Ticks and Tick-borne Diseases 5 (2014) 902–906
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Highly sensitive method for diagnosis of subclinical B. ovis infection夽 Sara Horta a,b , Maria C. Barreto a,b , Ana Pepe a , Joana Campos a,1 , Abel Oliva a,b,∗ a Biomolecular Diagnostic Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal b IBET, Apartado 12, 2781-901 Oeiras, Portugal
a r t i c l e
i n f o
Article history: Received 29 May 2014 Received in revised form 10 July 2014 Accepted 10 July 2014 Available online 7 August 2014 Keywords: Babesia ovis PCR Diagnostic Sheep and goats Ticks Portugal
a b s t r a c t Babesia ovis is a tick-transmitted protozoa parasite that infects small ruminants causing fever, anaemia, hemoglobinuria, anorexia and, in acute cases, death. Common in tropical and sub-tropical areas, the presence of this parasite in sheep herds has an economic impact on industry and therefore sensitive methods for the diagnosis and disease eradication are required. To achieve this goal, a semi-nested PCR for B. ovis specific identification was developed and consequent reaction conditions and enzymes were optimized and tested with field samples. 300 blood samples from small ruminants and 39 ticks from Rhipicephalus genus were collected from different regions of Portugal. Afterwards, DNA extraction was performed and conventional and semi-nested PCR were accomplished for all samples. The results obtained from both methodologies were compared and the sensitivity was evaluated. Employing the semi-nested PCR it was possible to identify a higher number of positive cases among the evaluated samples than using the conventional PCR, namely 38/300 blood samples and 7/39 ticks. However, fragment amplification was only observed in 5 out of 300 blood samples and in none of the 39 ticks when a conventional PCR was employed. The validation of the results was achieved by sequencing the DNA fragments corresponding to the hypervariable v4 region of the 18S ribosomal RNA gene and performing an alignment with sequences already published on GenBank® . The ticks collected in this study belong to the Rhipicephalus genus, although other species could be involved as a vector in the Babesia spread. The diagnostic assay here described is presently the most effective and sensitive method for detection of B. ovis in field blood samples and ticks, enabling the detection up to 1 parasite into 109 erythrocytes. © 2014 Elsevier GmbH. All rights reserved.
Introduction Ovine babesiosis is a tick-borne disease of small ruminants caused by intraerythrocytic parasites of the genus Babesia in tropical and subtropical regions. One of the responsible agent for this disease is B. ovis, an apicomplexan protozoa parasite mainly transmitted by Rhipicephalus ticks causing clinical signs of fever, anaemia, hemoglobinuria, anorexia and possible death (Homer et al., 2000; Uilenberg, 2006). Hence the presence of this parasite has an economic significance on industry in several parts of the world mainly related to the decrease of meat, milk and wool
夽 Nucleotide sequence data reported in this paper are available in GenBankTM database under the accession number KJ829366. ∗ Corresponding author at: Biomolecular Diagnostic Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. Tel.: +351 21446 9427; fax: +351 214421161. E-mail address: [email protected] (A. Oliva). 1 Present address: Paul O’Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, United Kingdom. http://dx.doi.org/10.1016/j.ttbdis.2014.07.005 1877-959X/© 2014 Elsevier GmbH. All rights reserved.
production. In the particular case of mainland Portugal, there is an estimated livestock of ∼2 million sheep and ∼400 thousand goats with a livestock slaughtering of ∼770 and ∼130 thousand, respectively (Estatística, 2013). Although the diagnosis of ovine babesiosis reveals to be an important tool for its control and prevention, a comprehensive picture of B. ovis prevalence and distribution along the country is still unknown. Previous studies performed in the Portuguese tick fauna in 1994–2009, with a sampling of 15 044 specimens, demonstrate that Rhipicephalus sanguineus (33.9%) is the most common in the country followed by Ixodes ricinus (25.6%), Dermacentor marginatus (10.3%) and Rhipicephalus bursa (4.3%) (Santos-Silva et al., 2011). All of them are vectors of B. ovis and are commonly found in sheep and goats in Portugal (Caeiro, 1999; Friedhoff, 1997; Uilenberg, 2006). Besides the symptomatology, a confirmatory diagnosis of the disease relates to the observation in microscope of parasites within erythrocyte through Giemsa-stained blood smears (Yabsley and Shock, 2013). In cases of low-level carriers or early stages of infection the microscopic detection becomes cumbersome, being difficult to discriminate among parasites infecting the erythrocytes
S. Horta et al. / Ticks and Tick-borne Diseases 5 (2014) 902–906
and therefore making this method inadequate for large scale prevalence studies (Silva et al., 2010). On the other hand, immunological and molecular techniques such as Enzyme-Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR) offer a higher sensitivity and specificity, enabling the diagnosis of Babesia’s infection. In terms of immunological methods, the diagnosis is based on the presence of specific antibodies against those parasites, with the disadvantage that they usually take days or weeks to develop in an infected animal or are present for long periods after infection or after vaccination (Mosqueda et al., 2012). In addition, the requirement of greater amounts of antigen as well as cross reactions against other Babesia species limits the specificity of serological tests (Zintl et al., 2003). Due to the aforementioned reasons, molecular methods such as PCR, based on nucleic acids detection, became the main diagnostic techniques. An adaptation of this technique is nested-PCR, described as more specific and sensitive by enabling the identification of subclinical cases where the animal is not clinically ill but still remains as a Babesia’s carrier. Its high specificity and sensitivity was already described for B. equi, B. bovis and B. bigemina (Costa-Junior et al., 2006; Nicolaiewsky et al., 2001). For B. ovis molecular detection, a pair of primers specific for the hypervariable v4 region of the 18S ribosomal RNA gene (SSU rRNA) was first described by Gubbels et al., and later by Schnittger et al. for small ruminants (Gubbels et al., 1999; Schnittger et al., 2004), which enables the simultaneous identification of different species of Babesia and Theileria genus in a single experiment. One year later Aktas et al. (2005) proposed a pair of complementary primers for the same hypervariable region, although specific for B. ovis, yielding a 549-bp fragment towards a positive case. In this study, we described a semi-nested PCR developed for B. ovis detection on blood samples from small ruminants and ticks. Reaction conditions were optimized, and comparison between single PCR and semi-nested PCR was performed in blood samples and ticks collected over mainland Portugal, evaluating the feasibility and sensitivity of the method.
