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Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India
Accepted Manuscript Title: Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India Author: Deepak Sumbria L.D. Singla Amrita Sharma M.S. Bal Sanjay Kumar PII: DOI: Reference:
S0304-4017(15)00267-8 http://dx.doi.org/doi:10.1016/j.vetpar.2015.05.018 VETPAR 7641
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
31-12-2014 20-5-2015 22-5-2015
Please cite this article as: Sumbria, Deepak, Singla, L.D., Sharma, Amrita, Bal, M.S., Kumar, Sanjay, Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2015.05.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short communication
Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India Deepak Sumbriaa, L.D. Singlaa1, Amrita Sharmaa, M. S. Bala and Sanjay Kumarb a Department of Veterinary Parasitology, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004 b National Research Centre on Equines, Haryana, India. Highlights ► of the study
•
Multiplex PCR was standardized for the first time targeting haemoprotozoan in equines.
•
Standardized PCR was applied on equine samples.
•
Geographic trend of equine piroplasmosis in infection prone area.
•
Association of disease preponderance with various related risk factors. . Abstract
Multiplex PCR for simultaneous detection of Trypanosoma evansi and Theileria equi in singlestep reaction was optimized and employed on 108 equids (99 horses and 9 donkeys/mules) blood samples collected from two agro-climatic zones (Sub-mountain undulating zone and Undulating plain zone) of Punjab to evaluate the status of concurrent infection and associated risk factors. The amplification products of 257 and 709 bp targeting repetitive nucleotide sequence of variable surface glycoproteins of T. evansi and 18S rRNA gene of T. equi, respectively expressed high fidelity of the primer pairs with sequence homology to neighboring geographic isolates. The overall prevalence of T. evansi and T. equi was 3.7 and 1.85%, with Undulating plain zone at higher infection risk for T. equi (OR=3.24, 95% CI=0.28-83.65); and Sub-mountain undulating zone (OR=∞, 95% CI=0.25-∞) for T. evansi. Multiplex PCR revealed higher risk of infection of both T. equi (OR=6.75, 95% CI=0.58-175.38) and T. evansi (OR=2.11, 95% CI=0.05-80.36) in the farms with inappropriate management system. The risk factor associated with the type of host 1
Corresponding author:
Dr. L. D. Singla, Professor-cum-Head, Department of Veterinary Parasitology, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004. Email: [email protected], Contact no.: +91- 9316061974
species had an odds ratio of 12.35 (95% CI=0.29-508.37) for donkeys/mules versus horses for T. evansi infection. This group was also at higher risk of infection with Odds ratio (OR) of 4 (95% CI=0.14-53.99) for T. equi. The current investigation brings out various commodities at risk of infection pertaining to equid trypanosomosis and theileriosis evaluated by a rapid and sensitive multiplex PCR assay. Keywords: Trypanosoma evansi, Theileria equi, multiplex PCR, prevalence, risk factors, Punjab 1.
Introduction Trypanosomosis and theileiriosis are two economically important vector-borne diseases
of equids (horses, donkeys and mules) in tropical and subtropical parts of the world including India. In the Indian subcontinent trypanosomosis caused by Trypanosoma evansi [Kinetoplastid; haemoprotozoan] is known as ‘surra’, (a Hindi word meaning ‘rotten’) (Soulsby, 1982). Surra is characterized by a fluctuating parasitaemia with periods of paroxysms and intermissions, anemia, edema of the legs, dehydration, fever, abortion, and incoordination, followed by paralysis of the hind limbs (Gill, 1991). Theileiriosis, caused by an apicomplexan parasite (Theileria equi), is clinically characterised by fever, haemolytic anemia, icterus, petechial haemorrhages of the mucous membranes etc., but these are variable and are non-specific (Baptista et al., 2013). The disease renders negative effects on the health of equids, decreasing their productivity and work efficacy. T. equi infection lasts for 7-12 days, but can be peracute with death occurring in 1-2 days, or can be chronic, lasting for weeks. Mortality rate associated with this disease can vary for 10%-50% (Taylor et al., 2007). Imidocarb dipropionate @ 4mg/kg intra-muscularly on four occasions at 72 hrs intervals eliminates T. equi from horses (Berlin et al., 2010) but this dose is lethal to donkeys (Soulsby, 1982). A single dose of diminazene aceturate @ 3.5 mg/kg intramuscularly eliminates T. evansi from equids (Berlin et al., 2010). T. evansi can also be successfully managed with Quinapyramine sulphate and a chloride combination [Triquin] (Kumar et al., 2012). In northern India, T. evansi is transmitted mechanically by biting hematophagous flies (Tabanids) (Sumba et al., 1998) and T. equi is mainly transmitted by longistrate tick, Hyalomma anatolicum anatolicum, causing significant morbidity and mortality in equids (Kumar et al., 2007). As the vectors of both the diseases are habitual to hot plain areas of south-western Punjab, the epidemiological status of these infections in north-eastern parts of the province still remains unexplored.
T. evansi and T. equi are routinely diagnosed by conventional parasitological techniques (stained blood smears), serological (CATT/T. evansi, LATEX/T.evansi, IFAT, ELISA, etc) and molecular techniques. For the demonstration of parasite in blood, examination of stained thin blood smears is not a sensitive method, as the parasite has a periodically cryptic nature. Serological methods proffer the limitation of cross reactivity and failure to differentiate between past and current infection. For the diagnosis of latent infection, molecular techniques (PCR) give a promising result with greater levels of sensitivity and specificity (Bashir et al., 2014). Since both T. equi and T. evansi infections have almost similar types of signs and their vectors co-exist in suitable tropical climatic conditions, as in Punjab, it becomes necessary to optimize an economical, specific, and sensitive technique for simultaneous detection of these two diseases in suspected equines. Further, to evade the expenses on epidemiology of two important haemoparasites in comparatively quiescent zone, their simultaneous detection in a single reaction by multiplex PCR will be both time and cost effective. Hence, the purpose of the present study was to employ multiplex PCR assay for one-step detection of clinical, and latent forms of T. evansi and T. equi infections, for assessing the prevalence of these infections in a Sub-mountain undulating zone and Undulating plain zone of Punjab state and to further evaluate the risk factors contributing to the prevalence of these infections in equids for their proper control strategies. 2.
