Comparative evaluation of different diagnostic tests for B. gibsoni in dogs

B-3413 [1-7] Indian J. Anim. Res., Print ISSN:0367-6722 / Online ISSN:0976-0555 AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.arccjournals.com/www.ijaronline.in Comparative evaluation of different diagnostic tests for B. gibsoni in dogs Neelam Kushwaha 1*, Debabrata Mondal 2, Karam Pal Singh 3 and Rashmi Ranjan Mahapatra 4 Division of Veterinary Medicine, ICAR-Indian Veterinary Research Institute, Bareilly- 243 122, Uttar Pradesh, India. Received: 23-03-2017 Accepted: 07-09-2017 DOI: 10.18805/ijar.B-3413 ABSTRACT Babesia gibsoni is the common cause of canine babesiosis in India. Its diagnosis by conventional diagnostic methods result in false negative condition, particularly in those cases in which parasitemia is low. Therefore the present study employed IFAT and PCR along with microscopic examination to overcome this situation. Microscopic examination of Giemsastained blood smears revealed presence of small pear-shaped, oval or signet ring shaped B. gibsoni within the erythrocytes of 1.34 % dogs. IFAT and PCR detected a total of 1.80 % and 1.82 % dogs positive for B. gibsoni, respectively. The sensitivity and specificity of IFAT was 93.10% and 88.89%, respectively whereas, sensitivity and specificity of microscopic examination was 71.26% and 95.56%, respectively. The agreement of IFAT and microscopic examination with PCR was 82.30% and 60.73%, respectively. The agreement between IFAT and microscopy was 60.99%. Therefore, IFAT is considered to be highly sensitive test for diagnosing B. gibsoni in subclinically or chronically infected dogs with significantly low level of parasitemia. Key words: Babesiosis, B. gibsoni, Dog, IFAT, PCR. INTRODUCTION Babesiosis causes economic loss in form of morbidity and mortality of affected hosts and has zoonotic potential also (Ayoob and Prittie, 2010). More research efforts have been given in recent decades because it causes infections worldwide (Yao et al., 2014) in domestic animals, humans and some wildlife species (Schnittger et al., 2012). Canine babesiosis is a tick-borne protozoal disease caused by Babesia canis, and Babesia gibsoni (Brandao et al., 2003). B. gibsoni is endemic in many regions of Asia, Africa, Europe, and the Americas (Irizarry-Rovira et al., 2001; Macintire et al., 2002). Clinical signs in dogs are lethargy, anorexia, pyrexia, pale mucous membranes, hepatomegaly, dehydration, lymph node enlargement, vomiting, diarrhoea, malena, dark yellow urine, anemia, thrombocytopenia and icteric mucus membrane (Rene-Martellet et al., 2013; Yao et al., 2014). *Corresponding author s e-mail: kushneils@gmail.com 1 Teaching Veterinary Clinical Complex, College of Veterinary and Animal Sciences, Udgir-413 517, India 2 Division of Veterinary Medicine, ICAR-Indian Veterinary Research Institute, Bareilly (India) 3 CADRAD,ICAR-IVRI, Bareilly (India) 4 Veterinary officer, Fisheries & Animal Resources Development Department, Govt. of Odisha, India There is a relative paucity of studies on canine Babesia, Ehrlichia, Anaplasma, Hepatozoon and haemotropic Mycoplasma infections in Indian subcontinent (Megat Abd-Rani et al., 2011). The diagnosis of babesiosis in dogs is based on the identification of the parasite in stained blood smears but it is difficult to find the parasite in carrier animal and in subclinical infection since the level of parasitemia is very low (Caccio et al., 2002). Serological tests like, indirect fluorescent antibody tests (IFAT), Indirectenzyme linked immunosorbent test are considered to be highly sensitive (Kaur et al., 2016), but only moderately specific because of antigenic cross-reactions to other Babesia spp (Yamane et al., 1993). The recent development of recombinant ELISA assays has improved test specificity (Verdida et al., 2004). Several PCR methods have been reported to diagnose B. gibsoni infection with high sensitivity and specificity (Birkenheuer et al., 2003; Inokuma et al., 2004). In Indian scenario, although there are several reports of canine babesiosis based on conventional diagnostic methods, but the true status of canine babesiosis is still obscure. Therefore, the present work was carried out to know the status of canine babesiosis caused by B. gibsoni by employing blood smear examination, IFAT and PCR as well as the comparison of these tests was made to know their diagnostic efficacy for B. gibsoni. MATERIALS AND METHODS The study was carried out in Referral Veterinary Polyclinic (RVP) and Division of Medicine, ICAR- Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly (UP), over the period of April 2014 to April 2015.The dogs presented at Outpatient Department of Medicine (OPD- Medicine) with clinical signs of fever, diarrhoea, lethargy, staggering gait, anaemia, debilitated condition or presence of ticks were screened for B. gibsoni.

