Trop Anim Health Prod DOI 10.1007/s11250-014-0710-6
SHORT COMMUNICATIONS
Detection and assessment of risk factors associated with natural concurrent infection of Trypanosoma evansi and Anaplasma marginale in dairy animals by duplex PCR in eastern Punjab Amrita Sharma & Lachhman Das Singla & Ashuma Tuli & Paramjit Kaur & Mandeep Singh Bal
Received: 4 September 2014 / Accepted: 17 October 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Duplex PCR consisting of two primer sets within a single mixture for the simultaneous detection of Anaplasma marginale and Trypanosoma evansi was standardized and employed on 219 blood samples collected from cattle (165) and buffaloes (54) from eastern Punjab to evaluate the status of concurrent infection and associated risk factors. The reaction produced 257- and 407-bp amplification products targeting repetitive nucleotide sequence of T. evansi and msp1β gene of A. marginale, respectively. The nucleotide sequence analysis of individual amplicons expressed the fidelity of the primer pairs used; duplex PCR was 100 % sensitive and 92.66 % specific with conventional microscopy for the detection of mixed infections. Among the agroclimatic zones of interest, undulating zone was at higher risk of T. evansi infection (odds ratio (OR)=1.75, 95 % confidence interval (CI)=0.94–3.27), and submountain zone (OR=1.89, 95 % CI=1.11–3.33) for A. marginale. For the concurrent infection, the relative risk among the two zones was almost unity. The cross-bred cattle population was at the highest risk of infection, may it be solo infection of T. evansi (OR=∞, 95 % CI=1.18–∞)/A. marginale (OR=6.39, 95 % CI=1.14– 125.3) or dual infection (OR=∞, 95 % CI=0.39–∞) of both as the indigenous cattle are resistant to the infection. Cross-bred cattle were at approximately three times the risk than buffaloes. For the dual infection, the cattle calves were at about 2.5 times higher risk than buffalo calves. Results indicate the endemic status of these infections in the region and mark out the commodities at great risk and requiring better surveillance.
A. Sharma : L. D. Singla (*) : A. Tuli : P. Kaur : M. S. Bal Department of Veterinary Parasitology, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, Punjab, India e-mail: [email protected]
Keywords Anaplasma marginale . Dairy animals . Duplex PCR . Prevalence . Punjab risk factors . Trypanosoma evansi Abbreviations PCR Polymerase chain reaction DNA Deoxyribonucleic acid
Introduction Haemoparasitic diseases are a major constraint to livestock health and production worldwide, primarily in tropical and subtropical areas (Juyal et al. 1994; Ananda et al. 2009). Their impact depends on the region, the species involved, the host population(s) involved, and the socio-economic and technological advances in control measures (Nair et al. 2011; Chaudhri et al. 2013; Sharma et al. 2013). Punjab state located in latitude 29″ 30′ N–32″ 32′ N and longitude 73″ 55′ E–76″ 50′ E is conducive for the endemic stability of these diseases with an estimation of more than 300 million of cattle and buffaloes at the risk (Livestock census 2007). Anaplasmosis and trypanosomosis are among the major haemoparasitic and rickettsial diseases of domestic animals. Rhipicephalus (Boophilus) microplus is the major vector of Anaplasma marginale in various countries including India, South Africa and Brazil (Guglielmone 1995; Potgieter 1996; Ghosh et al. 2007). A conservative estimate of the annual losses due to anaplasmosis in the USA alone amounts to US$100 million with 50,000 to 1,000,000 cattle deaths (McCallon 1973). Trypanosoma evansi infection, commonly known as surra, is one of the potent fatal haemoprotozoan diseases mechanically transmitted by biting flies such as tabanids (Claes et al. 2004). Dairy animals are potentially viable host to the infection. T. evansi infection results into acute or mild or subclinical form of the disease.