Materials and Methods
2.1 Ethical aspects The study has the approval (IAEC/2014/46-73) of the ethics committee for animal experiments duly constituted by the Guru Angad Dev Veterinary and Animal Sciences University. Blood samples were collected in a humanly manner, so as to avoid any accidental injury to the equids. A prior consent of the equines keepers was also sought. 2.2 Study area and sampling The province of Punjab covers a total area of 50,362 square kilometres between 29"30’N to 32"32’N latitude and 73”55’E to 76”50’E longitudes. In Punjab state lie areas between altitudes
213
meters
and
959
meters
above
sea
level
(http://punjabjudiciary.gov.in/index.php?trs=gurdist). There are about 34,000 equids which are at risk of infection due to haemoparasite in Punjab (Fazili and Kirmani, 2011). The present study was conducted in north-eastern Punjab comprising of Sub-mountains undulating zone (SMZ) and Undulating plain zone (UZ) lying in the foothill areas of the Zaskar Range, the Pir Panjal Range,
and part of the Siwalik Range. To study the status of molecular prevalence of the concurrent infection of T. equi and T. evansi, the expected prevalence was considered 10% with confidence limits of 95% and a desired absolute precision of 5% to collect maximum number of samples (Thrusfield, 2005). The number of samples thus calculated was adjusted for finite population and was correlated with 108 samples (99 horses and 9 donkeys/mules) that were collected for 36 discrete premises of four districts of Punjab (Table 2). Blood (~3 ml) was drawn from jugular vein of each animal into anticoagulant-coated vacutainers for microscopy and nucleic acid extraction. The information related with sampled animal (species, age, sex, health status and purchase history, if any) and to the farm (management, and presence of vectors and other in contact domestic ruminants) was collected from the owner on pre-designed questionnaire. The equine keepers following improper managemental practices of rearing their stock viz. stables with kacha floor, poor sanitation and unbalanced feeding program were classified as ‘unorganised farms’ while those pursuing appropriate scientific managemental schedule were considered ‘organised farms’. The animals were thoroughly screened for the presence of ticks especially in area around inner flap of ears, brisket, groin and dock regions. 2.3 Blood film Two thin blood films of each blood sample were prepared, dried and fixed with absolute methyl alcohol for 1-2 min. These blood smears were immersed for 30-45 min into diluted Giemsa stain, and thereafter washed with distilled water to remove excess of stain. The slides were air dried and examined under oil immersion lens (Coles, 1986) for the presence of any haemoprotozoan parasite (T. evansi and T. equi). 2.4 Micro Aerophilus stationary phase technique (MASP) for Theileria equi The laboratory at ICAR-National Research Centre on Equines, Hisar, India has developed the facilities for in-vitro propagation of T. equi by MASP technique (Avarzed et al., 1998; Kumar et al, 2013) and T. equi positive cultured red blood cells were collected and processed for DNA extraction. This DNA was used in the present study as a reference T. equi positive DNA template in PCR reaction. The detailed description of MASP technique for T. equi in vitro cultivation has been described elsewhere (Kumar et al., 2013). 2.5 Culturing for Trypanosome evansi
Blood was collected from a T. evansi clinically infected equid and transfused intraperitonially to Swiss albino mice. Mice were sacrificed when parasitaemia was maximum (107 to 109 parasites ml-1) and T. evansi parasites were purified from the collected blood by adsorbing it on to DEAE (Diethyl amino ethane cellulose) columns chromatography (Lanham and Godfrey, 1970; Bal et al., 2012). DNA was extracted from purified T. evansi parasites and was used as reference positive control in this study. 2.6 Oligonucleotide Primers for Multiplex PCR The details of oligonucleotide primers used for establishing multiplex PCR are mentioned in Table 1. 2.7 DNA Extraction and Multiplex PCR Amplification Genomic DNA was extracted from the equine blood samples as per the protocol of HiPuraTM Blood Genomic DNA Miniprep Purification Spin Kit. A total of 25 µl PCR reaction mixture was prepared which contained 12.5 µl of master ready mix (1X containing KAPA2G Fast HotStart DNA polymerase, KAPA 2G Fast HotStart PCR buffer, 0.2mM dNTP each, and 1.5mM MgCl2); 1.5 µl of each 10 pmol BeqF1/ BeqR1 primers (T. equi specific), 1.25 µl of each 10 pmol TR3/TR4 primers (T.evansi specific), additional 1mM MgCl2 suspended in nuclease free water and 5 µl DNA template of field equid samples. Positive control reaction was set up by taking, 2.5 µl each of T. evansi and T. equi refrence DNA as template instead of field sample DNA. Negative control reaction was also put with DNA obtained from a day old horse foal. The notemplate-control reaction contained 5µl of nuclease free water in place of DNA template. The PCR suspension was pre-heated for 5 min at 94 °C to activate the Taq polymerase, and 32 cycles were repeated - denaturation for 30 s at 94 °C, annealing for 30 s at 57 °C, extension for 1 min at 72°C; and a final extension for 10 min at 72 °C. The amplified DNA samples were electrophoresed on 1.5 % agarose gel, stained with ethidium bromide and visualized under ultraviolet light using gel documentation instrument (UVP Instruments, USA) for observation of 257 bp and 709 bp PCR products in the samples. 2.8
Nucleotide sequence analysis Amplicon of the PCR products were custom sequenced from Xcelris Genomics,
Ahmedabad, India. The nucleotide sequences were subjected to blast analysis (Altschul et al.,
1990) for determining the similarity with the sequence present in the nucleotide database. Phylogenetic analysis was conducted on MEGA4 (Tamura et al., 2007). The evolutionary history (Fig. 2, 3) of haemoparasite was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and was expressed in the units of the number of base substitutions per site. The bootstrap consensus tree inferred from 500 replicates (Felsenstein, 1985) was taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) was shown next to the branches (Felsenstein, 1985). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. 2.9 Statistical analysis The prevalence of haemoparasites with respect to various physical and biological factors was statistically analyzed employing Pearson’s chi-square test at P≤0.05. Analysis of risk factors and agreement between two techniques was assessed on WinEpiscope software V 0.1. The sensitivity and specificity of PCR assay with respect to blood film [gold standard] (Muieed et al., 2010, Ibrahim et al., 2011, Alanazi et al., 2012) were calculated as follows: Sensitivity of PCR with respect to blood film =
Specificity of PCR with respect to blood film =
3.
