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Haematological and immunophenotypic evaluation of peripheral blood cells of cattle naturally infected with bovine papillomavirus
The Veterinary Journal 244 (2019) 112–115
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short communication
Haematological and immunophenotypic evaluation of peripheral blood cells of cattle naturally infected with bovine papillomavirus Paula B. Bassi1, Fernanda F. Araujo1,2 , Guilherme C. Garcia1, Matheus F. Costa e Silva1,2 , Eustaquio R. Bittar1 , Candice M. Bertonha1, Olindo A. Martins-Filho2 , Marcio Sobreira Silva Araujo1,2,* , Joely F. Bittar1 1 Universidade de Uberaba (UNIUBE), Medicina Veterinária, Mestrado em Sanidade e Produção Animal nos Trópicos - Avenida Nenê Sabino 1697/1698, 38055-500, Uberaba-MG, Brazil. 2 Grupo Integrado de Pesquisa em Biomarcadores, Instituto René Rachou - Fundação Oswaldo Cruz. Avenida Augusto de Lima no 1715, 30190-002, Barro Preto, Belo Horizonte-MG, Brazil.
A R T I C L E I N F O
Keywords: Cattle Cytokines Immune response Papillomavirus
A B S T R A C T
Papillomaviruses are among the most widespread animal viruses, with many hosts harbouring multiple virus types. The present study aimed to evaluate the haematological and immunophenotypic profile of cattle infected with bovine papillomavirus (BPV). Blood samples were collected from 10 animals with clinical cutaneous BPV and without clinical papillomatosis (control). Haematological analysis demonstrated a significant reduction in haemoglobin and haematocrit for BPV-infected animals. The results also showed an increase of natural killer cells and a decrease of gd+ T-cells and the CD4+/CD8+ ratio for the BPV group when compared to the control group. The infection was also found to stimulate a pro-inflammatory profile with the participation of CD8+T cells producing elevated IFN-g and IL-17. These findings, although preliminary, provide a better understanding of the immune response of cattle infected with BPV. © 2018 Elsevier Ltd. All rights reserved.
Papillomaviruses are oncogenic viruses that cause benign or malignant lesions, depending on environmental factors, virus oncogenicity and the location of infection. Teat papillomatosis, which affects dairy cows worldwide, is a neglected health problem of cattle that results in economic losses. There are currently 23 recognized types of bovine papillomaviruses (BPVs), two of which (BPV22 and 23) were just recently described (Daudt et al., 2018). Subtypes 1 and 2 have been more widely studied than the other types, for which there have been only a few reports on their prevalence (Rojas-Anaya et al., 2016). Papillomavirus infections are usually eliminated by a cellmediated immune responagainst viral antigens (O’Brien and Campo, 2002). Even in the absence of malignant transformation, BPV infection can persist for a significant period of time before activation of the host immune system. Roperto et al. (2011) proposed that CD4+ and CD8+ T-cells are the main circulating targets of the virus, indicating that they may represent the most important reservoir of active BPV- 2 in blood. Indeed, authors have long suggested that cellular immunity plays an important
* Correspondence: E-mail address: sobreira@cpqrr.fiocruz.br (M.S.S. Araujo). https://doi.org/10.1016/j.tvjl.2018.12.004 1090-0233/© 2018 Elsevier Ltd. All rights reserved.