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homogenized before DNA extraction. For DNA extraction, a commercial kit (DNeasy® Blood & Tissue Kit, Qiagen) was used and the procedure performed according to manufacturer’s instructions. Polymerase chain reaction and semi-nested PCR For the preparation of the conventional PCR a pair of oligonucleotide primers specific for ssu rRNA gene of B. ovis, as described by Aktas et al. (2005), were used for the PCR amplification, yielding a fragment of 549-bp: forward strand primer Bbo-F 5 TGGGCAGGACCTTGGTTCTTCT-3 and reverse strand primer Bbo-R 5 -CCGCGTAGCGCCGGCTAAATA-3 . The reaction was performed in a Biometra® T3000 thermocycler in a total reaction volume of 25 L, containing 2,5 L of Pfu DNA polymerase 10× reaction buffer with MgSO4 , 200 M of dNTPs, 0.20 M of each primer, 1.25 U Pfu DNA polymerase (Promega) and 2 L of template DNA. The reaction was repeated for 35 cycles under the following conditions: 1 min at 94 ◦ C, 1 min at 62 ◦ C and 1 min at 75 ◦ C. Regarding the semi-nested PCR, for the first reaction was used the oligonucleotide sequence RLB-F (5 -GAGGTAGTGACAAGAAATAACAATA-3 ) as forward, as described by Schnittger et al. (2004), and the Bbo-R as reverse. The reaction mixture was the same as in the conventional PCR, although repeated for 30 cycles under the following conditions: 1 min at 94 ◦ C, 1 min at 57 ◦ C and 1 min at 75 ◦ C. The resultant amplified fragment has a length of 846-bp. After the reaction, a 1:50 dilution of the PCR products in Milli-Q water was performed. On the second reaction, the pair of primers Bbo-F and -R were used under the same conditions as the previous reaction. The diluted PCR products obtained on the first reaction were used as a template. All PCR products were visualized by UV transilluminator in a 1.5% agarose gel following electrophoresis and staining with SYBR® safe DNA Gel stain (Invitrogen). All samples had a negative control on which water was added instead of DNA. As a positive control, DNA extracted from Israeli B. ovis was used. This strain was kindly provided by Dr. Varda Shkap, Kimron Veterinary Institute, Israel. DNA sequencing
Material and methods Samples collection and DNA extraction Since 2010, 300 blood samples from randomized sheep and goats were collected in EDTA tubes (Vacutest system) from farms in different regions of Portugal (14 districts). The information related to sex, age, breed and putative treatment with antiparasitic drugs of the donor animal were registered and, with the exception of 2 samples from Montemor-o-Novo, none of the sampled animals showed piroplasmosis symptoms. From the total of samples received, 65.3% were sheep and 34.7% goats; 95.2% were females and, in total, are at an average age of 4.15 ± 2.61 years. DNA extraction from blood was performed using a commercial kit (ArchivePure DNA Purification, 5Prime), accordingly with manufacturer’s instructions. DNA quantification was performed using Thermo Scientific Nanodrop ND-2000c. When the DNA concentration was above 100 ng/L, a dilution in Milli-Q water was performed, aiming for a final concentration of 100 ng/L. DNA extraction was repeated in samples where its concentration was below 15 ng/L. All DNA samples were stored at −20 ◦ C until further use. In some of the places where blood samples were obtained, 39 ticks were collected from small ruminants and stored in vials with humidified paper for transport. In the laboratory the ticks were washed on ethanol 96% and stored in cryotubes at −80 ◦ C. Each tick was examined in terms of gender and species and the tissues
To confirm and validate the results, randomly selected positive samples for B. ovis (15 DNA samples extracted from blood and 2 DNA samples extracted from Ticks) identified by the semi-nested PCR were sequenced. The purification of PCR products was performed using the commercial kit Wizard® SV Gel and PCR Clean-up System (Promega), following the instructions of the manufacturer. After lyophilization, all samples were sent to GATC Biotech Company (Constance, Germany) for fragment’s sequencing. Portuguese B. ovis isolation and determination of PCR and semi-nested PCR sensitivity In order to test the sensitivity and determinate the detection limit of both PCR assay, cultivation of B. ovis was performed. Blood collected on EDTA tubes and identified as a positive through seminested PCR was suspended in HEPES-buffered Medium 199 (1×) supplemented with Earle’s Salts, l-glutamine and l-Amino acids (Gibco, Life technologies) supplemented with 20% sheep serum and 10% of sheep uninfected erythrocytes. The complete medium was also buffered with 20 mM TES and antibiotic–antimycotic (100×) (Gibco, Invitrogen) was added at a final concentration of 0.25 mg/mL amphotericin, 100 U/mL penicillin and 100 g/mL streptomycin to prevent contaminations. The culture was added in a 24-well plate and incubated at 37 ◦ C in a humidified chamber filled with a gas mixture of 5% CO2 , 2% O2 and 93% N2 . Every 24 h half of the supernatant was replaced for fresh medium and the parasitaemia monitored by preparing thin blood smears stained with
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Giemsa. Intraerythrocytic parasites were observed under a magnification of 1000× on a Nikon Eclipse TE2000-S (Japan) inverted microscope. After reaching a parasitaemia of 1% (50 000 parasites/L), B. ovis-infected erythrocytes were subjected to 10-fold serial dilutions (from 10−1 to 10−10 ) using uninfected lamb erythrocytes as diluent (Gama et al., 2007). DNA was extracted from each diluted sample and processed for PCR as previously described. For specificity test, DNA was extracted from isolates of B. bigemina (kindly provided by Silvina Wilkowsky (Instituto Nacional de tecnologia Agropecuaria, Argentina)), B. bovis mo7 (kindly provided by Erik de Vries (Faculty of Veterinary Medicine, Netherlands)), T. ovis, T. uilenberg (Kindly provided by Yin Hong (Lanzhou Veterinary Research Institute, China)) and Anaplasma ovis (kindly provided by Tulin Karagenc (Faculty of Veterinary Medicine, Turkey)) and semi-nested PCR was applied as previously described.