Results PCR amplification of individual DNA sample (T. evansi and T. equi) using their specific
primers produced expected fragments of size 257 and 709 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. In the phylogenetic analysis, there were a total of 209 positions in the
final dataset in case of T. evansi and 642 positions for T. equi. The amplification product for T. equi (accession number LC008132) obtained in the present study using BeqF1/BeqR1 primers showed significant difference from T. equi obtained from D. nuttalli in Mongolia (JQ657703.1) (P=0.009) in Codon-based Z test. It fell in the same node with isolated of T. equi/B. equi from Brazil, Spain and South Africa, while was quite distant from other species of Theileria (Fig. 2). The amplicon obtained in the present study for T. evansi (accession number LC008133) by TR3/TR4 primers showed significant difference from T. evansi strain RoTot1.2 from Palestine (EU931247.1) (P=0.050) and T. evansi Strain C6 of Camel from Israel (HM209054.1). The sequence showed closest homology with the local isolate obtained from cattle [AB979445] (Fig. 3). Parasitologically, only one sample (4.54%) from district SBS Nagar was found positive in blood film for T. equi. Molecular diagnosis by multiplex PCR revealed highest prevalence in district Mohali for T. equi (6.45%) and in SBS Nagar for T. evansi (4.45%), while no case of concurrent infection was reported from the two zones under study (Fig. 1). Statistically, there was no difference in the prevalence of these two infections among various districts (P<0.05) understudy. The overall prevalence of T. evansi and T. equi was 3.7% and 1.85%, respectively as diagnosed in multiplex PCR assay (Table 2) with no animal displaying clinical signs of infection. The pattern of prevalence was found decreasing southwards for T. evansi and vice-versa for T. equi (Fig. 1). Various risk factors considered in this study (Table 3) had distinct Odds ratios. The farms with improper managemental practices (stables with kacha floor, poor sanitation and unbalanced feeding program) displayed higher risk of latent infection of T. equi (OR=6.75, 95% CI=0.58175.38) and similar was the trend in the solo infection of T. evansi (OR=2.11, 95% CI=0.0580.36) (Table 3). The risk associated with the type of host species appeared to have marked effect on the prevalence of T. evansi infection with OR=12.35(95% CI=0.29-508.37), but due to the small sample size of the donkeys/mules population, their susceptibility to T. evansi infection remains inconclusive. This group was also at higher risk of infection with OR of 4 (95% CI=0.14-53.99). There was fair agreement between the blood film examination technique and PCR assay for the detection of T. equi (Kappa=0.39) whereas there was no agreement between these techniques for the detection of T. evansi (Kappa=0).
4.
Discussion PCR based diagnosis showed the overall 1.85% and 3.70% prevalence of T. evansi and T.
equi respectively, while only 0.93% T. equi prevalence by blood film was recorded. These results justify the greater sensitivity of multiplex PCR in detecting the latent infection of both the haemoprotozoans. In T. evansi, cryptic infections and antigenic variations are usually noticed which may lead to immunosuppression and non-detectable parasitaemia (Singla et al., 2010). Trend in geographic distribution and epidemiological findings of theileiriosis can be corroborated with the higher preponderance of tick vector, H. a. anatolicum (Kumar et al., 2007). The molecular prevalence of T. evansi was lower than that of T. equi because there is inconsistent occurrence of the tabanid flies as compared to the ticks. The incidence of coinfection of both protozoa was also lesser which may be due to different vectors (tick and hematophagus flies) responsible for their transmission. Due to the breech in management practices (no proper deworming and vaccination schedule, kacha flooring with cracks and crevices, no use of mesh, etc), unorganized farms had high risk of infection (OR=6.75, 2.11) for both the parasites as the chances of direct contact with vectors in these farms is higher (Moretti et al., 2010; Sumbria et al., 2014). Higher occurrence of infection in female equids in comparison to their male counterparts may be due to high preference of equine keepers due to their utilization for both draught and breeding (Moretti et al., 2010; Sumbria et al., 2014). As donkeys/mules were mainly kept in open yard under poor managemental conditions, hence they are at more risk with respect to vector (OR=4, 12.35) resulting in haemoparastic infection. Moreover, due to the small sample size of donkeys/mules, the higher prevalence of infection in donkeys/mules compared to Thoroughbred horses need more investigations on larger sample size (Kouam et al., 2010; Dos Santos et al., 2011). In this study a marked difference in infection rate of equids less than 2 year of age as compared to adults was observed. Maternal antibodies provide passive immunity to the young ones till the age of 3-6 months (Kumar et al., 2008), but as this immunity diminishes from 6 months to 2 years of age, their chances of encountering the infection increases (OR=3, 0.33) due to exposure of the animals to sports and/or to pull carts. Moreover, as sexual life cycle of T. equi completes in tick vector, ticks maintain cyclicity of parasite which helps in continuous spread of infection to natural host. Equids kept with domestic ruminants showed high prevalence of infection (OR=1.6, 1.58). Similar results were documented by Garcia-Bocanegra et al. (2013). As multiplex PCR
has 100% sensitivity and 95.32% specificity, so it was able to detect both the parasites irrespective of whether the amplification was against single or dual haemoparasites and with low level of infections, thus it can be suitable for epidemiological study of T. evansi and T. equi infection in equids. Acknowledgements Thanks are due to the Director of Research, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana for the facilities provided to carry out the research work. The funds for conducting research work were utilized from University Grant Commission (UGC) project entitled “Development of control strategies based on molecular epidemiology and drug efficacy for equine piroplasmosis in Punjab” (C-VPS-UGC-24). Thanks are also due to Dr. Sumedha Bhandari (Assistant Professor English, Department of Agricultural Journalism, Languages & Culture Punjab Agricultural University, Ludhiana) for editing the manuscript for the correct use of the English language.