2 INDIAN JOURNAL OF ANIMAL RESEARCH Sample collection: Three milliliter blood samples were collected from sephanous/cephalic vein in clean dry vial containing sterilized ethylene diamine tetracetate (EDTA) for DNA isolation. Microscopic examination: Thin blood smears were prepared from the ear margin capillary bed, fixed in methanol and stained with Giemsa (Coles, 1986). Indirect fluorescent antibody test (IFAT): The test was performed according to the method described by Fukumoto et al. (2001) with suitable modifications. A thin blood smears were fixed with acetone for 10 min and incubated with successive dilution of serum (1:1, 1:2, 1:4, 1:6, 1:8 and 1:10) at 37 C for 1 hr. The slides were washed three times with phosphate buffered saline (PBS) for 10 min each time and incubated with FITC-conjugated anti-dog IgG antibody (Anti-Dog IgG (whole molecule) FITC antibody produced in rabbit, Sigma- Aldrich) at 1:30 dilution in PBS at 37 C for 1 hr. The slides were again washed three times with PBS for 10 min each time, and then the stained cells were observed by fluorescent microscopy. PCR (Polymerase chain reaction): DNA from EDTAblood (200 µl) was extracted using the Purelink Genomic DNA Mini Kit (Invitrogen). A primer set viz. Gib599F (5 CTC GGC TAC TTG CCT TGT C 3 ) and Gib1270R (5 GCC GAA ACT GAA ATA ACG GC 3 ) was used to amplify a 670 bp fragment of the 18S rrna gene region (Inokuma et al., 2004). The PCR mix containing 12.5 pmol of each primer, 100 µm of each dntp (Invitrogen), 1X PCR reaction buffer, 1.25U Taq DNA polymerase (Invitrogen) and 5 µl DNA in a final volume of 25 µl was prepared. The cycling conditions in thermocycler were as follows: 95 C for 5 min, followed by 35 cycles at 95 C for 30 s, 55 C for 30 s, 72 C for 90 s, and a final extension step of 72 C for 10 min. A negative sample control (canine blood DNA only) and a negative DNA control (Milli- Q water) were also included in the PCR reaction. The PCR products were run on 1.5% agarose gel containing ethidium bromide (Aysul et al., 2013). Statistical analysis: Kappa (k) statistics were used to know the level of agreement between different diagnostics. RESULTS AND DISCUSSION During the study period a total of 4,774 dogs were presented to OPD-Medicine of RVP, ICAR-IVRI. Microscopic examination of Giemsa-stained blood smears revealed presence of small pear-shaped or oval or signet ring shaped B. gibsoni (Figure a and b) within the erythrocytes of 1.34 % dogs whereas, IFAT and PCR detected a total of 1.80 % (figure c) and 1.82 % (figure d) dogs positive for B. gibsoni, respectively. Studies of canine babesiosis in India are limited to epidemiological aspect with the prevalence of infection ranging from 0.1-22% on the morphological criteria by microscopic examination (Sundar et al., 2004; Chaudhari, 2006). The prevalence of canine babesiosis from various parts of northern India has been reported to be ranging from 0.66 to 8.9% (Varshney and Dey, 1998; Chaudhuri, 2006; Singh et al., 2011). Only 0.1% (n = 5832) of dogs were found positive for B. gibsoni microscopically in Chennai (Sundar et al., 2004) whereas, 9 % in Uttar Pradesh (Chaudhuri, 2006) and 22% (n = 2104) in Assam (Saud and Hazarika, 2000). Singh et al. (2012) recorded 5.45% (29/532) of canines positive for B. gibsoni by Giemsa stained peripheral blood smears at Small Animal Clinics, GADVASU, Ludhiana, (Punjab). A total of 6.54% (14/214) positivity for B. gibsoni has been reported by microscopic examination from Ludhiana region (Singh et al., 2014). Kumar et al. (2015) found 5.78 % B. gibsoni positive cases (25/432) microscopically at veterinary Polyclinic Jalandhar, (Punjab). A total of 9.83% (412/4190) dogs were found positive for B. gibsoni by peripheral blood smears examination at Madras Veterinary College Teaching Hospital (Kumar et al., 2009). Eljadar (2010) examined a total of Fig-a: Giemsa stained blood smear (100 X) showing oval shaped B. gibsoni Fig-b: Giemsa stained blood smear (100 X) showing signet ring shaped B. gibsoni

Vol. Issue, () Fig-c: B. gibsoni within RBC (40 X) in IFAT 951 suspected dog samples and reported 3.17% samples to be positive for B. gibsoni. The pathogenicity of Babesia is also known to differ in different parts of India which might be due to host factor (Megat Abd-Rani et al., 2010), but other factors such as the age and immune status of the host and the presence of concurrent infections also influence clinical outcome. Climatic conditions in India are quite favorable for the growth and multiplication of arthropod vectors (Jadhav et al., 2011). India s dog population is estimated to 11,673 thousands (DADF, 2012) and near about 5 million dogs within the pet category are exposed to tick infestation (Sudarshan et al., 2006). Due to urbanization, industrialization and changing social structure, people are increasingly keeping dogs as pets for security as well as companion animal. Growing population of dogs has increased the chance of disease transmission due to readily available susceptible host. Out of 4774 cases, 132 dogs were suspected for babesiosis on the basis of clinical signs. A total of 64 dogs were found positive by light microscopic examination whereas, 86 and 87 were found positive by FAT and PCR, respectively. A comparison of microscopic examination, IFAT and PCR is shown in Table 1. B. gibsoni infection is often subclinical therefore it goes unnoticed in microscopic examination. Direct blood PCR detected 26 % (39/150) positivity for babesiosis whereas, 18% (27/150) of samples were positive under microscopic examination (Ganguly et al., 2017). In comparison to microscopy, PCR detected more infection which is in agreement with the findings of other workers (Gotsch et al., 2009; O Dwyer et al., 2009; Amuta et al., 2010; Ionita et al., 2012; Laha et al., 2014). The prevalence studies of B. gibsoni with PCR based assays were variable worldwide and ranging from 3.3 to 55% (Macintire et al., 2002; Inokuma et al., 2004;Talukder et al., 2012; Mokhtar et al., 2013; Laha et al., 2014; Singh et al., 2014). Fig-d: Amplicons corresponding to 18S rrna gene of B. gibsoni Lane 1: 100 bp DNA marker Lane 2: Positive DNA control