The Veterinary Journal 202 (2014) 182–183
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Short Communication
Acute phase proteins in Andalusian horses infected with Theileria equi Rocío Rodríguez a, José J. Cerón b, Cristina Riber a,c, Francisco Castejón a, Manuel Gómez-Díez a, Juan M. Serrano-Rodríguez d, Ana Muñoz a,c,* a
Equine Sport Medicine Centre, CEMEDE, School of Veterinary Medicine, University of Córdoba, 14004 Córdoba, Spain Department of Animal Medicine and Surgery, School of Veterinary Medicine, Campus of Excellence Mare Nostrum, University of Murcia, 30100 Murcia, Spain c Department of Animal Medicine and Surgery, School of Veterinary Medicine, University of Córdoba, Córdoba, Spain d Department of Pharmacology, Toxicology, and Legal and Forensic Medicine, School of Veterinary Medicine, University of Córdoba, Córdoba, Spain b
A R T I C L E
I N F O
Article history: Accepted 11 July 2014 Keywords: Equine Theileria equi Acute phase proteins Clinical pathology
A B S T R A C T
Clinical and laboratory findings were determined in 23 Andalusian horses in southern Spain that were positive for Theileria equi by PCR, including 16 mares at pasture (group A1) and seven stabled stallions (group B1). Five healthy mares at pasture (group A2) and five stabled stallions (group B2), all of which were negative for T. equi in Giemsa stained blood smears and by PCR, were used as controls. The most frequent clinical signs were anorexia, anaemia, depression and icterus (group A1), along with loss of performance or failure to train and depression (group B1). Thrombocytopoenia was evident in 5/7 horses in group B1. Lower serum iron concentrations were observed in both diseased groups compared with their respective control groups. There were no significant differences in APP concentrations between diseased and control groups; all affected horses had APP concentrations within reference limits. Serum haptoglobin, serum amyloid A and plasma fibrinogen concentrations were higher than the reference limits in 5/23, 3/23 and 1/23 diseased horses, respectively. It was concluded that horses with theileriosis exhibited only a mild systemic inflammatory response. © 2014 Elsevier Ltd. All rights reserved.
Equine piroplasmosis is a tick-borne disease caused by the apicomplexan protozoa Theileria equi and Babesia caballi. Seroprevalences of 13.1% for B. caballi and 56.1% for T. equi have been reported in southern Spain using a competitive antibody ELISA (García-Bocanegra et al., 2013). Horses kept outside on pasture are more likely to be exposed to ticks and thus are more likely to be infected with T. equi and B. caballi than horses kept indoors (Kouam et al., 2010). Clinical signs associated with equine theileriosis are non-specific, ranging from mild inappetence, weight loss, transient fever, poor exercise tolerance, weakness and anaemia to sudden death (Muñoz et al., 2013). The aim of this study was to evaluate the clinical and laboratory findings in horses infected with T. equi in Córdoba, in the south of Spain, an area endemic for equine piroplasmosis. The study included 23 Andalusian horses on the same farm that were positive for T. equi by real-time quantitative PCR (qPCR, TaqMan; Kim et al., 2008) and negative for B. caballi and Anaplasma phagocytophilum by qPCR (AnaPha dtec qPCR test). The T. equi positive horses were divided into two groups: (1) group A1, mares at pasture (n = 16; age 8.5 ± 4.5 years) and (2) group B1, stabled stallions (n = 7; age 4.7 ± 1.1 years). In addition, 10 Andalusian horses
* Corresponding author. Tel.: +34 957 211068. E-mail address: [email protected] (A. Muñoz). http://dx.doi.org/10.1016/j.tvjl.2014.07.003 1090-0233/© 2014 Elsevier Ltd. All rights reserved.
