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Molecular fluorescent approach to assessing intraerythrocytic hemoprotozoan Babesia canis infection in dogs
Veterinary Parasitology 125 (2004) 221–235 www.elsevier.com/locate/vetpar
Molecular fluorescent approach to assessing intraerythrocytic hemoprotozoan Babesia canis infection in dogs Kelly Alves Bicalhoa,b,c, Mu´cio F. Barbosa Ribeirob, Olindo Assis Martins-Filhoa,* a
Laborato´rio de Doenc¸a de Chagas, Centro de Pesquisas Rene´ Rachou, FIOCRUZ, Av. Augusto de Lima 1715, 30 190-002, Belo Horizonte, Brazil b Departamento de Parasitologia, ICB, Universidade Federal de Minas Gerias, Belo Horizonte, Brazil c Biote´rio de Produc¸a˜o, Centro de Pesquisas Rene´ Rachou, FIOCRUZ, Av. Augusto de Lima 1715, 30 190-002, Belo Horizonte, Brazil Accepted 4 August 2004
Abstract The development of recent flow cytometry-based protocols for the diagnosis of canine babesiosis, Babesia gibsoni in particular, has encouraged us to investigate its applicability to detect B. canisinfected erythrocytes as well as optimize the hydroethidine-flow cytometry methodology (HE-FC), using peripheral blood samples from naturally and experimentally infected dogs. Our data demonstrated that HE at 25 mg/ml provided the most outstanding fluorescence profile, able to discriminate between infected and uninfected dogs with no alterations in cell properties such as forward scatter and unspecific fluorescence. The results were expressed as the percentage of positive fluorescent erythrocytes (PPFE) for each individual sample, with 1.53% of PPFE as the cut-off determined between infected and uninfected animals. B. canis-infected erythrocytes during both acute and chronic experimental infection were identified through HE-FC, validating its use for diagnosis purposes in endemic areas for canine babesiosis. In a clinical trial, 22.8% out of 162 dogs showed to be positive to Babesia infection through this approach. Such prevalence was similar to that estimated for altered hematological profiles (HT) 30% (29%), but highly distinct from the prevalence provided by direct blood smear (BS) examination (1.8%) or immunofluorescent assay (IFA) (60.5%). Furthermore, our findings indicate that positive PPFE data was associated with HT 30%, * Corresponding author. Tel.: +55 31 3295 3566; fax: +55 31 3295 3115. E-mail address: [email protected] (O.A. Martins-Filho). 0304-4017/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.08.009
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emphasizing that, in clinical practice, the haematocrit should be used as a screening test followed by HE-FC, suitable to confirm hypotheses of canine babesiosis. # 2004 Elsevier B.V. All rights reserved. Keywords: Flow cytometry; Babesia canis; Diagnosis
1. Introduction Canine babesiosis is one of the most important tickborne, protozoal hemoparasitic diseases. The intraerythrocytic hemoparasite, Babesia canis, invades and replicates within canine red blood cells, causing varying degrees of hemolytic anemia and multiple-organ dysfunction (Lobetti, 1998). Prompt and accurate diagnosis is difficult because the signs and symptoms are non-specific and thus, correctly identifying the infectious agent is important for treatment and prognosis. Although hemolytic anemia is the distinctive feature of canine babesiosis, definitive laboratory diagnosis of active babesiosis requires the visualization of Babesia organisms within infected erythrocytes (Lobetti, 1998). The available serological tests to detect anti-Babesia specific antibodies have restricted applicability since detectable antibody levels persist after parasite clearance due to the residual humoral immunity. In addition to the limited suitability of serological tests for endemic areas, in early stages of infection, when false-negative results may occur, they have shown not be sensitive. (Taboada and Merchant, 1991). One of the major challenges regarding the diagnosis of canine babesiosis is the lack of accurate laboratory approaches to elucidating cases of babesiosis in clinical practice. Despite its high specificity, the blood smear examination, using conventional optical microscopy with panoptic or GIEMSA staining protocol, presents low sensitivity (Breitschwerdt et al., 1983). Moreover, the detection of parasites in most atypical or chronic cases may become difficult due to scarce parasitemia (Anderson et al., 1980; Wlosniewski et al., 1997). Exceptional advances have been made in the diagnosis of canine babesiosis after the development of an easy protocol to detect Babesia gibsoni-infected erythrocytes in experimentally infected dogs by flow cytometry, using a single color staining for peripheral blood samples (Fukata et al., 1996). The rapid multiparametric examination of individual red blood cells has made the flow cytometry an useful tool for quantitative diagnosis of canine babesiosis. The basis of this methodology is the use of hydroethidine (HE), a compound that is taken up by viable cells and metabolically converted to ethidium bromide – a DNA-binding fluorochrome. The present investigation was designed to determine whether the HE-FC methodology was applicable to diagnose B. canis-infection using peripheral blood samples from naturally or experimentally infected dogs.
2. Material and methods 2.1. Parasites and animals B. canis organisms were obtained, at the peak of parasitemia, from peripheral blood of splenectomized mongrel pups (4–5-months old), with positive results by immunofluor-
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escent assay (IFA). Samples of B. canis infected blood were used to inoculate seven splenectomized mongrel IFA-negative dogs, confined in a holding kennel after birth at Escola de Veterina´ ria of Universidade Federal de Minas Gerais (EV/UFMG) (Veterinary School of the Federal University of Minas Gerais State), in Belo Horizonte, State of Minas Gerais, Brazil, in order to be out of tick contact. These animals were used as positive controls during the HE-FC standardization procedures. Other seven uninfected IFAnegative pups (4–5 months old), used as negative controls, were also confined in the holding kennel after birth. Peripheral blood samples were collected at days 3–5 after inoculation to search for B. canis-infected erythrocytes. A follow-up was daily carried out, during 28 consecutive weeks, to search for persistent B. canis-infected erythrocytes during chronic disease cases. A clinic trial was also performed; including dogs (1 month–15 years old) attended for ordinary clinical appointments, at the EV/UFMG and at two private veterinary clinics. Therefore, a randomized sample was used for the clinic–laboratory trial. The samples size was calculated as: n = p(100 p)z2/(pd)2, where n is the sample size, p the expected prevalence of positive B. canis IFA at veterinary clinics in Belo Horizonte, Minas Gerais (p = 40%), z the freedom degree (1.96 for 95%) and d the expected error (d = 0.2) (Spiewak, 1992). In order to increase confidence degree, the original sample size of 144 was increased in 12%. A total of 162 EDTA–peripheral blood samples for parasitological examination, hematological evaluation, HE-FC and serological studies were obtained by venipuncture. 2.2. Parasitological, serological and hematological tests Parasitological diagnosis of B. canis infection was performed by blood smears (BS), applied over a grease-free glass slide and stained through the panoptic staining kit (Laborclin, Pinhais, Parana), and examined under a total of 40 conventional microscopy bright fields with oil immersion (1000). Data were expressed as percentage of infected erythrocytes. Serological tests were carried out using frozen samples maintained at 20 8C for 2 months. Each other serial dilution (1:40–2.560 in phosphate buffered saline) was tested for anti-B. canis IgG antibody by indirect immunofluorescence assay (IFA), as recommended by Instituto Interamericano de Cooperacio´ n para la Agricultura-IICA (1987) and modified by Ribeiro et al. (1990). The results were found to be positive when antibody titer was 1:40. Negative and positive controls were also included. The hematological profile was assessed by using a micro-haematocrit protocol. Haematocrit percentage (HT) was determined for each sample macroscopically. Results were interpreted using normal reference values ranging from 30 < HT 55%. Thus, HT values 30% were considered to be positive for anemia. 2.3. Hydroethidine-flow cytometry (HE-FC) One millilitre of EDTA–whole blood samples were washed with 10 ml of PBS by centrifugation at 600 g for 7 min, RT. The supernatant and buffy-coat were discarded. Packed red blood cells (RBC) were resuspended and washed once with 10 ml of PBS at 600 g for 7 min, RT 25 ml of packed RBC were transferred to 5 ml of PBS in order to
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obtain a 0.005% suspension. Hydroethidine (Polysciences, Warrigton, PA, USA) was solubilized in anhydrous DMSO (Sigma Chemical, St. Louis, MI, USA) at 10 mg/ml as stock solution and diluted into PBS to obtain the final concentrations used, including 12.5, 25.0 and 50.0 mg/ml. A volume of 50 ml of RBC suspension in PBS was incubated in 96 well round bottom plates in the presence of 150 ml of HE solution in PBS at final concentrations. Suspensions were incubated at 37 8C for 20 min in a dark humidified incubator and then centrifuged at 600 g for 7 min, RT. Supernatants were discarded and PBS was added to turn HE-stained erythrocyte suspension to a total volume of 300 ml. Internal control samples were incubated in PBS with no HE. Flow cytometric measurements were performed on a Becton Dickinson FACScalibur interfaced to a digital apple computer. The CellQuest software package was used for data acquisition, storage and analysis. Stained erythrocytes were run in the cytometer and 30,000 events per sample were counted. Erythrocytes were first identified on the basis of their forward specific and side specific light scattering (FSC and SSC, respectively) properties, after adjustments of size and granularity gains with values of E00 and 300, respectively, both on log scale (Fig. 1a). After adjustments, erythrocytes were found by assuming a characteristic of homogeneous distribution, using FSC versus SSC dot plot. A gate surrounding the erythrocyte population was set to exclude noise from debris and platelets. Averages of 28,000–29,000 gated erythrocytes were analyzed for their relative ethidium bromide fluorescence intensity, recognized as fluorescence type 2 (FL-2). This protocol allows us to identify Babesia-infected erythrocytes on FSC versus FL-2 dot plots (Fig. 1b and c). A marker was initially set up using the internal control, that received 50 ml of RBC suspension and 150 ml of PBS (Fig. 1b) and determination of the percentage of fluorescent positive erythrocytes (PPFE) for a given sample (Fig. 1c). 2.4. Statistical analysis Comparative analysis between the performance of BS, IFA, HT and HE-FC was performed by analysis of variance (ANOVA) followed by Student’s t-test. Comparative analysis of prevalence at individual level as well as the scattering of B. canis infection detected by HE-FC as regards semi-quantitative HT was carried out by the chi-square test. Significance was defined in both test as P < 0.05.