role in the regression of papillomas and other papillomavirusinduced lesions; however, the precise immunological involvement necessary for the rejection of papillomas is still poorly understood. The aim of the present study was to evaluate the haematological and immunophenotypic profile of cattle infected with BPV. Ten crossbreed Girolando animals from the city of Uberaba, Brazil, were divided into two groups with the same gender and age composition: (i) BPV group — animals with clinical cutaneous bovine papillomatosis (n = 5); and (ii) control group — animals without clinical symptoms and with normal body condition scoring (BCS 3) (n = 5). The BPV group presented low body condition scoring (BCS 1-2), with autologous vaccination treatment without any lesions regression and the presence of pedunculated papillomas mainly on the neck. The study protocol was previously approved by the Ethics Committee for Animal Experimentation of Universidade de Uberaba, Brazil (CEEA/ UNIUBE, protocol number 016/2017, date of approval 22 June 2017), and follows all ethical principles for cattle experimentation procedures. Blood samples were collected for the analysis of red blood cells (RBC), which included erythrocyte count, haemoglobin concentration, haematocrit, mean corpuscular volume (MCV)
P.B. Bassi et al. / The Veterinary Journal 244 (2019) 112–115
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Table 1 Haematological parameters for BPV-infected cattle and non-infected cattle (control group). Parameter
Study groups Control (n = 5)
BPV (n = 5)
Red blood cells (1012/L) Haematocrit (L/L) Haemoglobin (g/L)
9.3 (0.99) 0.36 (0.02) 101.6 (3.28)
8.7 (0.92) 0.31 (0.02) 92.6 (7.23)
MCV (fL) MCH (fmol) MCHC (g/L)
37.8 (3.53) 0.68 (0.06) 292.6 (5.64)
35.9 (3.08) 0.66 (0.04) 298.8 (2.99)
Platelets (109/L) Fibrinogen (g/L) Proteins (g/L)
867.0 (278.4.0) 4.4 (2.19) 74.2 (3.49)
684.6 (157.58) 6.4 (1.67) 74.4 (3.58)
White blood cells (109/L) Neutrophils (109/L) Monocytes (109/L) Lymphocytes (109/L) Eosinophils (109/L) Basophils (109/L)
14.64 (3.34) 3.76 (0.65) 1.24 (0.80) 9.21 (3.45) 0.33 (0.48) 0.97 (0.92)
16.06 (2.86) 4.34 (1.78) 1.19 (0.55) 10.45 (2.83) 0.37 (0.27) 0.10 (0.94)
*
Data was tested for normality (Kolmogorov–Smirnov test with Dallal–Wilkinson–Lillie for P test) and are reported as mean standard deviation. Significant differences (P < 0.05 by Student’s T test) from the control group are underlined in bold. Statistical analyses were carried out using Prism Graphpad Software, version 5.0.3.
and mean corpuscular haemoglobin concentration (MCHC). All haematological measurements were performed with a haematology analyser (ABC VET — Horiba ABX Diagnostics) (Table 1). Immunophenotyping analyses of anti-CD4, anti-CD8, anti-CD21, anti-CD335 and anti-WC1, all labelled with FITC and ex vivo immunostained for intracellular cytokine (IL-8, IL-1α, IL-6, IL-17, IFNg, IL-4 and IL-10), were performed according to the protocol described by Dorneles et al. (2015). Distinct gating strategies were employed for data analysis as shown in Supplementary Fig. 1. The results of the haematological analysis demonstrated a significant reduction of haemoglobin and haematocrit for BPVinfected animals (Table 1). Indeed, Palanivel et al. (2017) demonstrated a reduction in the concentration of haemoglobin, packed cell volume (PCV), neutrophils and lymphocyte values, suggesting the release of endogenous corticosteroids in response to stress. The results revealed an increase of natural killer (NK) cells and a decrease of gd+ T-cells and the CD4+/CD8+ ratio for BPV-infected animals when compared with the control group (Fig. 1A). Evaluation of the immunological microenviroment through the analysis of pro-inflammatory and regulatory cytokines demonstrated that CD8+T cells from infected animals had a significantly increase in the pro-inflammatory cytokines IL-17 and IFN-g when compared to the control group (Fig. 1B). Chen et al. (2004) demonstrated the importance of CD8+IFN-g+ T-cells for reducing the growth of tumours in mice. Our results showed the involvement of CD8+ T-cells producing IFN-g, and an increase of NK cells in BPV-infected animals, suggesting that this exacerbated pro-inflammatory immune response, without an effective immunoregulatory response, may lead to an unbalanced immune response that may contribute to the persistence of the lesions observed in the BPV group. Indeed, IL-17 actively participates at sites of inflammation, largely from autoimmune diseases but also from a wide spectrum of chronic conditions and varied etiologies (O’Connor et al., 2010); IL-17 also exhibits anti- and protumorigenic effects, depending on the disease context (Zou and Restifo, 2010).