Lane 3-4: Test sample Lane 5: Negative control Taking PCR as more accurate test for diagnosing B. gibsoni infection, the sensitivity of IFAT was found to be of 93.10% while specificity was 88.89% whereas, sensitivity of microscopic examination was 71.26%, while specificity was 95.56% (Table 2). In other words, IFAT was found to be more sensitive but slightly less specific than that of microscopic examination. The diagnosis of B. gibsoni in dogs by microscopic examination of blood smear requires expertise because parasites are often overlooked when parasitemia is significantly low. The antigenic cross- reaction of B. gibsoni with other Babesia spp. (Yamane et al., 1994) and normal dog erythrocytes (Adachi and Makimura, 1993; Yamane et al., 1993, Adachi et al., 1994) probably lowers the specificity of IFAT. Further in early stage of infection, the possibility of detection of B. gibsoni infection by IFAT has been reported less (Yamane et al., 1993). IFAT is useful for the diagnosis of chronically infected dogs with significantly low level of parasitemia. PCR detects parasite DNA from earlier stages of infection which go unnoticed by IFAT and microscopic examination. Moreover, PCR detects parasite DNA in the late stages of infection with a significantly low level of parasitemia or with no apparent parasite shown in blood smear. These results suggest that the PCR has the potential to detect the parasite DNA in very early stages of infection, before the host animal shows clinical signs. The agreement of IFAT for detection of B. gibsoni in dogs, with PCR was 82.30% (k, 0.823), whereas it was 60.73% (k, 0.6073) for microscopic examination of blood smear with PCR. The agreement between IFAT and microscopy was 60.99% (k, 0.6099). Furthermore PCR detected parasite DNA in 25 negative samples examined by

4 INDIAN JOURNAL OF ANIMAL RESEARCH Table 1: Comparison of Microscopic examination, IFAT and PCR for detection of B. gibsoni in dogs Sample no. Microscopy a IFAT b PCR c 1 + + + 2 + + + 3 + + + 4 + + + 5 + + + 6 + + + 7 + + + 8 + - + 9 + + + 10 - - + 11 - - + 12 + + + 13 + + + 14 - - + 15 - + + 16 + + + 17 - + - 18 + + + 19 - + + 20 + + + 21 + + + 22 + + + 23 - + + 24 - + - 25 + + + 26 - + + 27 + + + 28 + + + 29 + + + 30 + + + 31 + + + 32 + + + 33 + + + 34 + + + 35 - + + 36 + + + 37 + + + 38 + + + 39 - + + 40 + + + 41 + + + 42 + + + 43 + + + 44 - + + 45 + + + 46 + + + 47 + + + 48 + + + 49 + + + 50 - + + 51 - - + 52 - + + 53 + - - 54 - + - 55 + + + 56 + + + 57 + + + Sample no. Microscopy a IFAT b PCR c 58 + + + 58 - + + 60 + + + 61 + + + 62 + + + 63 - - - 64 + + + 65 + + + 66 + + + 67 - + + 68 - + + 69 + + + 70 + + + 71 + + + 72 - + + 73 - - - 74 - + + 75 + + + 76 + + + 77 + + + 78 + + + 79 + + + 80 + + + 81 - + + 82 - + + 83 - - - 84 - - - 85 - - - 86 - + - 87 - + + 88 - + + 89 + + + 90 - - - 91 - - - 92 - + + 93 + + - 94 + + + 95 + + + 96 + + + 97 - - - 98 - + + 99 - - - 100 - - + 101 + + + 102 + + + 103 - - - 104 - - - 105 - - - 106 - - - 107 - - - 108 - - - 109 - - - 110 - - - 111 - - - 112 - - - 113 - - - 114 - - - 115 - - - continue Table 1...

