Comparison between conventional and molecular methods for diagnosis of bovine babesiosis (Babesia bovis infection) in tick infested cattle in upper Egypt

Abstract

Ticks and tick-borne diseases are the main problems affecting the livestock production in Egypt. Bovine babesiosis has adverse effects on the animal health and production. A comparison of Giemsa stained blood smears, polymerase chain reaction (PCR) and nested PCR (nPCR) assays for detection of Babesia bovis infection in Egyptian Baladi cattle (Bos taurus) in reference to reverse line blot was carried out. The sensitivity of PCR and nested PCR (nPCR) assays were 65 and 100 % respectively. Giemsa stained blood smears showed the lowest sensitivity (30 %). According to these results using of PCR and nPCR target for B. bovis, [BBOV-IV005650 (BV5650)] gene are suitable for diagnosis of B. bovis infection. The 18Ss rRNA partial sequence confirmed that all the positive samples were Babesia bovis and all of them were deposited in the GenBank databases (Accession No: KM455548, KM455549 and KM455550).

Introduction

Babesia bovis infection is a tick-borne parasite of cattle transmitted by Rhipicephalus annulatus ticks. The first description of this disease in Egypt was in 1947 by Nagati (1947). B. bovis infection is a worldwide distributed and considered as one of the most important destructive diseases of cattle (McCosker, 1981; Ibrahim et al. 2013 and Elsify et al. 2015). The clinical signs include fever, hemoglobinuria, acute anemia, and nervous signs (Mosqueda et al. 2012; Radwan et al. 2013). Low parasitemia carrier state is usually developed after survival of the animals; they serve as a reservoir of the parasite (Mahoney, 1969). The diagnosis of bovine babesiosis depends mainly on clinical and microscopic examination of Giemsa stained blood smear which is the conventional laboratory technique. Presence of piroplasms is the gold standard for the diagnosis, especially during the acute stage of the disease. Indeed, during this stage the number of parasites inside the erythrocytes is high enough to be detected easier by microscopical examination (Bose et al. 1995). In carrier animals this method is less sensitive since the parasitaemia is low. In this case, molecular tools using PCR and nested PCR increase the sensitivity. They are more sensitive and specific, providing rapid and accurate results (Oliveira-Sequeira et al. 2005; Costa-Junior et al. 2006; Martins et al. 2008). This study aim to compare four diagnostic techniques, namely Giemsa stained blood smears, PCR and nested PCR for detection of Babesia bovis infection in Egyptian Baladi cattle (Bos taurus). The Reverse line blot (RLB) was the reference technique.

Materials and methods

Animals

Baladi breed cattle belong to different localities in Assuit governorate in Upper Egypt (Egypt) were admitted to the Veterinary Teaching Hospital. They were subjected to clinical examination according to Radostits et al. (2000).

Parasitological diagnosis

Blood samples were collected directly from the ear vein and used for preparation of blood smears (Coles, 1986).

Molecular diagnosis

Whole blood samples were collected directly from the jugular vein on vacutainer tube containing EDTA and stored at −20 °C until used. DNA extraction was performed with QIAamp DNA blood Mini kit, (Qiagen, Ltd, UK) according to manufacturer’s instruction. PCR and nested PCR (nPCR) amplifications were carried out by using specific primers to B. bovis, [BBOV-IV005650 (BV5650)] F, 5′-CCGGAATTCCAAATGGCAACAAAGGTTGA-3′R, 5′-CCGCTCGAGGGAGCAGCGTATTACTTCCTCACGT-3′, F1, 5′-CGAGGATTTGGTAGACCTCATC-3′, R2 5′-CGTAAAATGTGTACAACTATTT-3′, respectively (Figueroa et al. 1993; Aboulaila, et al. 2010a, b). RLB was performed with a set of 18Ss rRNA Babesia spp.-catchall primers (RLB-F2 5′-GAC ACA GGG AGG TAG TGA CAA G-3′, Nested RLB-F 5′-GAC AAG AAA TAA CAA TAC RGG GC-3′, RLB-R2 5′CTA AGA ATT TCA CCT CTG ACA GT-3′) (Gubbels et al. 1999). The products were subjected to electrophoresis in 2 % agarose gel and then visualized under ultraviolet (UV) light after staining with ethidium bromide (Sigma-Aldrich). Positive DNA products from 18Ss rRNA primer were purified and then cloned into pDrive Cloning vector using QIAGEN Kit (Qiagen, Ltd, UK) according to manufacturer’s instructions. The amplicons were sequenced in both directions (Molecular Biology Research Unite, Assiut University, Egypt), and subjected to BLAST similarity searches.

Statistical analysis

Giemsa stained blood smears, PCR and nested PCR were compared with RLB as a reference test. Evaluation parameters included sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and combined predictive value (CPV) (Thrusfield, 2005).