Results Blood samples randomly collected from sheep and goats from 14 districts of Portugal were examined in terms of B. ovis prevalence by both semi-nested and conventional PCR. Out of 300 samples, 12.7% (38/300) were marked as positive by a semi-nested PCR and 1.67% (5/300) were marked as positive by conventional PCR. Those 5 positive samples were confirmed by semi-nested PCR. The sampling distribution and the positive cases along the country are presented in Fig. 1. Regarding the DNA extracted from ticks, it was possible to identify 7 out of 39 (17.9%) as carriers of B. ovis after semi-nested PCR methodology. However, it was not possible to identify any carrier on a conventional PCR. The ticks included in the study belong to the genus Rhipicephalus and 28 were adults (19 females and 9 males), 10 nymphs and 1 larva (Fig. 2). From those 4 females, 2 males and 1 nymph were positive for B. ovis. For results validation, 17 randomly selected positive samples were sequenced. The resultant sequence is available in GenBankTM database under the accession number KJ829366 and the amplified DNA fragments showed 100% of identity with the sequences AY150058 and AY533146, previously published. B. ovis from a positive sample was isolated and used to establish an in vitro cell culture. By adding the infected blood to the culture media, its presence was confirmed after 4 days of growth by microscopic examination of thin blood smears, Fig. 3. After the in vitro culture reached a parasitaemia of 1%, the detection limit of the assay was calculated and the results are presented in Fig. 4. It has been
Fig. 1. Distribution of blood samples collected along Portugal continental from 2010 to 2014. Blue circles: number of tested samples for B. ovis presence using seminested PCR; green circles: number of positive samples for B. ovis. Image adapted from Covas et al. (2013). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
observed that parasitaemia down to 10−7 % can be detected using the proposed semi-nested PCR. Although, when a conventional PCR is applied B. ovis can only be detected down to a parasitaemia of 10−3 %. For specificity test, semi-nested PCR using DNA extracted from isolates of different organisms (B. ovis, B. bovis, B. bigemina, T. ovis, T. uilenberg, A. ovis) was performed and only DNA of Babesia ovis was amplified. No unexpected amplification was observed (Fig. 5). Discussion B. ovis is a tick-transmitted protozoa parasite that infects sheep and goats and causes host-mediated pathology. Common in tropical and sub-tropical areas, the presence of this parasite has an economical impact on industry and the early diagnosis and successful therapy are the key steps for its control (Ranjbar-Bahadori et al.,
Fig. 2. Microscopic image of an adult male Rhipicephalus sanguineus collected from one of the field animals, 40× magnification. The left image shows the ventral view; the right image shows the dorsal view.
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Fig. 3. Thin blood smears showing erythrocytes infected with Portuguese isolate of B. ovis stained with Giemsa 1000× magnification.
2012). Unfortunately, the epidemiological knowledge on this disease is often impaired by the lack of a diagnostic tool available to the vets. The detection of Babesia infection in carrier animals and ticks by DNA amplification is a powerful tool for epidemiological investigation. A PCR specific for B. ovis was published for the first time in 2005 by Aktas et al. (Aktas et al., 2005), using a pair of oligonucleotide primers which target a 549-bp fragment. However, a high number of false negatives matching to a subclinical state of the animal were observed. Recognition of such cases is important given their potential role on vector-mediated disease spreading and, ultimately, loss of herd productivity (Alessandra and Santo, 2012). The hypervariable region V4 of 18S rRNA has been extensively used as a marker for taxonomic classification and phylogenetic analysis of Babesia and Theileria species (Salim et al., 2010; Schnittger et al., 2003). Due to the high degree of conservation associated with this region, it is possible to achieve higher sensitivity and specificity on hemoparasites identification towards multiple infections and low parasitaemia cases. In a total of 300 blood samples, 38 infected animals were identified by the semi-nested PCR whereas only 5 of them were positively diagnosed by a conventional PCR. Related to the diagnosis of Babesia
Fig. 4. Sensitivity of semi-nested and conventional PCR. 1.5% agarose gel electrophoresis of the amplicons from DNA extracted from in vitro culture with 10-fold serial dilutions (10−1 to 10−10 ). (A) Products obtained on conventional PCR with B. ovis specific primers. (B) Products obtained on the 2nd round of semi-nested PCR with B. ovis specific primers. Lane M, 100-bp Ladder DNA marker; lane 1, DNA extracted from blood with 1% parasitaemia; lane 2, 0.1% parasitaemia; lane 3, 0.01% parasitaemia; lane 4, 0.001% parasitaemia; lane 5, 10−4 % parasitaemia; lane 6, 10−5 % parasitaemia; lane 7, 10−6 % parasitaemia; lane 8, 10−7 % parasitaemia; lane 9, 10−8 % parasitaemia; lane 10, 10−9 % parasitaemia; lane 11, 10−10 % parasitaemia; lane 12, uninfected sheep blood (negative control).