Levkutová et al. (1998) demonstrated the lymphocyte profile of cattle infected with papillomavirus, and found a lower number of CD4+ T-cells and a low CD4+/CD8+ T-cell ratio in animals with tumours compared to a group of papilloma-free cattle. These findings were corroborated by the present study, in which a decrease of the CD4+/CD8+ ratio was observed for the BPV group, compared to the control group. Additionally, the low number of gd+ T lymphocytes observed may contribute to the persistence of lesions of infected cattle, since the functions of these cells have been associated with the protection of ruminant epithelial surfaces. Categorical data for cytokine-producing lymphocyte subsets, CD4+ T-cells, CD8+ T-cells and CD21+ B-cells are displayed as radar graphs plotted with the frequency of high producers (%) from each group (control and BPV-infected animals) (Fig. 1C). It was possible to observe that the cytokine profile of control group can be seen to delineate an area of the graph, which is confined below the 75% threshold; however, gd+ T-cells and the CD4+/CD8+ ratio for the control group exhibited a slight expansion of graphed area. Moreover, the involvement of adaptive immunity with the production of the cytokines IL-17 and IFN-g by CD8+ T-cells can be seen for BPV-infected animals but not for the control group. In general, our results suggest that papillomavirus triggers adaptive immunity (mainly CD8+ T cells) with a pro-inflammatory profile (Fig. 1C). In conclusion, BPV infection causes a weakening of animals, which leads to decreased feeding, anaemia and a reduction in some haematological parameters. Moreover, BPV induces a reduction of gd+ T-cells, which play a critical role in bridging the innate and adaptive arms of the immune system. The infection also stimulates a pro-inflammatory profile with the participation of CD8+T cells producing elevated IFN-g and IL-17. Our findings provide a better understanding of the immune response in BPV-infected cattle. It is important to note that this is preliminary in nature, and the results found here should be validated in further research that includes a greater number of animals.
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P.B. Bassi et al. / The Veterinary Journal 244 (2019) 112–115
Fig. 1. Phenotypic and cytokine profile of peripheral blood lymphocyte subsets from BPV-infected animals. (A) Peripheral blood lymphocyte subsets (natural killer, gd+, total T, CD4+, CD8+ and CD21+ cells) as well the T/B and CD4/CD8 ratios, are shown in box plot format (median with minimum and maximum values) for the control group (n = 5) and BPV-infected group (n = 5). (B) Cytokines IL-8, IL-1α, IL-6, IL-17, IFN-g, IL-10 and IL-4 produced by peripheral blood lymphocyte subsets (CD4+, CD8+ T cells and CD21+ cells) are shown in box plot format (median with minimum and maximum values) from the control group (n = 5) and BPV-infected animals (n = 5). In all cases, significant differences at p < 0.05 are highlighted by a grey background and identified by arrows to indicate an increase (") or a decrease (#) relative to the control group. (C) Overall pattern of high cytokine producers triggered by BPV infection. Categorical data for cytokine-producing subsets of lymphocytes are displayed as radar graphs plotted with the frequency of high producers (75%) for the control group and the BPV-infected group. Left wing indicates the surface markers CD4, CD8, CD21, CD335, and WC1, and the ratios T/B and CD4/ CD8. The right wing indicates the cytokines profile (IL-8, IL-1α, IL-6, IL-17, IFN-g, IL-10 and IL-4) of CD4+, CD8+ and CD21+ cells.