3. Results 3.1. HE-FC optimization procedures 3.1.1. Effect of HE concentration on the PPFE values In order to evaluate the effect of HE concentration on the performance of ethidium bromide binding to B. canis DNA, we carried out a parallel study with three HE final concentrations, 12.5, 25.0 and 50.0 mg/ml (Fig. 2). Our data demonstrated that in spite of HE concentration, PPFE values from B. canis infected dogs differ significantly from those of unifected dogs. In addition, our results showed that incubation of erythrocytes with 50 mg/ml of HE significantly increases PPFE values for uninfected dogs in comparison to
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Fig. 1. Density plot analysis of B. canis infected erythrocytes by flow cytometry. Erythrocytes were first identified on the basis of their specific forward (FSC size) and side (SSC granularity) light scattering properties. A gate surrounding the erythrocyte population was set to exclude noise and platelets (Fig. 1a). Gated erythrocytes were analyzed for their relative HE fluorescence intensity allowing the identification of Babesia-infected erythrocytes on FSC versus FL-2 dot plots. A marker was set up using the internal control, which received only PBS (Fig. 1b) and used to determine the percentage of fluorescent positive erythrocytes (PPFE) for a given sample (Fig. 1c).
the other concentrations of 25.0 and 12.5 mg/ml (P < 0,05). However, no effect was observed on PPFE values for B. canis-infected animals (P = 0.561). Analysis of density plot distributions of labeled erythrocytes based on their size (FSC) and fluorescence properties revealed that 50 mg/ml altered the FSC property and led to increased autofluorescence of erythrocytes from B. canis-infected animals (Fig. 3). No changes in red blood cell scattering properties were observed for uninfected dogs (Fig. 3b–d). Since no changes on PPFE values were detected for samples from B. canis-infected pups, regardless HE concentration (Fig. 3e–g), we have chosen HE at 25.0 mg/ml as the ideal condition to be used in further investigations in order to guarantee the sensitivity of HE-FC methodology, unlikely to provide false positive results. Moreover, as illustrated in Fig. 3, the concentration of 25.0 mg/ml HE (Fig. 3f) led to the most outstanding fluorescence profile for B. canis-infected erythrocytes, without modifying forward scatter property and with minor interference on autofluorescence, in comparison to HE at 50 mg/ml (Fig. 3g).
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Fig. 2. Effect of HE concentration on the PPFE values. Samples of 50 ml of RBC suspension 0.005% in PBS from uninfected (*) and B. canis infected dogs (*) were incubated in the presence of HE solution at final concentrations of 12.5, 25.0 and 50.0 mg/ml, as described in Section 1. Results are presented in scattering graph as percentage of fluorescent positive erythrocytes (PPFE) for each individual test. Statistical analysis showed that PPFE values from B. canis infected dogs differ significantly from those from uninfected dogs (P < 0.05). No effect was observed on PPFE values for B. canis-infected animals (P = 0.561).
3.1.2. Determination of a cut-off value for PPFE between uninfected and infected dogs In order to determine a cut-off between positive and negative samples, we have performed a parallel analysis of 14 blood samples from pups, from which seven were uninfected and seven experimentally infected, confined in a holding kennel after birth at the EV/UFMG, out of tick contact. HE-FC methodology was applied using HE at 25.0 mg/ ml, as described in Section 1.1. Our data demonstrated that PPFE from uninfected pups ranged from 0.31 to 1.27% whereas PHFP from B. canis-infected dogs varied from 10.8 to 22.5% (Fig. 4). Following the detected cut-off as PPFE mean value from uninfected group (1.01 0.265) added to two standard deviations (cutoff = [¯x + 2s]), data analysis allowed us to determine 1.53% as being the best PPFE cut-off between negative and positive results. 3.2. Performance of BS and HE-FC for detection of B. canis chronic infection A follow-up study was carried-out to determine the sensitivity of BS and HE-FC for identifying B. canis infected erythrocytes in experimentally infected dogs over 28 weeks after challenge. EDTA–whole blood samples were collected daily, during 28 consecutive weeks. Two different laboratory approaches were used for evaluating the status of B. canis chronic infection cases. Data from this follow-up are presented in Fig. 5 expressed as weekly mean percentage of infected erythrocytes.
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Fig. 3. Density plots distributions of labeled erythrocytes based on their size (FSC) and fluorescence (FL-2) properties. No changes on RBC scattering properties were observed for uninfected dogs despite HE conventration of 12.5, 25.0 and 50.0 mg/ml (a, b and c, respectively). Incubation of RBC from B. canis infected dogs with 50 mg/ml of HE disturbed the FSC property and led to increased autofluorescence of erythrocytes from B. canis-infected animals (Fig. 3f). On the other hand, incubation of RBC with 12.5 mg/ml of HE led to lower PPFE (Fig. 3d). For further analysis we have chosen HE at 25.0 mg/ml (e) as the ideal condition to be used on further investigations, in order to guarantee the sensitivity of HE-FC methodology, and led to a more outstanding fluorescence profile for B. canis-infected erythrocytes.
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Fig. 4. Establishment of a cut-off of PPFE between uninfected and B. canis-infected dogs. Parallel analysis of blood samples from uninfected (*, n = 7) and experimentally infected (*, n = 7) dogs were tested in HE-FC using HE at 25.0 mg/ml. Data are expressed as scattering of PPFE values for each group. Taking the cut-off definition as [¯x + 2s], PPFE = 1.53% was found as an edge between negative and positive results.
Thin blood smear (BS), labelled with panoptic staining to demonstrate the organism, revealed that B. canis infected erythrocytes could be found as early as only 2 days after infection, with BS = 0.24% as average parasitemia level for the first week. Parasitemia was oscillating between 0 and 0.01% as average parasitemia level from 2nd to 16th week, becoming undetectable after the 17th week post-infection (Fig. 5). Our data demonstrated that HE-FC approach was also able to identify B. canis infected erythrocytes 2 days after infection, with HE-FC = 2.60% as average for parasitemia level during the first week. However, this prevalence was higher than that observed by BS. Further, HE-FC could also detect B. canis infected erythrocytes from the 1st week throughout the whole follow-up study, with average parasitemia levels oscillating between 3.87 and 21.63%, but never absent as observed by BS. This marked difference between HE-FC and BS demonstrated the higher sensitivity of flow cytometry to identify chronic B. canis infection (Fig. 5). 3.3. HE-FC application in clinical trials 3.3.1. Performances of BS, IFA 1:40, HT 30% and HE-FC for evaluating the prevalence of B. canis infection on clinical survey Blood samples were collected from 162 randomly selected dogs in order to determine the prevalence of B. canis infection in dogs attended at the EV/UFMG and at two Private Veterinary Clinics in Belo Horizonte. Four different laboratory approaches were used to evaluate the status of B. canis infection. Data from this clinical trial are presented in Fig. 6. Thin blood smears (BS), labeled with panoptic staining, were examined for the organism determination and revealed that only 3 out of 162 dogs (1.8%) were positive for B. canis infection. On the other hand, serological tests by indirect immunofluorescence antibody
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Fig. 5. Performance of blood smear (BS *) and Flow Cytometry (HE-FC *) for detection of B. canis in acute and chronic experimental infection. Sensitivity of BS and HE-FC for identifying B. canis infected erythrocytes in experimentally infected dog was evaluated over 28 weeks after challenge. Parasitaemia by BS was oscillating between 0 and 0.01% from 2nd to 16th week, becoming undetectable after the 17th week post-infection. Parasitaemia by HE-FC was detect from 2nd week to the end of the follow-up study, with average parasitaemia level oscillating between 3.87 and 21.63%, but never absent as observed by BS.
assay (IFA), considered positive when titers were higher or equal than 1:40, demonstrated the presence of antibodies against B. canis in 98 out of 162 animals (60.5%). The prevalence of B. canis infection was also estimated by interpreting values of haematocrit 30% as suggestive of hemoparasite infection. Results of laboratory screening considering HT 30% suggested that babesiosis would be prevalent in 47 out of 162 animals (29%). Our data demonstrated that HE-FC approach was able to identify 22.8% of dogs as being positive to Babesia infection (37 out of 162 animals). This prevalence was similar to that estimated through hematological profile analysis – HT 30%, but markedly different from that identified by direct BS or IFA (P < 0.05).