in carrier ticks, the identification of positive bearers was only possible using semi-nested methodology (7/39). In addition, a sensitivity assay performed for both PCR methodologies demonstrates the higher sensibility of semi-nested when compared to the conventional one. Using this method it is possible to identify 1 parasite in 109 erythrocytes. Furthermore, when DNA of parasites phylogenetically related as B. bovis, B. bigemina, T. ovis, T. uilenberg and A. ovis was added, the amplification of the expected fragment only occurred when B. ovis DNA was present. Although being more laborious, the semi-nested PCR significantly enhances the identification of B. ovis’ carriers, can also be applied in the identification of parasites on ticks and is specific for B. ovis even during multiple infections. The development of this methodology also enabled us to initiate the first survey of B. ovis in Portugal and the isolation of the Portuguese strain, as observed in Fig. 3. With an epidemiologic study in sight, further investigation will be required in order to increase sample size and establish a correlation with positive vectors. However, obtained data from the current field work clearly shows a high prevalence (12.7%) of babesiosis mainly distributed in the southern and littoral regions of Portugal and suggests that B. ovis is endemic in most regions of the country. Interestingly, the majority of the sampled animals did not show symptoms of babesiosis at the time of blood collection, nor had disease track. Furthermore, no correlation between species, age and gender were found between the prevalence of positive cases although it was already proposed that B. ovis causes disease almost exclusively in sheep, rarely in goats (Aktas et al., 2007). Given the mentioned absence of symptoms, it can be concluded that either the B. ovis present in Portugal is not as pathogenic as reported in other countries, or the level of virulence may be related with mixed infections. Concluding two consecutive PCR rounds instead of one improves the specificity and sensitivity of the B. ovis detection in small ruminants and therefore contributes to a better control and monitoring of the disease.
Fig. 5. Specificity of the semi-nested PCR. 1.5% agarose gel electrophoresis of the amplicons from Babesia, Theileria and Anaplasma species using B. ovis-specific primers. Lane M, 100-bp Ladder DNA Marker; lane 1, B. ovis; lane 2, B. bovis; lane 3, B. bigemina; lane 4, Theileria ovis; lane 5, Theileria uilenberg; lane 6, Anaplasma ovis; lane 7, Babesia species without B. ovis; lane 8, Babesia species with B. ovis; lane 9, Theileria species without B. ovis; lane 10, Theileria species with B. ovis; lane 11, uninfected sheep blood (negative control).
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Acknowledgments This work was financed by the European Project PIROVAC (KBBE-3-245145-PIROVAC). Thanks to all collaborators who kindly provided the isolates used in this work, and special thanks to Dr. Varda Shkap, Kimron Veterinary Institute, Israel, for providing the Israeli strain of B. ovis. Thanks to Dr. Isabel Mariano of COPRAPEC, Montemor-o-Novo, for the advice and the biological material provided. To all veterinary associations and vets that gently cooperated with this study by collecting and sending samples from the field. References Aktas, M., Altay, K., Dumanli, N., 2005. Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats. Vet. Parasitol. 133, 277–281. Aktas, M., Altay, K., Dumanli, N., 2007. Determination of prevalence and risk factors for infection with Babesia ovis in small ruminants from Turkey by polymerase chain reaction. Parasitol. Res. 100, 797–802. Alessandra, T., Santo, C., 2012. Tick-borne diseases in sheep and goats: clinical and diagnostic aspects. Small Rumin. Res. 106 (Suppl.), S6–S11. Caeiro, V., 1999. General review of tick species present in Portugal. Parassitologia 41 (Suppl. 1), 11–15. Costa-Junior, L.M., Rabelo, E.M., Martins Filho, O.A., Ribeiro, M.F., 2006. Comparison of different direct diagnostic methods to identify Babesia bovis and Babesia bigemina in animals vaccinated with live attenuated parasites. Vet. Parasitol. 139, 231–236. Covas, M.T., Silva, C.A., Dias, L.C., 2013. On locating sustainable data centers in Portugal: problem structuring and GIS-based analysis. Sustain. Comput.: Inf. Syst. 3, 27–35. Estatística, I.N.D., 2013. Statistical Yearbook of Portugal 2012, Lisbon, Portugal. Friedhoff, K.T., 1997. Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp. Parassitologia 39, 99–109. Gama, B.E., Silva-Pires Fdo, E., Lopes, M.N., Cardoso, M.A., Britto, C., Torres, K.L., de Mendonca Lima, L., de Souza, J.M., Daniel-Ribeiro, C.T., Ferreira-da-Cruz, M.,
2007. Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia. Exp. Parasitol. 116, 427–432. Gubbels, J.M., de Vos, A.P., van der Weide, M., Viseras, J., Schouls, L.M., de Vries, E., Jongejan, F., 1999. Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J. Clin. Microbiol. 37, 1782–1789. Homer, M.J., Aguilar-Delfin, I., Telford III, S.R., Krause, P.J., Persing, D.H., 2000. Babesiosis. Clin. Microbiol. Rev. 13, 451–469. Mosqueda, J., Olvera-Ramirez, A., Aguilar-Tipacamu, G., Canto, G.J., 2012. Current advances in detection and treatment of babesiosis. Curr. Med. Chem. 19, 1504–1518. Nicolaiewsky, T.B., Richter, M.F., Lunge, V.R., Cunha, C.W., Delagostin, O., Ikuta, N., Fonseca, A.S., da Silva, S.S., Ozaki, L.S., 2001. Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction. Vet. Parasitol. 101, 9–21. Ranjbar-Bahadori, S., Eckert, B., Omidian, Z., Shirazi, N.S., Shayan, P., 2012. Babesia ovis as the main causative agent of sheep babesiosis in Iran. Parasitol. Res. 110, 1531–1536. Salim, B., Bakheit, M.A., Kamau, J., Nakamura, I., Sugimoto, C., 2010. Nucleotide sequence heterogeneity in the small subunit ribosomal RNA gene within Theileria equi from horses in Sudan. Parasitol. Res. 106, 493–498. Santos-Silva, M.M., Beati, L., Santos, A.S., De Sousa, R., Nuncio, M.S., Melo, P., SantosReis, M., Fonseca, C., Formosinho, P., Vilela, C., Bacellar, F., 2011. The hard-tick fauna of mainland Portugal (Acari: Ixodidae): an update on geographical distribution and known associations with hosts and pathogens. Exp. Appl. Acarol. 55, 85–121. Schnittger, L., Yin, H., Gubbels, M.J., Beyer, D., Niemann, S., Jongejan, F., Ahmed, J.S., 2003. Phylogeny of sheep and goat Theileria and Babesia parasites. Parasitol. Res. 91, 398–406. Schnittger, L., Yin, H., Qi, B., Gubbels, M.J., Beyer, D., Niemann, S., Jongejan, F., Ahmed, J.S., 2004. Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting. Parasitol. Res. 92, 189–196. Silva, M.G., Marques, P.X., Oliva, A., 2010. Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method. Vet. Parasitol. 174, 199–205. Uilenberg, G., 2006. Babesia – a historical overview. Vet. Parasitol. 138, 3–10. Yabsley, M.J., Shock, B.C., 2013. Natural history of Zoonotic Babesia: role of wildlife reservoirs. Int. J. Parasitol. Parasites Wildl. 2, 18–31. Zintl, A., Mulcahy, G., Skerrett, H.E., Taylor, S.M., Gray, J.S., 2003. Babesia divergens, a bovine blood parasite of veterinary and zoonotic importance. Clin. Microbiol. Rev. 16, 622–636.
