Veterinary Parasitology 144 (2007) 242–250 www.elsevier.com/locate/vetpar

Evidence of an acute phase response in dogs naturally infected with Babesia canis Vesna Matijatko a,*, Vladimir Mrljak a, Ivana Kisˇ a, Nada Kucˇer a, Jadranka Forsˇek b, Tanja Zˇivicˇnjak c, Zˇeljko Romic´ d, Zoran Sˇimec e, Jose Joaquin Ceron f a

Clinic for Internal Diseases, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia Central Clinical Laboratory, Clinic for Internal Diseases, Faculty of Veterinary Medicine, University of Zagreb, Croatia c Department of Parasitology, Faculty of Veterinary Medicine, University of Zagreb, Croatia d Clinical Hospital Dubrava, Zagreb, Croatia e Maza Small Animal Veterinary Practice, Zagreb, Croatia f Department of Animal Medicine and Surgery, University of Murcia, Spain

b

Received 15 April 2005; received in revised form 27 September 2006; accepted 6 October 2006

Abstract The erythrocyte sedimentation rate (ESR), white blood cell count (WBC), haematocrit (HCT) and platelet number (PLT) were quantified and compared with the acute phase proteins (APPs) in dogs naturally infected with Babesia canis and healthy dogs. Both groups were treated with imidocarb dipropionate on the day of admission and both groups were monitored for all parameters on the admission day and on the first, second, third, fourth and seventh days in order to determine the presence of an acute phase reaction, to assess the diagnostic value of these markers in uncomplicated canine babesiosis and to evaluate the use of APPs in treatment monitoring. It was demonstrated that an acute phase response occurs in dogs naturally infected with Babesia canis, with significant increases in the concentration of major acute phase proteins. The serum concentration of C-reactive protein (CRP), serum amyloid A (SAA) and the erythrocyte sedimentation rate (ESR) decreased daily after treatment and approached reference range values by the eighth day. PLT and haematocrit (HCT) increased daily after treatment and approached reference range values by the fourth day. WBC and haptoglobin increased after treatment and then decreased from the third and fourth days, respectively, to the eighth day. The diagnostic sensitivity of CRP, SAA and PLT was significantly higher compared to haptoglobin, ESR, HCT and the WBC count. CRP and SAA were of clinical use in monitoring the response to antibabesial treatment. # 2006 Elsevier B.V. All rights reserved. Keywords: Babesia canis; Acute phase reaction; Pathology; Treatment; Imidocarb dipropionate; C-reactive protein; Serum amyloid A; Haptoglobin

1. Introduction Canine babesiosis is a tick-borne disease caused by the intra-erythrocytic protozoan parasites Babesia canis,

* Corresponding author. Tel.: +385 1 23 90 350. E-mail address: [email protected] (V. Matijatko). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.10.004

Babesia gibsoni and Babesia microti-like piroplasms (Uilenberg et al., 1989; Taboada and Merchant, 1991; Camacho et al., 2001). Previous studies have suggested that B. canis should be divided into three biologically and immunologically distinct subspecies: B. canis canis, B. canis vogeli and B. canis rossi (Uilenberg et al., 1989). However, additional molecular biological studies have indicated that these three groups of parasites do not

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cluster in a single clade, which suggests that they are not subspecies (Carret et al., 1999). Since the discussion about classification of the Babesia species is not the aim of this paper, the classical nomenclature (B. canis, B. rossi and B. vogeli) will be used as suggested by Schetters et al. (1997), Zahler et al. (1998), Carret et al. (1999), Passos et al. (2005), and Schetters (2005). Canine babesiosis caused by B. canis is a very common cause of morbidity and mortality of dogs in Croatia (Caccio et al., 2002). The disease can be clinically classified into uncomplicated and complicated forms. Dogs with uncomplicated babesiosis are typically presented with signs relating to acute haemolysis, including pale mucous membranes, fever, anorexia, depression, splenomegaly and water hammer pulse (Taboada and Merchant, 1991). The clinical manifestation of the complicated form is variable and related to the complications developed. The complications of canine babesiosis are acute renal failure, cerebral babesiosis, coagulopathy, icterus and hepatopathy, immune-mediated haemolytic anaemia (IMHA), peracute babesiosis, acute respiratory distress syndrome (ARDS), haemoconcentration and shock (Lobetti, 2000). The clinical presentation of canine babesiosis caused by B. canis is highly variable. The many and varied clinical manifestations of this disease are difficult to relate to an organism that is solely restricted to the erythrocytes. Some authors have proposed that although the clinical manifestations are diverse, the mechanism promoting them is probably uniform (Jacobson and Clark, 1994; Lobetti, 1998; Welzl et al., 2001). They consider systemic inflammation as a major feature in the pathophysiologic mechanisms of this disease and have suggested that systemic inflammatory response syndrome (SIRS) and subsequent multiple organ dysfunction syndrome (MODS) provide the underlying pathophysiologic mechanism within which apparently unrelated aspects of babesiosis form a predictable pattern. Acute phase reaction develops following any tissue inflammatory injury and is considered as a part of innate immunity characterized by profound changes in the concentration of acute phase proteins (APP) in the plasma (Moshage, 1997; Gabay and Kushner, 1999). The circulating concentration of acute phase proteins is related to the severity of the underlying condition and it provides a means for evaluating the presence and extent of the disease process as well as the efficacy of the disease management in human and veterinary medicine (Steel and Whitehead, 1994; Pannen and Robotham, 1995; Gruys et al., 1994; Martinez-Subiela et al., 2002, 2003).

