INTRODUCTION
Malaria has been one of the most devastating dis- eases described since antiquity whose references can be found in the Vedic documents of 1600 BC in India and in the descriptions of Hippocrates in ancient Greece some 2500 years ago1. It has become endemic and subject of concern in most part of the India. During 2014, about 1.1 million malaria cases (65.5% due to Plasmodium falciparum), with 0.05% deaths were recorded by the National Vector Borne Disease Control Programme2. According to the WHO world malaria report 2014, 22%
of Indian population lives in high transmission areas (≥ 1 case per 1000 population), 67% lives in low transmission areas (0–1 case per 1000 population) and rest of 11% lives in malaria-free areas. The incidence of malaria in India accounted for 58% of cases in the Southeast Asia Region3. The Northeastern states (NE states) namely
Arunachal Pradesh, Assam, Meghalaya, Manipur, Mizoram, Nagaland, Sikkim and Tripura together con- tribute about 4% of the country’s population and reported 1.3 lakh malaria cases during 2014. Assam, Meghalaya, Manipur and Sikkim are the most effected states accord- ing to WHO world malaria report 20074. These four states, together contribute 48.8% malaria cases of the NE states and about 6.7% P. falciparum cases. During 2010–14, the malaria incidence increased significantly in these states along with increase in percentages of P. falciparum cases.The earlier studies have reported Anopheles minimus, followed by An. dirus (in forests) and An.
fluviatilis as major malaria vectors in NE states5–12. In subsequent studies, the absence of An. minimus after ap- plication of DDT as residual insecticide has been reported from Burnihat area of Meghalaya13, Tirap district of Arunachal Pradesh14 and Terai region of Uttar Pradesh15. Our previous study on the impact of ecological and
Role of An. culicifacies as a vector of malaria in changing ecological scenario of Northeastern states of India
Nasreen Akhtar
1, BN Nagpal
1, Neera Kapoor
2, Aruna Srivastava
1& Neena Valecha
11National Institute of Malaria Research (ICMR); 2Indira Gandhi National Open University, New Delhi, India
ABSTRACT
Background & objectives: Malaria has become endemic and subject of concern in most part of the India especially Northeastern states of India. Surveys before 2000 revealed that Anopheles minimus was major vector responsible for transmission of malaria in this region followed by An. dirus and An. fluviatilis. However, recent studies indicate replacement of An. minimus vector by An. culicifacies due to different ecological changes and change in landuse pattern etc. The objective of present study was to explore the vectorial role of An. culicifacies in transmission of malaria in four malaria endemic states, viz. Assam, Meghalaya, Manipur and Sikkim of India.
Methods: The seven surveys were conducted in 176 selected villages belonging to eight districts of the four states in both pre-monsoon (March–April) and post-monsoon (September–October) seasons from 2010 to 2013. However, in 2011 surveys could not be carried out due to public inconvenience in pre-monsoon season. For vectorial role of all vector species collected, ELISA and PCR were assayed.
Results: A total of 19,173 specimens belonging to 30 anopheline species were collected, out of which 4315 belonged to four established vector species. In total, 4183 specimens were processed through ELISA, out of which 236 specimens were found positive for circumsporozoite (CS) protein. Further, infectivity was confirmed by PCR in 35 samples, of which 12 samples were found positive for Plasmodium falciparum and three for P.
vivax. Out of 12 Plasmodium falciparum positive samples, nine samples were positive for An. culicifacies, two for An. fluviatilis and one for An. minimus. While out of three Plasmodium vivax positive samples, two samples were positive for An. dirus and one sample was positive for An. culicifacies.
Interpretation & conclusion: Anopheles culicifacies replaced the An. minimus, the vector of malaria in Northeastern states of India, as it was found to be highly infected with malaria parasite as compared to An. minimus by ELISA and PCR analysis, and thus playing a major role in malaria transmission in this region. The ecological changes like deforestation, development of irrigation channels and change in landuse pattern, have helped in evolution of An. culicifacies in the study area. Therefore, modified vector control strategies are required on urgent basis.
Key words Anophelines; An. culicifacies; An. dirus; An. fluviatilis; An. minimus; ELISA; malaria; PCR
sulted in the change of anopheline composition and also in vector species16–17. These changes create favourable breeding sites for An. culicifacies.