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Legends to figures and Tables
Fig. 1: Relative prevalence of T. equi, T. evansi and their co-infection in equines among the districts of Sub-mountain undulating and Undulating plain zone of Punjab as diagnosed by multiplex PCR.
Fig. 2: Phylogenetic tree of Theileria equi. This tree was constructed by Neighbor-Joining method with MEGA 4 program. Numbers shown at branch nodes indicate bootstrap values. This
phylogenetic analysis showed high genetic diversity of 18S rRNA gene among different strains of T. equi. Consensus sequences obtained in this study is indicated as ‘‘PresentStudy”.
Fig. 3: Phylogenetic tree of Trypanosoma evansi. This tree was constructed by Neighbor-Joining method with MEGA 4 program. Numbers shown at branch nodes indicate bootstrap values. This phylogenetic analysis showed high genetic diversity of repetitive nucleotide of T. evansi gene among different strains of T. evansi. Consensus sequences obtained in this study is indicated as ‘PresentStudy”.
Table 1: Oligonucleotide Primers for multiplex PCR.
Table 2: Prevalence of T. evansi and T. equi in various districts and villages of Punjab by multiplex PCR.
Table 3: Assessment of risk factors associated with the concurrent infection of T. equi and T. evansi in Punjab.
Table 1: Oligonucleotide Primers for multiplex-PCR
Haemoprotozoan
Primer
Region amplified
Product size
T. evansi (Berlin et al ., TR3: 5’ GCG CGG ATT CTT TGC AGA Repetitive 257 bp CGA 3’ 2010 ) nucleotide TR4: 5’ TGC AGA CAC TGG AAT GTT sequences (VSG) ACT 3’ of T. evansi
T. equi (Rampersad et BeqF1: 5’ TCG TTG ACT GCG CTT
GGC G 3’ BeqR1:5’CTA AGA AGC GGA AAT GAA A 3’
al., 2003)
18S rRNA of T.
709 bp
equi
Table 2: Prevalence of T. evansi and T. equi in various districts and villages of Punjab by multiplex PCR.
Zone
Districts
Villages
Submountain undulating zone
Hoshiarpur
Chak Mirpur Namoli Amroh Beh Nangal Tajpur Gobindsar Balakipur Behram Abhipur Dafferpur Siayu
Pathankot Undulating plain zone
SBS Nagar Mohali
Total
No of farms/ premises 3
Samples
T. equi (%)
T. evansi (%)
10
1 (10)
0 (0)
3 4 5
9 9 10
0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
2 4 3 3 2 4 3 36
8 9 13 9 7 14 10 108
0 (0) 0 (0) 1 (7.69) 0 (0) 0 (0) 1 (7.14) 1 (10) 4 (3.70)
0 (0) 0 (0) 0 (0) 1 (11.11) 0 (0) 0 (0) 1 (10) 2 (1.85)
Microscopy
Animal
Ticks
Management
Sex
Age
Species
Zones
61
47
73
35
77
31
42
Male
Female
Organised
Unorganised
Ticks absent
Ticks present
Domestic ruminants present
Positive
66
80
More than 2 year
Domestic ruminants absent
28
Less than 2 year
99
Horses
9
53
Donkeys/mules
55
Undulating plain zone
Total
Sub-mountain undulating zone
Factors
1/3
2 (3.03)
2 (4.76)
2 (6.45)
2 (2.60)
3 (8.57)
1 (1.37)
3 (6.38)
1 (1.94)
2 (2.5)
2 (7.14)
1 (11.11)
3 (3.03)
3 (5.66)
1 (1.82)
Positive (%)
k= 0.39 (0.007-0.39)
1.6 (0.15-16.73 )
2.58 (0.24-27.34 )
6.75 (0.58-175.38)
4.09 (0.36-105.72)
3 (0.28-31.87)
4 (0.14-53.99)
3.24 (0.28-83.65)
Odds ratio (OR) 95% confidence interval (CI)
T. equi
0/2
1 (1.52)
1 (2.38)
0 (0)
2 (2.60)
1 (2.86)
1 (1.37)
2 (4.26)
0 (0)
2 (2.5)
0 (0)
1 (11.11)
1 (1.01)
2 (3.77)
0 (0)
Positive (%)
k =0
1.58 (0.04-59.96)
∞(0.09-∞)
2.11(0.05-80.36)
∞(0.31-∞)
0.33(0.03-3.53)
12.35 (0.29-508.37)
Odds ratio (OR) 95% confidence interval (CI) ∞ (0.25-∞)
T. evansi
Table 3: Assessment of risk factors associated with the concurrent infection of T. equi and T. evansi in Punjab
Negative
104/104
106/106
Figure 1 .
Figure 2 .
Figure 3 .
S0304-4017(15)00267-8 http://dx.doi.org/doi:10.1016/j.vetpar.2015.05.018 VETPAR 7641
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
31-12-2014 20-5-2015 22-5-2015
Please cite this article as: Sumbria, Deepak, Singla, L.D., Sharma, Amrita, Bal, M.S., Kumar, Sanjay, Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2015.05.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short communication
Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India Deepak Sumbriaa, L.D. Singlaa1, Amrita Sharmaa, M. S. Bala and Sanjay Kumarb a Department of Veterinary Parasitology, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004 b National Research Centre on Equines, Haryana, India. Highlights ► of the study
•
Multiplex PCR was standardized for the first time targeting haemoprotozoan in equines.
