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Determination of prevalence and risk factors of infection with Babesia in small ruminants from Greece by polymerase chain reaction amplification
Veterinary Parasitology 135 (2006) 99–104 www.elsevier.com/locate/vetpar
Determination of prevalence and risk factors of infection with Babesia in small ruminants from Greece by polymerase chain reaction amplification G. Theodoropoulos a,*, M. Gazouli a, J.A. Ikonomopoulos a, V. Kantzoura a, A. Kominakis b a
Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science, Agricultural University of Athens, 75 Iera Odos Street, Votanikos, 11855 Athens, Greece b Department of Animal Breeding and Husbandry, Faculty of Animal Science, Agricultural University of Athens, 75 Iera Odos Street, Votanikos, 11855 Athens, Greece Received 2 June 2005; received in revised form 22 July 2005; accepted 27 July 2005
Abstract A total of 124 blood samples were collected from 92 sheep and 32 goats from 21 randomly selected herds located in two regions of Greece. Data on the characteristics of the animals (species, gender, age, tick burden, presence of haemoglobinuria, prior treatment for babesiosis) and the herd (location, size, species of animals, dogs associated with the herds, tick burden of dogs associated with the herds) were collected through questionnaires. Nineteen animals (15%) produced the DNA fragment specific for Babesia of which 16 were sheep and three were goats. Nucleotide sequence of PCR products revealed 100% homology with Babesia ovis 18S rRNA gene. Nine farms (43%) were found positive for B. ovis. The percentage of positive animals in each farm varied between 10 and 61%. The relative risk of the presence of ticks in sheep and goats ( p < 0.01) and farm dogs ( p < 0.01) for PCR-positive results for B. ovis in sheep and goats was found 6.63 and 4.14, respectively. # 2005 Elsevier B.V. All rights reserved. Keywords: Piroplasms; PCR; Sheep; Goats
1. Introduction The members of the genus Babesia encompass close to a hundred species of tick-borne haemoprotozoan parasites that infect a wide variety of vertebrate * Corresponding author. Tel.: +30 2105294387; fax: +30 2105294388. E-mail address: [email protected] (G. Theodoropoulos).
hosts (Levine, 1985; Piesman, 1987). The economic losses in sheep and goat production due to babesiosis are significant in tropical and subtropical areas (Mehlhorn and Schein, 1984). Clinical manifestation of the disease is variable, including anaemia, fever, icterus, and haemoglobinuria (Yeruham et al., 1998). After acute or primary infections, recovered animals frequently sustain subclinical infections, which are microscopically undetectable (Calder et al., 1996).
0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.07.021
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This carrier state serves as reservoir for infection in the herds, since animals that are not clinically ill may continue to infect the tick vector. Diagnostic tests which depend on serology for detecting this carrier state lack sensitivity and specificity, especially for this infection status (Calder et al., 1996; Nagore et al., 2004); thus detection systems based on in vitro amplification of Babesia-specific DNA sequences may facilitate the clinical diagnosis of ruminant babesiosis (Criado-Fornelio et al., 2003; Nagore et al., 2004). The literature on babesiosis of small ruminants in Greece is very limited. Previous serological studies in the Macedonia area of Greece reported that among Babesia, Babesia ovis was found to be extremely widespread in both sheep and goats, while B. motasi and B. crassa were spread to a lesser degree (Papadopoulos et al., 1996a; Papadopoulos, 1999). Taking into account the limitations of the serological studies, the objectives of the present study were: (i) to estimate the prevalence of Babesia in sheep and goats from two major livestock production regions of Greece by polymerase chain reaction amplification (PCR), and (ii) to identify risk factors that favour infection of sheep and goats with Babesia.