Therefore, the objective of the present study was to explore the vectorial role of An. culicifacies in transmis- sion of malaria due to episodes of different ecological changes in malaria endemic regions of NE states of India.
MATERIAL & METHODS Study sites
In NE states, namely Assam, Meghalaya, Manipur and Sikkim, the districts and villages were selected on the basis of following criteria: (i) Change in landuse pat- tern using remote sensing classification of images, with difference of at least six years. For example in Assam state, Sonitpur district was selected for the years 1999 and 2008; Nagaon for 1999 and 2008; Lakhimpur for 1999, 2002 and 2009; Goalpara for 2002 and 2010. The rests of the districts were also selected by same technique;
and (ii) Availability of historical survey data (surveys car- ried out before 2000) on anophelines species.
A total of eight districts, viz. Sonitpur, Nagaon, Lakhimpur and Goalpara from Assam state, East Khasi Hill and East Garo Hill from Meghalaya, Chandel from Manipur and East district from Sikkim state were se- lected. During the study period, a total of 176 villages were covered during pre-monsoon (March–April) and post-monsoon (September–October) seasons which in- cluded 114 villages from Assam, 27 villages from Manipur, 25 villages from Meghalaya, and 10 villages from Sikkim (Table 1 and Fig. 1). During 2010–13, a total of seven surveys were conducted. In 2011, pre-mon- soon surveys could not be carried out due to local distur- bance in the selected states.
Mosquito collection and morphological identification Mosquitoes were collected with the help of an aspi- rator and torch light using WHO standard technique18 in pre-monsoon and post-monsoon seasons during the study period, in all selected districts. In both seasons, indoor resting mosquito collection, total catch (space spray col- lection), outdoor collection (using CDC light trap) and whole night collection were performed. The indoor rest- ing collections were made from human dwellings/
cattlesheds/mixed dwellings between 0500 and 0900 hrs
by two experienced Insect Collectors for 15 min. Adult mosquito resting on the walls, hanging cloths, and under cots/tables/chairs, etc. in houses were collected by suc- tion tube aided by torch light. In each village four fixed station and two randomly selected houses and cattlesheds were covered during each survey.
Outdoor morning collection was carried out in each village using hand catch method from boundary walls/
fencing, treeholes, bushes, etc. Outdoor night collection was carried out using one CDC light trap in each village where morning collections were done.
Morphological identification of wild caught adult mosquitoes was done by using the keys of Christophers19, Puri20, Nagpal and Sharma21 and Nagpal et al22, and the catalogue of Knight and Stone23. The collected vector spe- cies were further subjected to different molecular confir- mation techniques for the presence of malaria parasites.
Enzyme linked immunosorbent assay
Enzyme-linked immunosorbent assays (ELISAs) were carried out to detect P. falciparum, P. vivax-210, and P. vivax-247 circumsporozoite (CS) proteins in ma- laria-infected mosquitoes. The head and thorax of col- lected specimens of An. culicifacies, An. minimus, An.
dirus and An. fluviatilis were assayed for the presence of sporozoites by ELISA using monoclonal antibodies against P. falciparum and P. vivax CS proteins (P. vivax -210 and P. vivax- 247) with appropriate positive con- trols as per manufacturer’s protocol. A total of 4183 speci- mens were assayed through ELISA test.
Polymerase chain reaction
The method of amplification and sequencing of D3 domain of 28S rDNA were carried out as described by Singh et al24 and Johnstonet al25. The nested polymerase chain reaction (PCR) was performed with primers as de- scribed by Rubio et al26 with changes in the cycling pa-
Nagaon 33
Lakhimpur 30
Goalpara 14
2. Meghalaya East Khasi Hill 13
East Garo Hill 12
3. Manipur Chandel 27
4. Sikkim East Sikkim 10
Total 176
rameters given in Table 2 using VeritiTM 96 well thermal cycler (Applied Biosystems, MA, USA). Uninfected in- dividuals or male mosquito were used as controls in this assay.