on the same farm, free of clinical signs and negative for T. equi, B. caballi and A. phagocytophilum by qPCR and by microscopic examination of Giemsa stained blood smears, were included as negative controls: (1) group A2, mares at pasture (n = 5; age 6.3 ± 2.3 years); and (2) group B2, stabled stallions (n = 5; age 6.4 ± 1.2 years). The Animal Care and Ethics Committee of the University of Córdoba concluded that the study did not require ethical approval under Spanish Law (RD 53/2013). Written owner consent was obtained for all control horses. Clinical examinations were performed and blood samples were collected from each horse for haematology and biochemistry, including testing for acute phase proteins (APPs) and for preparation of Giemsa stained blood smears for microscopic examination (see Appendix: Supplementary Table S1). Samples were collected from affected horses before treatment. Differences between affected and control groups of horses (A1 vs. A2; B1 vs. B2) were assessed using the Mann–Whitney test (Statistica v. 9.0; Statsoft); parameters were not normally distributed. The level of significance was set at P < 0.05. Clinical signs in horses infected with T. equi were mild. In group A1, the most frequent clinical signs were anorexia or reduced appetite, anaemia, depression and icterus (see Appendix: Supplementary Tables S2 and S3). In group B1, the predominant clinical signs were loss of performance or failure to train and depression. Horses in groups A2 and B2 were clinically normal. Horses in
R. Rodríguez et al./The Veterinary Journal 202 (2014) 182–183
183
Table 1 Frequency of increase/decrease of iron and some acute phase proteins (APPs), and median, percentiles, and minimum and maximum values for APPs, in two groups of Andalusian horses naturally infected with Theileria equi at pasture (mares, group A1) and stabled (males, group B1) and in comparison with their respective controls (groups A2 and B2). APPs Hp > 3 mg/dL SAA > 10 mg/Lb FIB > 400 mg/dL b
Group A1 (n = 16)
Group A2 (n = 5)
Group B1 (n = 7)
Group B2 (n = 5)
4/16 (25%) 3/16 (18.75%) 3/16 (18.75%)
0/5 (5%) 0/5 (0%) 0/5 (0%)
1/7 (14.29%) 0/7 (0%) 1/7 (14.29%)
0/5 (0%) 0/5 (0%) 0/5 (0%)
Median Hp (mg/dL) SAA (mg/L) FIB (mg/dL) CRP (μg/mL) Fe (μg/dL)
2.041 0.48 400 36.30 153.0a
Percentiles
Min–Max
Median
Percentiles
Min–Max
Median
Percentiles
Min–Max
Median
Percentiles
Min–Max
1.205–2.866 0.48–0.49 250–400 28.25–52.85 132.0–178.0
0.323–3.259 0.48–308.0 200–600 22.80–185.9 44.0–261.0
1.830 0.48 300 33.80 316.0
1.320–2.030 0.48–0.50 300–300 27.6–33.8 220–346
0.200–2.410 0.480–0.600 300–300 14.90–50.0 182.0–385.0
2.128 0.48 300 25.8 159.0a
1.954–2.919 0.48–0.48 200–400 25.0–34.5 135.0–186.0
0.987–3.564 0.48–0.48 100–500 24.1–40.2 129.0–217.0
1.310 0.480 300 25.40 311.0
1.04–1.36 0.48–0.49 200–300 22.8–26.7 301–345
0.71–2.45 0.48–0.50 100–400 22.4–31.0 188–360
CRP, C-reactive protein; Fe, iron; FIB, fibrinogen; Hp, haptoglobin; SAA, serum A amyloid; Min, minimum; Max, maximum. a Significant differences between groups A1 and B1, and control groups, A2 and B2, respectively. P < 0.05. b Reference intervals established by the Veterinary Clinical Pathology Laboratory of the Faculty of Veterinary Medicine, University of Murcia, Spain.