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The acute phase reaction in naturally occurrring canine babesiosis has been poorly described. Lobetti et al. (2000) documented a marked acute phase reaction by the detection of increased concentrations of a1-acid glycoprotein in canine babesiosis caused by B. rossi, and Matijatko et al. (2002) described a significant increase of CRP in naturally occurring canine babesiosis. Moreover, to the authors’ knowledge no studies about the use of acute phase proteins to monitor the response to antibabesial treatment have been published. Additionally, although it has been demonstrated that haptoglobin and ceruloplasmin have higher diagnostic sensitivity than WBC counts to detect inflammation in dogs (Solter et al., 1991), there is a lack of studies in which the diagnostic sensitivity of various traditional markers of inflammation or sepsis (such as ESR, total and differential WBC counts or platelet counts), and various major (CRP and SAA) and moderate (haptoglobin) acute phase proteins are evaluated and compared. In this study, therefore, serum concentrations of Creactive protein, serum amyloid A and haptoglobin were investigated in dogs naturally infected with B. canis in order to document the acute phase reaction and its potential value in the diagnosis of the disease. In addition, the use of the sequential measurement of acute phase proteins in assessing the response to antibabesial treatment was also evaluated. During the entire study, acute phase proteins were compared to commonly used inflammatory markers such as the erythrocyte sedimentation rate, WBC count and platelet count. Haematocrit determinations were included in our work in order to quantify the degree of anaemia and better understand the pathogenesis of the disease. 2. Materials and methods 2.1. Animals The study was performed on two groups of animals. Group 1 consisted of 50 dogs naturally infected by B. canis, admitted to the Clinic for Internal Diseases, Faculty of Veterinary Medicine, University of Zagreb, Croatia, with clinical signs of acute babesiosis. The diagnosis was confirmed by demonstration of the parasites within the infected erythrocytes in Romanowsky-stained thin blood smears. One dose (6 mg/kg) of imidocarb dipropionat (Imizol1, Shering-Plough) was administered to all the dogs subcutaneously on the day of admission. On the basis of clinical presentation, laboratory data and response to antibabesial treatment, only uncomplicated cases of babesiosis were included

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in the group. All the dogs included in this study came from the Zagreb area, in which no cases of leishmaniois or ehrlichiosis have been reported. Therefore, serology for these diseases was not performed. Group 2 consisted of 20 healthy dogs, which had no signs of any disease. Imidocarb dipropionat was administered in the same dose to all of them as a preventive measure against babesiosis at the request of their owners. 2.2. Samples The blood samples for analysis were collected from the cephalic vein on the day of admission, and the first, second, third, fourth and seventh days after the administration of imidocarb dipropionat. The samples were placed in tubes with EDTA for haematological analysis and tubes with no anticoagulant which were centrifuged at 1200  g. The sera obtained were stored at 70 8C until they were analyzed for acute phase proteins. 2.3. Blood cell analysis White blood cell count (WBC), platelet count and haematocrit (HCT) were determined using an automatic haematology analyzer (System 9120; Serono Baker Diagnostic). Blood smears were observed for possible platelet clumps. The erythrocyte sedimentation rate was determined using the standard Westergreen method.

automatic analyzer (Olympus diagnostica GMBH). The human haptoglobin turbidimetric assay for the detection of haptoglobin in canine serum had been validated by Wiedmeyer and Solter (1996). In addition, the validation of the kit was performed in our laboratory with satisfactory results. 2.5. Statistical analysis The sensitivity in detecting babesiosis was calculated for all the tests used in our study. The sensitivity of each test was calculated as the percentage of true positive values in the diseased population (animals with babesiosis that had test values outside the reference range). In addition, the concentrations of C-reactive protein, SAA, haptoglobin, ESR, platelet count, WBC count and HCT were compared between the control dogs and the dogs with babesiosis using the Mann–Whitney U test. p values <0.05 were considered to be significant. The correlation between the investigated parameters was determined using the Spearman rank order test. The significance level was set at 0.01. 3. Results 3.1. Clinical parameters on admission and clinical babesiosis