Fig. 6. Performances of BS, IFA 1:40, HT 30% and HE-FC for evaluating the prevalence of B. canis infection on clinical survey. Data are expressed as prevalence of positive results at population level (n = 162) for each test used. Different letters (a–c) represent statistically significant differences at P < 0.05. Detection of B. canis infections through BS revealed that only 3 out of 162 dogs were positive. Serology by IFA demonstrated the presence of antibodies against B. canis in 98 out of 162 animals. HT 30% suggested that babesiosis was prevalent in 47 out of 162 animals. HE-FC identified 37 out of 162 dogs as positive to Babesia infection.
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Fig. 7. Comparative analysis of B. canis prevalence detected by BS, IFA and HT with HE-FC performance at individual level (n = 162) on clinical survey. Data are expressed as scattering PPFE. Different letters (a and b) represent statistically significant differences at P < 0.001. Samples were first categorized as negative or positive based on their performance on BS, IFA 1:40 and HT 30% and then analyzed for HE-FC. These data confirmed that positive HE-FC correlates better with HT 30% than BS and IFA, showing that 44.7% of animals with HT 30% were prone to be positive in HE-FC.
3.3.2. Comparative analysis of BS, IFA and HT with HE-FC performance at individual level on clinical survey In order to further characterize the high degree of similarity between B. canis infection prevalence estimated by HE-FC and HT 30%, we performed a comparative analysis of PPFE data with those obtained by BS, IFA 1:40 and HT 30% for each individual sample. For this purpose, a number of 162 samples were first categorized as negative or positive based on results provided by BS, IFA 1:40 and HT 30% and then analyzed by HE-FC, using PPFP cut-off of 1.53 between negative or positive results (Fig. 7). Our data confirmed, by analyzing individual samples, that positive HE-FC correlates better with HT 30%, showing that 44.7% of animals with HT 30% were prone to be positive in HE-FC with PPFE higher than 1.53 (P 0.001). No significant relationship was observed between HE-FC results when data were first categorized based on BS or IFA results. 3.3.3. Scatter of B. canis infection detected by HE-FC concerning semi-quantitative HT Our data have suggested a strong association between the results from HT 30% and the positivity of HE-FC. To further focus this issue, we selected the dogs based on their HT results, using a semi-quantitative distribution including six categories (05–09%, 10–14%, 15–19%, 20–24%, 25–30% and >30%), and then evaluated the prevalence of positive HEFC results within each HT category (Fig. 8). Data analysis demonstrated a higher prevalence of positive HE-FC as HT became lower than or equal to 24%, with prevalence ranging from 50% (05–09%) and 56 (10–14%) to 60 (15–19% and 20–24%). Lower prevalence was observed for HT from 25–30% (29%) but meaningful only when HT >30% (13.9%). Statistical analysis was based on Chi-square test at a significance level of P 0.01.
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Fig. 8. Scatter of B. canis infection detected by HE-FC as regards semi-quantitative HT on clinical survey. Samples were categorized based on their HT results using a semi-quantitative distribution and then evaluated for the prevalence of positive HE-FC results within each HT category. Data are expressed as mean prevalence of positive HE-FC results for each HT category. Different letters (a and b) represent statistically significant differences at P < 0.01, except for HT 25–30% in comparison to HT >30%. There was a higher prevalence of positive HE-FC as HT became 24% whereas lower prevalence was observed for HT at 25–30% (29%) but with significance only when HT >30% (13.9%).
4. Discussion Considering canine babesiosis prevalence in large urban centers, the lack of an accurate and sensitive laboratory method for definitive diagnosis is still a major challenge in veterinary clinics. In addition, chronic evolution of the disease makes the clinical and laboratory diagnoses difficult due to low parasitemia levels. Efforts to standardize protocols for new approaches to detect Babesia infected erythrocytes have brought out possible techniques for quantitative analysis of organisms accurately measured over a wide range of parasitemia. Flow cytometric methods using membrane-permeable fluorochrome, thiazole orange, have been described for the detection of parasite-infected erythrocytes (Howard and Rodwell, 1979; Makler et al., 1987). However, Uemura et al. (1990) remarked that thiazole orange was unable to identify B. gibsoni in those cells. On the other hand, when hydroethidine staining was used, B. bovisinfected erythrocytes were detected (by flow cytometry Wyatt et al., 1991). Hydroethidine (HE) is a relatively new vital dye, which is converted to ethidium when taken up by living cells (Davis et al., 1992; Gallop et al., 1984). Since ethidium is a fluorochrome excited at 488 nm and emits at 585 nm, when intercalated into DNA molecules, it can be used in flow cytometry. Thus, it is now possible to monitor the viability and growth features of protozoa parasites such as Babesia, following exposure of hosts to infection. In addition to providing an accurate evaluation of intracellular growth as a viability assay, HE also excludes reticulocytes staining, which is unable to have HE converted into ethidium dye. Therefore, HE has been pointed out as a new method for detection and analysis of parasite-infected erythrocytes due to its sensitivity and reproducibility, in addition to being a short laboratory procedure. Here, we have described an optimized methodology to identify B. canis infected erythrocytes in peripheral blood samples from mongrel dogs. After testing a range of HE
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concentrations, we found that 25 mg/ml HE provided the most outstanding fluorescence profile able to discriminate between infected and uninfected dogs, without changing other cell properties such as forward scatter and auto-fluorescence. Using this standardized protocol in further analyses, we expressed our results as the percentage of positive fluorescent erythrocytes (PPFE) for each individual sample, establishing 1.53% of PPFE as the cut-off between infected and uninfected animals. The high performance of HE-FC method described here is in agreement with those reported by Wyatt et al. (1991), Davis et al. (1992) and Fukata et al. (1996) who identified other Babesia species by flow cytometry using HE/ethidium as a fluorescent dye. In this study, sensitivity of BS and HE-FC for identifying B. canis infected erythrocytes, in acute and chronic experimentally infected dogs, was assessed over 28 weeks after challenge. Our data have confirmed the low sensitivity of blood smear examination to diagnose chronic babesiosis, which is consistent with previous reports (Anderson et al., 1980; Breitschwerdt et al., 1983; Levy et al., 1987; Wlosniewski et al., 1997). Furthermore, we demonstrated that HE-FC was able to identify B. canis infected erythrocytes during both acute and chronic experimental infection, validating its use for diagnosis purposes in endemic areas for canine babesiosis. One of the major advantages of using flow cytometry, besides automation, is the speed with which data can be obtained. HE-FC is a rapid methodology that yields final data within 2–3 h, even when processing a large number of samples. Animals with signs of B. canis infection, usually raging from anorexia, central nervous system depression and lethargy to weakness and anemia, require a differential diagnosis for an accurate interpretation and preclusion of other diseases, as previously mentioned, which is important for early treatment planning and prognosis. By using the standardized HE-FC methodology, we performed a clinical trial and identified 37 out of 162 dogs with positive results, showing higher sensitivity (22.8%) in comparison to blood smear examination that detected only 1.8% of Babesia infection. This low prevalence of infection detected by BS, in dogs attended at the two private veterinary clinics, supports previous reports regarding low sensitivity of conventional parasitological methods (BS) to detect B. canis infected erythrocytes, mainly in