Trop Anim Health Prod
In North India, 20 outbreaks of anaplasmosis occurred during the period of January to June 2013, out of which five were reported from Jammu and Kashmir, six from West Bengal and nine (45 %) from Punjab, indicating the threats posed on livestock by the disease (http://www.oie.int/wahis_2/ public/wahid.php/Diseaseinformation/statusdetail). Outbreaks of trypanosomiasis were also reported from various parts of India (Bidyanta et al. 1990; Singh and Joshi 1991; Batra et al. 1994; Aulakh et al. 2005). As both of these infections’ understudies have similar types of signs, moreover, arthropod vectors of these two diseases co-exist in suitable tropical climatic conditions; it becomes necessary that an economical, specific, and sensitive technique be standardized and employed for simultaneous detection of these two diseases in suspected animals. Hence, the purposes of the present study were to assess the concurrent infection of A. marginale and T. evansi by duplex PCR assay and to describe the trend of prevalence pertaining to the associated risk factors in different regions of Punjab state.
Materials and methods Punjab state is stratified into five major agro-climatic zones based on edaphic, agricultural and climatic indices. A representative cattle (165) and buffalo (54) blood sample collection was done from March 2011 to September 2013 from the hilly and undulating regions, viz., undulating and submountain zones comprising eastern Punjab. A total of 219 samples (123 cattle and 33 buffalo; 42 cattle calves and 21 buffalo calves) were randomly selected for the present study to screen the animals for concurrent infection of A. marginale and T. evansi. Blood (∼3 mL) was collected from jugular vein of each animal into anticoagulant-coated vacutainers for microscopy and nucleic acid extraction. The data related to sampled animals (species, age and health status) and farm (management and location) was also collected from the dairy farmers on pre-designed questionnaire. To study the status of molecular prevalence of the concurrent infection, the expected prevalence to be 50 % with confidence limits of 95 % and a desired absolute precision of 5 % to collect maximum number of samples was considered. The number of samples thus calculated was adjusted for finite population and was correlated with 219 samples collected. From the blood samples of all the selected animals, thin blood smears were made, air dried, fixed in methyl alcohol for 2 min and stained with working dilution of 10 % Giemsa stain for 30 min. The smears were then washed with tap water to remove extra stain, air dried and examined under oil immersion for demonstration of A. marginale and T. evansi. Genomic DNA was extracted from the whole blood collected in EDTA-coated vacutainers using HiPurA™ Blood Genomic DNA MiniPrep Purification Spin Kit as per the
given protocol (HiMedia® Laboratories, India). The DNA extracted from blood leucocytes from a 3-day-old calf was included as a negative control. DNA extracted from T. evansi parasites of cattle strain propagated in mice and purified by DEAE cellulose columns chromatography and DNA extracted from A. marginale-infected cattle blood were used as positive controls for respective parasites. Oligonucleotide primers used for the establishment of duplex PCR for A. marginale and T. evansi targeted msp1β gene (BAP-2 5′ GTA TGG CAC GTA GTC TTG GGA TCA 3′/AL34S -5′ CAG CAG CAG CAA GAC CTT CA 3′) (Ulrike et al. 2003) and repetitive nucleotide sequences (TR3 5′ GCG CGG ATT CTT TGC AGA CGA 3′/TR4 5′ TGC AGA CAC TGG AAT GTT ACT 3′) (Wuyts et al. 1994), respectively. A total of 25 μL PCR reaction mixture was constituted with KAPA2G™ Fast HotStart ReadyMix (1X containing KAPA2G™ Fast HotStart DNA polymerase, KAPA2G™ Fast HotStart PCR buffer, 0.2 mM dNTP each, 1.5 mM MgCl2), with 15 pmol of BAP-2/AL34S primers and 12.5 pmol of TR3/TR4 primers and additional 1 mM MgCl2. The reaction was set in automated thermocycler with the following programme: initial denaturation at 95 °C (5 min), 30 cycles of denaturation at 95 °C (30 s), annealing at 57 °C (1 min) and extension at 72 ° C (1.5 min) with final extension at 72 °C (10 min). The amplified PCR products were separated by electrophoresis on 1 % agarose gel and visualized under UV Transilluminator for detection of 407- and 257-bp amplified products. Primers were examined for their specificity for each individual parasite species by amplification of DNA samples of individual parasite species as well as from mixture of DNA samples derived from parasitologically positive samples of respective haemoprotozoan, with each set of primers. Host leucocytes DNA from a 3-day-old calf was used as a negative control. The individual 257- and 407-bp products obtained by the primers specific for T. evansi and A. marginale PCR, respectively, were custom sequenced from Xcelris Genomics, Ahmedabad, India. The nucleotide sequences were subjected to BLASTn analysis (Altschul et al. 1990) for determining the similarity with the sequences present in the nucleotide database. χ2 test was employed to compare prevalence of T. evansi, A. marginale and dual infections by conventional parasitological and molecular techniques among various districts of eastern Punjab. The agreement between the two techniques and assessment of relative risk was done on WinEpiscope Software.