•
Standardized PCR was applied on equine samples.
•
Geographic trend of equine piroplasmosis in infection prone area.
•
Association of disease preponderance with various related risk factors. . Abstract
Multiplex PCR for simultaneous detection of Trypanosoma evansi and Theileria equi in singlestep reaction was optimized and employed on 108 equids (99 horses and 9 donkeys/mules) blood samples collected from two agro-climatic zones (Sub-mountain undulating zone and Undulating plain zone) of Punjab to evaluate the status of concurrent infection and associated risk factors. The amplification products of 257 and 709 bp targeting repetitive nucleotide sequence of variable surface glycoproteins of T. evansi and 18S rRNA gene of T. equi, respectively expressed high fidelity of the primer pairs with sequence homology to neighboring geographic isolates. The overall prevalence of T. evansi and T. equi was 3.7 and 1.85%, with Undulating plain zone at higher infection risk for T. equi (OR=3.24, 95% CI=0.28-83.65); and Sub-mountain undulating zone (OR=∞, 95% CI=0.25-∞) for T. evansi. Multiplex PCR revealed higher risk of infection of both T. equi (OR=6.75, 95% CI=0.58-175.38) and T. evansi (OR=2.11, 95% CI=0.05-80.36) in the farms with inappropriate management system. The risk factor associated with the type of host 1
Corresponding author:
Dr. L. D. Singla, Professor-cum-Head, Department of Veterinary Parasitology, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004. Email: [email protected], Contact no.: +91- 9316061974
species had an odds ratio of 12.35 (95% CI=0.29-508.37) for donkeys/mules versus horses for T. evansi infection. This group was also at higher risk of infection with Odds ratio (OR) of 4 (95% CI=0.14-53.99) for T. equi. The current investigation brings out various commodities at risk of infection pertaining to equid trypanosomosis and theileriosis evaluated by a rapid and sensitive multiplex PCR assay. Keywords: Trypanosoma evansi, Theileria equi, multiplex PCR, prevalence, risk factors, Punjab 1.
Introduction Trypanosomosis and theileiriosis are two economically important vector-borne diseases
of equids (horses, donkeys and mules) in tropical and subtropical parts of the world including India. In the Indian subcontinent trypanosomosis caused by Trypanosoma evansi [Kinetoplastid; haemoprotozoan] is known as ‘surra’, (a Hindi word meaning ‘rotten’) (Soulsby, 1982). Surra is characterized by a fluctuating parasitaemia with periods of paroxysms and intermissions, anemia, edema of the legs, dehydration, fever, abortion, and incoordination, followed by paralysis of the hind limbs (Gill, 1991). Theileiriosis, caused by an apicomplexan parasite (Theileria equi), is clinically characterised by fever, haemolytic anemia, icterus, petechial haemorrhages of the mucous membranes etc., but these are variable and are non-specific (Baptista et al., 2013). The disease renders negative effects on the health of equids, decreasing their productivity and work efficacy. T. equi infection lasts for 7-12 days, but can be peracute with death occurring in 1-2 days, or can be chronic, lasting for weeks. Mortality rate associated with this disease can vary for 10%-50% (Taylor et al., 2007). Imidocarb dipropionate @ 4mg/kg intra-muscularly on four occasions at 72 hrs intervals eliminates T. equi from horses (Berlin et al., 2010) but this dose is lethal to donkeys (Soulsby, 1982). A single dose of diminazene aceturate @ 3.5 mg/kg intramuscularly eliminates T. evansi from equids (Berlin et al., 2010). T. evansi can also be successfully managed with Quinapyramine sulphate and a chloride combination [Triquin] (Kumar et al., 2012). In northern India, T. evansi is transmitted mechanically by biting hematophagous flies (Tabanids) (Sumba et al., 1998) and T. equi is mainly transmitted by longistrate tick, Hyalomma anatolicum anatolicum, causing significant morbidity and mortality in equids (Kumar et al., 2007). As the vectors of both the diseases are habitual to hot plain areas of south-western Punjab, the epidemiological status of these infections in north-eastern parts of the province still remains unexplored.
T. evansi and T. equi are routinely diagnosed by conventional parasitological techniques (stained blood smears), serological (CATT/T. evansi, LATEX/T.evansi, IFAT, ELISA, etc) and molecular techniques. For the demonstration of parasite in blood, examination of stained thin blood smears is not a sensitive method, as the parasite has a periodically cryptic nature. Serological methods proffer the limitation of cross reactivity and failure to differentiate between past and current infection. For the diagnosis of latent infection, molecular techniques (PCR) give a promising result with greater levels of sensitivity and specificity (Bashir et al., 2014). Since both T. equi and T. evansi infections have almost similar types of signs and their vectors co-exist in suitable tropical climatic conditions, as in Punjab, it becomes necessary to optimize an economical, specific, and sensitive technique for simultaneous detection of these two diseases in suspected equines. Further, to evade the expenses on epidemiology of two important haemoparasites in comparatively quiescent zone, their simultaneous detection in a single reaction by multiplex PCR will be both time and cost effective. Hence, the purpose of the present study was to employ multiplex PCR assay for one-step detection of clinical, and latent forms of T. evansi and T. equi infections, for assessing the prevalence of these infections in a Sub-mountain undulating zone and Undulating plain zone of Punjab state and to further evaluate the risk factors contributing to the prevalence of these infections in equids for their proper control strategies. 2.