Results

Giemsa stained blood smears confirmed the infection in 7.9 % (6/76) of the examined animals. The positive animals showed specific clinical signs of babesiosis, including fever (>40 °C) and hemoglobin urea with different intensities of Boophilus (Rhipicephalus) annulatus ticks infestations. PCR and nested PCR detected the DNA target of Babesia bovis in 17.1 % (13/76) and 26.3 % (20/76) of examined cattle respectively. RLB assay confirmed the infection in 26.3 % (20/76) of the animals (Table 1, Figs. 1, 2, 3, 4, 5). The PCR showed 100 % specificity for detection of Babesia bovis infection but their sensitivity varied between 30 and 100 % (Table 2). Partial sequences were deposited in the GenBank databases under accession numbers (KM455548, KM455549 and KM455550).

Table 1.

Infection rates according to Giemsa stained blood smears, PCR, nPCR and RLB for detection of Babesia bovis in tick infested cattle

Diagnostic assays	Positive	±SD (%)
Blood smear	6/76	7.89 ± 25.32
BV5650 PCR	13/76	17.11 ± 25.23
BV5650 nPCR	20/76	26.31 ± 26.41
RLB	20/76	26.31 ± 26.41

Fig. 1.

Giemsa stained blood smear showing Babesia bovis piroplasms

Fig. 2.

Babesia bovis PCR products. Lane M: DNA ladder 100 bp, Lanes 1, 3, 5, 7 and 8: positive PCR with bands of 720 bp

Fig. 3.

Babesia bovis nested PCR products. Lane M: DNA ladder 100 bp, Lanes 1, 3, 5, 7, 8 and 10: positive PCR with bands of 561 bp

Fig. 4.

Babesia bovis RLB-PCR products. Lane M: DNA ladder 100 bp, Lanes 1, 4, 6, 7 and 10: positive PCR

Fig. 5.

Babesia bovis nested RLB-PCR products. Lane M: DNA ladder 100 bp, Lanes 1, 2, 4, 6, 7 and 10: positive PCRs both of them gave bands of 460 and 520 bp

Table 2.

Evaluation of Giemsa stained blood smears, PCR and nPCR against RLB as a reference test for Babesia bovis detection in tick infested cattle

Diagnostic assays	TP	TN	FP	FN	Sensitivity	Specificity	PPV	NPV	CPV
Blood film	6	56	0	14	30	100	100	80	81.58
BV5650 PCR	13	56	0	7	65	100	100	88.89	90.79
BV5650 nPCR	20	56	0	0	100	100	100	100	100

TP true positive, TN true negative, FP false positive, FN false negative

Discussion

Bovine babesiosis is one of the most important diseases in Egypt. It has an adverse effect on both production and reproduction of cattle. B. bovis and B. bigemina are the most common parasites in cattle (Ibrahim et al. 2013; Elsify et al. 2015). The prevalence of them usually becomes different according to the investigated locality. The prevalence of B. bovis is higher than B. bigemina in Dakahlia Governorate, Egypt it was 7.3 and 1.2 %, respectively according to El-Ashker a et al. (2015) while Ibrahimet et al. (2013) reported that the prevalence of B. bigemina is higher than B. bovis it was 5.30 and 3.97 %, respectively in Beheira and Faiyum, Governorates Egypt. In Upper Egypt PCR and nPCR of BV5650 gene are recently used in detection of B. bovis infection in randomly collected field samples from baladi breed cattle. The molecular assay, specially nested PCR of BV5650 gene was the most sensitive test (100 %) followed by BV5650 gene PCR (65 %) and blood smears examination (30 %) this came in agreement with El-Ashker et al. (2015) and Aboulaila, et al. (2010a, b) the first study reported that molecular assay was more sensitive than conventional method and both of them should be in combination to detect both clinically infected animals and carrier state; the second study reported that using of PCR and nPCR of gene BV5650 had the highest sensitivity rate when applied on field samples collected from cattle in Ghana, Mongolia and Brazil, respectively. This could be attributed to the presence of several gene copies in the genome; also the sequence conservation among strains may be playing an important role in these findings according to Aboulaila, et al. (2010a, b). Giemsa stained blood smears, PCR and nPCR of BV5650 gene revealed high PPV (100 %). These findings indicate that during further investigations, these tests will have the ability to detect the infected animals correctly. The NPVs of Giemsa stained blood smears and PCR were relatively low as 80 and 88.9 %, respectively; if compared with nPCR BV5650 which revealed 100 % NPV. These results indicate the ability of nPCR BV5650 to detect true negative if compared with Giemsa stained blood smears and BV5650 PCR assay. The combined predictive values were 81.6, 90.8 and 100 % for Giemsa stained blood film and BV5650 PCR and nPCR assays.

In conclusion, PCR and nPCR methods based on BV5650 gene detection should be recommended for the detection of B. bovis infection in cattle under field conditions and for epidemiological studies.

Future study

A recent type of molecular assay like Loop-Mediated isothermal amplification (LAMP) test is more applicable, not expensive and not need sophisticated instruments. This molecular assay is more sensitive than conventional method, especially in the carrier state. We will start to use it for confirmation of infection in randomly collected field samples and evaluate its validity to use in epidemiological studies.

Compliance with ethical standards

Conflict of interest

Author confirmed that there is no conflict of interest.