atypical and chronic cases, when scarce parasitemia is reported (Anderson et al., 1980; Breitschwerdt et al., 1983; Levy et al., 1987; Wlosniewski et al., 1997). In addition, despite its high specificity and straightforwardness, blood smear examination is laborious and time-consuming, especially when a large number of samples must be evaluated. Regarding the use of immunological methods for B. canis diagnosis, our data have confirmed the findings of early reports in endemic areas, where antibodies against B. canis are more prevalent than are current B. canis infection in dogs (Martinod et al., 1986). Our results demonstrated a high prevalence (60.5%) of seropositivity to B. canis, as detected by IFA. These data are markedly different from those obtained by BS, which probably also under estimated B. canis infection. Despite the fact that the IFA test appears to represent a reliable method to detect infected dogs with patent and sub patent parasitemia, due to its high sensitivity to detect antiBabesia antibodies during chronic disease, limitations regarding cross-reactivity and lack of association between a positive serology and active disease have led to criticism of IFA test interpretations (Breitschwerdt et al., 1983; Ristic, 1988). Moreover, the seronegativity in B. canis infection, mainly during acute disease, may also contribute limitations of the
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IFA test as a diagnostic tool for canine babesiosis. The reason why infected dogs failed to produce antibodies against the parasite may represent multiple phenomena (Weiland, 1982; Ristic, 1988; Assis, 1993; Vercammen et al., 1995). Herein, we have found that 14 out of 98 dogs with negative serology presented positive HE-FC results. Further analysis revealed that three out of these animals were less than 4 months of age, presenting low HT values and typical clinical signs of canine babesiosis, consistent with B. canis infection, and suggesting that they probably needed some latent period to produce antibodies. This is borne out by the findings of Bodabe et al. (1989) that most seronegative dogs in southwestern Nigeria were less than 1-year old and seroconvertion could be notified 2 weeks after initial examination. Similar results have also been reported by Farwell et al. (1982) and Breitschwerdt et al. (1983), showing seronegativity in B. canis-infected young pups. Another reason for seronegative results in infected animals is that some dogs require a booster challenge to produce specific antibodies against the parasite (Bodabe et al., 1989) In addition, IFA application has another restriction for use in endemic areas because of the presence of animals chronically infected with low antibody titers and those presenting residual serology after disease clearance. Herein we have detected a similar prevalence of B. canis-infected dogs estimated by either HT 30%, as suggestive of hemoparasite infection (29%), or PHFP 1.53%, as a positive result for B. canis infection (22.8%). The similarity between these findings was further confirmed, at an individual level, showing that positive HE-FC correlates better with HT 30%, showing 44.7% of animals with HT 30%, being prone to be positive in HE-FC, which displays PPFE higher than 1.53%. Since PPFE 1.53% was predominantly higher in animals with HT 30, low HT values might be due to the hemolytic anemia caused by B. canis multiplication and erythrocyte lysis (Reyers et al., 1998; Lobetti, 1998). Despite associations between hemolytic anemia and canine babesiosis that have been explained, at least in part, by the immune mediated hemolysis caused by antibodies associated to erythrocyte membrane, our findings did not show any association between IFA titers and HT values (data not shown). Most HE-FC positive results were found in the dogs with low levels of antibodies (IFA) = 1:40 (data not shown)—suggesting, as previously reported by Ristic (1988), that serological data may reflect parasite multiplication into the host but parasitemia levels are not associated with antibody titers. Thus, these results suggest that HT could be used as a screening approach to identify suspect dogs with a hematological profile of HT 30, which should be further evaluated by HE-FC to confirm the presence of B. canis infected erythrocytes.
5. Conclusion The technique described here provides an useful approach to quantify intraerythrocytic B. canis applicable to the diagnosis of infection. Furthermore, our findings indicate that a positive PPFE result was associated with HT 30%, emphasizing that, in clinical practice, the haematocrit should be used as a screening test followed by HE-FC, suitable to confirm diagnosis of canine babesiosis. Moreover, as HE is as a vital dye, this methodology also can be used to monitor parasite clearance after treatment. This feature is important because it will enable us to more effectively monitor the impact of treatment on intraerythrocytic
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parasite growth, a necessary prerequisite for cure assessment after specific chemotherapy. Evaluation of HE-FC performance as a cure criterion after treatment is currently under evaluation in our laboratory.
Acknowledgments This work was supported by Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil and Fundac¸a˜ o Oswaldo Cruz (FIOCRUZ-BH). We are grateful to Dr. Rodrigo Correa-Oliveira who offered us the opportunity to use the FACScalibur Flow Cytometer at the Immunology Laboratory, Rene Rachou Research Center, Belo Horizonte, Brazil. References Anderson, J.F., Magnarelli, L.A., Sulzer, A.J., 1980. Canine babesiosis: indirect fluorescent antibody test for a North American isolate of Babesia gibsoni. Am. J. Vet. Res. 41, 2102–2105. Assis, C.B. Estudo de aspectos hematolo´ gico, bioquı´mico e imunolo´ gico em ca˜ es na˜ o esplenectomizados e esplenectomizados inoculados com Babesia canis [tese]. Botucatu (SP): UNESP; 1993. Bodabe, P.A., Oduye, O.O., Aghomo, H.O., 1989. Prevalence of antibodies against Babesia canis in dogs in an endemic area. Revue Elev. Med. Vet. Pays Trop. 42, 211–217. Breitschwerdt, E.B., Malone, J.B., Macwilliams, P., Levy, M.G., Qualls-JR, C.W., Prudich, M.J., 1983. Babesiosis in the Greyhound. J. Am. Vet. Med. Assoc. 182, 978–982. Davis, W.C., Wyatt, C.R., Hamilton, M.J., Goff, W.L., 1992. A rapid, reliable method of evaluating growth and viability of intaeritrocytic protozoan hemoparasites using fluorescence flow cytometry. Mem. Inst. Oswaldo Cruz. 87, 235–239. Farwell, G.E., Legrand, E.K., Cobb, C.C., 1982. Clinical observations on Babesia gibsoni and Babesia canis infections in dogs. J. Am. Vet. Med. Assoc. 180, 507–511. Fukata, T., Ohnishi, T., Okuda, S., Sasai, K., Baba, E., Arakawa, A., 1996. Detection of canine erytrocytes infected with Babesia gibsoni by flow cytometry. J. Parasitol. 82, 641–642. Gallop, P., Paz, M., Henson, E., 1984. Dynamic approaches to the delivery of reporter reagents into living cells. BioTechniques 1, 32. Howard, R.J., Rodwell, B.J., 1979. Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow flourimetry using 33258 Hoechst. Exp. Parasitol. 8, 421–431. Instituto Interamericano de Cooperacio´ n para la Agricultura-IICA. 1987. Te´ cnicas para el diagno´ stico de babesiosis y anaplasmosis bovina. San Jose´ , 79 pp. Levy, M.G., Breitschwerdt, E.B., Moncol, D.J., 1987. Antibody activity to Babesia canis in dogs in North Caroline. Am. J. Vet. Res. 48, 339–341. Lobetti, R.G., 1998. Canine babesiosis. Comp. Contin. Educ. Pract. Vet. 20, 418–430. Makler, M.T., Lee, L.G., Recktenwald, D., 1987. Thiazole orange: a new dye for Plasmodium species analysis. Cytometry 8, 568–570. Martinod, S., Laurent, N., Moreau, Y., 1986. Resistance and immunity of dogs against Babesia canis in an endemic area. Vet. Parasitol. 19, 245–254. Reyers, F., Leisewitz, A.L., Lobetti, R.G., Milner, R.J., Jacobson, L.S., Van Zyl, M., 1998. Canine babesioses in South Africa: more than one disease. Does this serve as a model for falciparum malaria? Ann. Trop. Med. Parasitol. 92, 503–511. Ribeiro, M.F.B., Lima, J.D., Passos, L.M.F., Guimara˜ es, A.M., 1990. Frequ¨ eˆ ncia de anticorpos fluorescentes antiBabesia canis em ca˜ es de Belo Horizonte. Minas Gerais. Arq. Bras. Med. Vet. Zootec. 42, 511–517. Ristic, M., 1988. Babesioses of Domestic Animals and Man. CRC Press, Florida.