Conflict of interest statement None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgements This work was financially supported by Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and Programa de Apoio a Pesquisa da Universidade de Uberaba, Minas Gerais (PAPE-UNIUBE). OAMF is thankful to CNPq for the PQ fellowship program. FFA thanks PNPD/CAPES fellowship program. The authors thank the program for technological
development in tools for health-FIOCRUZ for the use of Flow Cytometry Platform. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.tvjl.2018.12.004. References Chen, Y.F., Lin, C.W., Tsao, Y.P., Chen, S.L., 2004. Cytotoxic-T-lymphocyte human papillomavirus type 16 E5 peptide with CpG-oligodeoxynucleotide can eliminate tumour growth in C57BL/6 mice. J. Virol. 78, 1333–1343. Daudt, C., Da Silva, F.R., Lunardi, M., Alves, C.B.D.T., Weber, M.N., Cibulski, S.P., Alfieri, A.F., Alfieri, A.A., Canal, C.W., 2018. Papillomaviruses in ruminants: an update. Transbound. Emerg. Dis. doi:http://dx.doi.org/10.1111/tbed.12868. Dorneles, E.M., Araújo, M.S., Teixeira-Carvalho, A., Martins-Filho, O.A., Lage, A.P., 2015. Cross-reactivity of anti-human cytokine monoclonal antibodies used as a
P.B. Bassi et al. / The Veterinary Journal 244 (2019) 112–115 tool to identify novel immunological biomarkers in domestic ruminants. Genet. Mol. Res. 14, 940–951. doi:http://dx.doi.org/10.4238/2015. Levkutová, M., Revajová, V., Levkut, M., Leng, L., 1998. Subpopulations of lymphocytes in cattle naturally infected with papillomavirus. Acta Vet. Hung. 46, 13–18. O’Brien, P.M., Campo, M.S., 2002. Evasion of host immunity directed by papillomavirus-encoded proteins. Virus Res. 88, 103–117. O’Connor Jr, W., Zenewicz, L.A., Flavell, R.A., 2010. The dual nature of Th17 cells: shifting the focus to function. Nat. Immunol. 11, 471–476. Palanivel, K.M., Surendar, A.P., Kanimozhi, P., 2017. Sero biochemical profile of crossbred calves naturally infected by bovine papilloma virus. Int. J. Curr.
115
Microbiol. Appl. Sci. 6 (8), 366–371. doi:http://dx.doi.org/10.20546/ ijcmas.2017.608.049. Rojas-Anaya, E., Cantú-Covarrubias, A., Álvarez, J.F., Loza-Rubio, E., 2016. Detection and phylogenetic analysis of bovine papillomavirus in cutaneous warts in cattle in Tamaulipas, Mexico. Can. J. Vet. Res. 80, 262–268. Roperto, S., Comazzi, S., Ciusani, E., Paolini, F., Borzacchiello, G., Esposito, I., Lucà, R., Russo, V., Urraro, C., Venuti, A., Roperto, F., 2011. PBMCs are additional sites of productive infection of bovine papillomavirus type 2. J. Gen. Virol. 92, 1787– 1794. Zou, W., Restifo, N.P., 2010. Th17 cells in tumour immunity and immunotherapy. Nat. Rev. Immunol. 10, 248–256.
Contents lists available at ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Short communication
Haematological and immunophenotypic evaluation of peripheral blood cells of cattle naturally infected with bovine papillomavirus Paula B. Bassi1, Fernanda F. Araujo1,2 , Guilherme C. Garcia1, Matheus F. Costa e Silva1,2 , Eustaquio R. Bittar1 , Candice M. Bertonha1, Olindo A. Martins-Filho2 , Marcio Sobreira Silva Araujo1,2,* , Joely F. Bittar1 1 Universidade de Uberaba (UNIUBE), Medicina Veterinária, Mestrado em Sanidade e Produção Animal nos Trópicos - Avenida Nenê Sabino 1697/1698, 38055-500, Uberaba-MG, Brazil. 2 Grupo Integrado de Pesquisa em Biomarcadores, Instituto René Rachou - Fundação Oswaldo Cruz. Avenida Augusto de Lima no 1715, 30190-002, Barro Preto, Belo Horizonte-MG, Brazil.
A R T I C L E I N F O
Keywords: Cattle Cytokines Immune response Papillomavirus
A B S T R A C T
Papillomaviruses are among the most widespread animal viruses, with many hosts harbouring multiple virus types. The present study aimed to evaluate the haematological and immunophenotypic profile of cattle infected with bovine papillomavirus (BPV). Blood samples were collected from 10 animals with clinical cutaneous BPV and without clinical papillomatosis (control). Haematological analysis demonstrated a significant reduction in haemoglobin and haematocrit for BPV-infected animals. The results also showed an increase of natural killer cells and a decrease of gd+ T-cells and the CD4+/CD8+ ratio for the BPV group when compared to the control group. The infection was also found to stimulate a pro-inflammatory profile with the participation of CD8+T cells producing elevated IFN-g and IL-17. These findings, although preliminary, provide a better understanding of the immune response of cattle infected with BPV. © 2018 Elsevier Ltd. All rights reserved.