2.4. Acute phase protein determinations

Fifty cases fulfilled the selection criteria for uncomplicated acute canine babesiosis and were included in the study. On the day of admission, all of

The CRP concentration was determined with a solid sandwich immunoassay (Tridelta Phase range canine CRP kit; Tridelta development). The absorbance of the samples was measured in a microtitre plate reader (Multiscan EX 355; Labsystem) at 450 nm. The concentration of SAA was determined with a solid sandwich immunoassay (Tridelta Phase range Serum Amyloid A Assay; Tridelta development). The absorbance of the samples was measured in a microtitre plate reader (Multiscan EX 355; Labsystem) at 450 nm. Both assays had been previously validated in canine samples with satisfactory results when all the samples were analyzed in the same batch in order to avoid high between-run imprecision (Kjelgaard-Hansen et al., 2003; Martinez-Subiela et al., 2005). The concentration of haptoglobin was determined by an immunoturbidimetric assay (Haptoglobin OSR 6165; Olympus diagnostica GMBH). The absorbance of the samples was measured in an Olympus AU 600

Fig. 1. CRP concentration in control group and in canine babesiosis before (first day) and after antibabesial treatment (second, third, fourth, fifth and eighth day). (*) Indicates a significant difference ( p < 0.01) between uninfected (control) and infected (canine babesiosis) group by the Mann–Whitney test for significance. (") Indicates time of antibabesial treatment. The plots show the mean (dot within box), median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) minimum and maximum values (x).

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Fig. 2. SAA concentration in control group and in canine babesiosis before (first day) and after antibabesial treatment (second, third, fourth, fifth and eighth day). (*) Indicates a significant difference ( p < 0.01) between uninfected (control) and infected (canine babesiosis) group by the Mann–Whitney test for significance. (") Indicates time of antibabesial treatment. The plots show the mean (dot within box), median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) minimum and maximum values (x).

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Fig. 4. Erythrocyte sedimentation rate in control group and in canine babesiosis before (first day) and after antibabesial treatment (second, third, fourth, fifth and eighth day). (*) Indicates a significant difference ( p < 0.01) between uninfected (control) and infected (canine babesiosis) group by the Mann–Whitney test for significance. (") Indicates time of antibabesial treatment. The plots show the mean (dot within box), median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) minimum and maximum values (x).

the dogs included in this study presented with one or more of the following clinical signs: depression (50/50), anorexia (44/50), pale mucous membranes (34/50), fever (29/50), splenomegaly (22/50) and water hammer pulse (12/50). All the dogs with babesiosis showed improvement in their clinical symptoms within 24 h after the antibabesial treatment. The treatment was successful in all the cases. The values for the investigated acute phase markers (range, mean and significance between the dogs

naturally infected with B. canis and the healthy control group) in all the samples taken during the study are shown in Figs. 1–7. On the day of admission, the values for CRP, SAA, haptoglobin, erythrocyte sedimentation rate, WBC, platelet count and HCT were significantly different ( p < 0.01) in dogs with babesiosis in comparison to the control group. The concentrations of CRP, HPT and SAA were significantly increased while the platelet count and HCT were significantly decreased.

Fig. 3. Haptoglobin concentration in control group and in canine babesiosis before (first day) and after antibabesial treatment (second, third, fourth, fifth and eighth day). (*) Indicates a significant difference ( p < 0.01) between uninfected (control) and infected (canine babesiosis) group by the Mann–Whitney test for significance. (") Indicates time of antibabesial treatment. The plots show the mean (dot within box), median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) minimum and maximum values (x).

Fig. 5. Platelet count in control group and in canine babesiosis before (first day) and after antibabesial treatment (second, third, fourth, fifth and eighth day). (*) Indicates a significant difference ( p < 0.01) between uninfected (control) and infected (canine babesiosis) group by the Mann–Whitney test for significance. (") Indicates time of antibabesial treatment. The plots show the mean (dot within box), median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) minimum and maximum values (x).