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Spiewak, G. Aspectos epidemiolo´ gicos, clı´nicos e de diagno´ stico da infecc¸a˜ o por Babesia canis, em ca˜ es atendidos em clı´nicas veterina´ rias, em Belo Horizonte [dissertac¸a˜ o]. Belo Horizonte (MG): UFMG, 1992. Taboada, J., Merchant, S.R., 1991. Babesioses of companion animals and man. Vet. Clin. North Am. Small Anim. Pract. 21, 103–123. Uemura, T., Suzuki, S., Ohnishi, T., 1990. Flow cytometric enumeration of reticulocyte in the pheriferal blood from canine infected with Babesia gibsoni. J. Vet. Med. 37, 468–472. Vercammen, F., Deken, R., Maes, L., 1995. Clinical and serological observations on experimental infections with Babesia canis and its diagnosis using IFAT. Parasite 2, 407–410. Weiland, G., 1982. Moglichkeiten des serologischen Nachweises von Babesien infektionen bei Hund und Rind. Zentbl. VetMed. 35, 286–289. Wlosniewski, A., Leriche, M.A., Chavigny, C., Ulmer, P., Donnay, V., Boulouis, H.J., Mahl, P., Druilhe, P., 1997. Asymptomatic carriers of Babesia canis in an enzootic area. Comp. Immunol. Microbiol. Infect. Dis. 20, 75–86. Wyatt, C.R., Goff, W., Davis, W.C., 1991. A flow cytometric method for assessing viability of intraerytrocytic hemoparasites. J. Immunol. Methods 140, 23–30.
Molecular fluorescent approach to assessing intraerythrocytic hemoprotozoan Babesia canis infection in dogs Kelly Alves Bicalhoa,b,c, Mu´cio F. Barbosa Ribeirob, Olindo Assis Martins-Filhoa,* a
Laborato´rio de Doenc¸a de Chagas, Centro de Pesquisas Rene´ Rachou, FIOCRUZ, Av. Augusto de Lima 1715, 30 190-002, Belo Horizonte, Brazil b Departamento de Parasitologia, ICB, Universidade Federal de Minas Gerias, Belo Horizonte, Brazil c Biote´rio de Produc¸a˜o, Centro de Pesquisas Rene´ Rachou, FIOCRUZ, Av. Augusto de Lima 1715, 30 190-002, Belo Horizonte, Brazil Accepted 4 August 2004
Abstract The development of recent flow cytometry-based protocols for the diagnosis of canine babesiosis, Babesia gibsoni in particular, has encouraged us to investigate its applicability to detect B. canisinfected erythrocytes as well as optimize the hydroethidine-flow cytometry methodology (HE-FC), using peripheral blood samples from naturally and experimentally infected dogs. Our data demonstrated that HE at 25 mg/ml provided the most outstanding fluorescence profile, able to discriminate between infected and uninfected dogs with no alterations in cell properties such as forward scatter and unspecific fluorescence. The results were expressed as the percentage of positive fluorescent erythrocytes (PPFE) for each individual sample, with 1.53% of PPFE as the cut-off determined between infected and uninfected animals. B. canis-infected erythrocytes during both acute and chronic experimental infection were identified through HE-FC, validating its use for diagnosis purposes in endemic areas for canine babesiosis. In a clinical trial, 22.8% out of 162 dogs showed to be positive to Babesia infection through this approach. Such prevalence was similar to that estimated for altered hematological profiles (HT) 30% (29%), but highly distinct from the prevalence provided by direct blood smear (BS) examination (1.8%) or immunofluorescent assay (IFA) (60.5%). Furthermore, our findings indicate that positive PPFE data was associated with HT 30%, * Corresponding author. Tel.: +55 31 3295 3566; fax: +55 31 3295 3115. E-mail address: [email protected] (O.A. Martins-Filho). 0304-4017/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2004.08.009
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emphasizing that, in clinical practice, the haematocrit should be used as a screening test followed by HE-FC, suitable to confirm hypotheses of canine babesiosis. # 2004 Elsevier B.V. All rights reserved. Keywords: Flow cytometry; Babesia canis; Diagnosis
1. Introduction Canine babesiosis is one of the most important tickborne, protozoal hemoparasitic diseases. The intraerythrocytic hemoparasite, Babesia canis, invades and replicates within canine red blood cells, causing varying degrees of hemolytic anemia and multiple-organ dysfunction (Lobetti, 1998). Prompt and accurate diagnosis is difficult because the signs and symptoms are non-specific and thus, correctly identifying the infectious agent is important for treatment and prognosis. Although hemolytic anemia is the distinctive feature of canine babesiosis, definitive laboratory diagnosis of active babesiosis requires the visualization of Babesia organisms within infected erythrocytes (Lobetti, 1998). The available serological tests to detect anti-Babesia specific antibodies have restricted applicability since detectable antibody levels persist after parasite clearance due to the residual humoral immunity. In addition to the limited suitability of serological tests for endemic areas, in early stages of infection, when false-negative results may occur, they have shown not be sensitive. (Taboada and Merchant, 1991). One of the major challenges regarding the diagnosis of canine babesiosis is the lack of accurate laboratory approaches to elucidating cases of babesiosis in clinical practice. Despite its high specificity, the blood smear examination, using conventional optical microscopy with panoptic or GIEMSA staining protocol, presents low sensitivity (Breitschwerdt et al., 1983). Moreover, the detection of parasites in most atypical or chronic cases may become difficult due to scarce parasitemia (Anderson et al., 1980; Wlosniewski et al., 1997). Exceptional advances have been made in the diagnosis of canine babesiosis after the development of an easy protocol to detect Babesia gibsoni-infected erythrocytes in experimentally infected dogs by flow cytometry, using a single color staining for peripheral blood samples (Fukata et al., 1996). The rapid multiparametric examination of individual red blood cells has made the flow cytometry an useful tool for quantitative diagnosis of canine babesiosis. The basis of this methodology is the use of hydroethidine (HE), a compound that is taken up by viable cells and metabolically converted to ethidium bromide – a DNA-binding fluorochrome. The present investigation was designed to determine whether the HE-FC methodology was applicable to diagnose B. canis-infection using peripheral blood samples from naturally or experimentally infected dogs.
2. Material and methods 2.1. Parasites and animals B. canis organisms were obtained, at the peak of parasitemia, from peripheral blood of splenectomized mongrel pups (4–5-months old), with positive results by immunofluor-
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escent assay (IFA). Samples of B. canis infected blood were used to inoculate seven splenectomized mongrel IFA-negative dogs, confined in a holding kennel after birth at Escola de Veterina´ ria of Universidade Federal de Minas Gerais (EV/UFMG) (Veterinary School of the Federal University of Minas Gerais State), in Belo Horizonte, State of Minas Gerais, Brazil, in order to be out of tick contact. These animals were used as positive controls during the HE-FC standardization procedures. Other seven uninfected IFAnegative pups (4–5 months old), used as negative controls, were also confined in the holding kennel after birth. Peripheral blood samples were collected at days 3–5 after inoculation to search for B. canis-infected erythrocytes. A follow-up was daily carried out, during 28 consecutive weeks, to search for persistent B. canis-infected erythrocytes during chronic disease cases. A clinic trial was also performed; including dogs (1 month–15 years old) attended for ordinary clinical appointments, at the EV/UFMG and at two private veterinary clinics. Therefore, a randomized sample was used for the clinic–laboratory trial. The samples size was calculated as: n = p(100 p)z2/(pd)2, where n is the sample size, p the expected prevalence of positive B. canis IFA at veterinary clinics in Belo Horizonte, Minas Gerais (p = 40%), z the freedom degree (1.96 for 95%) and d the expected error (d = 0.2) (Spiewak, 1992). In order to increase confidence degree, the original sample size of 144 was increased in 12%. A total of 162 EDTA–peripheral blood samples for parasitological examination, hematological evaluation, HE-FC and serological studies were obtained by venipuncture. 2.2. Parasitological, serological and hematological tests Parasitological diagnosis of B. canis infection was performed by blood smears (BS), applied over a grease-free glass slide and stained through the panoptic staining kit (Laborclin, Pinhais, Parana), and examined under a total of 40 conventional microscopy bright fields with oil immersion (1000). Data were expressed as percentage of infected erythrocytes. Serological tests were carried out using frozen samples maintained at 20 8C for 2 months. Each other serial dilution (1:40–2.560 in phosphate buffered saline) was tested for anti-B. canis IgG antibody by indirect immunofluorescence assay (IFA), as recommended by Instituto Interamericano de Cooperacio´ n para la Agricultura-IICA (1987) and modified by Ribeiro et al. (1990). The results were found to be positive when antibody titer was 1:40. Negative and positive controls were also included. The hematological profile was assessed by using a micro-haematocrit protocol. Haematocrit percentage (HT) was determined for each sample macroscopically. Results were interpreted using normal reference values ranging from 30 < HT 55%. Thus, HT values 30% were considered to be positive for anemia. 2.3. Hydroethidine-flow cytometry (HE-FC) One millilitre of EDTA–whole blood samples were washed with 10 ml of PBS by centrifugation at 600 g for 7 min, RT. The supernatant and buffy-coat were discarded. Packed red blood cells (RBC) were resuspended and washed once with 10 ml of PBS at 600 g for 7 min, RT 25 ml of packed RBC were transferred to 5 ml of PBS in order to
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obtain a 0.005% suspension. Hydroethidine (Polysciences, Warrigton, PA, USA) was solubilized in anhydrous DMSO (Sigma Chemical, St. Louis, MI, USA) at 10 mg/ml as stock solution and diluted into PBS to obtain the final concentrations used, including 12.5, 25.0 and 50.0 mg/ml. A volume of 50 ml of RBC suspension in PBS was incubated in 96 well round bottom plates in the presence of 150 ml of HE solution in PBS at final concentrations. Suspensions were incubated at 37 8C for 20 min in a dark humidified incubator and then centrifuged at 600 g for 7 min, RT. Supernatants were discarded and PBS was added to turn HE-stained erythrocyte suspension to a total volume of 300 ml. Internal control samples were incubated in PBS with no HE. Flow cytometric measurements were performed on a Becton Dickinson FACScalibur interfaced to a digital apple computer. The CellQuest software package was used for data acquisition, storage and analysis. Stained erythrocytes were run in the cytometer and 30,000 events per sample were counted. Erythrocytes were first identified on the basis of their forward specific and side specific light scattering (FSC and SSC, respectively) properties, after adjustments of size and granularity gains with values of E00 and 300, respectively, both on log scale (Fig. 1a). After adjustments, erythrocytes were found by assuming a characteristic of homogeneous distribution, using FSC versus SSC dot plot. A gate surrounding the erythrocyte population was set to exclude noise from debris and platelets. Averages of 28,000–29,000 gated erythrocytes were analyzed for their relative ethidium bromide fluorescence intensity, recognized as fluorescence type 2 (FL-2). This protocol allows us to identify Babesia-infected erythrocytes on FSC versus FL-2 dot plots (Fig. 1b and c). A marker was initially set up using the internal control, that received 50 ml of RBC suspension and 150 ml of PBS (Fig. 1b) and determination of the percentage of fluorescent positive erythrocytes (PPFE) for a given sample (Fig. 1c). 2.4. Statistical analysis Comparative analysis between the performance of BS, IFA, HT and HE-FC was performed by analysis of variance (ANOVA) followed by Student’s t-test. Comparative analysis of prevalence at individual level as well as the scattering of B. canis infection detected by HE-FC as regards semi-quantitative HT was carried out by the chi-square test. Significance was defined in both test as P < 0.05.
