Immune response and biochemistry of calves immunized with rMSP1<i>a</i> ( <i>Anaplasma marginale</i>) using carbon nanotubes as carrier molecules

Silvestre, Bruna Torres; Silveira, Júlia Angélica Gonçalves da; Facury-Filho, Elias Jorge; Carvalho, Antônio Último de; Versiani, Alice Freitas; Estevam, Letícia Gracielle Tôrres de Miranda; Araújo, Márcio Sobreira Silva; Martins-Filho, Olindo Assis; Negrão-Corrêa, Deborah Aparecida; Ribeiro, Múcio Flávio Barbosa

doi:10.1590/S1984-296120180029

Bruna Torres Silvestre

Departamento de Parasitologia, Instituto de Ciências Biológicas – ICB, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil

Júlia Angélica Gonçalves da Silveira ^** Corresponding author: Júlia Angélica Gonçalves da Silveira. Departamento de Parasitologia, Instituto de Ciências Biológicas – ICB, Universidade Federal de Minas Gerais – UFMG, Av. Presidente Antônio Carlos, 6627, Pampulha, CEP 31270-901, Belo Horizonte, MG, Brasil. e-mail: juliaags@yahoo.com.br

Departamento de Parasitologia, Instituto de Ciências Biológicas – ICB, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil

Elias Jorge Facury-Filho

Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil

Antônio Último de Carvalho

Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil

Alice Freitas Versiani

Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil

Letícia Gracielle Tôrres de Miranda Estevam

Departamento de Parasitologia, Instituto de Ciências Biológicas – ICB, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil

Márcio Sobreira Silva Araújo

Laboratório de Biomarcadores de Diagnóstico e Monitoração, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz – FIOCRUZ, Belo Horizonte, MG, Brasil

Olindo Assis Martins-Filho

Laboratório de Biomarcadores de Diagnóstico e Monitoração, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz – FIOCRUZ, Belo Horizonte, MG, Brasil

Deborah Aparecida Negrão-Corrêa

Departamento de Parasitologia, Instituto de Ciências Biológicas – ICB, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil

Múcio Flávio Barbosa Ribeiro

Departamento de Parasitologia, Instituto de Ciências Biológicas – ICB, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, MG, Brasil

* Corresponding author: Júlia Angélica Gonçalves da Silveira. Departamento de Parasitologia, Instituto de Ciências Biológicas – ICB, Universidade Federal de Minas Gerais – UFMG, Av. Presidente Antônio Carlos, 6627, Pampulha, CEP 31270-901, Belo Horizonte, MG, Brasil. e-mail: juliaags@yahoo.com.br

Figures (4)
Tables (2)

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Figure 1
Median values of leukocyte counts at days 0 (pre-immunization), 21, 42 and 63. (*) Represents statistically significant differences within each group compared to day 0 (p<0.05); (**) Statistically significant difference within group G2 compared to day 21, according to the Friedman test (p<0.05). Letters represent statistically significant differences among groups according to the Kruskal-Wallis test (p<0.05): (a) = represent statistically significant difference in relation to the control group - G1; (b) = statistically significant difference in relation to the G2 group - AmUFMG2; (c) = in relation to the G3 group - MWNT+rMSP1 a; (d) = in relation to the G4 group - AmUFMG2 associated with MWNT+rMSP1 a (n=6).

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Figure 2
(A) Lymphocytes were identified based on their size and granularity using flow cytometry. Analysis of CD335⁺ (NK cells) was performed within the selected lymphocyte region based on the relative fluorescence intensity observed in FL1 versus FL2 dot plot distributions (arrow); (B) Absolute number of CD335⁺ in whole-blood of calves at days 0 (pre-immunization), 42 and 63. (*) Represents statistically significant differences within each group compared to day 0, according to the Friedman test (p<0.05). Letters represent statistically significant differences among groups according to the Kruskal-Wallis test (p<0.05): (a) = represent statistically significant difference in relation to the control group - G1; (b) = statistically significant difference in relation to the G2 group - AmUFMG2; (c) = in relation to the G3 group - MWNT+rMSP1a ; (d) = in relation to the G4 group - AmUFMG2 associated with MWNT+rMSP1a (n=6).

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Figure 3
Analysis of the lymphocytes in whole-blood of calves at days 0 (pre-immunization), 42 and 63. (A) Kinetics of the absolute number of CD4⁺ T lymphocytes; (B) CD8 ⁺ T lymphocytes; (C) CD21⁺ lymphocytes (B cells); and (D) CD25 ⁺ T lymphocytes (activated T lymphocytes). (*) Represent statistically significant differences within each group compared to day 0 (p<0.05); (**) Statistically significant difference within group G3 compared to day 42, according to the Friedman test (p<0.05). Letters represent statistically significant differences among groups according to the Kruskal-Wallis test (p<0.05): (a) = represent statistically significant difference in relation to the control group - G1; (b) = statistically significant difference in relation to the G2 group - AmUFMG2; (c) = in relation to the G3 group - MWNT+rMSP1 a; (d) = in relation to the G4 group - AmUFMG2 associated with MWNT+rMSP1 a (n=6).