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V. Matijatko et al. / Veterinary Parasitology 144 (2007) 242–250 Table 1 Sensitivity (taking the reference limit as mean (2S.D.) of control value) in dogs naturally infected with B. canis at the day of the presentation Group

Number of dogs tested

N

Sensitivity (%)

S.E. WBC Platelets C-reactive protein Haptoglobin Serum amyloid A

50 50 50 50 50 50

44 9 50 50 30 50

88 18 100 100 60 100

N number of animal outside control limits (mean [2S.D.] of control value). Fig. 6. White blood cell count in control group and in canine babesiosis before (first day) and after antibabesial treatment (second, third, fourth, fifth and eighth day). (*) Indicates a significant difference ( p < 0.01) between uninfected (control) and infected (canine babesiosis) group by the Mann–Whitney test for significance. (") Indicates time of antibabesial treatment. The plots show the mean (dot within box), median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) minimum and maximum values (x).

Significant differences ( p < 0.01) for CRP, HPT, SAA, WBC count and platelet count between the control and the babesiosis group could be detected during the entire study. The sensitivity of the various diagnostic tests applied in our study on the day of admission is presented in Table 1. Platelet count, CRP and SAA showed a sensitivity of 100%.

3.2. Dynamics of blood cell parameters upon curative treatment The mean values of ESR showed a consistent downward trend from the first day to the eighth day of the investigation (Fig. 4). The mean platelet count showed a consistent upward trend from the first day to the eighth day (Fig. 5). The mean white blood cell count was significantly lower before antibabesial treatment and significantly higher after antibabesial treatment (Fig. 6). The mean HCT value showed a consistent upward trend from first day to the eighth day, reaching normal values on the fifth day (Fig. 7). 3.3. The dynamics of APP values upon curative treatment The highest mean concentration of CRP was obtained before antibabesial treatment, while for SAA the highest mean concentration was obtained the day after antibabesial treatment (Figs. 1 and 2). The highest mean concentration of haptoglobin was in the samples taken three days after antibabesial treatment (Fig. 3). The mean concentrations of CRP showed a consistent downward trend from the first day to the eighth day, and the mean concentrations of SAA showed a downward trend from the second day to the eighth day. Individual values showed variation but with general downward trend in parallel with clinical recovery. 3.4. Correlation between inflammatory markers

Fig. 7. Haematocrit in control group and in canine babesiosis before (first day) and after antibabesial treatment (second, third, fourth, fifth and eighth day). (*) Indicates a significant difference ( p < 0.01) between uninfected (control) and infected (canine babesiosis) group by the Mann–Whitney test for significance. (") Indicates time of antibabesial treatment. The plots show the mean (dot within box), median (line within box), 25th and 75th percentiles (box), 5th and 95th percentiles (whiskers) minimum and maximum values (x).

Correlations between investigated inflammatory markers are shown in Table 2. There were significant positive correlations ( p < 0.01) between SAA and CRP, ESR and CRP, ESR and SAA, WBC and HPT, WBC and PLT, and WBC and HCT, while significant negative correlations were detected between CRP and PLT, SAA

V. Matijatko et al. / Veterinary Parasitology 144 (2007) 242–250 Table 2 Correlation between inflammatory markers (Spearman rank order) Diagnostic HCT test (rho) CRP SAA HPT ESR PLT WBC

WBC

PLT

ESR

HPT

SAA

0.219* 0.252* 0.725* 0.381* 0.027 0.508* 0.263* 0.192 0.700* 0.331* 0.112 0.158 0.336* 0.179 0.107 0.211 0.281* 0.275* 0.224 0.230* 0.245*

rho—Spearman’s coefficient of rank correlation. * p < 0.01.

and PLT, ESR and PLT, WBC and ESR, WBC and CRP, and HCT and SAA. These results demonstrated the strongest positive correlation between CRP and SAA (rho 0.508) and the strongest negative correlations between CRP and PLT (rho 0.725) and between SAA and PLT (rho 0.700) (Table 2). 4. Discussion Canine babesiosis is an economically important and potentially life-threatening disease which has worldwide distribution, with numerous cases reported throughout the United States, South Africa, Asia and Europe (Anderson et al., 1979; Lobetti, 1998; Zahler et al., 2000; Caccio et al., 2002; Macintire et al., 2002), and which involves several pathophysyological mechanisms that are still not completely understood (Jacobson and Clark, 1994; Boozer and Macintire, 2003). Complicated canine babesiosis has been suggested to be a consequence of the development of SIRS and MODS, both of which are cytokine-mediated phenomena (Jacobson and Clark, 1994; Welzl et al., 2001). Several different haematological tests such as WBC, platelets and ESR have been routinely used in monitoring canine babesiosis (Abdullahi et al., 1990; Page´s et al., 1990; Clark and Jacobson, 1997; Jacobson et al., 2000). These markers were also determined in our study, with the objective of comparing their diagnostic sensitivity and use in treatment monitoring with the acute phase proteins. ESR has been routinely used as a non-specific marker in the diagnosis of infections in human medicine (Sox and Liang, 1986; Gabay and Kushner, 1999) since an accelerated ESR is caused by an increased concentration of acute phase proteins, especially fibrinogen (Ruhenstroth-Bauer et al., 1990). The positive correlation found in our study between ESR and the two major APPs measured (CRP and SAA) could indicate some influence from both APPs in this test and could explain in part the ESR’s relatively high sensitivity (88%) demonstrated in