3. Results 3.1. HE-FC optimization procedures 3.1.1. Effect of HE concentration on the PPFE values In order to evaluate the effect of HE concentration on the performance of ethidium bromide binding to B. canis DNA, we carried out a parallel study with three HE final concentrations, 12.5, 25.0 and 50.0 mg/ml (Fig. 2). Our data demonstrated that in spite of HE concentration, PPFE values from B. canis infected dogs differ significantly from those of unifected dogs. In addition, our results showed that incubation of erythrocytes with 50 mg/ml of HE significantly increases PPFE values for uninfected dogs in comparison to
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Fig. 1. Density plot analysis of B. canis infected erythrocytes by flow cytometry. Erythrocytes were first identified on the basis of their specific forward (FSC size) and side (SSC granularity) light scattering properties. A gate surrounding the erythrocyte population was set to exclude noise and platelets (Fig. 1a). Gated erythrocytes were analyzed for their relative HE fluorescence intensity allowing the identification of Babesia-infected erythrocytes on FSC versus FL-2 dot plots. A marker was set up using the internal control, which received only PBS (Fig. 1b) and used to determine the percentage of fluorescent positive erythrocytes (PPFE) for a given sample (Fig. 1c).
the other concentrations of 25.0 and 12.5 mg/ml (P < 0,05). However, no effect was observed on PPFE values for B. canis-infected animals (P = 0.561). Analysis of density plot distributions of labeled erythrocytes based on their size (FSC) and fluorescence properties revealed that 50 mg/ml altered the FSC property and led to increased autofluorescence of erythrocytes from B. canis-infected animals (Fig. 3). No changes in red blood cell scattering properties were observed for uninfected dogs (Fig. 3b–d). Since no changes on PPFE values were detected for samples from B. canis-infected pups, regardless HE concentration (Fig. 3e–g), we have chosen HE at 25.0 mg/ml as the ideal condition to be used in further investigations in order to guarantee the sensitivity of HE-FC methodology, unlikely to provide false positive results. Moreover, as illustrated in Fig. 3, the concentration of 25.0 mg/ml HE (Fig. 3f) led to the most outstanding fluorescence profile for B. canis-infected erythrocytes, without modifying forward scatter property and with minor interference on autofluorescence, in comparison to HE at 50 mg/ml (Fig. 3g).
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Fig. 2. Effect of HE concentration on the PPFE values. Samples of 50 ml of RBC suspension 0.005% in PBS from uninfected (*) and B. canis infected dogs (*) were incubated in the presence of HE solution at final concentrations of 12.5, 25.0 and 50.0 mg/ml, as described in Section 1. Results are presented in scattering graph as percentage of fluorescent positive erythrocytes (PPFE) for each individual test. Statistical analysis showed that PPFE values from B. canis infected dogs differ significantly from those from uninfected dogs (P < 0.05). No effect was observed on PPFE values for B. canis-infected animals (P = 0.561).
3.1.2. Determination of a cut-off value for PPFE between uninfected and infected dogs In order to determine a cut-off between positive and negative samples, we have performed a parallel analysis of 14 blood samples from pups, from which seven were uninfected and seven experimentally infected, confined in a holding kennel after birth at the EV/UFMG, out of tick contact. HE-FC methodology was applied using HE at 25.0 mg/ ml, as described in Section 1.1. Our data demonstrated that PPFE from uninfected pups ranged from 0.31 to 1.27% whereas PHFP from B. canis-infected dogs varied from 10.8 to 22.5% (Fig. 4). Following the detected cut-off as PPFE mean value from uninfected group (1.01 0.265) added to two standard deviations (cutoff = [¯x + 2s]), data analysis allowed us to determine 1.53% as being the best PPFE cut-off between negative and positive results. 3.2. Performance of BS and HE-FC for detection of B. canis chronic infection A follow-up study was carried-out to determine the sensitivity of BS and HE-FC for identifying B. canis infected erythrocytes in experimentally infected dogs over 28 weeks after challenge. EDTA–whole blood samples were collected daily, during 28 consecutive weeks. Two different laboratory approaches were used for evaluating the status of B. canis chronic infection cases. Data from this follow-up are presented in Fig. 5 expressed as weekly mean percentage of infected erythrocytes.
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Fig. 3. Density plots distributions of labeled erythrocytes based on their size (FSC) and fluorescence (FL-2) properties. No changes on RBC scattering properties were observed for uninfected dogs despite HE conventration of 12.5, 25.0 and 50.0 mg/ml (a, b and c, respectively). Incubation of RBC from B. canis infected dogs with 50 mg/ml of HE disturbed the FSC property and led to increased autofluorescence of erythrocytes from B. canis-infected animals (Fig. 3f). On the other hand, incubation of RBC with 12.5 mg/ml of HE led to lower PPFE (Fig. 3d). For further analysis we have chosen HE at 25.0 mg/ml (e) as the ideal condition to be used on further investigations, in order to guarantee the sensitivity of HE-FC methodology, and led to a more outstanding fluorescence profile for B. canis-infected erythrocytes.
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Fig. 4. Establishment of a cut-off of PPFE between uninfected and B. canis-infected dogs. Parallel analysis of blood samples from uninfected (*, n = 7) and experimentally infected (*, n = 7) dogs were tested in HE-FC using HE at 25.0 mg/ml. Data are expressed as scattering of PPFE values for each group. Taking the cut-off definition as [¯x + 2s], PPFE = 1.53% was found as an edge between negative and positive results.
