: immunized with clone 6; and Dim 1: immunized with clone 17. Four immunizations were performed (0, 7, 14 and 21 days) with 5x10¹¹ phage particles displaying mimotopes of H. contortus peptides - Clone 6 displayed a mimotope of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the clone 17 a mimotope of a Disorganized muscle family member (Dim 1).

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Table 1
Hemoglobin, total leukocyte number and percentage of lymphocyte, neutrophil and eosinophil cells in blood are shown as means ± SD. Twelve 8-10 month-old Suffolk sheep were alocated into 3 groups of 4 animals as follows: Control: infected but not immunized; GAPDH: immunized with clone 6; and Dim 1: immunized with clone 17. Four immunizations were performed (0, 7, 14 and 21 days) with 5x10¹¹ phage particles displaying mimotopes of H. contortus peptides - On day 28 all groups were orally challenged with 2500 infective H. contortus infective larvae. All groups demonstrated similar results (P>0.05) by the Kruskall-Wallis test.

Day of experiment	GAPDH	Dim 1	Control
Hemoglobin (g/dl)
Day 0	10.64 ± 2.90	11.90 ± 2.92	11.03 ± 4.05
Day 14	14.50 ± 2.23	11.66 ± 3.82	13.37 ± 3.03
Day 28	11.75 ± 2.42	13.52 ± 3.97	13.20 ± 2.02
Day 44	12.56 ± 2.68	14.73 ± 1.68	14.28 ± 3.05
Day 50	13.89 ± 1.96	14.41 ± 2.06	13.10 ± 0.81
Total leukocytes (x 10³/µL)
Day 0	11.85 ± 2.06	12.00 ± 1.48	12.12 ± 1.42
Day 14	12.20 ± 2.14	10.97 ± 0.28	12.12 ± 1.38
Day 28	11.07 ± 1.96	10.70 ± 2.09	12.50 ± 1.58
Day 44	11.37 ± 1.15	12.47 ± 0.75	12.27 ± 1.61
Day 50	12.05 ± 2.04	10.92 ± 1.90	12.57 ± 1.19
Lymphocytes (%)
Day 0	58.75 ± 3.86	59.50 ± 4.72	57.50 ± 2.38
Day 14	68.00 ± 8.16	64.50 ± 6.35	66.00 ± 6.22
Day 28	61.00 ± 4.08	65.00 ± 0.82	61.50 ± 1.91
Day 44	59.25 ± 3.30	53.75 ± 0.50	56.75 ± 6.95
Day 50	64.25 ± 3.77	62.00 ± 6.22	60.50 ± 2.65
Neutrophil (%)
Day 0	36.75 ± 3.30	36.75 ± 3.77	39.00 ± 2.44
Day 14	26.75 ± 7.80	31.00 ± 5.35	30.00 ± 6.48
Day 28	34.25 ± 3.59	31.00 ± 1.56	32.75 ± 1.72
Day 44	36.00 ± 3.37	41.25 ± 1.73	37.00 ± 5.71
Day 50	30.75 ± 2.75	32.75 ± 5.44	34.50 ± 2.38
Eosinophil (%)
Day 0	2.25 ± 0.50	1.25 ± 0.50	1.50 ± 0.58
Day 14	2.25 ± 0.50	2.25 ± 0.50	1.75 ± 0.96
Day 28	1.75 ± 0.50	1.75 ± 0.96	2.75 ± 1.26
Day 44	1.75 ± 0.50	1.50 ± 0.58	2.25 ± 0.50
Day 50	2.50 ± 0.58	1.75 ± 0.96	1.75 ± 0.50
Monocytes (%)
Day 0	1.50 ± 1.00	1.75 ± 0.50	1.50 ± 0.58
Day 14	1.50 ± 0.58	1.25 ± 0.50	1.75 ± 0.50
Day 28	2.00 ± 0.82	1.50 ± 0.58	2.00 ± 0.82
Day 44	2.00 ± 0.82	1.75 ± 0.50	1.75 ± 0.96
Day 50	1.75 ± 0.50	1.50 ± 0.58	1.25 ± 0.50
Basophils (%)
Day 0	0.25 ± 0.50	0.50 ± 0.58	0.50 ± 0.58
Day 14	1.25 ± 0.96	0.75 ± 0.96	0.50 ± 0.58
Day 28	0.75 ± 0.50	0.50 ± 0.58	0.00 ± 0.00
Day 44	0.50 ± 0.58	0.75 ± 0.96	1.50 ± 0.58
Day 50	0.25 ± 0.50	1.50 ± 0.58	1.25 ± 0.96

[1] * Corresponding author: Andréia Buzatti. Laboratório de Doenças Parasitárias, Departamento de Medicina Veterinária, Universidade Federal do Paraná – UFPR, Rua dos Funcionários, 1540, CEP 80035-050, Curitiba, PR, Brasil. e-mail: deiabuzatti@gmail.com

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Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library

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Abstract