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our work. However, the results obtained with the ESR test should be interpreted cautiously because a reduced haematocrit can increase the ESR. Therefore, it could not be considered to be an optimal laboratory test for monitoring a haemolytic disease such as canine babesiosis. Regarding other haematological tests, the WBC count was not demonstrated to be adequate in detecting and monitoring canine babesiosis since it had very low sensitivity in our study. Various results with leukopenia as well as leukocytosis have been frequently reported in canine babesiosis (Page´s and Trouillet, 1984; Abdullahi et al., 1990; Reyers et al., 1998; Lobetti, 1998).The very low platelet counts found in 100% of the dogs infected with B. canis in our study (around a 24-fold decrease compared to the mean values of healthy dogs) would support the assertion that severe thrombocytopenia is one of the most striking and consistent haematological abnormalities in babesiosis and it is a routine finding in both complicated and uncomplicated cases (Moore and Williams, 1979; Guelfi et al., 1984; Page´s and Trouillet, 1984; Page´s et al., 1990; Kettner et al., 2003). Although haemolytic anaemia is a well-known haematological abnormality in babesiosis (Lobetti, 1998), not all cases become anaemic enough in clinical practice to explain the severe clinical signs (Jacobson and Clark, 1994; Reyers et al., 1998). It has been suggested that acute phase response could lead to vasodilatation, hypotension and dilution of the blood (Jacobson and Clark, 1994). Our results with significantly decreased haematocrit and increased acute phase protein concentrations in dogs naturally infected with B. canis support the hypothesis postulated by Reyers et al. (1998) that the pathogenesis of babesiosis caused by B. canis consists of two parallel processes: a haemolytic process and an inflammatory process. Measurements of acute phase proteins are a potentially useful clinical tool in veterinary medicine but further studies are required to assure their value in particular diseases, because the acute phase response varies in different species and in different pathological processes (Kent, 1992; Gruys et al., 1994; Murata et al., 2004). In human uncomplicated Plasmodium falciparum malaria, which can be classified as protozoal sepsis like canine babesiosis (Jacobson et al., 2002), clinical studies demonstrated elevated concentration of CRP (Graninger et al., 1992) and Gillespie et al. (1991) reported that sequential measurement of C-reactive protein proved valuable in assessment of the clinical course in this disease. In our study the concentration of CRP was increased in all the dogs with babesiosis. Similarly, increases in CRP have also been reported in

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dogs with other infectious diseases such leptospirosis (Caspi et al., 1987), trypanosomiasis (Ndung’u et al., 1991), parvovirus infection, ehrlichiosis (Rikihisa et al., 1994) and leishmaniasis (Martinez-Subiela et al., 2002). The mean serum concentration of CRP in the canine babesiosis group decreased from day 1 to 8 as a response to treatment. These results were in concordance with a study monitoring short-term treatment in dogs with leishmaniosis (Martinez-Subiela et al., 2003). All the dogs with babesiosis in our study had elevated SAA values on the day of admission, which was not the case for the dogs with leishmaniosis (Martinez-Subiela et al., 2003). Moreover, the concentrations of SAA obtained were significantly higher than in the leishmaniosis study, which could be due to the fact that leishmaniosis is not an acute illness like babesiosis. SAA decreased significantly in response to treatment in our study, which was in concordance with a study monitoring short-term treatment of dogs with leishmaniosis (Martinez-Subiela et al., 2003) and supports the use of this APP for treatment monitoring in cases of canine babesiosis. Haptoglobin in dogs is recognized as a constitutive protein and moderate APP (Conner et al., 1988). The irregular behaviour of the haptoglobin concentrations seen in our study could have been influenced by the mixed effect of the following: (a) inflammation, which produces moderate increases in haptoglobin (Conner et al., 1988; Solter et al., 1991; Martinez-Subiela et al., 2002) and (b) haemolysis which reduces the haptoglobin concentration (Wiedmeyer and Solter, 1996). Therefore, haptoglobin would not be an ideal laboratory test for monitoring haemolytic diseases such as canine babesiosis. Unlike other drugs such as glucocorticoids (Martinez-Subiela et al., 2004), imidocarb dipropionate does not seem to produce an increase in haptoglobin concentration. Overall, the results of this study indicate that B. canis induces marked acute phase response in a host. SAA and CRP (considered major APPs in the dog), are the markers which showed the highest response, with increases of 800-fold and 30-fold, respectively. It is interesting to note that concentrations of CRP and SAA decreased daily after treatment and approached normal by the eighth day. This information suggests the measurement of these parameters may be useful in monitoring response to treatment. Further studies should be performed in order to determine if there could be a correlation among persistently elevated APP, the inability to eradicate the parasite and a persistent carrier state. In addition, both APPs (CRP and SAA)