Results PCR amplification employed on each individual DNA sample (T. evansi and A. marginale) using their specific primers lead
Trop Anim Health Prod
to the detection of the expected fragments of sizes 257 and 407 bp, respectively. Each set of the primers was specific for the respective parasite DNA, and non-target DNA amplification was not seen in negative controls (Fig. 1). The sequence obtained for the 257-bp product showed the closest alignments (99 %) with T. evansi isolate H4 VSG gene (GenBank No. HM209055.1). The 407-bp sequence showed homology with (100 %) A. marginale clone LDH1 major surface protein 1-beta (msp1β) gene (GenBank No. KF696857).
Geographic pattern
Fig. 2 Giemsa-stained thin blood smear showing concurrent occurrence of A. marginale (arrows) and T. evansi
The parasitological prevalence was found to be highest in district Nawanshahr for T. evansi (2.38 %) and in Hoshiarpur for A. marginale (16.28), while the only case of concurrent infection by thin blood smear examination was reported from Hoshiarpur (Fig. 2). Statistically, there was no difference in the prevalence of the three types of infections among various district (P1 year
SHORT COMMUNICATIONS
Detection and assessment of risk factors associated with natural concurrent infection of Trypanosoma evansi and Anaplasma marginale in dairy animals by duplex PCR in eastern Punjab Amrita Sharma & Lachhman Das Singla & Ashuma Tuli & Paramjit Kaur & Mandeep Singh Bal
Received: 4 September 2014 / Accepted: 17 October 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Duplex PCR consisting of two primer sets within a single mixture for the simultaneous detection of Anaplasma marginale and Trypanosoma evansi was standardized and employed on 219 blood samples collected from cattle (165) and buffaloes (54) from eastern Punjab to evaluate the status of concurrent infection and associated risk factors. The reaction produced 257- and 407-bp amplification products targeting repetitive nucleotide sequence of T. evansi and msp1β gene of A. marginale, respectively. The nucleotide sequence analysis of individual amplicons expressed the fidelity of the primer pairs used; duplex PCR was 100 % sensitive and 92.66 % specific with conventional microscopy for the detection of mixed infections. Among the agroclimatic zones of interest, undulating zone was at higher risk of T. evansi infection (odds ratio (OR)=1.75, 95 % confidence interval (CI)=0.94–3.27), and submountain zone (OR=1.89, 95 % CI=1.11–3.33) for A. marginale. For the concurrent infection, the relative risk among the two zones was almost unity. The cross-bred cattle population was at the highest risk of infection, may it be solo infection of T. evansi (OR=∞, 95 % CI=1.18–∞)/A. marginale (OR=6.39, 95 % CI=1.14– 125.3) or dual infection (OR=∞, 95 % CI=0.39–∞) of both as the indigenous cattle are resistant to the infection. Cross-bred cattle were at approximately three times the risk than buffaloes. For the dual infection, the cattle calves were at about 2.5 times higher risk than buffalo calves. Results indicate the endemic status of these infections in the region and mark out the commodities at great risk and requiring better surveillance.