Papillomaviruses are oncogenic viruses that cause benign or malignant lesions, depending on environmental factors, virus oncogenicity and the location of infection. Teat papillomatosis, which affects dairy cows worldwide, is a neglected health problem of cattle that results in economic losses. There are currently 23 recognized types of bovine papillomaviruses (BPVs), two of which (BPV22 and 23) were just recently described (Daudt et al., 2018). Subtypes 1 and 2 have been more widely studied than the other types, for which there have been only a few reports on their prevalence (Rojas-Anaya et al., 2016). Papillomavirus infections are usually eliminated by a cellmediated immune responagainst viral antigens (O’Brien and Campo, 2002). Even in the absence of malignant transformation, BPV infection can persist for a significant period of time before activation of the host immune system. Roperto et al. (2011) proposed that CD4+ and CD8+ T-cells are the main circulating targets of the virus, indicating that they may represent the most important reservoir of active BPV- 2 in blood. Indeed, authors have long suggested that cellular immunity plays an important
* Correspondence: E-mail address: sobreira@cpqrr.fiocruz.br (M.S.S. Araujo). https://doi.org/10.1016/j.tvjl.2018.12.004 1090-0233/© 2018 Elsevier Ltd. All rights reserved.
role in the regression of papillomas and other papillomavirusinduced lesions; however, the precise immunological involvement necessary for the rejection of papillomas is still poorly understood. The aim of the present study was to evaluate the haematological and immunophenotypic profile of cattle infected with BPV. Ten crossbreed Girolando animals from the city of Uberaba, Brazil, were divided into two groups with the same gender and age composition: (i) BPV group — animals with clinical cutaneous bovine papillomatosis (n = 5); and (ii) control group — animals without clinical symptoms and with normal body condition scoring (BCS 3) (n = 5). The BPV group presented low body condition scoring (BCS 1-2), with autologous vaccination treatment without any lesions regression and the presence of pedunculated papillomas mainly on the neck. The study protocol was previously approved by the Ethics Committee for Animal Experimentation of Universidade de Uberaba, Brazil (CEEA/ UNIUBE, protocol number 016/2017, date of approval 22 June 2017), and follows all ethical principles for cattle experimentation procedures. Blood samples were collected for the analysis of red blood cells (RBC), which included erythrocyte count, haemoglobin concentration, haematocrit, mean corpuscular volume (MCV)
P.B. Bassi et al. / The Veterinary Journal 244 (2019) 112–115
113
Table 1 Haematological parameters for BPV-infected cattle and non-infected cattle (control group). Parameter
Study groups Control (n = 5)
BPV (n = 5)
Red blood cells (1012/L) Haematocrit (L/L) Haemoglobin (g/L)
9.3 (0.99) 0.36 (0.02) 101.6 (3.28)
8.7 (0.92) 0.31 (0.02) 92.6 (7.23)
MCV (fL) MCH (fmol) MCHC (g/L)
37.8 (3.53) 0.68 (0.06) 292.6 (5.64)
35.9 (3.08) 0.66 (0.04) 298.8 (2.99)
Platelets (109/L) Fibrinogen (g/L) Proteins (g/L)
867.0 (278.4.0) 4.4 (2.19) 74.2 (3.49)
684.6 (157.58) 6.4 (1.67) 74.4 (3.58)
White blood cells (109/L) Neutrophils (109/L) Monocytes (109/L) Lymphocytes (109/L) Eosinophils (109/L) Basophils (109/L)
14.64 (3.34) 3.76 (0.65) 1.24 (0.80) 9.21 (3.45) 0.33 (0.48) 0.97 (0.92)
16.06 (2.86) 4.34 (1.78) 1.19 (0.55) 10.45 (2.83) 0.37 (0.27) 0.10 (0.94)
*
Data was tested for normality (Kolmogorov–Smirnov test with Dallal–Wilkinson–Lillie for P test) and are reported as mean standard deviation. Significant differences (P < 0.05 by Student’s T test) from the control group are underlined in bold. Statistical analyses were carried out using Prism Graphpad Software, version 5.0.3.