Thin blood smear (BS), labelled with panoptic staining to demonstrate the organism, revealed that B. canis infected erythrocytes could be found as early as only 2 days after infection, with BS = 0.24% as average parasitemia level for the first week. Parasitemia was oscillating between 0 and 0.01% as average parasitemia level from 2nd to 16th week, becoming undetectable after the 17th week post-infection (Fig. 5). Our data demonstrated that HE-FC approach was also able to identify B. canis infected erythrocytes 2 days after infection, with HE-FC = 2.60% as average for parasitemia level during the first week. However, this prevalence was higher than that observed by BS. Further, HE-FC could also detect B. canis infected erythrocytes from the 1st week throughout the whole follow-up study, with average parasitemia levels oscillating between 3.87 and 21.63%, but never absent as observed by BS. This marked difference between HE-FC and BS demonstrated the higher sensitivity of flow cytometry to identify chronic B. canis infection (Fig. 5). 3.3. HE-FC application in clinical trials 3.3.1. Performances of BS, IFA 1:40, HT 30% and HE-FC for evaluating the prevalence of B. canis infection on clinical survey Blood samples were collected from 162 randomly selected dogs in order to determine the prevalence of B. canis infection in dogs attended at the EV/UFMG and at two Private Veterinary Clinics in Belo Horizonte. Four different laboratory approaches were used to evaluate the status of B. canis infection. Data from this clinical trial are presented in Fig. 6. Thin blood smears (BS), labeled with panoptic staining, were examined for the organism determination and revealed that only 3 out of 162 dogs (1.8%) were positive for B. canis infection. On the other hand, serological tests by indirect immunofluorescence antibody
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Fig. 5. Performance of blood smear (BS *) and Flow Cytometry (HE-FC *) for detection of B. canis in acute and chronic experimental infection. Sensitivity of BS and HE-FC for identifying B. canis infected erythrocytes in experimentally infected dog was evaluated over 28 weeks after challenge. Parasitaemia by BS was oscillating between 0 and 0.01% from 2nd to 16th week, becoming undetectable after the 17th week post-infection. Parasitaemia by HE-FC was detect from 2nd week to the end of the follow-up study, with average parasitaemia level oscillating between 3.87 and 21.63%, but never absent as observed by BS.
assay (IFA), considered positive when titers were higher or equal than 1:40, demonstrated the presence of antibodies against B. canis in 98 out of 162 animals (60.5%). The prevalence of B. canis infection was also estimated by interpreting values of haematocrit 30% as suggestive of hemoparasite infection. Results of laboratory screening considering HT 30% suggested that babesiosis would be prevalent in 47 out of 162 animals (29%). Our data demonstrated that HE-FC approach was able to identify 22.8% of dogs as being positive to Babesia infection (37 out of 162 animals). This prevalence was similar to that estimated through hematological profile analysis – HT 30%, but markedly different from that identified by direct BS or IFA (P < 0.05).
Fig. 6. Performances of BS, IFA 1:40, HT 30% and HE-FC for evaluating the prevalence of B. canis infection on clinical survey. Data are expressed as prevalence of positive results at population level (n = 162) for each test used. Different letters (a–c) represent statistically significant differences at P < 0.05. Detection of B. canis infections through BS revealed that only 3 out of 162 dogs were positive. Serology by IFA demonstrated the presence of antibodies against B. canis in 98 out of 162 animals. HT 30% suggested that babesiosis was prevalent in 47 out of 162 animals. HE-FC identified 37 out of 162 dogs as positive to Babesia infection.
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Fig. 7. Comparative analysis of B. canis prevalence detected by BS, IFA and HT with HE-FC performance at individual level (n = 162) on clinical survey. Data are expressed as scattering PPFE. Different letters (a and b) represent statistically significant differences at P < 0.001. Samples were first categorized as negative or positive based on their performance on BS, IFA 1:40 and HT 30% and then analyzed for HE-FC. These data confirmed that positive HE-FC correlates better with HT 30% than BS and IFA, showing that 44.7% of animals with HT 30% were prone to be positive in HE-FC.
3.3.2. Comparative analysis of BS, IFA and HT with HE-FC performance at individual level on clinical survey In order to further characterize the high degree of similarity between B. canis infection prevalence estimated by HE-FC and HT 30%, we performed a comparative analysis of PPFE data with those obtained by BS, IFA 1:40 and HT 30% for each individual sample. For this purpose, a number of 162 samples were first categorized as negative or positive based on results provided by BS, IFA 1:40 and HT 30% and then analyzed by HE-FC, using PPFP cut-off of 1.53 between negative or positive results (Fig. 7). Our data confirmed, by analyzing individual samples, that positive HE-FC correlates better with HT 30%, showing that 44.7% of animals with HT 30% were prone to be positive in HE-FC with PPFE higher than 1.53 (P 0.001). No significant relationship was observed between HE-FC results when data were first categorized based on BS or IFA results. 3.3.3. Scatter of B. canis infection detected by HE-FC concerning semi-quantitative HT Our data have suggested a strong association between the results from HT 30% and the positivity of HE-FC. To further focus this issue, we selected the dogs based on their HT results, using a semi-quantitative distribution including six categories (05–09%, 10–14%, 15–19%, 20–24%, 25–30% and >30%), and then evaluated the prevalence of positive HEFC results within each HT category (Fig. 8). Data analysis demonstrated a higher prevalence of positive HE-FC as HT became lower than or equal to 24%, with prevalence ranging from 50% (05–09%) and 56 (10–14%) to 60 (15–19% and 20–24%). Lower prevalence was observed for HT from 25–30% (29%) but meaningful only when HT >30% (13.9%). Statistical analysis was based on Chi-square test at a significance level of P 0.01.
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Fig. 8. Scatter of B. canis infection detected by HE-FC as regards semi-quantitative HT on clinical survey. Samples were categorized based on their HT results using a semi-quantitative distribution and then evaluated for the prevalence of positive HE-FC results within each HT category. Data are expressed as mean prevalence of positive HE-FC results for each HT category. Different letters (a and b) represent statistically significant differences at P < 0.01, except for HT 25–30% in comparison to HT >30%. There was a higher prevalence of positive HE-FC as HT became 24% whereas lower prevalence was observed for HT at 25–30% (29%) but with significance only when HT >30% (13.9%).
4. Discussion Considering canine babesiosis prevalence in large urban centers, the lack of an accurate and sensitive laboratory method for definitive diagnosis is still a major challenge in veterinary clinics. In addition, chronic evolution of the disease makes the clinical and laboratory diagnoses difficult due to low parasitemia levels. Efforts to standardize protocols for new approaches to detect Babesia infected erythrocytes have brought out possible techniques for quantitative analysis of organisms accurately measured over a wide range of parasitemia. Flow cytometric methods using membrane-permeable fluorochrome, thiazole orange, have been described for the detection of parasite-infected erythrocytes (Howard and Rodwell, 1979; Makler et al., 1987). However, Uemura et al. (1990) remarked that thiazole orange was unable to identify B. gibsoni in those cells. On the other hand, when hydroethidine staining was used, B. bovisinfected erythrocytes were detected (by flow cytometry Wyatt et al., 1991). Hydroethidine (HE) is a relatively new vital dye, which is converted to ethidium when taken up by living cells (Davis et al., 1992; Gallop et al., 1984). Since ethidium is a fluorochrome excited at 488 nm and emits at 585 nm, when intercalated into DNA molecules, it can be used in flow cytometry. Thus, it is now possible to monitor the viability and growth features of protozoa parasites such as Babesia, following exposure of hosts to infection. In addition to providing an accurate evaluation of intracellular growth as a viability assay, HE also excludes reticulocytes staining, which is unable to have HE converted into ethidium dye. Therefore, HE has been pointed out as a new method for detection and analysis of parasite-infected erythrocytes due to its sensitivity and reproducibility, in addition to being a short laboratory procedure. Here, we have described an optimized methodology to identify B. canis infected erythrocytes in peripheral blood samples from mongrel dogs. After testing a range of HE