Contents lists available at ScienceDirect
Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Highly sensitive method for diagnosis of subclinical B. ovis infection夽 Sara Horta a,b , Maria C. Barreto a,b , Ana Pepe a , Joana Campos a,1 , Abel Oliva a,b,∗ a Biomolecular Diagnostic Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal b IBET, Apartado 12, 2781-901 Oeiras, Portugal
a r t i c l e
i n f o
Article history: Received 29 May 2014 Received in revised form 10 July 2014 Accepted 10 July 2014 Available online 7 August 2014 Keywords: Babesia ovis PCR Diagnostic Sheep and goats Ticks Portugal
a b s t r a c t Babesia ovis is a tick-transmitted protozoa parasite that infects small ruminants causing fever, anaemia, hemoglobinuria, anorexia and, in acute cases, death. Common in tropical and sub-tropical areas, the presence of this parasite in sheep herds has an economic impact on industry and therefore sensitive methods for the diagnosis and disease eradication are required. To achieve this goal, a semi-nested PCR for B. ovis specific identification was developed and consequent reaction conditions and enzymes were optimized and tested with field samples. 300 blood samples from small ruminants and 39 ticks from Rhipicephalus genus were collected from different regions of Portugal. Afterwards, DNA extraction was performed and conventional and semi-nested PCR were accomplished for all samples. The results obtained from both methodologies were compared and the sensitivity was evaluated. Employing the semi-nested PCR it was possible to identify a higher number of positive cases among the evaluated samples than using the conventional PCR, namely 38/300 blood samples and 7/39 ticks. However, fragment amplification was only observed in 5 out of 300 blood samples and in none of the 39 ticks when a conventional PCR was employed. The validation of the results was achieved by sequencing the DNA fragments corresponding to the hypervariable v4 region of the 18S ribosomal RNA gene and performing an alignment with sequences already published on GenBank® . The ticks collected in this study belong to the Rhipicephalus genus, although other species could be involved as a vector in the Babesia spread. The diagnostic assay here described is presently the most effective and sensitive method for detection of B. ovis in field blood samples and ticks, enabling the detection up to 1 parasite into 109 erythrocytes. © 2014 Elsevier GmbH. All rights reserved.
Introduction Ovine babesiosis is a tick-borne disease of small ruminants caused by intraerythrocytic parasites of the genus Babesia in tropical and subtropical regions. One of the responsible agent for this disease is B. ovis, an apicomplexan protozoa parasite mainly transmitted by Rhipicephalus ticks causing clinical signs of fever, anaemia, hemoglobinuria, anorexia and possible death (Homer et al., 2000; Uilenberg, 2006). Hence the presence of this parasite has an economic significance on industry in several parts of the world mainly related to the decrease of meat, milk and wool
夽 Nucleotide sequence data reported in this paper are available in GenBankTM database under the accession number KJ829366. ∗ Corresponding author at: Biomolecular Diagnostic Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. Tel.: +351 21446 9427; fax: +351 214421161. E-mail address: [email protected] (A. Oliva). 1 Present address: Paul O’Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, United Kingdom. http://dx.doi.org/10.1016/j.ttbdis.2014.07.005 1877-959X/© 2014 Elsevier GmbH. All rights reserved.