groups A1 and B1 had higher respiratory rates than those in groups A2 and B2, respectively. Many of these clinical signs could be attributed to anaemia. Pyrexia was not observed in any horses, possibly because cases were examined in the chronic stage of disease, whereas pyrexia is usually observed around the time of peak parasitaemia (Guimaraes et al., 1998). Horses in groups A1 and B1 had significantly lower packed cell volumes than their respective control groups, probably due to haemolytic anaemia (see Appendix: Supplementary Tables S4 and S5). Thrombocytopoenia was evident in 5/7 horses in group B1 and in 1/16 horses in group A1. Thrombocytopoenia has been reported previously in one horse with piroplasmosis (Diana et al., 2007). Horses in group A1 had significantly longer prothrombin times than horses in control group A2, but values remained within reference ranges and no coagulopathies were observed. There were no significant differences in APP concentrations between diseased horses and their corresponding control groups (Table 1). None of the control horses had APP concentrations out of our reference ranges for healthy horses. Haptoglobin and fibrinogen are ‘moderate’ APPs that show a slow kinetic, needing more time to increase and to return to normal values, whereas serum amyloid A, a ‘major’ APP, has a more rapid kinetic (Jacobsen et al., 2005). More diseased horses had increased haptoglobin and fibrinogen concentrations than increased serum amyloid A concentrations. These data indicate that most of the horses infected with T. equi did not have a severe or active inflammatory process at the time of sampling. Horses in infected groups had lower serum iron concentrations than their respective control groups (Table 1). A decrease in iron concentration is a host defence mechanism to restrict iron availability during infection (Borges et al., 2007). Serum phosphorus concentrations, and creatine kinase and aspartate aminotransferase activities, were within reference limits in both diseased and control horses. However, diseased mares in group A1 had higher phosphorus concentrations than mares in control group A2; elevated phosphorus concentrations may have been a consequence of intravascular haemolysis (see Appendix: Supplementary Table S5). Horses in control group A1 had higher creatine kinase activities than horses in infected group A2 and those in control group B1 had higher aspartate aminotransferase activities than horses in infected group B2. In conclusion, Andalusian horses naturally infected with T. equi in an endemic area had mild clinical signs, moderate haematological
and biochemical changes, and moderate acute phase responses. These findings are interpreted as indicating an inflammatory response of low intensity, probably as a consequence of the chronic status of the disease at the time of sampling.
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
Appendix: Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.tvjl.2014.07.003.
References Borges, A.S., Divers, T.J., Stokol, T., Mohammed, O.H., 2007. Serum iron and plasma fibrinogen concentrations as indicators of systemic inflammatory diseases in horses. Journal of Veterinary Internal Medicine 21, 489–494. Diana, A., Gugliemini, C., Candini, D., Pietra, M., Cipone, M., 2007. Cardiac arrhythmias associated with piroplasmosis in the horse: A case report. The Veterinary Journal 174, 193–195. García-Bocanegra, I., Arenas-Montes, A., Hernández, E., Adaszek, L., Carbonero, A., Almería, S., Jaén-Tellez, J.Á., Gutierrez-Palomino, P., Arenas, A., 2013. Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infection in equids. The Veterinary Journal 195, 172–178. Guimaraes, A.M., Lima, J.D., Ribeiro, M.F.B., 1998. Sporogony and experimental transmission of Babesia equi by Boophilus microplus. Parasitology Research 84, 323–327. Jacobsen, S., Jensen, J.C., Frei, S., Jensen, A.L., Thoefner, M.B., 2005. Use of serum amyloid A and other acute phase reactants to monitor the inflammatory response after castration in horses: A field study. Equine Veterinary Journal 37, 552–556. Kim, C.M., Blanco, L.B., Alhassan, A., Iseki, H., Yokoyama, N., Xuan, X., Igarashi, I., 2008. Diagnostic real-time PCR assay for the quantitative detection of Theileria equi from equine blood samples. Veterinary Parasitology 151, 158–163. Kouam, M.K., Kantzoura, V., Gajadhar, A.V., Theis, J.H., Papadopoulos, E., Theodoropoulos, G., 2010. Seroprevalence of equine piroplasms and host-related factors associated with infection in Greece. Veterinary Parasitology 169, 273–278. Muñoz, A., Rodríguez, R.G.M., Riber, C., Trigo, P., Gómez-Díez, M., Castejón, F., 2013. Subclinical Theileria equi infection and rhabdomyolysis in three endurance horses. Pakistan Veterinary Journal 33, 257–259.
