Determination of Prevalence and Risk Factors of Infection with Babesia ovis in Small Ruminants from West Azerbaijan Province, Iran by Polymerase Chain Reaction

Abstract

Small ruminants' babesiosis caused by Babesia ovis, is transmitted during blood feeding by infected ticks and is the most economically important tick-borne disease in tropical and subtropical areas. This study was carried out to to estimate the infection rate of B. ovis in sheep and goats by PCR. We have analysed risk factors that might influence infection of sheep and goats with B. ovis. A total 402 blood samples were examined microscopically for the presence of Babesia infection. All samples were tested by PCR. During sampling, whole body of each animal and farm dogs was examined for the presence of ticks. Forty-two animals (10.4%) were positive for Babesia spp. upon microscopic examination, whereas 67 animals (16.7%) yielded the specific DNA for B. ovis of which 52 animals were sheep and 15 animals were goats. Twenty-nine farms (72.5%) were found positive for B. ovis. The percentage of positive animals in each location varied from 13 % to 20 %. 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 forB. ovis in sheep and goats was found 3.8 and 2.9, respectively. A total of 747 ticks identified as Rhipicephalus bursa, R. sanguineus and R. turanicus on the basis of morphological features. Other animal species besides dogs may also be risk factors for babesiosis in sheep and goats. Also, R. bursa may play an important role as a vector of the parasite in Iran.

Headnote

Abstract

Background: Small ruminants' babesiosis caused by Babesia ovis, is transmitted during blood feeding by infected ticks and is the most economically important tick-borne disease in tropical and subtropical areas. This study was carried out to to estimate the infection rate of B. ovis in sheep and goats by PCR. We have analysed risk factors that might influence infection of sheep and goats with B. ovis.

Methods: A total 402 blood samples were examined microscopically for the presence of Babesia infection. All samples were tested by PCR. During sampling, whole body of each animal and farm dogs was examined for the presence of ticks.

Results: Forty-two animals (10.4%) were positive for Babesia spp. upon microscopic examination, whereas 67 animals (16.7%) yielded the specific DNA for B. ovis of which 52 animals were sheep and 15 animals were goats. Twenty-nine farms (72.5%) were found positive for B. ovis. The percentage of positive animals in each location varied from 13 % to 20 %. 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 forB. ovis in sheep and goats was found 3.8 and 2.9, respectively. A total of 747 ticks identified as Rhipicephalus bursa, R. sanguineus and R. turanicus on the basis of morphological features.

Conclusion: Other animal species besides dogs may also be risk factors for babesiosis in sheep and goats. Also, R. bursa may play an important role as a vector of the parasite in Iran.

Keywords: Babesia, Rhipicephalus, Small ruminant, Iran, PCR

Introduction

The genus Babesia contains tick-borne hemoprotozoan parasites that infect a wide variety of vertebrate hosts. The economic losses in small ruminant production due to babesiosis are significant in tropical and subtropical regions of the world (Theodoropoulos et al. 2006). Babesia spp. are transmitted by Ixidid ticks and causes, anemia, jaundice, haemoglobinuria and in some cases mortality may occur (Sevinc et al. 2007). The disease is caused by B. ovis, B. motasi and B. crassa (Hashemi-Fesharki 1997, Razmi et al. 2003, Uilenberg 2006). Babesia ovis, a small Babesia (< 2.5 µ m), is the most common species that causes sheep babesiosis in Iran (Rahbari et al. 2008, Shayan et al. 2008). The pathogenicity of B. motasi is not high and appears to be moderately virulent (Soulsby 1982). Whereas, B. crassa is considered as being non-pathogenic to samall ruminants (Uilenberg 2006).

The diagnosis of babesiosis can be achieved by microscopic examination of Giemsa-stained blood smears and clinical signs in acute phase of the disease, but after acute or primary infections, recovered animals frequently sustain subclinical infections, which are microscopically undetectable (Aktas et al. 2005). This carrier state serves as reservoir for infection in the herds, since animals that are not clinically ill may continue it infect the tick vector (Aktas et al. 2007). Diagnostic tests which depend on serology for detecting this carrier state, but serological methods are not specific for any Babesia spp. due to cross-reactivity with other Babesia spp. and false positive and negative results are commonly observed in these tests (Aktas et al. 2005). The use of alternative techniques, such as DNA amplification methods, has become necessary to detect and identify Babesia infections effectively and has been reported in numerous recent studies (Jefferies et al. 2003). Molecular techniques are more sensitive and specific than other traditional diagnostic methods (Aktas et al. 2007).

Studies on small ruminants' babesiosis in Iran are very limited. Previous microscopic and serological studies state that the disease caused by B. ovis is endemic in the country (Tavassoli and Rahbari 1998, Razmi et al. 2003).

Taking into account the limitation of serological studies, the objective of the present study was to determine the prevalence of the infection in northwest of Iran by polymerase chain reaction (PCR). PCR was compared with the examination of thin blood smear. We have identified risk factors that favour infection of sheep and goats with B. ovis. We have also identified tick species found on sheep, goats and farm dogs in the region.