2. Materials and methods 2.1. Collection of samples and data Blood samples were collected from 21 randomly selected herds located in the important livestock production regions of Thessaly (15) and Epirus (6) during August–October of 2002 following codification of the herds in the two regions. Some farms were excluded on grounds of accessibility. At an average level of 20% prevalence of babesiosis in small ruminants in Greece (Papadopoulos et al., 1996a), the minimum sample size required for detecting a difference of at least 20%, for a level of a = 0.05 (Type I error) and b = 0.10 (Type II error) was estimated as high as n = 106. A total of 124 whole blood samples in heparin were collected from 92 sheep and 32 goats that belonged to eight herds with sheep only, two herds with goats only, and 11 herds with sheep and goats together. Data on the characteristics of the animals (species, gender, age,
tick burden, presence of haemoglobinuria, prior treatment for babesiosis) and the herd (location, size, species of animals, dogs associated with the herds, tick burden of dogs associated with the herds) were collected through questionnaires completed by the investigators on location during sample collection. Blood samples from cattle naturally infected with B. bovis and B. bigemina, obtained from Prof. Sonia Almeria at the Autonomous University of Barcelona in Spain, served as positive controls in our study. 2.2. DNA isolation DNA was isolated from 200 ml of blood with the NucleoSpin Blood Kit (Macherey-Nagel, Germany). The quality of the DNA extract in regard to purity and integrity was assessed with optical density counts at 260/280 nm and submerged gel electrophoresis. 2.3. Oligonucleotide design and PCR amplification A pair of oligonucleotide primers was designed based on the 18SrRNA sequence of Babesia sp. (GenBank accession numbers AY150058, AY534883, AY150061, AY048113) using the software package OLIGO (National Biosciences Inc., MN, USA). The nucleotide sequence of the primer-pair was: BABF: 50 GCGATGGCCCATTCAAGTTT-30 and BABR: 50 CGCCTGCTGCCTTCCTTAGA-30 and targets a 146 bp fragment of 18SrRNA sequence of Babesia. The primers’ specificity was assessed with extended GenBank database search with NCBI BLAST. PCR was performed in 50 ml of a mixture containing about 500 ng of template DNA, 10 pmol of each primer, 0.2 mM of dNTPs, 1.5 mM MgCl2, 1% betaine solution and 1 U of Taq DNA polymerase TM (GoTaq DNA Polymerase, Promega, Madison, WI, TM USA) in 1 Taq DNA polymerase buffer (GoTaq Reaction Buffer, Promega, Madison, WI, USA). PCR was performed for 5 min at 95 8C to initial denaturation, and then the reaction was repeated for 35 cycles under the following conditions: 1 min of denaturation at 95 8C, 1 min of annealing at 56 8C and 1 min of extension at 72 8C following by a 5 min completion step at 72 8C. In each PCR assay, 10% of the samples tested were positive and negative controls, the latter aiming to assess cross contamination. Negative control
G. Theodoropoulos et al. / Veterinary Parasitology 135 (2006) 99–104
samples consisted of the PCR mixture containing distilled water instead of DNA or DNA isolated from blood samples collected from healthy sheep and goats belonging to a herd maintained in the Agricultural University premises under conditions that did not allow direct or indirect contact with other animals. This herd had no history of Babesiosis or other infectious disease and was monitored routinely by clinical evaluation and blood testing to establish health status. Ten microliters of the PCR products were analysed by 2% agarose gel electrophoresis followed by ethidium bromide staining and photography. The results were confirmed by nucleotide sequence of PCR products. Sequencing was performed on both strands using the dye terminator cycle sequencing kit supplied by Applied Biosystems (ABI, Foster City, CA, USA) with the two primers BABF and BABR, and an ABI PRISM1 377 automated DNA sequencer. 2.4. Statistical analysis For statistical purposes, animals were grouped into two age categories: less than 1-year-old and more than 1-year-old up to 6-years-old. Herds were divided into two size categories: herds with 20–150 animals and herds with more than 151 animals. Also, herds were divided according to their composition into three categories: herds with sheep only, herds with goats only, and herds with sheep and goats together. The tick burden of sheep, goats, and dogs associated with the
101
herds was scored as follows: 0 for no ticks and 1 for one or more ticks. Association between the presence (positive and negative blood samples) of Babesia and the various parameters, i.e. herd location, herd size, species, gender and age of animal, herd composition, tick burden of sheep and goats, presence and tick burden of dogs in the herd was assessed by contingency table analysis using the Fisher’s exact test (for 2 2 tables) and/or the Mantel-Haenszel test for tables with order higher than two (i.e. herd composition). Results were displayed as p values as well as relative risk values (with 95% confidence intervals).
3. Results The GeneBank data base search revealed that the primer-pair BABF–BABR, produces a 146 bp DNA fragment only from members of the genus Babesia. This product was produced in all positive control samples and none of the negative control samples (Fig. 1). The minimum amount of genomic DNA necessary for a clearly visible amplification product was estimated by the application of the employed PCR assay on serial dilutions of DNA isolated from blood samples of cattle infected with B. bovis, at 135 ng (Fig. 2). None of the study animals had haemoglobinuria and/or prior treatment for babesiosis. Table 1 presents
Fig. 1. PCR assay performed on representative control and study blood samples. Lane 1: 100 bp DNA ladder (Fermentas). Lanes 2 and 3: positive-control samples from cattle infected respectively with B. bigemina and B. bovis, showing a 146 bp DNA product. Lanes 4 and 5: study blood samples collected from sheep naturally infected with B. ovis showing a 146 bp DNA product. Lane 6: negative control (blood sample from healthy sheep) showing no amplification product.