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concentrations, we found that 25 mg/ml HE provided the most outstanding fluorescence profile able to discriminate between infected and uninfected dogs, without changing other cell properties such as forward scatter and auto-fluorescence. Using this standardized protocol in further analyses, we expressed our results as the percentage of positive fluorescent erythrocytes (PPFE) for each individual sample, establishing 1.53% of PPFE as the cut-off between infected and uninfected animals. The high performance of HE-FC method described here is in agreement with those reported by Wyatt et al. (1991), Davis et al. (1992) and Fukata et al. (1996) who identified other Babesia species by flow cytometry using HE/ethidium as a fluorescent dye. In this study, sensitivity of BS and HE-FC for identifying B. canis infected erythrocytes, in acute and chronic experimentally infected dogs, was assessed over 28 weeks after challenge. Our data have confirmed the low sensitivity of blood smear examination to diagnose chronic babesiosis, which is consistent with previous reports (Anderson et al., 1980; Breitschwerdt et al., 1983; Levy et al., 1987; Wlosniewski et al., 1997). Furthermore, we demonstrated that HE-FC was able to identify B. canis infected erythrocytes during both acute and chronic experimental infection, validating its use for diagnosis purposes in endemic areas for canine babesiosis. One of the major advantages of using flow cytometry, besides automation, is the speed with which data can be obtained. HE-FC is a rapid methodology that yields final data within 2–3 h, even when processing a large number of samples. Animals with signs of B. canis infection, usually raging from anorexia, central nervous system depression and lethargy to weakness and anemia, require a differential diagnosis for an accurate interpretation and preclusion of other diseases, as previously mentioned, which is important for early treatment planning and prognosis. By using the standardized HE-FC methodology, we performed a clinical trial and identified 37 out of 162 dogs with positive results, showing higher sensitivity (22.8%) in comparison to blood smear examination that detected only 1.8% of Babesia infection. This low prevalence of infection detected by BS, in dogs attended at the two private veterinary clinics, supports previous reports regarding low sensitivity of conventional parasitological methods (BS) to detect B. canis infected erythrocytes, mainly in atypical and chronic cases, when scarce parasitemia is reported (Anderson et al., 1980; Breitschwerdt et al., 1983; Levy et al., 1987; Wlosniewski et al., 1997). In addition, despite its high specificity and straightforwardness, blood smear examination is laborious and time-consuming, especially when a large number of samples must be evaluated. Regarding the use of immunological methods for B. canis diagnosis, our data have confirmed the findings of early reports in endemic areas, where antibodies against B. canis are more prevalent than are current B. canis infection in dogs (Martinod et al., 1986). Our results demonstrated a high prevalence (60.5%) of seropositivity to B. canis, as detected by IFA. These data are markedly different from those obtained by BS, which probably also under estimated B. canis infection. Despite the fact that the IFA test appears to represent a reliable method to detect infected dogs with patent and sub patent parasitemia, due to its high sensitivity to detect antiBabesia antibodies during chronic disease, limitations regarding cross-reactivity and lack of association between a positive serology and active disease have led to criticism of IFA test interpretations (Breitschwerdt et al., 1983; Ristic, 1988). Moreover, the seronegativity in B. canis infection, mainly during acute disease, may also contribute limitations of the
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IFA test as a diagnostic tool for canine babesiosis. The reason why infected dogs failed to produce antibodies against the parasite may represent multiple phenomena (Weiland, 1982; Ristic, 1988; Assis, 1993; Vercammen et al., 1995). Herein, we have found that 14 out of 98 dogs with negative serology presented positive HE-FC results. Further analysis revealed that three out of these animals were less than 4 months of age, presenting low HT values and typical clinical signs of canine babesiosis, consistent with B. canis infection, and suggesting that they probably needed some latent period to produce antibodies. This is borne out by the findings of Bodabe et al. (1989) that most seronegative dogs in southwestern Nigeria were less than 1-year old and seroconvertion could be notified 2 weeks after initial examination. Similar results have also been reported by Farwell et al. (1982) and Breitschwerdt et al. (1983), showing seronegativity in B. canis-infected young pups. Another reason for seronegative results in infected animals is that some dogs require a booster challenge to produce specific antibodies against the parasite (Bodabe et al., 1989) In addition, IFA application has another restriction for use in endemic areas because of the presence of animals chronically infected with low antibody titers and those presenting residual serology after disease clearance. Herein we have detected a similar prevalence of B. canis-infected dogs estimated by either HT 30%, as suggestive of hemoparasite infection (29%), or PHFP 1.53%, as a positive result for B. canis infection (22.8%). The similarity between these findings was further confirmed, at an individual level, showing that positive HE-FC correlates better with HT 30%, showing 44.7% of animals with HT 30%, being prone to be positive in HE-FC, which displays PPFE higher than 1.53%. Since PPFE 1.53% was predominantly higher in animals with HT 30, low HT values might be due to the hemolytic anemia caused by B. canis multiplication and erythrocyte lysis (Reyers et al., 1998; Lobetti, 1998). Despite associations between hemolytic anemia and canine babesiosis that have been explained, at least in part, by the immune mediated hemolysis caused by antibodies associated to erythrocyte membrane, our findings did not show any association between IFA titers and HT values (data not shown). Most HE-FC positive results were found in the dogs with low levels of antibodies (IFA) = 1:40 (data not shown)—suggesting, as previously reported by Ristic (1988), that serological data may reflect parasite multiplication into the host but parasitemia levels are not associated with antibody titers. Thus, these results suggest that HT could be used as a screening approach to identify suspect dogs with a hematological profile of HT 30, which should be further evaluated by HE-FC to confirm the presence of B. canis infected erythrocytes.
5. Conclusion The technique described here provides an useful approach to quantify intraerythrocytic B. canis applicable to the diagnosis of infection. Furthermore, our findings indicate that a positive PPFE result was associated with HT 30%, emphasizing that, in clinical practice, the haematocrit should be used as a screening test followed by HE-FC, suitable to confirm diagnosis of canine babesiosis. Moreover, as HE is as a vital dye, this methodology also can be used to monitor parasite clearance after treatment. This feature is important because it will enable us to more effectively monitor the impact of treatment on intraerythrocytic
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parasite growth, a necessary prerequisite for cure assessment after specific chemotherapy. Evaluation of HE-FC performance as a cure criterion after treatment is currently under evaluation in our laboratory.
Acknowledgments This work was supported by Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil and Fundac¸a˜ o Oswaldo Cruz (FIOCRUZ-BH). We are grateful to Dr. Rodrigo Correa-Oliveira who offered us the opportunity to use the FACScalibur Flow Cytometer at the Immunology Laboratory, Rene Rachou Research Center, Belo Horizonte, Brazil. References Anderson, J.F., Magnarelli, L.A., Sulzer, A.J., 1980. Canine babesiosis: indirect fluorescent antibody test for a North American isolate of Babesia gibsoni. Am. J. Vet. Res. 41, 2102–2105. Assis, C.B. Estudo de aspectos hematolo´ gico, bioquı´mico e imunolo´ gico em ca˜ es na˜ o esplenectomizados e esplenectomizados inoculados com Babesia canis [tese]. Botucatu (SP): UNESP; 1993. Bodabe, P.A., Oduye, O.O., Aghomo, H.O., 1989. Prevalence of antibodies against Babesia canis in dogs in an endemic area. Revue Elev. Med. Vet. Pays Trop. 42, 211–217. Breitschwerdt, E.B., Malone, J.B., Macwilliams, P., Levy, M.G., Qualls-JR, C.W., Prudich, M.J., 1983. Babesiosis in the Greyhound. J. Am. Vet. Med. Assoc. 182, 978–982. Davis, W.C., Wyatt, C.R., Hamilton, M.J., Goff, W.L., 1992. A rapid, reliable method of evaluating growth and viability of intaeritrocytic protozoan hemoparasites using fluorescence flow cytometry. Mem. Inst. Oswaldo Cruz. 87, 235–239. Farwell, G.E., Legrand, E.K., Cobb, C.C., 1982. Clinical observations on Babesia gibsoni and Babesia canis infections in dogs. J. Am. Vet. Med. Assoc. 180, 507–511. Fukata, T., Ohnishi, T., Okuda, S., Sasai, K., Baba, E., Arakawa, A., 1996. Detection of canine erytrocytes infected with Babesia gibsoni by flow cytometry. J. Parasitol. 82, 641–642. Gallop, P., Paz, M., Henson, E., 1984. Dynamic approaches to the delivery of reporter reagents into living cells. BioTechniques 1, 32. Howard, R.J., Rodwell, B.J., 1979. Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow flourimetry using 33258 Hoechst. Exp. Parasitol. 8, 421–431. Instituto Interamericano de Cooperacio´ n para la Agricultura-IICA. 1987. Te´ cnicas para el diagno´ stico de babesiosis y anaplasmosis bovina. San Jose´ , 79 pp. Levy, M.G., Breitschwerdt, E.B., Moncol, D.J., 1987. Antibody activity to Babesia canis in dogs in North Caroline. Am. J. Vet. Res. 48, 339–341. Lobetti, R.G., 1998. Canine babesiosis. Comp. Contin. Educ. Pract. Vet. 20, 418–430. Makler, M.T., Lee, L.G., Recktenwald, D., 1987. Thiazole orange: a new dye for Plasmodium species analysis. Cytometry 8, 568–570. Martinod, S., Laurent, N., Moreau, Y., 1986. Resistance and immunity of dogs against Babesia canis in an endemic area. Vet. Parasitol. 19, 245–254. Reyers, F., Leisewitz, A.L., Lobetti, R.G., Milner, R.J., Jacobson, L.S., Van Zyl, M., 1998. Canine babesioses in South Africa: more than one disease. Does this serve as a model for falciparum malaria? Ann. Trop. Med. Parasitol. 92, 503–511. Ribeiro, M.F.B., Lima, J.D., Passos, L.M.F., Guimara˜ es, A.M., 1990. Frequ¨ eˆ ncia de anticorpos fluorescentes antiBabesia canis em ca˜ es de Belo Horizonte. Minas Gerais. Arq. Bras. Med. Vet. Zootec. 42, 511–517. Ristic, M., 1988. Babesioses of Domestic Animals and Man. CRC Press, Florida.
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