production. In the particular case of mainland Portugal, there is an estimated livestock of ∼2 million sheep and ∼400 thousand goats with a livestock slaughtering of ∼770 and ∼130 thousand, respectively (Estatística, 2013). Although the diagnosis of ovine babesiosis reveals to be an important tool for its control and prevention, a comprehensive picture of B. ovis prevalence and distribution along the country is still unknown. Previous studies performed in the Portuguese tick fauna in 1994–2009, with a sampling of 15 044 specimens, demonstrate that Rhipicephalus sanguineus (33.9%) is the most common in the country followed by Ixodes ricinus (25.6%), Dermacentor marginatus (10.3%) and Rhipicephalus bursa (4.3%) (Santos-Silva et al., 2011). All of them are vectors of B. ovis and are commonly found in sheep and goats in Portugal (Caeiro, 1999; Friedhoff, 1997; Uilenberg, 2006). Besides the symptomatology, a confirmatory diagnosis of the disease relates to the observation in microscope of parasites within erythrocyte through Giemsa-stained blood smears (Yabsley and Shock, 2013). In cases of low-level carriers or early stages of infection the microscopic detection becomes cumbersome, being difficult to discriminate among parasites infecting the erythrocytes
S. Horta et al. / Ticks and Tick-borne Diseases 5 (2014) 902–906
and therefore making this method inadequate for large scale prevalence studies (Silva et al., 2010). On the other hand, immunological and molecular techniques such as Enzyme-Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR) offer a higher sensitivity and specificity, enabling the diagnosis of Babesia’s infection. In terms of immunological methods, the diagnosis is based on the presence of specific antibodies against those parasites, with the disadvantage that they usually take days or weeks to develop in an infected animal or are present for long periods after infection or after vaccination (Mosqueda et al., 2012). In addition, the requirement of greater amounts of antigen as well as cross reactions against other Babesia species limits the specificity of serological tests (Zintl et al., 2003). Due to the aforementioned reasons, molecular methods such as PCR, based on nucleic acids detection, became the main diagnostic techniques. An adaptation of this technique is nested-PCR, described as more specific and sensitive by enabling the identification of subclinical cases where the animal is not clinically ill but still remains as a Babesia’s carrier. Its high specificity and sensitivity was already described for B. equi, B. bovis and B. bigemina (Costa-Junior et al., 2006; Nicolaiewsky et al., 2001). For B. ovis molecular detection, a pair of primers specific for the hypervariable v4 region of the 18S ribosomal RNA gene (SSU rRNA) was first described by Gubbels et al., and later by Schnittger et al. for small ruminants (Gubbels et al., 1999; Schnittger et al., 2004), which enables the simultaneous identification of different species of Babesia and Theileria genus in a single experiment. One year later Aktas et al. (2005) proposed a pair of complementary primers for the same hypervariable region, although specific for B. ovis, yielding a 549-bp fragment towards a positive case. In this study, we described a semi-nested PCR developed for B. ovis detection on blood samples from small ruminants and ticks. Reaction conditions were optimized, and comparison between single PCR and semi-nested PCR was performed in blood samples and ticks collected over mainland Portugal, evaluating the feasibility and sensitivity of the method.
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homogenized before DNA extraction. For DNA extraction, a commercial kit (DNeasy® Blood & Tissue Kit, Qiagen) was used and the procedure performed according to manufacturer’s instructions. Polymerase chain reaction and semi-nested PCR For the preparation of the conventional PCR a pair of oligonucleotide primers specific for ssu rRNA gene of B. ovis, as described by Aktas et al. (2005), were used for the PCR amplification, yielding a fragment of 549-bp: forward strand primer Bbo-F 5 TGGGCAGGACCTTGGTTCTTCT-3 and reverse strand primer Bbo-R 5 -CCGCGTAGCGCCGGCTAAATA-3 . The reaction was performed in a Biometra® T3000 thermocycler in a total reaction volume of 25 L, containing 2,5 L of Pfu DNA polymerase 10× reaction buffer with MgSO4 , 200 M of dNTPs, 0.20 M of each primer, 1.25 U Pfu DNA polymerase (Promega) and 2 L of template DNA. The reaction was repeated for 35 cycles under the following conditions: 1 min at 94 ◦ C, 1 min at 62 ◦ C and 1 min at 75 ◦ C. Regarding the semi-nested PCR, for the first reaction was used the oligonucleotide sequence RLB-F (5 -GAGGTAGTGACAAGAAATAACAATA-3 ) as forward, as described by Schnittger et al. (2004), and the Bbo-R as reverse. The reaction mixture was the same as in the conventional PCR, although repeated for 30 cycles under the following conditions: 1 min at 94 ◦ C, 1 min at 57 ◦ C and 1 min at 75 ◦ C. The resultant amplified fragment has a length of 846-bp. After the reaction, a 1:50 dilution of the PCR products in Milli-Q water was performed. On the second reaction, the pair of primers Bbo-F and -R were used under the same conditions as the previous reaction. The diluted PCR products obtained on the first reaction were used as a template. All PCR products were visualized by UV transilluminator in a 1.5% agarose gel following electrophoresis and staining with SYBR® safe DNA Gel stain (Invitrogen). All samples had a negative control on which water was added instead of DNA. As a positive control, DNA extracted from Israeli B. ovis was used. This strain was kindly provided by Dr. Varda Shkap, Kimron Veterinary Institute, Israel. DNA sequencing