Contents lists available at ScienceDirect
The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Short Communication
Acute phase proteins in Andalusian horses infected with Theileria equi Rocío Rodríguez a, José J. Cerón b, Cristina Riber a,c, Francisco Castejón a, Manuel Gómez-Díez a, Juan M. Serrano-Rodríguez d, Ana Muñoz a,c,* a
Equine Sport Medicine Centre, CEMEDE, School of Veterinary Medicine, University of Córdoba, 14004 Córdoba, Spain Department of Animal Medicine and Surgery, School of Veterinary Medicine, Campus of Excellence Mare Nostrum, University of Murcia, 30100 Murcia, Spain c Department of Animal Medicine and Surgery, School of Veterinary Medicine, University of Córdoba, Córdoba, Spain d Department of Pharmacology, Toxicology, and Legal and Forensic Medicine, School of Veterinary Medicine, University of Córdoba, Córdoba, Spain b
A R T I C L E
I N F O
Article history: Accepted 11 July 2014 Keywords: Equine Theileria equi Acute phase proteins Clinical pathology
A B S T R A C T
Clinical and laboratory findings were determined in 23 Andalusian horses in southern Spain that were positive for Theileria equi by PCR, including 16 mares at pasture (group A1) and seven stabled stallions (group B1). Five healthy mares at pasture (group A2) and five stabled stallions (group B2), all of which were negative for T. equi in Giemsa stained blood smears and by PCR, were used as controls. The most frequent clinical signs were anorexia, anaemia, depression and icterus (group A1), along with loss of performance or failure to train and depression (group B1). Thrombocytopoenia was evident in 5/7 horses in group B1. Lower serum iron concentrations were observed in both diseased groups compared with their respective control groups. There were no significant differences in APP concentrations between diseased and control groups; all affected horses had APP concentrations within reference limits. Serum haptoglobin, serum amyloid A and plasma fibrinogen concentrations were higher than the reference limits in 5/23, 3/23 and 1/23 diseased horses, respectively. It was concluded that horses with theileriosis exhibited only a mild systemic inflammatory response. © 2014 Elsevier Ltd. All rights reserved.
Equine piroplasmosis is a tick-borne disease caused by the apicomplexan protozoa Theileria equi and Babesia caballi. Seroprevalences of 13.1% for B. caballi and 56.1% for T. equi have been reported in southern Spain using a competitive antibody ELISA (García-Bocanegra et al., 2013). Horses kept outside on pasture are more likely to be exposed to ticks and thus are more likely to be infected with T. equi and B. caballi than horses kept indoors (Kouam et al., 2010). Clinical signs associated with equine theileriosis are non-specific, ranging from mild inappetence, weight loss, transient fever, poor exercise tolerance, weakness and anaemia to sudden death (Muñoz et al., 2013). The aim of this study was to evaluate the clinical and laboratory findings in horses infected with T. equi in Córdoba, in the south of Spain, an area endemic for equine piroplasmosis. The study included 23 Andalusian horses on the same farm that were positive for T. equi by real-time quantitative PCR (qPCR, TaqMan; Kim et al., 2008) and negative for B. caballi and Anaplasma phagocytophilum by qPCR (AnaPha dtec qPCR test). The T. equi positive horses were divided into two groups: (1) group A1, mares at pasture (n = 16; age 8.5 ± 4.5 years) and (2) group B1, stabled stallions (n = 7; age 4.7 ± 1.1 years). In addition, 10 Andalusian horses
* Corresponding author. Tel.: +34 957 211068. E-mail address: [email protected] (A. Muñoz). http://dx.doi.org/10.1016/j.tvjl.2014.07.003 1090-0233/© 2014 Elsevier Ltd. All rights reserved.