Materials and Methods

Surveyed-area (West Azerbaijan Province, north-western Iran) was divided into three different geographical areas including north, center and south. Blood samples were collected from 40 randomly selected flocks located in four important livestock production regions of West Azerbaijan Province Iran (Maku, Khoy, Urmia, Piranshahr). Ecologically, this area is classified as a semi-arid zone. Small ruminants rising are economically a very important occupation in this province.

A total of 402 blood samples in EDTA tubes were collected from 280 sheep and 122 goats that belonged to twelve flocks with sheep only, four flocks with goats only, and 24 flocks with sheep and goats together. Data on the characteristics of the animals (species, gender and tick burden) and the flock (size, species of animals, dogs associated with the flocks and tick burden of dogs associated with the flocks) were collected through questionnaires completed by the investigators on location during sample collection.

The whole body of each sampled small ruminant and farm dog was inspected for the presence of ticks by palpation, mainly on their ears, along their nape, perineum, and udder/scrotum, between thigh, shoulder region and tail base. The ticks were manually removed and transferred to the parasitology laboratory in tubes containing 70 % ethanol solution. Thin blood smears were prepared immediately after blood sampling. The thin blood smears were fixed in methanol for 5 min and stained in 10 % Giemsa solution in phosphate buffer solution (PBS), pH 7.2, for 20 min and examined under an oil-immersion objective of a magnification of 1000 x for the presence of intracellular forms of the parasite with morphology compatible with B. ovis (Aktas et al. 2007). Parasitemia was expressed as the log number of red blood cells infected with Babesia parasites per 105 erythrocytes (Schetters et al. 2009). The smears were recorded as negative for Babesia spp. if no parasites were detected in observed oilimmersion fields. The collected adult ticks from animals were counted. Tick species were identified using standard taxonomic keys (Walker et al. 2003, Estrada-Pena et al. 2004).

DNA was extracted using a DNA purification kit (Fementas, Germany) according to the manufacturer's instruction. A pair of primers, Bbo-F 5'-TGGGCAGGACCTTGG TTCTTC T-3' and Bbo-R 5'-CCGCGTAG CGCCGGCTAAATA-3' were used to amplify a 549 bp fragment of the ssu rRNA gene of B. ovis. The primers' specificity and sensitivity was assessed by Aktas et al. (2005).

PCR was carried out in 50 µ l total reaction volume containing 5 µ l of 10 X PCR buffer, 2 mM MgCl2, 250 µ M of each of the four deoxynucleotide triphosphate, 1.25 U Taq DNA polymerase (Fermentas, Germany), 50 pmol of each primer and 50 ng of extracted DNA. Amplification of parasite DNA was done in a CP2-003 thermocycler (Corbett Research, Australia). Cycling conditions for B. ovis were 95 °C for 5 min, followed by 45 cycles at 94 °C for 45 sec, 63 °C for 45 sec and 72 °C for 1 min with a final extension step of 72 °C for 10 min. The PCR products were separated by electrophoresis on 1.5 % agarose gel in Tris-Borate-EDTA (TBE) buffer and visualized using ethidium bromide (1 µ g/ml) and UV transilluminator (BTS-20M, Japan).

The positive control for B. ovis was obtained from sheep with clinical babesiosis (diagnosis was done on the basis of clinical signs and light microscopic examination Giemsa-stained thin blood smear). Venous blood sample, taken from healthy lamb without contact with ticks, served as negative control in the study.

The Fisher's exact test and MantelHaenszel test was used to express association between the presence (positive and negative blood samples) of Babesia and the various parameters ie flock size, species, gender and age of animal, tick infestation of sheep and goats, presence of ticks in the flock and flock composition. Software SPSS version 17.0 was used to compare the data of blood smears with blood PCR method. Results were displayed as P values as well as relative risk values (with 95 % confidence intervals). P< 0.05 was accepted to be statistically significant.

Results

Microscopic examination of thin blood smears showed parasitaemia in infected animals ranging from 0.01 to 3 %. Piroplasms, detected inside the red blood cells, were polymorphous. The shapes of the parasites were pyriform and single ring. All of these forms classified as Babesia spp.

Of the 402 blood samples examined, 42/402 (10.4%) were positive for piroplasms upon microscopic examination, whereas, 67/ 402 (16.7%) were positive for presence of B. ovis by PCR. All of forty-two positive by microscopic examination were also positive by PCR (Fig. 1). Compared to microscopic examination results, PCR showed a significantly higher efficacy of detection of Babesia spp. (P< 0.05). Out of 40 examined farms, twentynine (72.5%) were found positive for B. ovis. The percentage of positive animals in each farm varied from 13 % to 20 %.