A. Sharma : L. D. Singla (*) : A. Tuli : P. Kaur : M. S. Bal Department of Veterinary Parasitology, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, Punjab, India e-mail: [email protected]
Keywords Anaplasma marginale . Dairy animals . Duplex PCR . Prevalence . Punjab risk factors . Trypanosoma evansi Abbreviations PCR Polymerase chain reaction DNA Deoxyribonucleic acid
Introduction Haemoparasitic diseases are a major constraint to livestock health and production worldwide, primarily in tropical and subtropical areas (Juyal et al. 1994; Ananda et al. 2009). Their impact depends on the region, the species involved, the host population(s) involved, and the socio-economic and technological advances in control measures (Nair et al. 2011; Chaudhri et al. 2013; Sharma et al. 2013). Punjab state located in latitude 29″ 30′ N–32″ 32′ N and longitude 73″ 55′ E–76″ 50′ E is conducive for the endemic stability of these diseases with an estimation of more than 300 million of cattle and buffaloes at the risk (Livestock census 2007). Anaplasmosis and trypanosomosis are among the major haemoparasitic and rickettsial diseases of domestic animals. Rhipicephalus (Boophilus) microplus is the major vector of Anaplasma marginale in various countries including India, South Africa and Brazil (Guglielmone 1995; Potgieter 1996; Ghosh et al. 2007). A conservative estimate of the annual losses due to anaplasmosis in the USA alone amounts to US$100 million with 50,000 to 1,000,000 cattle deaths (McCallon 1973). Trypanosoma evansi infection, commonly known as surra, is one of the potent fatal haemoprotozoan diseases mechanically transmitted by biting flies such as tabanids (Claes et al. 2004). Dairy animals are potentially viable host to the infection. T. evansi infection results into acute or mild or subclinical form of the disease.
Trop Anim Health Prod
In North India, 20 outbreaks of anaplasmosis occurred during the period of January to June 2013, out of which five were reported from Jammu and Kashmir, six from West Bengal and nine (45 %) from Punjab, indicating the threats posed on livestock by the disease (http://www.oie.int/wahis_2/ public/wahid.php/Diseaseinformation/statusdetail). Outbreaks of trypanosomiasis were also reported from various parts of India (Bidyanta et al. 1990; Singh and Joshi 1991; Batra et al. 1994; Aulakh et al. 2005). As both of these infections’ understudies have similar types of signs, moreover, arthropod vectors of these two diseases co-exist in suitable tropical climatic conditions; it becomes necessary that an economical, specific, and sensitive technique be standardized and employed for simultaneous detection of these two diseases in suspected animals. Hence, the purposes of the present study were to assess the concurrent infection of A. marginale and T. evansi by duplex PCR assay and to describe the trend of prevalence pertaining to the associated risk factors in different regions of Punjab state.