and mean corpuscular haemoglobin concentration (MCHC). All haematological measurements were performed with a haematology analyser (ABC VET — Horiba ABX Diagnostics) (Table 1). Immunophenotyping analyses of anti-CD4, anti-CD8, anti-CD21, anti-CD335 and anti-WC1, all labelled with FITC and ex vivo immunostained for intracellular cytokine (IL-8, IL-1α, IL-6, IL-17, IFNg, IL-4 and IL-10), were performed according to the protocol described by Dorneles et al. (2015). Distinct gating strategies were employed for data analysis as shown in Supplementary Fig. 1. The results of the haematological analysis demonstrated a significant reduction of haemoglobin and haematocrit for BPVinfected animals (Table 1). Indeed, Palanivel et al. (2017) demonstrated a reduction in the concentration of haemoglobin, packed cell volume (PCV), neutrophils and lymphocyte values, suggesting the release of endogenous corticosteroids in response to stress. The results revealed an increase of natural killer (NK) cells and a decrease of gd+ T-cells and the CD4+/CD8+ ratio for BPV-infected animals when compared with the control group (Fig. 1A). Evaluation of the immunological microenviroment through the analysis of pro-inflammatory and regulatory cytokines demonstrated that CD8+T cells from infected animals had a significantly increase in the pro-inflammatory cytokines IL-17 and IFN-g when compared to the control group (Fig. 1B). Chen et al. (2004) demonstrated the importance of CD8+IFN-g+ T-cells for reducing the growth of tumours in mice. Our results showed the involvement of CD8+ T-cells producing IFN-g, and an increase of NK cells in BPV-infected animals, suggesting that this exacerbated pro-inflammatory immune response, without an effective immunoregulatory response, may lead to an unbalanced immune response that may contribute to the persistence of the lesions observed in the BPV group. Indeed, IL-17 actively participates at sites of inflammation, largely from autoimmune diseases but also from a wide spectrum of chronic conditions and varied etiologies (O’Connor et al., 2010); IL-17 also exhibits anti- and protumorigenic effects, depending on the disease context (Zou and Restifo, 2010).
Levkutová et al. (1998) demonstrated the lymphocyte profile of cattle infected with papillomavirus, and found a lower number of CD4+ T-cells and a low CD4+/CD8+ T-cell ratio in animals with tumours compared to a group of papilloma-free cattle. These findings were corroborated by the present study, in which a decrease of the CD4+/CD8+ ratio was observed for the BPV group, compared to the control group. Additionally, the low number of gd+ T lymphocytes observed may contribute to the persistence of lesions of infected cattle, since the functions of these cells have been associated with the protection of ruminant epithelial surfaces. Categorical data for cytokine-producing lymphocyte subsets, CD4+ T-cells, CD8+ T-cells and CD21+ B-cells are displayed as radar graphs plotted with the frequency of high producers (%) from each group (control and BPV-infected animals) (Fig. 1C). It was possible to observe that the cytokine profile of control group can be seen to delineate an area of the graph, which is confined below the 75% threshold; however, gd+ T-cells and the CD4+/CD8+ ratio for the control group exhibited a slight expansion of graphed area. Moreover, the involvement of adaptive immunity with the production of the cytokines IL-17 and IFN-g by CD8+ T-cells can be seen for BPV-infected animals but not for the control group. In general, our results suggest that papillomavirus triggers adaptive immunity (mainly CD8+ T cells) with a pro-inflammatory profile (Fig. 1C). In conclusion, BPV infection causes a weakening of animals, which leads to decreased feeding, anaemia and a reduction in some haematological parameters. Moreover, BPV induces a reduction of gd+ T-cells, which play a critical role in bridging the innate and adaptive arms of the immune system. The infection also stimulates a pro-inflammatory profile with the participation of CD8+T cells producing elevated IFN-g and IL-17. Our findings provide a better understanding of the immune response in BPV-infected cattle. It is important to note that this is preliminary in nature, and the results found here should be validated in further research that includes a greater number of animals.