Material and methods Samples collection and DNA extraction Since 2010, 300 blood samples from randomized sheep and goats were collected in EDTA tubes (Vacutest system) from farms in different regions of Portugal (14 districts). The information related to sex, age, breed and putative treatment with antiparasitic drugs of the donor animal were registered and, with the exception of 2 samples from Montemor-o-Novo, none of the sampled animals showed piroplasmosis symptoms. From the total of samples received, 65.3% were sheep and 34.7% goats; 95.2% were females and, in total, are at an average age of 4.15 ± 2.61 years. DNA extraction from blood was performed using a commercial kit (ArchivePure DNA Purification, 5Prime), accordingly with manufacturer’s instructions. DNA quantification was performed using Thermo Scientific Nanodrop ND-2000c. When the DNA concentration was above 100 ng/L, a dilution in Milli-Q water was performed, aiming for a final concentration of 100 ng/L. DNA extraction was repeated in samples where its concentration was below 15 ng/L. All DNA samples were stored at −20 ◦ C until further use. In some of the places where blood samples were obtained, 39 ticks were collected from small ruminants and stored in vials with humidified paper for transport. In the laboratory the ticks were washed on ethanol 96% and stored in cryotubes at −80 ◦ C. Each tick was examined in terms of gender and species and the tissues
To confirm and validate the results, randomly selected positive samples for B. ovis (15 DNA samples extracted from blood and 2 DNA samples extracted from Ticks) identified by the semi-nested PCR were sequenced. The purification of PCR products was performed using the commercial kit Wizard® SV Gel and PCR Clean-up System (Promega), following the instructions of the manufacturer. After lyophilization, all samples were sent to GATC Biotech Company (Constance, Germany) for fragment’s sequencing. Portuguese B. ovis isolation and determination of PCR and semi-nested PCR sensitivity In order to test the sensitivity and determinate the detection limit of both PCR assay, cultivation of B. ovis was performed. Blood collected on EDTA tubes and identified as a positive through seminested PCR was suspended in HEPES-buffered Medium 199 (1×) supplemented with Earle’s Salts, l-glutamine and l-Amino acids (Gibco, Life technologies) supplemented with 20% sheep serum and 10% of sheep uninfected erythrocytes. The complete medium was also buffered with 20 mM TES and antibiotic–antimycotic (100×) (Gibco, Invitrogen) was added at a final concentration of 0.25 mg/mL amphotericin, 100 U/mL penicillin and 100 g/mL streptomycin to prevent contaminations. The culture was added in a 24-well plate and incubated at 37 ◦ C in a humidified chamber filled with a gas mixture of 5% CO2 , 2% O2 and 93% N2 . Every 24 h half of the supernatant was replaced for fresh medium and the parasitaemia monitored by preparing thin blood smears stained with
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Giemsa. Intraerythrocytic parasites were observed under a magnification of 1000× on a Nikon Eclipse TE2000-S (Japan) inverted microscope. After reaching a parasitaemia of 1% (50 000 parasites/L), B. ovis-infected erythrocytes were subjected to 10-fold serial dilutions (from 10−1 to 10−10 ) using uninfected lamb erythrocytes as diluent (Gama et al., 2007). DNA was extracted from each diluted sample and processed for PCR as previously described. For specificity test, DNA was extracted from isolates of B. bigemina (kindly provided by Silvina Wilkowsky (Instituto Nacional de tecnologia Agropecuaria, Argentina)), B. bovis mo7 (kindly provided by Erik de Vries (Faculty of Veterinary Medicine, Netherlands)), T. ovis, T. uilenberg (Kindly provided by Yin Hong (Lanzhou Veterinary Research Institute, China)) and Anaplasma ovis (kindly provided by Tulin Karagenc (Faculty of Veterinary Medicine, Turkey)) and semi-nested PCR was applied as previously described.
Results Blood samples randomly collected from sheep and goats from 14 districts of Portugal were examined in terms of B. ovis prevalence by both semi-nested and conventional PCR. Out of 300 samples, 12.7% (38/300) were marked as positive by a semi-nested PCR and 1.67% (5/300) were marked as positive by conventional PCR. Those 5 positive samples were confirmed by semi-nested PCR. The sampling distribution and the positive cases along the country are presented in Fig. 1. Regarding the DNA extracted from ticks, it was possible to identify 7 out of 39 (17.9%) as carriers of B. ovis after semi-nested PCR methodology. However, it was not possible to identify any carrier on a conventional PCR. The ticks included in the study belong to the genus Rhipicephalus and 28 were adults (19 females and 9 males), 10 nymphs and 1 larva (Fig. 2). From those 4 females, 2 males and 1 nymph were positive for B. ovis. For results validation, 17 randomly selected positive samples were sequenced. The resultant sequence is available in GenBankTM database under the accession number KJ829366 and the amplified DNA fragments showed 100% of identity with the sequences AY150058 and AY533146, previously published. B. ovis from a positive sample was isolated and used to establish an in vitro cell culture. By adding the infected blood to the culture media, its presence was confirmed after 4 days of growth by microscopic examination of thin blood smears, Fig. 3. After the in vitro culture reached a parasitaemia of 1%, the detection limit of the assay was calculated and the results are presented in Fig. 4. It has been
Fig. 1. Distribution of blood samples collected along Portugal continental from 2010 to 2014. Blue circles: number of tested samples for B. ovis presence using seminested PCR; green circles: number of positive samples for B. ovis. Image adapted from Covas et al. (2013). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
observed that parasitaemia down to 10−7 % can be detected using the proposed semi-nested PCR. Although, when a conventional PCR is applied B. ovis can only be detected down to a parasitaemia of 10−3 %. For specificity test, semi-nested PCR using DNA extracted from isolates of different organisms (B. ovis, B. bovis, B. bigemina, T. ovis, T. uilenberg, A. ovis) was performed and only DNA of Babesia ovis was amplified. No unexpected amplification was observed (Fig. 5). Discussion B. ovis is a tick-transmitted protozoa parasite that infects sheep and goats and causes host-mediated pathology. Common in tropical and sub-tropical areas, the presence of this parasite has an economical impact on industry and the early diagnosis and successful therapy are the key steps for its control (Ranjbar-Bahadori et al.,
Fig. 2. Microscopic image of an adult male Rhipicephalus sanguineus collected from one of the field animals, 40× magnification. The left image shows the ventral view; the right image shows the dorsal view.
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Fig. 3. Thin blood smears showing erythrocytes infected with Portuguese isolate of B. ovis stained with Giemsa 1000× magnification.