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Fig. 2. PCR performed on serial dilutions of a positive-control blood sample (cattle infected with B. bovis). Lane 1: 100 bp DNA ladder (Fermentas). Lanes 2–5: 146 bp DNA product amplified by the application of the employed PCR assay on 540, 270, 135 and 67.5 ng of DNA isolated from the positive control sample. Lane 6: negative control sample showing no amplification product.
the prevalence of Babesia in sheep and goats in relation to the parameters describing the characteristics of the animals and the herd. Out of 124 examined animals which represented 0.01% of all animals in the study areas (NSSG, 2002), 19 (15%) produced the
DNA fragment specific for Babesia of which 16 were sheep and three were goats (Fig. 3). Nucleotide sequence of PCR products revealed 100% homology with B. ovis 18S rRNA gene. Consequently, all the 19 positive animals were infected with B. ovis.
Table 1 Association between the presence (PCR-positive and negative blood samples) of Babesia spp. in sheep and goats and the studied parameters describing animal and herd characteristics
N Negative Positive p(F), p(MH) RR
Total sheep and goats
Herd location Thessaly
Epirus
20–150 animals
>151 animals
Sheep
Goats
124 105 (85%) 19 (15%)
93 78 (84%) 15 (16%) p(F) = 0.78 (NS)
31 27 (87%) 4 (13%)
73 63 (86%) 10 (14%) p(F) = 0.62 (NS)
51 42 (82%) 9 (18%)
92 76 (83%) 16 (17%) p(F) = 0.40 (NS)
32 29 (91%) 3 (9%)
Gender of animal Male
Herd size
Age of animal Female
<1 year
Species of animal
Herd composition >1 year
Sheep only Goats only
N 11 113 11 113 55 Negative 11 (100%) 94 (83%) 10 (91%) 95 (84%) 44 (80%) Positive 0 (0%) 19 (17%) 1 (9%) 18 (16%) 11 (20%) p(F), p(MH) p(F) = 0.21 (NS) p(F) = 1.00 (NS) RR
N Negative Positive p(F), p(MH) RR
Sheep and goats together
15 54 13 (87%) 48 (89%) 2 (13%) 6 (11%) p(MH) = 0.20 (NS)
Tick burden of sheep and goats
Presence of dogs in the herd
Tick burden of dogs in the herd
No ticks
Yes
No ticks
More than one tick
48 76 46 (96%) 59 (78%) 2 (4%) 17 (22%) p(F) = 0.009 6.63 (1.46–30.15)
No
101 23 88 (87%) 17 (74%) 13 (13%) 6 (26%) p(F) = 0.12 (NS)
More than one tick
61 40 57 (93%) 31 (78%) 4 (7%) 9 (22%) p(F) = 0.003 4.14 (1.18–14.53)
p(F), probability of Fisher’s exact test; p(MH), probability of Mantel–Haenszel test; RR, relative risk with 95% confidence intervals; NS, not statistically significant.
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Fig. 3. PCR performed on representative study blood samples. Lane 1: 100 bp DNA ladder (Fermentas). Lanes 2–5, 7, and 8: 146 bp DNA product from sheep (lanes 2 and 3) and goat (lanes 4, 5, 7, and 8) showing the 146 bp DNA product specific for Babesia spp. Lanes 6 and 9: sheep (lane 6) and goat (lane 9) PCR-negative blood samples. Lane 10: negative control consisted of the PCR-mixture without any DNA.
Out of 21 examined farms, which represented 0.2% of all sheep and goat farms in the study areas (NSSG, 2000), nine farms (43%) were found positive for B. ovis. The percentage of positive animals in each farm varied between 10 and 61%, but mostly between 10 and 20%. The statistical analysis of the data showed that the relative risk of the presence of ticks in sheep and goats ( p < 0.01) and farm dogs ( p < 0.01) for PCRpositive results for B. ovis in sheep and goats was 6.63 and 4.14, respectively.