Materials and Methods
2.1 Ethical aspects The study has the approval (IAEC/2014/46-73) of the ethics committee for animal experiments duly constituted by the Guru Angad Dev Veterinary and Animal Sciences University. Blood samples were collected in a humanly manner, so as to avoid any accidental injury to the equids. A prior consent of the equines keepers was also sought. 2.2 Study area and sampling The province of Punjab covers a total area of 50,362 square kilometres between 29"30’N to 32"32’N latitude and 73”55’E to 76”50’E longitudes. In Punjab state lie areas between altitudes
213
meters
and
959
meters
above
sea
level
(http://punjabjudiciary.gov.in/index.php?trs=gurdist). There are about 34,000 equids which are at risk of infection due to haemoparasite in Punjab (Fazili and Kirmani, 2011). The present study was conducted in north-eastern Punjab comprising of Sub-mountains undulating zone (SMZ) and Undulating plain zone (UZ) lying in the foothill areas of the Zaskar Range, the Pir Panjal Range,
and part of the Siwalik Range. To study the status of molecular prevalence of the concurrent infection of T. equi and T. evansi, the expected prevalence was considered 10% with confidence limits of 95% and a desired absolute precision of 5% to collect maximum number of samples (Thrusfield, 2005). The number of samples thus calculated was adjusted for finite population and was correlated with 108 samples (99 horses and 9 donkeys/mules) that were collected for 36 discrete premises of four districts of Punjab (Table 2). Blood (~3 ml) was drawn from jugular vein of each animal into anticoagulant-coated vacutainers for microscopy and nucleic acid extraction. The information related with sampled animal (species, age, sex, health status and purchase history, if any) and to the farm (management, and presence of vectors and other in contact domestic ruminants) was collected from the owner on pre-designed questionnaire. The equine keepers following improper managemental practices of rearing their stock viz. stables with kacha floor, poor sanitation and unbalanced feeding program were classified as ‘unorganised farms’ while those pursuing appropriate scientific managemental schedule were considered ‘organised farms’. The animals were thoroughly screened for the presence of ticks especially in area around inner flap of ears, brisket, groin and dock regions. 2.3 Blood film Two thin blood films of each blood sample were prepared, dried and fixed with absolute methyl alcohol for 1-2 min. These blood smears were immersed for 30-45 min into diluted Giemsa stain, and thereafter washed with distilled water to remove excess of stain. The slides were air dried and examined under oil immersion lens (Coles, 1986) for the presence of any haemoprotozoan parasite (T. evansi and T. equi). 2.4 Micro Aerophilus stationary phase technique (MASP) for Theileria equi The laboratory at ICAR-National Research Centre on Equines, Hisar, India has developed the facilities for in-vitro propagation of T. equi by MASP technique (Avarzed et al., 1998; Kumar et al, 2013) and T. equi positive cultured red blood cells were collected and processed for DNA extraction. This DNA was used in the present study as a reference T. equi positive DNA template in PCR reaction. The detailed description of MASP technique for T. equi in vitro cultivation has been described elsewhere (Kumar et al., 2013). 2.5 Culturing for Trypanosome evansi
Blood was collected from a T. evansi clinically infected equid and transfused intraperitonially to Swiss albino mice. Mice were sacrificed when parasitaemia was maximum (107 to 109 parasites ml-1) and T. evansi parasites were purified from the collected blood by adsorbing it on to DEAE (Diethyl amino ethane cellulose) columns chromatography (Lanham and Godfrey, 1970; Bal et al., 2012). DNA was extracted from purified T. evansi parasites and was used as reference positive control in this study. 2.6 Oligonucleotide Primers for Multiplex PCR The details of oligonucleotide primers used for establishing multiplex PCR are mentioned in Table 1. 2.7 DNA Extraction and Multiplex PCR Amplification Genomic DNA was extracted from the equine blood samples as per the protocol of HiPuraTM Blood Genomic DNA Miniprep Purification Spin Kit. A total of 25 µl PCR reaction mixture was prepared which contained 12.5 µl of master ready mix (1X containing KAPA2G Fast HotStart DNA polymerase, KAPA 2G Fast HotStart PCR buffer, 0.2mM dNTP each, and 1.5mM MgCl2); 1.5 µl of each 10 pmol BeqF1/ BeqR1 primers (T. equi specific), 1.25 µl of each 10 pmol TR3/TR4 primers (T.evansi specific), additional 1mM MgCl2 suspended in nuclease free water and 5 µl DNA template of field equid samples. Positive control reaction was set up by taking, 2.5 µl each of T. evansi and T. equi refrence DNA as template instead of field sample DNA. Negative control reaction was also put with DNA obtained from a day old horse foal. The notemplate-control reaction contained 5µl of nuclease free water in place of DNA template. The PCR suspension was pre-heated for 5 min at 94 °C to activate the Taq polymerase, and 32 cycles were repeated - denaturation for 30 s at 94 °C, annealing for 30 s at 57 °C, extension for 1 min at 72°C; and a final extension for 10 min at 72 °C. The amplified DNA samples were electrophoresed on 1.5 % agarose gel, stained with ethidium bromide and visualized under ultraviolet light using gel documentation instrument (UVP Instruments, USA) for observation of 257 bp and 709 bp PCR products in the samples. 2.8
Nucleotide sequence analysis Amplicon of the PCR products were custom sequenced from Xcelris Genomics,
Ahmedabad, India. The nucleotide sequences were subjected to blast analysis (Altschul et al.,
1990) for determining the similarity with the sequence present in the nucleotide database. Phylogenetic analysis was conducted on MEGA4 (Tamura et al., 2007). The evolutionary history (Fig. 2, 3) of haemoparasite was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and was expressed in the units of the number of base substitutions per site. The bootstrap consensus tree inferred from 500 replicates (Felsenstein, 1985) was taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) was shown next to the branches (Felsenstein, 1985). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. 2.9 Statistical analysis The prevalence of haemoparasites with respect to various physical and biological factors was statistically analyzed employing Pearson’s chi-square test at P≤0.05. Analysis of risk factors and agreement between two techniques was assessed on WinEpiscope software V 0.1. The sensitivity and specificity of PCR assay with respect to blood film [gold standard] (Muieed et al., 2010, Ibrahim et al., 2011, Alanazi et al., 2012) were calculated as follows: Sensitivity of PCR with respect to blood film =
Specificity of PCR with respect to blood film =
3.