on the same farm, free of clinical signs and negative for T. equi, B. caballi and A. phagocytophilum by qPCR and by microscopic examination of Giemsa stained blood smears, were included as negative controls: (1) group A2, mares at pasture (n = 5; age 6.3 ± 2.3 years); and (2) group B2, stabled stallions (n = 5; age 6.4 ± 1.2 years). The Animal Care and Ethics Committee of the University of Córdoba concluded that the study did not require ethical approval under Spanish Law (RD 53/2013). Written owner consent was obtained for all control horses. Clinical examinations were performed and blood samples were collected from each horse for haematology and biochemistry, including testing for acute phase proteins (APPs) and for preparation of Giemsa stained blood smears for microscopic examination (see Appendix: Supplementary Table S1). Samples were collected from affected horses before treatment. Differences between affected and control groups of horses (A1 vs. A2; B1 vs. B2) were assessed using the Mann–Whitney test (Statistica v. 9.0; Statsoft); parameters were not normally distributed. The level of significance was set at P < 0.05. Clinical signs in horses infected with T. equi were mild. In group A1, the most frequent clinical signs were anorexia or reduced appetite, anaemia, depression and icterus (see Appendix: Supplementary Tables S2 and S3). In group B1, the predominant clinical signs were loss of performance or failure to train and depression. Horses in groups A2 and B2 were clinically normal. Horses in
R. Rodríguez et al./The Veterinary Journal 202 (2014) 182–183
183
Table 1 Frequency of increase/decrease of iron and some acute phase proteins (APPs), and median, percentiles, and minimum and maximum values for APPs, in two groups of Andalusian horses naturally infected with Theileria equi at pasture (mares, group A1) and stabled (males, group B1) and in comparison with their respective controls (groups A2 and B2). APPs Hp > 3 mg/dL SAA > 10 mg/Lb FIB > 400 mg/dL b
Group A1 (n = 16)
Group A2 (n = 5)
Group B1 (n = 7)
Group B2 (n = 5)
4/16 (25%) 3/16 (18.75%) 3/16 (18.75%)
0/5 (5%) 0/5 (0%) 0/5 (0%)
1/7 (14.29%) 0/7 (0%) 1/7 (14.29%)
0/5 (0%) 0/5 (0%) 0/5 (0%)
Median Hp (mg/dL) SAA (mg/L) FIB (mg/dL) CRP (μg/mL) Fe (μg/dL)
2.041 0.48 400 36.30 153.0a
Percentiles
Min–Max
Median
Percentiles
Min–Max
Median
Percentiles
Min–Max
Median
Percentiles
Min–Max
1.205–2.866 0.48–0.49 250–400 28.25–52.85 132.0–178.0
0.323–3.259 0.48–308.0 200–600 22.80–185.9 44.0–261.0
1.830 0.48 300 33.80 316.0
1.320–2.030 0.48–0.50 300–300 27.6–33.8 220–346
0.200–2.410 0.480–0.600 300–300 14.90–50.0 182.0–385.0
2.128 0.48 300 25.8 159.0a
1.954–2.919 0.48–0.48 200–400 25.0–34.5 135.0–186.0
0.987–3.564 0.48–0.48 100–500 24.1–40.2 129.0–217.0
1.310 0.480 300 25.40 311.0
1.04–1.36 0.48–0.49 200–300 22.8–26.7 301–345
0.71–2.45 0.48–0.50 100–400 22.4–31.0 188–360
CRP, C-reactive protein; Fe, iron; FIB, fibrinogen; Hp, haptoglobin; SAA, serum A amyloid; Min, minimum; Max, maximum. a Significant differences between groups A1 and B1, and control groups, A2 and B2, respectively. P < 0.05. b Reference intervals established by the Veterinary Clinical Pathology Laboratory of the Faculty of Veterinary Medicine, University of Murcia, Spain.