4. Discussion Tick borne parasitic diseases like babesiosis remain an important impediment to meat and milk production (Caracappa, 1999) because infected animals exhibit high parasitaemia and mortality (Bai et al., 2002). PCR based diagnostic methods may prove very useful since they have been reported to detect as little as 10 infected cells per ml in blood, when parasitaemia is 107 (Figueroa et al., 1992; Fahrimal et al., 1992; Calder et al., 1996). In addition to sensitivity, molecular techniques offer an advantage over microscopic examinations of blood smears due to their higher specificity that allows identification and differentiation even among related species directly from clinical samples. The significance of serology on the other hand is often impaired by the fact that antibodies cannot be detected in long-term carriers and the presence of piroplasms and cross-reactivity of antibodies against other species may lead to false
positive results. Carriers are important contributors of transmission to susceptible animals by ticks in endemic areas and their detection is an important epidemiological parameter (Almeria et al., 2001). In the present study, the observation that 15% of sheep and goats were B. ovis asymptomatic carriers coupled with the observation that 43% of the small ruminant farms had 10–61% of their animals infected, shows the dynamic nature of the epizootic in the study areas where 14,090 small ruminant farms are located (NSSG, 2000). Collection of blood samples took place during summer and autumn when the highest prevalence of babesiosis is usually observed (Razmi et al., 2003). In a previous study in Greece where IFAT was used the prevalence in sheep and goats was 52.1 and 36.4% for B. ovis and 10.5 and 4.2% for B. motasi (Papadopoulos et al., 1996a), respectively. Although the results of the present and previous study cannot be compared due to the differences of the methods employed, they both demonstrate the broad dispersal of Babesia among the Greek population of small ruminants. Among the factors examined in the present study, the presence of ticks in sheep, goats, and farm dogs was associated with PCR-positive results, which indicate a high risk of infection with Babesia in sheep and goats. Ticks suitable for transmission of Babesia have been recorded in Greece (Papadopoulos et al., 1996b; Papazahariadou et al., 1995, 2003). In regard to the role of farm dogs as a risk factor for babesiosis in sheep and goats, ticks can transmit Babesia even when feeding on other hosts (Yeruham
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et al., 1996) and the tick Rhipicephalus sanguineus that usually infests dogs has been found also on ruminants (Bouattour et al., 1999). On the other hand, the fact that Babesia can infest a wide variety of animal hosts (Criado-Fornelio et al., 2003) indicates that other animal species besides dogs may be risk factors for babesiosis in sheep and goats.
Acknowledgement The investigators are grateful to Prof. Sonia Almeria at the Autonomous University of Barcelona in Spain for providing the positive control samples for this study.
References Almeria, S., Castella, J., Ferrer, D., Ortuno, A., Estrada-Pena, A., Gutierrez, J.F., 2001. Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection. Vet. Parasitol. 99, 249–259. Bai, Q., Liu, G., Liu, D., Ren, J., Li, X., 2002. Isolation and preliminary characterization of large Babesia sp. from sheep and goats in the eastern pat of Gansu Province. Chin. Parasitol. Res. 88 (13 Suppl. 1), 16–21. Bouattour, A., Darghouth, M.A., Daoud, A., 1999. Distribution and ecology of ticks (Acari: Ixodidae) infesting livestock in Tunisia: An overview of eighth years field collections. Parassitologia 41 (Suppl. 1), 5–10. Calder, J.A., Reddy, G.R., Chieves, L., Courtney, C.H., Littell, R., Livengood, J.R., Norval, R.A., Smith, C., Dame, J.B., 1996. Monitoring Babesia bovis infections in cattle by using PCRbased tests. J. Clin. Microbiol. 34, 2748–2755. Caracappa, S., 1999. Livestock production and animal health in Sicily, Italy. Parassitologia 41, 17–23. Criado-Fornelio, A., Martinez-Marcos, A., Buling-Sarana, A., Barba-Carretero, J.C., 2003. Molecular studies on Babesia, Theileria and Hepatozoon in southern Europe. Part I. Epizootiological aspects. Vet. Parasitol. 113, 189–201. Fahrimal, Y., Goff, W.L., Jasmer, D.P., 1992. Detection of Babesia bovis carrier cattle by using polymerase chain reaction