Materials and methods Punjab state is stratified into five major agro-climatic zones based on edaphic, agricultural and climatic indices. A representative cattle (165) and buffalo (54) blood sample collection was done from March 2011 to September 2013 from the hilly and undulating regions, viz., undulating and submountain zones comprising eastern Punjab. A total of 219 samples (123 cattle and 33 buffalo; 42 cattle calves and 21 buffalo calves) were randomly selected for the present study to screen the animals for concurrent infection of A. marginale and T. evansi. Blood (∼3 mL) was collected from jugular vein of each animal into anticoagulant-coated vacutainers for microscopy and nucleic acid extraction. The data related to sampled animals (species, age and health status) and farm (management and location) was also collected from the dairy farmers on pre-designed questionnaire. To study the status of molecular prevalence of the concurrent infection, the expected prevalence to be 50 % with confidence limits of 95 % and a desired absolute precision of 5 % to collect maximum number of samples was considered. The number of samples thus calculated was adjusted for finite population and was correlated with 219 samples collected. From the blood samples of all the selected animals, thin blood smears were made, air dried, fixed in methyl alcohol for 2 min and stained with working dilution of 10 % Giemsa stain for 30 min. The smears were then washed with tap water to remove extra stain, air dried and examined under oil immersion for demonstration of A. marginale and T. evansi. Genomic DNA was extracted from the whole blood collected in EDTA-coated vacutainers using HiPurA™ Blood Genomic DNA MiniPrep Purification Spin Kit as per the
given protocol (HiMedia® Laboratories, India). The DNA extracted from blood leucocytes from a 3-day-old calf was included as a negative control. DNA extracted from T. evansi parasites of cattle strain propagated in mice and purified by DEAE cellulose columns chromatography and DNA extracted from A. marginale-infected cattle blood were used as positive controls for respective parasites. Oligonucleotide primers used for the establishment of duplex PCR for A. marginale and T. evansi targeted msp1β gene (BAP-2 5′ GTA TGG CAC GTA GTC TTG GGA TCA 3′/AL34S -5′ CAG CAG CAG CAA GAC CTT CA 3′) (Ulrike et al. 2003) and repetitive nucleotide sequences (TR3 5′ GCG CGG ATT CTT TGC AGA CGA 3′/TR4 5′ TGC AGA CAC TGG AAT GTT ACT 3′) (Wuyts et al. 1994), respectively. A total of 25 μL PCR reaction mixture was constituted with KAPA2G™ Fast HotStart ReadyMix (1X containing KAPA2G™ Fast HotStart DNA polymerase, KAPA2G™ Fast HotStart PCR buffer, 0.2 mM dNTP each, 1.5 mM MgCl2), with 15 pmol of BAP-2/AL34S primers and 12.5 pmol of TR3/TR4 primers and additional 1 mM MgCl2. The reaction was set in automated thermocycler with the following programme: initial denaturation at 95 °C (5 min), 30 cycles of denaturation at 95 °C (30 s), annealing at 57 °C (1 min) and extension at 72 ° C (1.5 min) with final extension at 72 °C (10 min). The amplified PCR products were separated by electrophoresis on 1 % agarose gel and visualized under UV Transilluminator for detection of 407- and 257-bp amplified products. Primers were examined for their specificity for each individual parasite species by amplification of DNA samples of individual parasite species as well as from mixture of DNA samples derived from parasitologically positive samples of respective haemoprotozoan, with each set of primers. Host leucocytes DNA from a 3-day-old calf was used as a negative control. The individual 257- and 407-bp products obtained by the primers specific for T. evansi and A. marginale PCR, respectively, were custom sequenced from Xcelris Genomics, Ahmedabad, India. The nucleotide sequences were subjected to BLASTn analysis (Altschul et al. 1990) for determining the similarity with the sequences present in the nucleotide database. χ2 test was employed to compare prevalence of T. evansi, A. marginale and dual infections by conventional parasitological and molecular techniques among various districts of eastern Punjab. The agreement between the two techniques and assessment of relative risk was done on WinEpiscope Software.
Results PCR amplification employed on each individual DNA sample (T. evansi and A. marginale) using their specific primers lead
Trop Anim Health Prod
to the detection of the expected fragments of sizes 257 and 407 bp, respectively. Each set of the primers was specific for the respective parasite DNA, and non-target DNA amplification was not seen in negative controls (Fig. 1). The sequence obtained for the 257-bp product showed the closest alignments (99 %) with T. evansi isolate H4 VSG gene (GenBank No. HM209055.1). The 407-bp sequence showed homology with (100 %) A. marginale clone LDH1 major surface protein 1-beta (msp1β) gene (GenBank No. KF696857).
Geographic pattern
Fig. 2 Giemsa-stained thin blood smear showing concurrent occurrence of A. marginale (arrows) and T. evansi
The parasitological prevalence was found to be highest in district Nawanshahr for T. evansi (2.38 %) and in Hoshiarpur for A. marginale (16.28), while the only case of concurrent infection by thin blood smear examination was reported from Hoshiarpur (Fig. 2). Statistically, there was no difference in the prevalence of the three types of infections among various district (P1 year