About 1 μl supernatant, containing DNA of each vec- tor species, i.e. An. culicifacies, An. minimus, An. dirus and An. fluviatilis, was used for allele specific PCR (ASPCR) based species identification, and amplification of nuclear loci such as r-DNA-ITS2, and r-DNAD3.
A total of 35 samples of four vector species were pro- cessed through ELISA. The data were analysed through Excel program of MS Office and t-test (Chi-square test).
RESULTS
During the 2010–13 entomological surveys, a total of 19,173 specimens belonging to 30 anopheline species were collected from 176 villages of selected eight dis- tricts of Assam, Meghalaya, Manipur and Sikkim states.
Out of these 30 anopheline species collected, four were major vector species, viz. An. culicifacies, An. minimus, An. fluviatilis and An. dirus27. A total of 4315 specimens of these vector species were collected, which majorly com- prised An. culicifacies (92.9%) followed by An. mimimus (4.4%), An. fluviatilis (2%) and An. dirus (0.55%).
Table 2. The primers, annealing temperatures and number of cycles used for PCR amplifications of polymorphic segments of genes
Name of gene Sequence of primer Annealing temperature (°C) No. of cycles
PLF (Plasmodium—All species) 5'-AGTGTGTATCAATCGAGTTTCTG-3' 60 and 55 30
FAR (P. falciparum) 5'-GGAAGCGTATTAAAGCCAAAAGCTA-3' 60 and 55 30
VIR (P. vivax) 5'-AGGACTTCCAAGCCGAAGC-3' 60 and 55 30
Sequences taken from (Rubio et al26).
Fig. 1:Map of surveyed villages in Northeast of India (Red bullet indicate those villages where ecological changes occurred and black bullets are new villages surveyed).
positive for the presence of CS protein in in- fected mosquito. Out of 236 positive specimens, 227 specimens were of An. culicifacies, five specimens were of An. minimus and two speci- mens (each) were of An. dirus and An. fluviatilis.
The statewise results of ELISA are described below:
Assam: A total of 2048 specimens of vector species, were collected, comprising 1903 speci- mens of An. culicifacies, 109 specimens of An.
minimus, 25 specimens of An. fluviatilis and 11 specimens of An. dirus. Out of 2048 specimens, 143 specimens were found positive for CS in ELISA; consisting 138 specimens of An.
culicifacies, followed by An. minimus (3 speci- mens) and one specimen each of An. fluviatilis and An. dirus (Table 3).
Meghalaya: A total of 1564 specimens of vector species were collected and consisting 1497 specimens of An. culicifacies, 35 speci- mens of An. minimus, 29 specimens of An.
fluviatilis and three specimens of An. dirus. Out of 1564 specimens, 63 specimens were found positive for CS. From total ELISA positive specimens, 61 specimens were of An.
culicifacies, one specimen each of An. minimus and An. dirus, while An. fluviatilis was found negative for CS protein (Table 3).
Manipur: From this state, a total of 302 specimens of vector species were collected, out of which 273 specimens were of An.
culicifacies, 16 specimens of An. fluviatilis and 13 specimens were of An. minimus. The ELISA results showed that only 17 specimens were positive for CS protein among which 15 speci- mens belonged to An. culicifacies and one speci- men each to An. minimus and An. fluviatilis (Table 3).
Sikkim: A total of 269 specimens of two vec- tor species were collected in which 267 speci- mens were of An. culicifacies and two speci- mens were of An. fluviatilis. Only 13 specimens of An. culicifacies were found positive in ELISA result, while An. fluviatilis results were nega- tive (Table 3).
Table 3. Results of ELISA assay of vector mosquito in States of Assam, Meghalaya, Manipur and Sikkim Pre-monsoon season (March–April)Post-monsoon season (September–October) An. culicifaciesAn. minimusAn. fluviatilisAn. dirusAn. culicifaciesAn. minimusAn. fluviatilis StateYearsNo.ELISA+No.ELISA+No.ELISA+No.ELISA+No.ELISA+NoELISA+No.ELISA+ examinedexaminedexaminedsexaminedexaminedexaminedexaminedexamined Assam201276217020004451121150 20138989110106907770111 20141051920030104982135130 Total2702946160201633109632191 Meghalaya2012581201000280117090 2013942308010188168070 201417562010007022513130 Total32797010010117052281190 Manipur20121300020006711091 20131500000004112130 2014292001000108110010 Total57200300021613131131 Sikkim2012400000002504000 20132710010009533000 20149000100010793000 Total401002000227120000 Grand Total694415312103032461861044512
PCR amplification
The PCR was also performed for the confirmation of the ELISA results. The matched positivity in the mosqui- toes was included in the present study. The analysis of PCR products of 35 samples of the four vector species revealed that 26 samples were of An. culicifacies, five of An. minimus, and two of An. dirus and An. fluviatilis each.