amplification of parasite DNA. J. Clin. Microbiol. 30, 1374– 1379. Figueroa, J.V., Chieves, L.P., Johnson, G.S., Buening, G.M., 1992. Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification. J. Clin. Microbiol. 30, 2576– 2582. Levine, N.D. (Ed.), .1985. Apicomplexa: the piroplasms. Veterinary Protozoology. Iowa State University Press, Ames, pp. 291–328. Mehlhorn, H., Schein, E., 1984. The piroplasms: life cycle and sexual stages. Adv. Parasitol. 23, 37–103. Nagore, D., Garcia-Sanmartin, J., Garcia-Perez, A.L., Juste, R.A., Hurtado, A., 2004. Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain. Int. J. Parasitol. 34, 1059–1067. NSSG (National Statistical Service of Greece), 2000. Basic study of crop and livestock enterprises, 1999–2000 (Data in a table provided in a CD). NSSG (National Statistical Service of Greece), 2002. Number of domesticated animals. In: Agricultural Statistics of Greece Year 1998. pp. 94–95. Papadopoulos, B., Brossard, M., Perie, N.M., 1996a. Piroplasms of domestic animals in the Macedonia region of Greece. 3. Piroplasms of small ruminants. Vet. Parasitol. 63, 67–74. Papadopoulos, B., Morel, P.C., Aeschlimann, A., 1996b. Ticks of domestic animals in the Macedonia region of Greece. Vet. Parasitol. 63, 25–40. Papadopoulos, B., 1999. Cattle and small ruminant piroplasmosis in Macedonia, Greece. Parassitologia 41 (Suppl. 1), 81–84. Papazahariadou, M.G., Papadopoulos, E.G., Himonas, C.A., 1995. Seasonal activity of ixodid ticks on goats in northern Greece. Vet. Parasitol. 136, 585–588. Papazahariadou, M.G., Saridomichelakis, M.N., Koutinas, A.F., Papadopoulos, E.G., Leontides, L., 2003. Tick infection of dogs in Thessaloniki, northern Greece. Med. Vet. Entomol. 17, 110– 113. Piesman, J., 1987. Emerging tick-borne diseases in temperate climates. Parasitol. Today 3, 197–199. Razmi, G.R., Naghibi, A., Aslani, M.R., Dastjerdi, K., Hossieni, H., 2003. An epidemiological study on Babesia infection in small ruminants in Mashhad suburb, Khorasan province. Iran. Small Ruminant Res. 50, 39–44. Yeruham, I., Hamdami, A., Galker, F., 1996. Effect of passage of Babesia ovis in the gerbil (Acomys cahirinus) on the course of infection in splenectomized lambs. Vet. Parasitol. 65, 157–161. Yeruham, I., Hadani, A., Galker, F., 1998. Some epizootiological and clinical aspects of ovine babesiosis caused by Babesia ovis—a review. Vet. Parasitol. 74, 153–163.
Determination of prevalence and risk factors of infection with Babesia in small ruminants from Greece by polymerase chain reaction amplification G. Theodoropoulos a,*, M. Gazouli a, J.A. Ikonomopoulos a, V. Kantzoura a, A. Kominakis b a
Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science, Agricultural University of Athens, 75 Iera Odos Street, Votanikos, 11855 Athens, Greece b Department of Animal Breeding and Husbandry, Faculty of Animal Science, Agricultural University of Athens, 75 Iera Odos Street, Votanikos, 11855 Athens, Greece Received 2 June 2005; received in revised form 22 July 2005; accepted 27 July 2005
Abstract A total of 124 blood samples were collected from 92 sheep and 32 goats from 21 randomly selected herds located in two regions of Greece. Data on the characteristics of the animals (species, gender, age, tick burden, presence of haemoglobinuria, prior treatment for babesiosis) and the herd (location, size, species of animals, dogs associated with the herds, tick burden of dogs associated with the herds) were collected through questionnaires. Nineteen animals (15%) produced the DNA fragment specific for Babesia of which 16 were sheep and three were goats. Nucleotide sequence of PCR products revealed 100% homology with Babesia ovis 18S rRNA gene. Nine farms (43%) were found positive for B. ovis. The percentage of positive animals in each farm varied between 10 and 61%. The relative risk of the presence of ticks in sheep and goats ( p < 0.01) and farm dogs ( p < 0.01) for PCR-positive results for B. ovis in sheep and goats was found 6.63 and 4.14, respectively. # 2005 Elsevier B.V. All rights reserved. Keywords: Piroplasms; PCR; Sheep; Goats
1. Introduction The members of the genus Babesia encompass close to a hundred species of tick-borne haemoprotozoan parasites that infect a wide variety of vertebrate * Corresponding author. Tel.: +30 2105294387; fax: +30 2105294388. E-mail address: [email protected] (G. Theodoropoulos).
hosts (Levine, 1985; Piesman, 1987). The economic losses in sheep and goat production due to babesiosis are significant in tropical and subtropical areas (Mehlhorn and Schein, 1984). Clinical manifestation of the disease is variable, including anaemia, fever, icterus, and haemoglobinuria (Yeruham et al., 1998). After acute or primary infections, recovered animals frequently sustain subclinical infections, which are microscopically undetectable (Calder et al., 1996).