groups A1 and B1 had higher respiratory rates than those in groups A2 and B2, respectively. Many of these clinical signs could be attributed to anaemia. Pyrexia was not observed in any horses, possibly because cases were examined in the chronic stage of disease, whereas pyrexia is usually observed around the time of peak parasitaemia (Guimaraes et al., 1998). Horses in groups A1 and B1 had significantly lower packed cell volumes than their respective control groups, probably due to haemolytic anaemia (see Appendix: Supplementary Tables S4 and S5). Thrombocytopoenia was evident in 5/7 horses in group B1 and in 1/16 horses in group A1. Thrombocytopoenia has been reported previously in one horse with piroplasmosis (Diana et al., 2007). Horses in group A1 had significantly longer prothrombin times than horses in control group A2, but values remained within reference ranges and no coagulopathies were observed. There were no significant differences in APP concentrations between diseased horses and their corresponding control groups (Table 1). None of the control horses had APP concentrations out of our reference ranges for healthy horses. Haptoglobin and fibrinogen are ‘moderate’ APPs that show a slow kinetic, needing more time to increase and to return to normal values, whereas serum amyloid A, a ‘major’ APP, has a more rapid kinetic (Jacobsen et al., 2005). More diseased horses had increased haptoglobin and fibrinogen concentrations than increased serum amyloid A concentrations. These data indicate that most of the horses infected with T. equi did not have a severe or active inflammatory process at the time of sampling. Horses in infected groups had lower serum iron concentrations than their respective control groups (Table 1). A decrease in iron concentration is a host defence mechanism to restrict iron availability during infection (Borges et al., 2007). Serum phosphorus concentrations, and creatine kinase and aspartate aminotransferase activities, were within reference limits in both diseased and control horses. However, diseased mares in group A1 had higher phosphorus concentrations than mares in control group A2; elevated phosphorus concentrations may have been a consequence of intravascular haemolysis (see Appendix: Supplementary Table S5). Horses in control group A1 had higher creatine kinase activities than horses in infected group A2 and those in control group B1 had higher aspartate aminotransferase activities than horses in infected group B2. In conclusion, Andalusian horses naturally infected with T. equi in an endemic area had mild clinical signs, moderate haematological
and biochemical changes, and moderate acute phase responses. These findings are interpreted as indicating an inflammatory response of low intensity, probably as a consequence of the chronic status of the disease at the time of sampling.
Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.
Appendix: Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.tvjl.2014.07.003.
References Borges, A.S., Divers, T.J., Stokol, T., Mohammed, O.H., 2007. Serum iron and plasma fibrinogen concentrations as indicators of systemic inflammatory diseases in horses. Journal of Veterinary Internal Medicine 21, 489–494. Diana, A., Gugliemini, C., Candini, D., Pietra, M., Cipone, M., 2007. Cardiac arrhythmias associated with piroplasmosis in the horse: A case report. The Veterinary Journal 174, 193–195. García-Bocanegra, I., Arenas-Montes, A., Hernández, E., Adaszek, L., Carbonero, A., Almería, S., Jaén-Tellez, J.Á., Gutierrez-Palomino, P., Arenas, A., 2013. Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infection in equids. The Veterinary Journal 195, 172–178. Guimaraes, A.M., Lima, J.D., Ribeiro, M.F.B., 1998. Sporogony and experimental transmission of Babesia equi by Boophilus microplus. Parasitology Research 84, 323–327. Jacobsen, S., Jensen, J.C., Frei, S., Jensen, A.L., Thoefner, M.B., 2005. Use of serum amyloid A and other acute phase reactants to monitor the inflammatory response after castration in horses: A field study. Equine Veterinary Journal 37, 552–556. Kim, C.M., Blanco, L.B., Alhassan, A., Iseki, H., Yokoyama, N., Xuan, X., Igarashi, I., 2008. Diagnostic real-time PCR assay for the quantitative detection of Theileria equi from equine blood samples. Veterinary Parasitology 151, 158–163. Kouam, M.K., Kantzoura, V., Gajadhar, A.V., Theis, J.H., Papadopoulos, E., Theodoropoulos, G., 2010. Seroprevalence of equine piroplasms and host-related factors associated with infection in Greece. Veterinary Parasitology 169, 273–278. Muñoz, A., Rodríguez, R.G.M., Riber, C., Trigo, P., Gómez-Díez, M., Castejón, F., 2013. Subclinical Theileria equi infection and rhabdomyolysis in three endurance horses. Pakistan Veterinary Journal 33, 257–259.