Results PCR amplification of individual DNA sample (T. evansi and T. equi) using their specific
primers produced expected fragments of size 257 and 709 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. In the phylogenetic analysis, there were a total of 209 positions in the
final dataset in case of T. evansi and 642 positions for T. equi. The amplification product for T. equi (accession number LC008132) obtained in the present study using BeqF1/BeqR1 primers showed significant difference from T. equi obtained from D. nuttalli in Mongolia (JQ657703.1) (P=0.009) in Codon-based Z test. It fell in the same node with isolated of T. equi/B. equi from Brazil, Spain and South Africa, while was quite distant from other species of Theileria (Fig. 2). The amplicon obtained in the present study for T. evansi (accession number LC008133) by TR3/TR4 primers showed significant difference from T. evansi strain RoTot1.2 from Palestine (EU931247.1) (P=0.050) and T. evansi Strain C6 of Camel from Israel (HM209054.1). The sequence showed closest homology with the local isolate obtained from cattle [AB979445] (Fig. 3). Parasitologically, only one sample (4.54%) from district SBS Nagar was found positive in blood film for T. equi. Molecular diagnosis by multiplex PCR revealed highest prevalence in district Mohali for T. equi (6.45%) and in SBS Nagar for T. evansi (4.45%), while no case of concurrent infection was reported from the two zones under study (Fig. 1). Statistically, there was no difference in the prevalence of these two infections among various districts (P<0.05) understudy. The overall prevalence of T. evansi and T. equi was 3.7% and 1.85%, respectively as diagnosed in multiplex PCR assay (Table 2) with no animal displaying clinical signs of infection. The pattern of prevalence was found decreasing southwards for T. evansi and vice-versa for T. equi (Fig. 1). Various risk factors considered in this study (Table 3) had distinct Odds ratios. The farms with improper managemental practices (stables with kacha floor, poor sanitation and unbalanced feeding program) displayed higher risk of latent infection of T. equi (OR=6.75, 95% CI=0.58175.38) and similar was the trend in the solo infection of T. evansi (OR=2.11, 95% CI=0.0580.36) (Table 3). The risk associated with the type of host species appeared to have marked effect on the prevalence of T. evansi infection with OR=12.35(95% CI=0.29-508.37), but due to the small sample size of the donkeys/mules population, their susceptibility to T. evansi infection remains inconclusive. This group was also at higher risk of infection with OR of 4 (95% CI=0.14-53.99). There was fair agreement between the blood film examination technique and PCR assay for the detection of T. equi (Kappa=0.39) whereas there was no agreement between these techniques for the detection of T. evansi (Kappa=0).
4.
Discussion PCR based diagnosis showed the overall 1.85% and 3.70% prevalence of T. evansi and T.
equi respectively, while only 0.93% T. equi prevalence by blood film was recorded. These results justify the greater sensitivity of multiplex PCR in detecting the latent infection of both the haemoprotozoans. In T. evansi, cryptic infections and antigenic variations are usually noticed which may lead to immunosuppression and non-detectable parasitaemia (Singla et al., 2010). Trend in geographic distribution and epidemiological findings of theileiriosis can be corroborated with the higher preponderance of tick vector, H. a. anatolicum (Kumar et al., 2007). The molecular prevalence of T. evansi was lower than that of T. equi because there is inconsistent occurrence of the tabanid flies as compared to the ticks. The incidence of coinfection of both protozoa was also lesser which may be due to different vectors (tick and hematophagus flies) responsible for their transmission. Due to the breech in management practices (no proper deworming and vaccination schedule, kacha flooring with cracks and crevices, no use of mesh, etc), unorganized farms had high risk of infection (OR=6.75, 2.11) for both the parasites as the chances of direct contact with vectors in these farms is higher (Moretti et al., 2010; Sumbria et al., 2014). Higher occurrence of infection in female equids in comparison to their male counterparts may be due to high preference of equine keepers due to their utilization for both draught and breeding (Moretti et al., 2010; Sumbria et al., 2014). As donkeys/mules were mainly kept in open yard under poor managemental conditions, hence they are at more risk with respect to vector (OR=4, 12.35) resulting in haemoparastic infection. Moreover, due to the small sample size of donkeys/mules, the higher prevalence of infection in donkeys/mules compared to Thoroughbred horses need more investigations on larger sample size (Kouam et al., 2010; Dos Santos et al., 2011). In this study a marked difference in infection rate of equids less than 2 year of age as compared to adults was observed. Maternal antibodies provide passive immunity to the young ones till the age of 3-6 months (Kumar et al., 2008), but as this immunity diminishes from 6 months to 2 years of age, their chances of encountering the infection increases (OR=3, 0.33) due to exposure of the animals to sports and/or to pull carts. Moreover, as sexual life cycle of T. equi completes in tick vector, ticks maintain cyclicity of parasite which helps in continuous spread of infection to natural host. Equids kept with domestic ruminants showed high prevalence of infection (OR=1.6, 1.58). Similar results were documented by Garcia-Bocanegra et al. (2013). As multiplex PCR
has 100% sensitivity and 95.32% specificity, so it was able to detect both the parasites irrespective of whether the amplification was against single or dual haemoparasites and with low level of infections, thus it can be suitable for epidemiological study of T. evansi and T. equi infection in equids. Acknowledgements Thanks are due to the Director of Research, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana for the facilities provided to carry out the research work. The funds for conducting research work were utilized from University Grant Commission (UGC) project entitled “Development of control strategies based on molecular epidemiology and drug efficacy for equine piroplasmosis in Punjab” (C-VPS-UGC-24). Thanks are also due to Dr. Sumedha Bhandari (Assistant Professor English, Department of Agricultural Journalism, Languages & Culture Punjab Agricultural University, Ludhiana) for editing the manuscript for the correct use of the English language.