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Figure 4
Induction of the humoral response after immunizations. Measurements of antibodies (A) IgG anti-rMSP1a; (B) IgG1 anti-rMSP1a; (C) IgG2 anti-rMSP1a; (D) IgG anti-A. marginale ; (E) IgG1 anti-A. marginale; and (F) IgG2 anti-A. marginale by ELISA. Each bar represents the median value of each group (n=6). (*) Represents statistically significant differences within each group compared to day 0 (pre-immunization), according to the Friedman test (p<0.05). Letters represent statistically significant differences among groups according to the Kruskal-Wallis test (p<0.05): (a) = represent statistically significant difference in relation to the control group - G1; (b) = statistically significant difference in relation to the G2 group - AmUFMG2; (c) = in relation to the G3 group - MWNT+rMSP1a; (d) = in relation to the G4 group - AmUFMG2 associated with MWNT+rMSP1a (n=6).

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Table 1
Immunization protocol of the calves.

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Table 2
Biochemical profile of the G1 (non-carboxylated MWNTs / Control group), G2 (AmUFMG2), G3 (MWNT+rMSP1a) and G4 (AmUFMG2 associated with MWNT+rMSP1 a) groups.

Group	Immunogen
G1	PBS + non-carboxylated MWNTs (66 µg/calf) - Control group
G2	AmUFMG2 (400 µg/calf)
G3	MWNT+rMSP1a (200 µg/calf)
G4	AmUFMG2 (400 µg/calf) and MWNT+rMSP1a (200 µg/calf)

PARAMETERS	Reference Values Bovine ^* * Reference values bovine according to Kaneko et al. (2008) ; GGT values according to Bouda et al. (1980) .	GROUP 1 (Control)				GROUP 2 (AmUFMG2)				GROUP 3 (MWNT+rMSP1a)				GROUP 4 (AmUFMG2 and MWNT+rMSP1a)
PARAMETERS		Day 0	Day 21	Day 42	Day 63	Day 0	Day 21	Day 42	Day 63	Day 0	Day 21	Day 42	Day 63	Day 0	Day 21	Day 42	Day 63
Glucose mg/dL	40-100	57.14	65.98	82.34	103.30	61.23	56.61	83.79	98.96	55.29	64.13	84.72	95.39	49.09	60.04	77.46	102.82
Creatinine mg/dL	0.5-2.2	1.06	1.42	1.32	1.72	0.96	1.44	1.22	1.74	1.06	1.37	1.18	1.49	1.29	1.49	1.23	1.62
Albumin g/dL	2.5-3.8	2.74	2.78	2.48	4.09	2.57	2.56	2.41	4.05	2.61	2.66	2.47	3.84	2.64	2.66	2.42	3.96
ALP U/L	0-488	164.80	177.33	157.87	278.45	240.67	253.97	186.74	311.67	170.15	158.44	142.78	189.68	175.76	180.12	178.24	289.33
Total Protein g/dL	6.7-7.5	7.46	7.31	6.83	9.22	7.40	7.30	6.86	9.04	7.64	7.79	7.12	9.46	7.23	7.39	6.86	9.44
GGT U/L	6-32.4	16.25	24.24	18.43	26.52	12.04	18.92	10.37	23.94	13.04	20.25	12.42	23.11	14.40	16.28	16.37	21.60
ALT U/L	11-40	18.62	19.74	24.80	32.56	23.20	20.86	25.30	40.20	22.03	19.74	26.71	35.48	20.06	22.27	27.65	40.50
AST U/L	60-125	57.71	48.67	77.33	82.51	68.60	52.34	68.52	95.33	62.86	52.06	73.88	92.58	63.60	57.51	68.89	94.71

[1] Values represent the median of each group. Statistical analysis among groups did not show significant change between calves immunized with MWNT (G1, G3 and G4) and calves from G2 group (MWNT non-immunized) (Kruskal-Wallis test, p>0.05).

[TFN1] ^*
Reference values bovine according to Kaneko et al. (2008) ; GGT values according to Bouda et al. (1980) .

Immune response and biochemistry of calves immunized with rMSP1a ( Anaplasma marginale) using carbon nanotubes as carrier molecules

Resposta imunológica e bioquímica de bezerros imunizados com rMSP1 a (Anaplasma marginale) utilizando nanotubos de carbono como moléculas carreadoras

Abstract