0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.07.021
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G. Theodoropoulos et al. / Veterinary Parasitology 135 (2006) 99–104
This carrier state serves as reservoir for infection in the herds, since animals that are not clinically ill may continue to infect the tick vector. Diagnostic tests which depend on serology for detecting this carrier state lack sensitivity and specificity, especially for this infection status (Calder et al., 1996; Nagore et al., 2004); thus detection systems based on in vitro amplification of Babesia-specific DNA sequences may facilitate the clinical diagnosis of ruminant babesiosis (Criado-Fornelio et al., 2003; Nagore et al., 2004). The literature on babesiosis of small ruminants in Greece is very limited. Previous serological studies in the Macedonia area of Greece reported that among Babesia, Babesia ovis was found to be extremely widespread in both sheep and goats, while B. motasi and B. crassa were spread to a lesser degree (Papadopoulos et al., 1996a; Papadopoulos, 1999). Taking into account the limitations of the serological studies, the objectives of the present study were: (i) to estimate the prevalence of Babesia in sheep and goats from two major livestock production regions of Greece by polymerase chain reaction amplification (PCR), and (ii) to identify risk factors that favour infection of sheep and goats with Babesia.
2. Materials and methods 2.1. Collection of samples and data Blood samples were collected from 21 randomly selected herds located in the important livestock production regions of Thessaly (15) and Epirus (6) during August–October of 2002 following codification of the herds in the two regions. Some farms were excluded on grounds of accessibility. At an average level of 20% prevalence of babesiosis in small ruminants in Greece (Papadopoulos et al., 1996a), the minimum sample size required for detecting a difference of at least 20%, for a level of a = 0.05 (Type I error) and b = 0.10 (Type II error) was estimated as high as n = 106. A total of 124 whole blood samples in heparin were collected from 92 sheep and 32 goats that belonged to eight herds with sheep only, two herds with goats only, and 11 herds with sheep and goats together. Data on the characteristics of the animals (species, gender, age,
tick burden, presence of haemoglobinuria, prior treatment for babesiosis) and the herd (location, size, species of animals, dogs associated with the herds, tick burden of dogs associated with the herds) were collected through questionnaires completed by the investigators on location during sample collection. Blood samples from cattle naturally infected with B. bovis and B. bigemina, obtained from Prof. Sonia Almeria at the Autonomous University of Barcelona in Spain, served as positive controls in our study. 2.2. DNA isolation DNA was isolated from 200 ml of blood with the NucleoSpin Blood Kit (Macherey-Nagel, Germany). The quality of the DNA extract in regard to purity and integrity was assessed with optical density counts at 260/280 nm and submerged gel electrophoresis. 2.3. Oligonucleotide design and PCR amplification A pair of oligonucleotide primers was designed based on the 18SrRNA sequence of Babesia sp. (GenBank accession numbers AY150058, AY534883, AY150061, AY048113) using the software package OLIGO (National Biosciences Inc., MN, USA). The nucleotide sequence of the primer-pair was: BABF: 50 GCGATGGCCCATTCAAGTTT-30 and BABR: 50 CGCCTGCTGCCTTCCTTAGA-30 and targets a 146 bp fragment of 18SrRNA sequence of Babesia. The primers’ specificity was assessed with extended GenBank database search with NCBI BLAST. PCR was performed in 50 ml of a mixture containing about 500 ng of template DNA, 10 pmol of each primer, 0.2 mM of dNTPs, 1.5 mM MgCl2, 1% betaine solution and 1 U of Taq DNA polymerase TM (GoTaq DNA Polymerase, Promega, Madison, WI, TM USA) in 1 Taq DNA polymerase buffer (GoTaq Reaction Buffer, Promega, Madison, WI, USA). PCR was performed for 5 min at 95 8C to initial denaturation, and then the reaction was repeated for 35 cycles under the following conditions: 1 min of denaturation at 95 8C, 1 min of annealing at 56 8C and 1 min of extension at 72 8C following by a 5 min completion step at 72 8C. In each PCR assay, 10% of the samples tested were positive and negative controls, the latter aiming to assess cross contamination. Negative control
G. Theodoropoulos et al. / Veterinary Parasitology 135 (2006) 99–104