The prevalence of B. ovis in sheep and goats in relation to the parameters describing the characteristics of the animals and the flock is shown in Table 1. Out of the 280 sheep and 122 goats examined, 18.5 % (52/ 280) and 12.2 % (15/122) were infected with B. ovis. The difference between the prevalence of B. ovis infection in sheep and goats were statistically significant (P< 0.05). The prevalence of B. ovis infection in age groups and different gender were not significantly different (P> 0.05). Frequency of B. ovis infection was significantly higher in flocks with tick burden (P< 0.05).

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 PCR-positive results for B. ovis in sheep and goats was found 3.8 and 2.9, respectively.

During this survey, a total of 747 adult Ixodidae ticks were collected from different body areas, i.e. external ear, perineum, between thigh and udder/scrotum of sampled small ruminants and farm dogs. The following ticks were identified, Rhipicephalus bursa 49.9 % (373/747), R. sanguineus 43.9 % (328/747) and R. turanicus 6.2 % (46/747). The main attachment site of ticks was the perineum region.

Discussion

Microscopic examination, such as Giemsastained blood smears is mostly used as a confirmatory diagnosis of vertebrate host suffering of piroplasm infections. However, the method requires expertise because these parasites have similar morphological features and therefore, may confuse the examiner when mixed infections occur. Serological tests were also used, but there are some difficulties with specificity and sensitivity (Passos et al. 1998). An exact differentiation between these parasites is crucial to understanding their epidemiology. The detection of Babesia infection in carrier animals by DNA amplification has been a powerful tool for epidemiological investigation, since these animals represent an important source of alimentary infection of ixodid ticks (Aktas et al. 2005, Altay et al. 2008). The present study is the first molecular diagnostic technique that was employed to determine the epidemiology of small ruminant's babesiosis in Iran. The results showed more than 16 % of small ruminants in northwest of Iran were infected with B. ovis.

In present study, as expected, the prevalence of B. ovis infection in small ruminants detected by PCR (16.7%) was significantly higher than obtained in microscopic examination of thin blood smears (10.4%). Due to main drawbacks for microscopic detection of Babesia spp. in the early stage of infection and the long-term carrier status are difficult to detect of the parasites in very low parasitemia, therefore PCR method has higher efficiency than microscopic examination for detection of B. ovis. The results agree with a previous report about B. bovis (Calder et al. 1996).

In the microscopic examination of blood smears, we found that parasitaemia ranges from 0.01 to 3 %, in sheep and goats. In similar studies, Razmi et al. (2003) and Papadopoulos et al. (1996) has observed that sheep infected with B. ovis commonly had low parasitemia.

In previous studies in Iran, serological tests employing Indirect Fluorescent Antibody Test (IFAT) was used and the prevalence of seropositive animals varied from 12 % to 58.8 % in different regions of the country (Tavassoli and Rahbari 1998). In the present study, covering four different regions of West-Azerbaijan Province, Iran, the prevalence ranged from 13 % to 20 % on the farms that were examined. Although the results of the present and previous study cannot be compared due to the different methods employed, they both indicate that ovine babesiosis is seen in almost each region of Iran and prevalence of the disease shows difference among the provinces located in regions with different endemic features.

The finding that the prevalence of ovine babesiosis was higher in herds with tick burden indicates the presence of a positive correlation between the prevalence of the disease and the presence of vector ticks. The result is accordance with the find of Theodorpoulos et al. (2005).

Among the factors examined in the present study, the presence of ticks in sheep, goats and farm dogs were associated with PCR-positive results, which indicate a high risk of infection with B. ovis in sheep and goats. Ticks suitable for transmission of Babesia have been reported in Iran (Razmi et al. 2003). Concerning the species of animals (sheep or goats), it is stated that in the field, B. ovis causes disease exclusively in sheep, rarely in goats (Papadopoulos et al. 1996). In regard to the role of farm dogs as a risk factor for babesiosis in sheep and goats, ticks can transmit Babesia even after feeding on other hosts (Yeruham et al. 1996) and R. sanguineus that usually infests dogs has been found also on ruminants (Bouattour et al. 1999). However, the role of dogs in ovine or caprine babesiosis needs to be further studies. On the other hand, according to CriadoFornelio et al. (2003) the same Babesia species can infect a wide variety of animal hosts. This finding indicates that other animal species besides dogs may also be risk factors for babesiosis in sheep and goats.

Conclusion

The epidemiology of ovine babesiosis due to B. ovis is closely related to the ecology of vector ticks. The disease occurs yearly in the Rhipicephalus spp. infested areas, during the activity period of the adult tick stage (Yeruham et al. 1995). In the present study, R. bursa exhibited the highest (49.9%) frequency of infection. According to Tavassoli and Haji-Ghahremani (2004) in a tick's survey carried out in northwester Iran, the majority of the ticks found on sheep and goats were identified as R. bursa. Thus, the tick may play an important role as a vector of the parasite in Iran. Work is currently underway to provide data on the tick vector competency of B. ovis.

Acknowledgements

This project was supported by Urmia University (No.1-67). The authors would like to express their gratitude to Mr Esmaeil Aghapour and Mr Armen Badali for their technical assistance. The authors declare that there is no conflict of interests.

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