2012). Unfortunately, the epidemiological knowledge on this disease is often impaired by the lack of a diagnostic tool available to the vets. The detection of Babesia infection in carrier animals and ticks by DNA amplification is a powerful tool for epidemiological investigation. A PCR specific for B. ovis was published for the first time in 2005 by Aktas et al. (Aktas et al., 2005), using a pair of oligonucleotide primers which target a 549-bp fragment. However, a high number of false negatives matching to a subclinical state of the animal were observed. Recognition of such cases is important given their potential role on vector-mediated disease spreading and, ultimately, loss of herd productivity (Alessandra and Santo, 2012). The hypervariable region V4 of 18S rRNA has been extensively used as a marker for taxonomic classification and phylogenetic analysis of Babesia and Theileria species (Salim et al., 2010; Schnittger et al., 2003). Due to the high degree of conservation associated with this region, it is possible to achieve higher sensitivity and specificity on hemoparasites identification towards multiple infections and low parasitaemia cases. In a total of 300 blood samples, 38 infected animals were identified by the semi-nested PCR whereas only 5 of them were positively diagnosed by a conventional PCR. Related to the diagnosis of Babesia
Fig. 4. Sensitivity of semi-nested and conventional PCR. 1.5% agarose gel electrophoresis of the amplicons from DNA extracted from in vitro culture with 10-fold serial dilutions (10−1 to 10−10 ). (A) Products obtained on conventional PCR with B. ovis specific primers. (B) Products obtained on the 2nd round of semi-nested PCR with B. ovis specific primers. Lane M, 100-bp Ladder DNA marker; lane 1, DNA extracted from blood with 1% parasitaemia; lane 2, 0.1% parasitaemia; lane 3, 0.01% parasitaemia; lane 4, 0.001% parasitaemia; lane 5, 10−4 % parasitaemia; lane 6, 10−5 % parasitaemia; lane 7, 10−6 % parasitaemia; lane 8, 10−7 % parasitaemia; lane 9, 10−8 % parasitaemia; lane 10, 10−9 % parasitaemia; lane 11, 10−10 % parasitaemia; lane 12, uninfected sheep blood (negative control).
in carrier ticks, the identification of positive bearers was only possible using semi-nested methodology (7/39). In addition, a sensitivity assay performed for both PCR methodologies demonstrates the higher sensibility of semi-nested when compared to the conventional one. Using this method it is possible to identify 1 parasite in 109 erythrocytes. Furthermore, when DNA of parasites phylogenetically related as B. bovis, B. bigemina, T. ovis, T. uilenberg and A. ovis was added, the amplification of the expected fragment only occurred when B. ovis DNA was present. Although being more laborious, the semi-nested PCR significantly enhances the identification of B. ovis’ carriers, can also be applied in the identification of parasites on ticks and is specific for B. ovis even during multiple infections. The development of this methodology also enabled us to initiate the first survey of B. ovis in Portugal and the isolation of the Portuguese strain, as observed in Fig. 3. With an epidemiologic study in sight, further investigation will be required in order to increase sample size and establish a correlation with positive vectors. However, obtained data from the current field work clearly shows a high prevalence (12.7%) of babesiosis mainly distributed in the southern and littoral regions of Portugal and suggests that B. ovis is endemic in most regions of the country. Interestingly, the majority of the sampled animals did not show symptoms of babesiosis at the time of blood collection, nor had disease track. Furthermore, no correlation between species, age and gender were found between the prevalence of positive cases although it was already proposed that B. ovis causes disease almost exclusively in sheep, rarely in goats (Aktas et al., 2007). Given the mentioned absence of symptoms, it can be concluded that either the B. ovis present in Portugal is not as pathogenic as reported in other countries, or the level of virulence may be related with mixed infections. Concluding two consecutive PCR rounds instead of one improves the specificity and sensitivity of the B. ovis detection in small ruminants and therefore contributes to a better control and monitoring of the disease.
Fig. 5. Specificity of the semi-nested PCR. 1.5% agarose gel electrophoresis of the amplicons from Babesia, Theileria and Anaplasma species using B. ovis-specific primers. Lane M, 100-bp Ladder DNA Marker; lane 1, B. ovis; lane 2, B. bovis; lane 3, B. bigemina; lane 4, Theileria ovis; lane 5, Theileria uilenberg; lane 6, Anaplasma ovis; lane 7, Babesia species without B. ovis; lane 8, Babesia species with B. ovis; lane 9, Theileria species without B. ovis; lane 10, Theileria species with B. ovis; lane 11, uninfected sheep blood (negative control).
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Acknowledgments This work was financed by the European Project PIROVAC (KBBE-3-245145-PIROVAC). Thanks to all collaborators who kindly provided the isolates used in this work, and special thanks to Dr. Varda Shkap, Kimron Veterinary Institute, Israel, for providing the Israeli strain of B. ovis. Thanks to Dr. Isabel Mariano of COPRAPEC, Montemor-o-Novo, for the advice and the biological material provided. To all veterinary associations and vets that gently cooperated with this study by collecting and sending samples from the field. References Aktas, M., Altay, K., Dumanli, N., 2005. Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats. Vet. Parasitol. 133, 277–281. Aktas, M., Altay, K., Dumanli, N., 2007. Determination of prevalence and risk factors for infection with Babesia ovis in small ruminants from Turkey by polymerase chain reaction. Parasitol. Res. 100, 797–802. Alessandra, T., Santo, C., 2012. Tick-borne diseases in sheep and goats: clinical and diagnostic aspects. Small Rumin. Res. 106 (Suppl.), S6–S11. Caeiro, V., 1999. General review of tick species present in Portugal. Parassitologia 41 (Suppl. 1), 11–15. Costa-Junior, L.M., Rabelo, E.M., Martins Filho, O.A., Ribeiro, M.F., 2006. Comparison of different direct diagnostic methods to identify Babesia bovis and Babesia bigemina in animals vaccinated with live attenuated parasites. Vet. Parasitol. 139, 231–236. Covas, M.T., Silva, C.A., Dias, L.C., 2013. On locating sustainable data centers in Portugal: problem structuring and GIS-based analysis. Sustain. Comput.: Inf. Syst. 3, 27–35. Estatística, I.N.D., 2013. Statistical Yearbook of Portugal 2012, Lisbon, Portugal. Friedhoff, K.T., 1997. Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp. Parassitologia 39, 99–109. Gama, B.E., Silva-Pires Fdo, E., Lopes, M.N., Cardoso, M.A., Britto, C., Torres, K.L., de Mendonca Lima, L., de Souza, J.M., Daniel-Ribeiro, C.T., Ferreira-da-Cruz, M.,
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