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P.B. Bassi et al. / The Veterinary Journal 244 (2019) 112–115
Fig. 1. Phenotypic and cytokine profile of peripheral blood lymphocyte subsets from BPV-infected animals. (A) Peripheral blood lymphocyte subsets (natural killer, gd+, total T, CD4+, CD8+ and CD21+ cells) as well the T/B and CD4/CD8 ratios, are shown in box plot format (median with minimum and maximum values) for the control group (n = 5) and BPV-infected group (n = 5). (B) Cytokines IL-8, IL-1α, IL-6, IL-17, IFN-g, IL-10 and IL-4 produced by peripheral blood lymphocyte subsets (CD4+, CD8+ T cells and CD21+ cells) are shown in box plot format (median with minimum and maximum values) from the control group (n = 5) and BPV-infected animals (n = 5). In all cases, significant differences at p < 0.05 are highlighted by a grey background and identified by arrows to indicate an increase (") or a decrease (#) relative to the control group. (C) Overall pattern of high cytokine producers triggered by BPV infection. Categorical data for cytokine-producing subsets of lymphocytes are displayed as radar graphs plotted with the frequency of high producers (75%) for the control group and the BPV-infected group. Left wing indicates the surface markers CD4, CD8, CD21, CD335, and WC1, and the ratios T/B and CD4/ CD8. The right wing indicates the cytokines profile (IL-8, IL-1α, IL-6, IL-17, IFN-g, IL-10 and IL-4) of CD4+, CD8+ and CD21+ cells.
Conflict of interest statement None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgements This work was financially supported by Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and Programa de Apoio a Pesquisa da Universidade de Uberaba, Minas Gerais (PAPE-UNIUBE). OAMF is thankful to CNPq for the PQ fellowship program. FFA thanks PNPD/CAPES fellowship program. The authors thank the program for technological
development in tools for health-FIOCRUZ for the use of Flow Cytometry Platform. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.tvjl.2018.12.004. References Chen, Y.F., Lin, C.W., Tsao, Y.P., Chen, S.L., 2004. Cytotoxic-T-lymphocyte human papillomavirus type 16 E5 peptide with CpG-oligodeoxynucleotide can eliminate tumour growth in C57BL/6 mice. J. Virol. 78, 1333–1343. Daudt, C., Da Silva, F.R., Lunardi, M., Alves, C.B.D.T., Weber, M.N., Cibulski, S.P., Alfieri, A.F., Alfieri, A.A., Canal, C.W., 2018. Papillomaviruses in ruminants: an update. Transbound. Emerg. Dis. doi:http://dx.doi.org/10.1111/tbed.12868. Dorneles, E.M., Araújo, M.S., Teixeira-Carvalho, A., Martins-Filho, O.A., Lage, A.P., 2015. Cross-reactivity of anti-human cytokine monoclonal antibodies used as a
P.B. Bassi et al. / The Veterinary Journal 244 (2019) 112–115 tool to identify novel immunological biomarkers in domestic ruminants. Genet. Mol. Res. 14, 940–951. doi:http://dx.doi.org/10.4238/2015. Levkutová, M., Revajová, V., Levkut, M., Leng, L., 1998. Subpopulations of lymphocytes in cattle naturally infected with papillomavirus. Acta Vet. Hung. 46, 13–18. O’Brien, P.M., Campo, M.S., 2002. Evasion of host immunity directed by papillomavirus-encoded proteins. Virus Res. 88, 103–117. O’Connor Jr, W., Zenewicz, L.A., Flavell, R.A., 2010. The dual nature of Th17 cells: shifting the focus to function. Nat. Immunol. 11, 471–476. Palanivel, K.M., Surendar, A.P., Kanimozhi, P., 2017. Sero biochemical profile of crossbred calves naturally infected by bovine papilloma virus. Int. J. Curr.
115
Microbiol. Appl. Sci. 6 (8), 366–371. doi:http://dx.doi.org/10.20546/ ijcmas.2017.608.049. Rojas-Anaya, E., Cantú-Covarrubias, A., Álvarez, J.F., Loza-Rubio, E., 2016. Detection and phylogenetic analysis of bovine papillomavirus in cutaneous warts in cattle in Tamaulipas, Mexico. Can. J. Vet. Res. 80, 262–268. Roperto, S., Comazzi, S., Ciusani, E., Paolini, F., Borzacchiello, G., Esposito, I., Lucà, R., Russo, V., Urraro, C., Venuti, A., Roperto, F., 2011. PBMCs are additional sites of productive infection of bovine papillomavirus type 2. J. Gen. Virol. 92, 1787– 1794. Zou, W., Restifo, N.P., 2010. Th17 cells in tumour immunity and immunotherapy. Nat. Rev. Immunol. 10, 248–256.