showed higher sensitivity compared to other routinely used acute phase markers such as the erythrocyte sedimentation rate and WBC count. However, it should be indicated that increases in these proteins have been described in many other conditions such as infectious or parasitic diseases, trauma, surgery and other inflammatory processes, so their specificity is very low, thereby limiting the use of APPs in the diagnostics of babesiosis. Considering that the platelet count and concentrations of CRP and SAA had 100% sensitivity in detecting dogs naturally infected with B. canis; the concurrent presence of thrombocytopenia and increased concentrations of CRP and SAA should raise the suspicion of babesiosis, and multiple peripheral smears or a more sensitive test for the detection of parasite-specific antigen levels should be performed. In a work by Inokuma et al. (2004), cases of babesiosis carriers without clinical signs, supportive laboratory findings such as anaemia or thrombocytopenia and negative blood smear findings but positive PCR were identified. Additional studies should be developed to determine if APPs could be an additional tool in differentiating active infection from the passive carrier state and if in cases when only serology titres are used to diagnose babesiosis, measurement of SAA and CRP could help differentiate exposure titres from active infection. It could be concluded that canine babesiosis caused by B. canis produces an acute phase response with increased concentrations of acute phase proteins. CRP and SAA demonstrated higher sensitivity in detecting canine babesiosis than traditional inflammatory markers such as WBC and ESR. Sequential measurement of Creactive protein and serum amyloid A concentrations proved valuable in monitoring response to antibabesial treatment in uncomplicated canine babesiosis. Acknowledgments This work was partially supported by the Ministry of Science of the Republic of Croatia (Project 0053336). The authors would like to thank to Silvia MartinezSubiela for her friendly contribution in preparing this manuscript. References Abdullahi, S.U., Mohammed, A.A., Trimnell, A.R., Sannusi, A., Alafitayo, R., 1990. Clinical and haematological findings in 70 naturally occurring cases of canine babesiosis. J. Small Anim. Pract. 31, 145–147.

V. Matijatko et al. / Veterinary Parasitology 144 (2007) 242–250 Anderson, J.F., Magnarelli, L.A., Donner, C.S., Spielman, A., Piesman, J., 1979. Canine babesia new to North America. Science 204, 1431–1432. Boozer, A.L., Macintire, D.K., 2003. Canine babesiosis. Vet. Clin. North. Am. Small Anim. Pract. 33, 885–904. Caccio, S.M., Antunovic, B., Moretti, A., Mangili, V., Marinculic, A., Baric-Rafaj, R., Slemenda, S.B., Pieniazek, N.J., 2002. Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs. Vet. Parasitol. 106, 285– 292. Camacho, A.T., Pallas, E., Gestal, J.J., Guitian, F.J., Olmeda, A.S., Goethert, H.K., Telford, S.R., 2001. Infection of dogs in northwest Spain with a Babesia microti-like agent. Vet. Rec. 3 (149), 552–555. Carret, C., Walas, F., Carey, B., Grande, N., Precigout, E., Moubri, K., Schetters, T.P., Gorenflot, A., 1999. Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes. J. Eukaryot. Microbiol. 46, 298–303. Caspi, D., Snell, F.W.J.J., Batt, R.M., Benett, D., Rutteman, G.R., Hartman, E.G., Baltz, M.L., Gruys, E., Pepys, M.B., 1987. Creactive protein in dogs. Am. J. Vet. Res. 48, 919–921. Clark, I.A., Jacobson, L.S., 1997. Do babesiosis and malaria share a common disease process? Ann. Trop. Med. Parasitol. 92, 483– 488. Conner, J.G., Eckersall, P.D., Ferguson, J., Douglas, T.A., 1988. Acute phase response in the dog following surgical trauma. Res. Vet. Sci. 45, 107–110. Gabay, C., Kushner, I., 1999. Acute-phase proteins and other systemic responses to inflammation. N. Engl. J. Med. 340, 448–454. Gillespie, S.H., Dow, C., Raynes, J.G., Behrens, R.H., Chiodini, P.I., McAdam, K.P.W.J., 1991. Measurement of acute phase proteins for assessing severity of Plasmodium falciparum malaria. J. Clin. Pathol. 33, 228–231. Graninger, W., Thalhammer, F., Hollenstein, U., Zotter, G.M., Kremsner, P.G., 1992. Serum protein concentrations in Plasmodium falciparum malaria. Acta Trop. 52, 121–128. Gruys, E., Obwolo, J.M., Toussaint, M.J.M., 1994. Diagnostic significance of the major acute phase proteins in veterinary clinical chemistry: a review. Vet. Bull. 64, 1009–1018. Guelfi, J.F., Dubois, P., Boneu, B., 1984. Exploration de l’he´mostase des chiens atteints de babe´siose. Revue de Me´d. Ve´t. 135, 699– 703. Inokuma, H., Yoshizaki, Y., Matsumoto, K., Okuda, M., Onishi, T., Nakagome, K., Kosugi, R., Hirakawa, M., 2004. Molecular survey of Babesia infection in dogs in Okinawa, Japan. Vet. Parasitol. 121, 341–346. Jacobson, L.S., Clark, I., 1994. The pathophysiology of canine babesiosis: new approaches to an old puzzle. J. S. Afr. Vet. Assoc. 65, 134–145. Jacobson, L.S., Lobetti, R.G., Waughan-Scott, T., 2000. Blood pressure changes in dogs with babesiosis. J. S. Afr. Vet. Assoc. 71, 14–20. Jacobson, L.S., Lobetti, R.G., Becker, P., Reyes, F., Waughan-Scott, T., 2002. Nitric oxid metabolites in naturally occuring canine babesiosis. Vet. Parasitol 104, 27–41. Kent, J., 1992. Acute phase proteins: their use in veterinary diagnosis. Br. Vet. J. 148, 279–282. Kettner, F., Reyers, F., Miller, D., 2003. Thrombocytopenia in canine babesiosis and its clinical usefulness. J. S. Afr. Vet. Assoc. 74, 63– 68.