Sample no. Microscopy a IFAT b PCR c 116 - - - 117 - - - 118 - - - 119 - - - 120 - - - 121 - - - 122 - - - 123 - - - 124 - - - 125 - - - 126 - - - 127 - - - 128 - - - 129 - - - 130 - - - 131 - - - 132 - - - a) Microscopy was considered positive when B. gibsoni was detected in Giemsa stained thin blood smear b) IFAT was considered positive when florescence was observed at dilution of 1:10 c) PCR was considered positive when specific PCR product was detected in agarose gel electrophoresis Vol. Issue, () microscopic examination and 6 negative samples examined by IFAT indicating that the sensitivity of PCR was higher than that of IFAT and microscopic examination for diagnosing B. gibsoni. The similar observation has been reported by Fukumoto et al. (2001). IFAT detected the parasite in 24 negative samples examined by microscopy. Therefore the efficacy of IFAT for diagnosing B. gibsoni was comparable with PCR. CONCLUSION As far as the diagnosis of canine babesiosis is concerned, direct microscopic examination of the stained blood smear is the most commonly used method as it is conclusive, feasible, and cost effective but not necessarily detects parasites in dogs with unapparent or chronic infection since the level of parasitemia is very low. IFAT for B. gibsoni, are considered to be highly sensitive, but slightly less specific. PCR detects and identifies piroplasms with greater sensitivity and specificity than traditional methods, but it is time consuming and requires expertise. Great care must be taken to avoid contamination with the PCR, which can lead to false positive results. Thus, PCR data should always be Table 2: Sensitivity and Specificity of IFAT and microscopic examination vis a vis PCR for detection of B. gibsoni Tests Results PCR Sensitivity Specificity Positive Negative Total IFAT Positive 81 5 86 93.10% 88.89% Negative 6 40 46 Total 87 45 132 Microscopy Positive 62 2 64 71.26% 95.56% Negative 25 43 68 Total 87 45 132 corroborated by immunologic tests. As the efficacy of IFAT is comparable to PCR for detection of B. gibsoni in dogs therefore, IFAT could be used in place of PCR for diagnosis of B. gibsoni as it is less time consuming, have high sensitivity and easy to perform. ACKNOWLEDGEMENT The authors are thankful to the Director, ICAR- IVRI, Izatnagar and Indian Council of Agricultural Research, New Delhi for providing the necessary facilities/fund to carry out the study. REFERENCES Adachi, K., Tateishi, M., Horii, Y., Nagatomo, H., Shimizu, T. and Makimura, S. (1994). Reactivity of serum antierythrocyte membrane antibody in Babesia gibsoni infected dogs. J. Vet. Med. Sci., 56: 997-999. Adachi, K., Ueno, C. and Makimura, S. (1993). Immunosuppression in dogs naturally infected with Babesia gibsoni. J. Vet. Med. Sci., 55:503 505. Amuta, E. U., Atu, B. O., Houmsou, R. S. and Ayashar, J. G. (2010). Rhipicephalus sanguineus infestation and Babesia canis infection among domestic dogs in Makurdi, Benue State-Nigeria. Int. J. Acad. Res., 2: 170 172. Ayoob, A. L. and Prittie, J. (2010). Clinical management of canine babesiosis. J. Vet. Emerg. Crit. Care., 20: 77-89. Aysul, N., Ural, K., Ulutas, B., Eren, H. and Karagenc, T. (2013). First detection and molecular identification of Babesia gibsoni in two dogs from the Aydyn Province of Turkey. Turk. J. Vet. Anim. Sci., 37: 226-229. Birkenheuer, A. J., Levy, M. G., Stebbins, M., Poore, M. and Breitschwerdt, E. (2003). Serosurvey of anti-babesia antibodies in stray dogs and American pit bull terriers and American Staffordshire terriers from North Carolina. J. Am. Anim. Hosp. Assoc., 39: 551-557. Brandao, L. P., Hagiwara, M. K. and Myashiro, S. I. (2003). Humoral immunity and reinfection resistance in dogs experimentally inoculated with Babesia canis and either treated or untreated with imidocarb dipropionate. Vet. Parasitol., 114: 253-265. Caccio, S. M., Antunovic, B., Moretti, A., Mangili, V., Marinculic, A., Baric, R. R., et al. (2002). Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs. Vet. Parasitol., 106:285-292.

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