samples consisted of the PCR mixture containing distilled water instead of DNA or DNA isolated from blood samples collected from healthy sheep and goats belonging to a herd maintained in the Agricultural University premises under conditions that did not allow direct or indirect contact with other animals. This herd had no history of Babesiosis or other infectious disease and was monitored routinely by clinical evaluation and blood testing to establish health status. Ten microliters of the PCR products were analysed by 2% agarose gel electrophoresis followed by ethidium bromide staining and photography. The results were confirmed by nucleotide sequence of PCR products. Sequencing was performed on both strands using the dye terminator cycle sequencing kit supplied by Applied Biosystems (ABI, Foster City, CA, USA) with the two primers BABF and BABR, and an ABI PRISM1 377 automated DNA sequencer. 2.4. Statistical analysis For statistical purposes, animals were grouped into two age categories: less than 1-year-old and more than 1-year-old up to 6-years-old. Herds were divided into two size categories: herds with 20–150 animals and herds with more than 151 animals. Also, herds were divided according to their composition into three categories: herds with sheep only, herds with goats only, and herds with sheep and goats together. The tick burden of sheep, goats, and dogs associated with the
101
herds was scored as follows: 0 for no ticks and 1 for one or more ticks. Association between the presence (positive and negative blood samples) of Babesia and the various parameters, i.e. herd location, herd size, species, gender and age of animal, herd composition, tick burden of sheep and goats, presence and tick burden of dogs in the herd was assessed by contingency table analysis using the Fisher’s exact test (for 2 2 tables) and/or the Mantel-Haenszel test for tables with order higher than two (i.e. herd composition). Results were displayed as p values as well as relative risk values (with 95% confidence intervals).
3. Results The GeneBank data base search revealed that the primer-pair BABF–BABR, produces a 146 bp DNA fragment only from members of the genus Babesia. This product was produced in all positive control samples and none of the negative control samples (Fig. 1). The minimum amount of genomic DNA necessary for a clearly visible amplification product was estimated by the application of the employed PCR assay on serial dilutions of DNA isolated from blood samples of cattle infected with B. bovis, at 135 ng (Fig. 2). None of the study animals had haemoglobinuria and/or prior treatment for babesiosis. Table 1 presents
Fig. 1. PCR assay performed on representative control and study blood samples. Lane 1: 100 bp DNA ladder (Fermentas). Lanes 2 and 3: positive-control samples from cattle infected respectively with B. bigemina and B. bovis, showing a 146 bp DNA product. Lanes 4 and 5: study blood samples collected from sheep naturally infected with B. ovis showing a 146 bp DNA product. Lane 6: negative control (blood sample from healthy sheep) showing no amplification product.
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Fig. 2. PCR performed on serial dilutions of a positive-control blood sample (cattle infected with B. bovis). Lane 1: 100 bp DNA ladder (Fermentas). Lanes 2–5: 146 bp DNA product amplified by the application of the employed PCR assay on 540, 270, 135 and 67.5 ng of DNA isolated from the positive control sample. Lane 6: negative control sample showing no amplification product.
the prevalence of Babesia in sheep and goats in relation to the parameters describing the characteristics of the animals and the herd. Out of 124 examined animals which represented 0.01% of all animals in the study areas (NSSG, 2002), 19 (15%) produced the
DNA fragment specific for Babesia of which 16 were sheep and three were goats (Fig. 3). Nucleotide sequence of PCR products revealed 100% homology with B. ovis 18S rRNA gene. Consequently, all the 19 positive animals were infected with B. ovis.
Table 1 Association between the presence (PCR-positive and negative blood samples) of Babesia spp. in sheep and goats and the studied parameters describing animal and herd characteristics
N Negative Positive p(F), p(MH) RR
Total sheep and goats
Herd location Thessaly
Epirus
20–150 animals
>151 animals
Sheep
Goats
124 105 (85%) 19 (15%)
93 78 (84%) 15 (16%) p(F) = 0.78 (NS)
31 27 (87%) 4 (13%)
73 63 (86%) 10 (14%) p(F) = 0.62 (NS)
51 42 (82%) 9 (18%)
92 76 (83%) 16 (17%) p(F) = 0.40 (NS)
32 29 (91%) 3 (9%)
Gender of animal Male
Herd size
Age of animal Female
<1 year
Species of animal
Herd composition >1 year
Sheep only Goats only
N 11 113 11 113 55 Negative 11 (100%) 94 (83%) 10 (91%) 95 (84%) 44 (80%) Positive 0 (0%) 19 (17%) 1 (9%) 18 (16%) 11 (20%) p(F), p(MH) p(F) = 0.21 (NS) p(F) = 1.00 (NS) RR
N Negative Positive p(F), p(MH) RR
Sheep and goats together
15 54 13 (87%) 48 (89%) 2 (13%) 6 (11%) p(MH) = 0.20 (NS)
Tick burden of sheep and goats
Presence of dogs in the herd
Tick burden of dogs in the herd
No ticks
Yes
No ticks
More than one tick
48 76 46 (96%) 59 (78%) 2 (4%) 17 (22%) p(F) = 0.009 6.63 (1.46–30.15)
No
101 23 88 (87%) 17 (74%) 13 (13%) 6 (26%) p(F) = 0.12 (NS)
More than one tick
61 40 57 (93%) 31 (78%) 4 (7%) 9 (22%) p(F) = 0.003 4.14 (1.18–14.53)
p(F), probability of Fisher’s exact test; p(MH), probability of Mantel–Haenszel test; RR, relative risk with 95% confidence intervals; NS, not statistically significant.