Out of 35 samples, 15 samples were found positive by PCR, in which 12 samples were of P. falciparum con- taining ~205-bp band and three samples were of P. vivax containing ~120-bp band while rest of the samples were found negative with no parasite DNA. Out of 12 P.
falciparum positive samples, nine samples were positive for An. culicifacies, two for An. fluviatilis and one for An.
minimus. While out of three P. vivax positive samples, two samples were positive for An. dirus and one sample was positive for An. culicifacies (Figs. 2 a and b).
Human blood index
Anopheles culicifacies is a zoophilic species and pre- fer to rest in cattlesheds as compared to human dwell- ings. A total of 360 specimens of An. culicifacies were checked for human blood out of which only eight speci- mens were found positive, while only five specimens of An. minimus were analyzed and no specimen was found positive (Table 4).
DISCUSSION
In the recent years, NE states of India have under- gone severe ecological modifications, for example, re- duction in forest cover. The forest cover recorded in Assam was 1419 thousand hectare in 2011–12 compared to 2114 thousand hectare in 1974, a decrease by 32.87%16.
In Meghalaya, it was recorded 111 thousand hectare in 2003 as compared to 740 thousand hectare in 1973–74, thereafter, it gradually increased to 948 thousand hectare in 2008–0916. In Manipur, forest cover reduced to 1699 thousand hectare in 2013 as compared to 1741 thousand hectares in 1997, i.e. decrease by 2.46%. In Sikkim state, forest cover was reported to be 336 thousand hectare in 2013 as compared to 709 thousand hectare in 1988, i.e.
decrease by 52.66%16. In our previous study, it has been reported that due to these ecological changes, species composition of the region also changed, for example An.
ahomi, An. aitkenii, An. benglalensis, An. insulaeflorum, An. lindsayi and An. umbrosus collected in earlier sur- veys, were not recorded. Due to these ecological changes, replacement of malaria vector species occurred like An.
minimus was replaced by An. culicifacies. The sporozo- ite rate of all four vector species in selected NE state of India was estimated in the present study. During previ- ous studies, i.e. 1900–2000 and 2000–10, the sporozoite positivity of An. minimus was 4.06% (1742 specimens in 42,804 specimens) and 2.85% (5 specimens out of 105) respectively7–8,12,28–36 (Table 5), whereas the sporozoite positivity in present study was found to be 3.18% (5 speci-
Table 4. Human blood index (HBI) of An. culicifacies in NE states of India
S. No. Name of the No. of No. of state An. culicifacies positive
processed
1. Assam 150 3
2. Meghalaya 110 2
3. Manipur 50 1
4. Sikkim 50 2
Fig. 2:(a) Ethidium bromide-stained gel eletrophoresis of multiplex PCR products of the An. culicifacies species complex. Lane M–100-bp DNA ladder; a~205 bp band appears in sample 1–4, 6, 8–10 and 16 indicating P. falciparum positive samples; Lanes 5, 7, 11–15 and 17 are negative control with no DNA; and (b) a ~120 bp band appears in samples 5 indicating P. vivas positive sample; Lanes 1–4 and 6–8 are negative control with no DNA.
Another vector, viz. An. fluviatilis recorded in previ- ous studies was found in the current survey as well. One study in NE states by Singh et al37 has revealed that few subtypes of An. fluviatilis were misidentified as An.
minimus due to similarity in morphology of rDNA data of spacer 2 (ITS2) and D3 domain of 28S rDNA (28S-D3).
According to the previous studies, i.e. during 1900–2000, the percentage of sporozoite positivity was 3% (16 speci- mens out of 520 specimens)11 (Table 5), while in present study, the percent positivity of infection through ELISA was 2.7% (2 specimens out of 72 specimens) (Table 3).