249

Kjelgaard-Hansen, M., Kristensen, A.T., Jensen, A.L., 2003. Evaluation of a commercially available enzyme-linked immunosorbent assay (ELISA) for the determination of C-reactive protein in canine serum. J. Vet. Med. 50, 164–168. Lobetti, R.G., 1998. Canine babesiosis. Comp. Cont. Educ. Pract. Vet. 20, 418–431. Lobetti, R.G., 2000. Canine babesiosis. In: Day, M., Mackin, A., Littlewood, J. (Eds.), Manual of Canine and Feline Haematology and Transfusion Medicine. British Small Animal Veterinary Association, Gloucester, pp. 85–91. Lobetti, R.G., Mohr, A.J., Dipenaar, T., Myburgh, E., 2000. A preliminary study on the serum protein response in canine babesiosis. J. S. Afr. Vet. Assoc. 71, 38–42. Macintire, D.K., Boudreaux, M.K., West, G.D., Bourne, C., Wright, J.C., Conrad, P.A., 2002. Babesia gibsoni infection among dogs in the southeastern United States. J. Am. Vet. Med. Assoc. 220, 325– 329. Martinez-Subiela, S., Tecles, F., Eckersall, P.D., Ceron, J.J., 2002. Serum concentrations of acute phase proteins in dogs with leishmaniasis. Vet. Rec. 150, 241–244. Martinez-Subiela, S., Bernal, L.J., Ceron, J.J., 2003. Serum concentrations of acute-phase proteins in dogs with leishmaniosis during short-term treatment. Am. J. Vet. Res. 64, 1021–1026. Martinez-Subiela, S., Ginel, P.J., Ceron, J.J., 2004. Effects of different glucocorticoid treatments on serum acute phase proteins in dogs. Vet. Rec. 154, 814–817. Martinez-Subiela, S., Tecles, F., Parra, M.D., Ceron, J.J., 2005. Validacio´n analı´tica de te´cnicas comerciales para la determinacio´n de haptoglobina, proteı´na C reactiva y amiloide A se´rico en caninos [Analytical validation of commercial techniques for haptoglobin, C reactive protein and serum amyloid A determinations in dogs]. Arch. Med. Vet. 37, 61–66. Matijatko, V., Kucer, N., Baric Rafaj, R., Forsek, J., Kis, I., Potocnjak, D., Razdorov, G., Mrljak, V., 2002. CRP concentration in dogs with uncomplicated and complicated babesiosis. In: Proceedings of the Third European Colloquium on Food Safety and Acute Phase Proteins, Doorn, 2002. Moore, D.J., Williams, M.C., 1979. Disseminated intravascular coagulation: a complication of Babesia canis infection in the dog. J. S. Afr. Vet. Assoc. 50, 265–275. Moshage, H.J., 1997. Cytokines and the hepatic acute phase response. J. Pathol. 181, 257–266. Murata, H., Shimada, N., Yoshioka, M., 2004. Current research on acute phase proteins in veterinary diagnosis: an overview. Vet. J. 168, 28–40. Ndung’u, J.M., Eckersall, P.D., Jennings, F.W., 1991. Elevation of the concentration of acute phase proteins in dogs infected with Trypanosoma brucei. Acta Trop. 49, 77–86. Page´s, J.P., Trouillet, J.L., 1984. Thrombocytope´nie dans la babe´siose du chien: a` propos de 153 observations. Pratique Me´dicale et Chirurgicale de l’Animal de Compagnie 19, 222–227. Page´s, J.P., Vidor, E., Trouillet, J.L., Bissuel, G., Lecointre, O., Moreau, Y., 1990. Description clinique, he´matologique et serologique de 133 cas de babe´siose canine. Pratique Me´dicale et Chirurgicale de l’Animal de Compagnie 25, 89–97. Pannen, B.H.J., Robotham, J.L., 1995. The acute-phase response. New Horiz. 3, 183–197. Passos, L.M.F., Geiger, S.M., Ribeiro, M.F.B., Pfister, K., ZahlerRinder, M., 2005. First molecular detection of Babesia vogeli in dogs from Brasil. Vet. Parasitol. 127, 81–85. Reyers, F., Leisewitz, A.L., Lobetti, R.G., Milner, R.J., Jacobson, L.S., 1998. Canine babesiosis in South Africa: more than one disease.