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Fig. 3. PCR performed on representative study blood samples. Lane 1: 100 bp DNA ladder (Fermentas). Lanes 2–5, 7, and 8: 146 bp DNA product from sheep (lanes 2 and 3) and goat (lanes 4, 5, 7, and 8) showing the 146 bp DNA product specific for Babesia spp. Lanes 6 and 9: sheep (lane 6) and goat (lane 9) PCR-negative blood samples. Lane 10: negative control consisted of the PCR-mixture without any DNA.
Out of 21 examined farms, which represented 0.2% of all sheep and goat farms in the study areas (NSSG, 2000), nine farms (43%) were found positive for B. ovis. The percentage of positive animals in each farm varied between 10 and 61%, but mostly between 10 and 20%. The statistical analysis of the data showed that the relative risk of the presence of ticks in sheep and goats ( p < 0.01) and farm dogs ( p < 0.01) for PCRpositive results for B. ovis in sheep and goats was 6.63 and 4.14, respectively.
4. Discussion Tick borne parasitic diseases like babesiosis remain an important impediment to meat and milk production (Caracappa, 1999) because infected animals exhibit high parasitaemia and mortality (Bai et al., 2002). PCR based diagnostic methods may prove very useful since they have been reported to detect as little as 10 infected cells per ml in blood, when parasitaemia is 107 (Figueroa et al., 1992; Fahrimal et al., 1992; Calder et al., 1996). In addition to sensitivity, molecular techniques offer an advantage over microscopic examinations of blood smears due to their higher specificity that allows identification and differentiation even among related species directly from clinical samples. The significance of serology on the other hand is often impaired by the fact that antibodies cannot be detected in long-term carriers and the presence of piroplasms and cross-reactivity of antibodies against other species may lead to false
positive results. Carriers are important contributors of transmission to susceptible animals by ticks in endemic areas and their detection is an important epidemiological parameter (Almeria et al., 2001). In the present study, the observation that 15% of sheep and goats were B. ovis asymptomatic carriers coupled with the observation that 43% of the small ruminant farms had 10–61% of their animals infected, shows the dynamic nature of the epizootic in the study areas where 14,090 small ruminant farms are located (NSSG, 2000). Collection of blood samples took place during summer and autumn when the highest prevalence of babesiosis is usually observed (Razmi et al., 2003). In a previous study in Greece where IFAT was used the prevalence in sheep and goats was 52.1 and 36.4% for B. ovis and 10.5 and 4.2% for B. motasi (Papadopoulos et al., 1996a), respectively. Although the results of the present and previous study cannot be compared due to the differences of the methods employed, they both demonstrate the broad dispersal of Babesia among the Greek population of small ruminants. Among the factors examined in the present study, the presence of ticks in sheep, goats, and farm dogs was associated with PCR-positive results, which indicate a high risk of infection with Babesia in sheep and goats. Ticks suitable for transmission of Babesia have been recorded in Greece (Papadopoulos et al., 1996b; Papazahariadou et al., 1995, 2003). In regard to the role of farm dogs as a risk factor for babesiosis in sheep and goats, ticks can transmit Babesia even when feeding on other hosts (Yeruham
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et al., 1996) and the tick Rhipicephalus sanguineus that usually infests dogs has been found also on ruminants (Bouattour et al., 1999). On the other hand, the fact that Babesia can infest a wide variety of animal hosts (Criado-Fornelio et al., 2003) indicates that other animal species besides dogs may be risk factors for babesiosis in sheep and goats.
Acknowledgement The investigators are grateful to Prof. Sonia Almeria at the Autonomous University of Barcelona in Spain for providing the positive control samples for this study.
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