In previous studies (before 2000), An. culicifacies played a supporting role in malaria transmission with An.
minimus, but gradually due to different ecological
study, the percentage of CS protein positivity was 5.76%
(227 specimens out of 3940 specimens) (Table 3). There- fore, in the present study the percentage infection of An.
culicifacies was high as compared to An. minimus and it may be concluded that An. culicifacies play a major role in malaria transmission with the support of An. minimus, An. dirus and An. fluviatilis.
The reducing trend of malaria in the NE region is evident from this study. The replacement of highly an- thropophilic species, i.e. An. minimus by a zoophilic spe- cies viz. An. culicifacies, might have helped in reducing the cases of malaria in recent years38–39. Therefore, there is an urgent need of planning the modified vector control strategies for these regions.
Table 5. Vector incrimination of An. minimus, An. dirus, An. fluviatilis and An. culicifacies in NE states of India
S. No. Locality Year References Number Number positive Percentage
dissected Gut Gland Total (%)
An. minimus
1. Assam 1929 Strickland28 1489 39 19 58 3.89
2. Assam 1930 Ramsay29 3847 59 27 86 2.23
3. Assam 1931 Ramsay30 1221 26 38 64 5.24
4. Assam 1932 Gupta et al31 1119 91 9 100 8.93
5. Assam 1933 Gupta et al32 934 41 12 53 5.67
6. Assam 1941 Anderson and Viswanathan33 14092 393 391 784 5.56
7. Assam 1941 Viswanathan et al7 5102 83 72 155 3.03
8. Assam 1941 Clark and Choudhury8 408 0 0 13 3.18
9. Assam 1942 Khan34 94 2 3 5 5.32
10. Assam 1984 Annual Report, NMEP 1256 0 45 45 3.58
11. Assam 1989 S & T Project Report, NIMR 3543 2 121 123 3.47
12. Assam 1990 S & T Project Report, NIMR 5019 3 148 151 3
13. Assam 1996 Dev V12 4680 0 155 155 3.31
14. Assam 2004 Dev V et al35 17 0 1 1 5.88
15. Meghalaya 2010 Dev V et al36 88 0 2 2 2.27
An. dirus
16. Digboi area (Assam) 1941 Clark and Choudhury8 859 8 21 29 3.37
17. Assam 1948 Macan9 92 – – 1 1.08
18. Boko (Assam) 1984 Annual Report, NMEP 199 0 1 1 0.5
19. Dibrugarh (Assam) 1989 Dutta et al10 74 0 1 1 1.35
An. fluviatilis
20. Assam 1923 Challam11 315 0 12 12 3.80
21. Sonapur (Assam) 1990 S & T Project Report, NIMR 205 0 4 4 1.95
An. culicifacies
22. Assam 1941 Anderson and Viswanathan33 1232 2 5 7 0.56
Source: Indian Anophelines (Nagpal & Sharma)21.
CONCLUSION
Due to change in vector ecology, the composition of two major vectors An. culicifacies and An. minimus were altered. Pair-wise comparison of An. culicifacies and An.
minimus using chi-square with Yates correction (χ2 = 11902.252; p<0.0001) was found statistically significant indicating that An. culicifacies is replacing An. minimus.
The study indicates that An. culicifacies is playing a ma- jor role in malaria transmission in selected NE states of India. Therefore, modified vector control strategies are required on urgent basis.
ACKNOWLEDGEMENTS
The authors are thankful to the Indian Council of Medical Research, New Delhi for providing funds for this study under NE Task Force and to the State Health De- partments for providing malaria epidemiological data of the districts. Mr. Kumar Vikram, Mr. Hari Om and Mr.
Arshad Shamim are acknowledged for help in mosquito collections.
Conflict of interest
The authors declare that they don’t have any conflict of interest.
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Correspondence to: Dr B.N. Nagpal, Scientist ‘G’, GIS, Taxonomy and OVBD Division, National Institute of Malaria Research, Dwarka, Sector–8, New Delhi–110 077, India.
E-mail: b_n_nagpal@hotmail.com
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