250

V. Matijatko et al. / Veterinary Parasitology 144 (2007) 242–250

Does this serve as a model for falciparum malaria? Ann. Trop. Med. Parasitol. 92, 503–511. Rikihisa, Y., Yamamoto, S., Kwak, I., Iqbal, Z., Kociba, G., Mott, J., Chichanasiriwithaya, W., 1994. C-reactive protein and alpha-1acid-glycoprotein levels in dogs infected with Ehrlichia canis. J. Clin. Microbiol. 32, 912–917. Ruhenstroth-Bauer, G., Schedler, K., Scherer, R., Vesterberg, O., 1990. On the possibility of differential diagnosis at elevated erythrocyte sedimentation rate by analysis of the concentrations of blood plasma proteins—a model study. J. Clin. Chem. Clin. Biochem. 28, 845–850. Schetters, T.P., Moubri, K., Precigout, E., Kleuskens, J., Scholtes, N.C., Gorenflot, A., 1997. Different Babesia canis isolates, different diseases. Parasitology 115, 485–493. Schetters, T., 2005. Vaccination against canine babesiosis. Trends Parasitol. 21, 179–184. Solter, P.F., Hoffmann, W.E., Hungerford, L.L., Siegel, J.P., St. Denis, S.H., Dorner, J.L., 1991. Haptoglobin and ceruloplasmin as determinants of inflammation in dogs. Am. J. Vet. Res. 52, 1738–1742. Sox, H.C., Liang, M.H., 1986. The erythrocyte sedimentation rate. Guidelines for rational use. Ann. Inter. Med. 104, 515–523.

Steel, D.M., Whitehead, A.S., 1994. The major acute phase reactants: C-reactive protein, serum amyloid P component and serum amyloid A protein. Immunol. Today 15, 81–87. Taboada, J., Merchant, S.R., 1991. Babesiosis of companion animals and man. Vet. Clin. North Am. Small Anim. Pract. 21, 103–123. Uilenberg, G., Franssen, F.F.J., Perrie, N.M., 1989. Three groups of Babesia canis distinguished and a proposal for nomenclature. Vet. Q. 11, 33–40. Welzl, C., Leisewitz, A.L., Jacobson, L.S., Vaughan-Scott, T., Myburgh, E., 2001. Systemic inflammatory response syndrome and multiple-organ damage/dysfunction in complicated canine babesiosis. J. S. Afr. Vet. Assoc. 72, 158–162. Wiedmeyer, C.E., Solter, P.F., 1996. Validation of human haptoglobin immunoturbidimetric assay for detection of haptoglobin in equine and canine serum and plasma. Vet. Clin. Pathol. 25, 141–146. Zahler, M., Schein, E., Rinder, H., Gothe, R., 1998. Characteristic genotypes discriminate between Babesia canis isolates of differing vector specificity and pathogenicity to dogs. Parasitol. Res. 84, 544–548. Zahler, M., Rinder, H., Zweygarth, E., Fukata, T., Maede, Y., Schein, E., Gothe, R., 2000. ‘‘Babesia Gibsoni’’ of dogs from North America and Asia belong to different species. Parasitology 120, 359–365.