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Soulsby, E.T.L., 1982. Helminthes, Arthropods, and Protozoa of Domesticated Animals.7th; ed. Bailliere and Tindal, London. Sumba, A.L., Mihok, S., Oyieke, F.A, 1998. Mechanical transmission of Trypanosoma evansi and T. congolense by Stomoxy sniger and S. taeniatus in a laboratory mouse model. Med. Vet. Entomol. 12, 417-422. Sumbria, D., Singla, L.D., Sharma, A., Moudgil, A.D., Bal, M.S., 2014. Equine trypanosomosis in central and western Punjab: Prevalence, haemato-biochemical response and associated risk factors. Acta Trop. 138, 44-50. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596-1599. Tamura, K., Nei, M., Kumar, S., 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Nat. Acad. Sci. USA 101, 11030-11035. Taylor, M.A., Coop, R.L., Wall, R.L., 2007. Veterinary Parasitology, third edition, Blackwell publication, 297pp. Thrusfield, M., 2005. Veterinary Epidemiology, second edition. Blackwell Science Ltd United Kingdom.
Legends to figures and Tables
Fig. 1: Relative prevalence of T. equi, T. evansi and their co-infection in equines among the districts of Sub-mountain undulating and Undulating plain zone of Punjab as diagnosed by multiplex PCR.
Fig. 2: Phylogenetic tree of Theileria equi. This tree was constructed by Neighbor-Joining method with MEGA 4 program. Numbers shown at branch nodes indicate bootstrap values. This
phylogenetic analysis showed high genetic diversity of 18S rRNA gene among different strains of T. equi. Consensus sequences obtained in this study is indicated as ‘‘PresentStudy”.
Fig. 3: Phylogenetic tree of Trypanosoma evansi. This tree was constructed by Neighbor-Joining method with MEGA 4 program. Numbers shown at branch nodes indicate bootstrap values. This phylogenetic analysis showed high genetic diversity of repetitive nucleotide of T. evansi gene among different strains of T. evansi. Consensus sequences obtained in this study is indicated as ‘PresentStudy”.
Table 1: Oligonucleotide Primers for multiplex PCR.
Table 2: Prevalence of T. evansi and T. equi in various districts and villages of Punjab by multiplex PCR.
Table 3: Assessment of risk factors associated with the concurrent infection of T. equi and T. evansi in Punjab.
Table 1: Oligonucleotide Primers for multiplex-PCR
Haemoprotozoan
Primer
Region amplified
Product size
T. evansi (Berlin et al ., TR3: 5’ GCG CGG ATT CTT TGC AGA Repetitive 257 bp CGA 3’ 2010 ) nucleotide TR4: 5’ TGC AGA CAC TGG AAT GTT sequences (VSG) ACT 3’ of T. evansi
T. equi (Rampersad et BeqF1: 5’ TCG TTG ACT GCG CTT
GGC G 3’ BeqR1:5’CTA AGA AGC GGA AAT GAA A 3’
al., 2003)
18S rRNA of T.
709 bp
equi
Table 2: Prevalence of T. evansi and T. equi in various districts and villages of Punjab by multiplex PCR.
Zone
Districts
Villages
Submountain undulating zone
Hoshiarpur
Chak Mirpur Namoli Amroh Beh Nangal Tajpur Gobindsar Balakipur Behram Abhipur Dafferpur Siayu
Pathankot Undulating plain zone
SBS Nagar Mohali
Total
No of farms/ premises 3
Samples
T. equi (%)
T. evansi (%)
10
1 (10)
0 (0)
3 4 5
9 9 10
0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0)
2 4 3 3 2 4 3 36
8 9 13 9 7 14 10 108
0 (0) 0 (0) 1 (7.69) 0 (0) 0 (0) 1 (7.14) 1 (10) 4 (3.70)
0 (0) 0 (0) 0 (0) 1 (11.11) 0 (0) 0 (0) 1 (10) 2 (1.85)
Microscopy
Animal
Ticks
Management
Sex
Age
Species
Zones
61
47
73
35
77
31
42
Male
Female
Organised
Unorganised
Ticks absent
Ticks present
Domestic ruminants present
Positive
66
80
More than 2 year
Domestic ruminants absent
28
Less than 2 year
99
Horses
9
53
Donkeys/mules
55
Undulating plain zone
Total
Sub-mountain undulating zone
Factors
1/3
2 (3.03)
2 (4.76)
2 (6.45)
2 (2.60)
3 (8.57)
1 (1.37)
3 (6.38)
1 (1.94)
2 (2.5)
2 (7.14)
1 (11.11)
3 (3.03)
3 (5.66)
1 (1.82)
Positive (%)
k= 0.39 (0.007-0.39)
1.6 (0.15-16.73 )
2.58 (0.24-27.34 )
6.75 (0.58-175.38)
4.09 (0.36-105.72)
3 (0.28-31.87)
4 (0.14-53.99)
3.24 (0.28-83.65)
Odds ratio (OR) 95% confidence interval (CI)
T. equi
0/2
1 (1.52)
1 (2.38)
0 (0)
2 (2.60)
1 (2.86)
1 (1.37)
2 (4.26)
0 (0)
2 (2.5)
0 (0)
1 (11.11)
1 (1.01)
2 (3.77)
0 (0)
Positive (%)
k =0
1.58 (0.04-59.96)
∞(0.09-∞)
2.11(0.05-80.36)
∞(0.31-∞)
0.33(0.03-3.53)
12.35 (0.29-508.37)
Odds ratio (OR) 95% confidence interval (CI) ∞ (0.25-∞)
T. evansi
Table 3: Assessment of risk factors associated with the concurrent infection of T. equi and T. evansi in Punjab
Negative
104/104
106/106
Figure 1 .
Figure 2 .
Figure 3 .