International Journal of Mosquito Research 2016; 3(2): 39-46 ISSN: 2348-5906 CODEN: IJMRK2 IJMR 2016; 3(2): 39-46 © 2016 IJMR Received: 06-01-2016 Accepted: 08-02-2016 BK Tyagi (a) Visiting Professor, Department of Environmental Biotechnology, School of Environmental Sciences, Bharathidasan University, Tiruchirapalli - 620 024 (TN), India. (b) Centre for Research in Medical Entomology (ICMR), 4, Sarojini Street, Chinna Chokkikulam, Madurai 625002 (TN), India. P. Philip Samuel Centre for Research in Medical Entomology (ICMR), 4, Sarojini Street, Chinna Chokkikulam, Madurai 625002 (TN), India. V. Thenmozhi Centre for Research in Medical Entomology (ICMR), 4, Sarojini Street, Chinna Chokkikulam, Madurai 625002 (TN), India. J. Nagaraj Centre for Research in Medical Entomology (ICMR), 4, Sarojini Street, Chinna Chokkikulam, Madurai 625002 (TN), India. D. Ramesh Centre for Research in Medical Entomology (ICMR), 4, Sarojini Street, Chinna Chokkikulam, Madurai 625002 (TN), India. S. Karthigai Selvi Centre for Research in Medical Entomology (ICMR), 4, Sarojini Street, Chinna Chokkikulam, Madurai 625002 (TN), India. A. Venkatesh Centre for Research in Medical Entomology (ICMR), 4, Sarojini Street, Chinna Chokkikulam, Madurai 625002 (TN), India. Correspondence BK Tyagi (a) Visiting Professor, Department of Environmental Biotechnology, School of Environmental Sciences, Bharathidasan University, Tiruchirapalli - 620 024 (TN), India. (b) Centre for Research in Medical Entomology (ICMR), 4, Sarojini Street, Chinna Chokkikulam, Madurai 625002 (TN), India. Determination of critical density and vectorial capacity for Culex tritaeniorhynchus Giles, 1901 (Diptera: Culicidae), the primary vector for Japanese encephalitis in southern India BK Tyagi, P Philip Samuel, V Thenmozhi, J Nagaraj, D Ramesh, S Karthigai Selvi and A Venkatesh Abstract Of all the kinds of mosquito-borne viral encephalitides, Japanese encephalitis (JE) is the most important viral infection as it causes irreversible neuropsychiatric sequelae in the paediatric population mostly below 15 years of age. The disease, caused by a flavivirus, the Japanese Encephalitis Virus (JEV), is transmitted by an array of more than a dozen species of mosquitoes, mainly belonging to Culex vishnui subgroup comprising Culex tritaeniorhynchus, Cx. pseudovishnui, and Cx. vishnui, all of these prefer breeding in flooded rice fields. The JE virus prevalent in most Asian countries including India, is highly endemic to southern states particularly Tamil Nadu. The JE infection has been endemic to Cuddalore district of Tamil Nadu for several decades in past. Although much information is available on biology and ecology of the principal vector, Culex tritaeniorhynchus, absolutely nothing is known as to its critical density (CD) and vectorial capacity (VC) which are the basic requirements to define transmission potential of the vector. The Centre for Research in Medical Entomology (CRME) has been vertically pursuing for past three decades the various facets of transmission dynamics of JE in Cuddalore district, and in course mined huge data on the transmission potential of the principal vector, Culex tritaeniorhynchus. This paper attempts to assimilate all the possible information with respect to ecology and biology of the vector, Culex tritaeniorhynchus and postulates that a critical density of 430 mosquitoes/human/day is necessary to effect an infective bite in order to transmit virus during the transmission season. We have accordingly calibrated the vectorial capacity of Culex tritaeniorhynchus which varied marginally from 0.01 in 2011 to 0.002 in 2012. Such estimates will be helpful not only to understand the biology of vector in great detail but also its potential to transmit the infection. Keywords: Culex tritaeniorhynchus, critical density, vectorial capacity, Cuddalore. Introduction The mosquito-borne Japanese encephalitis (JE) is one of the world’s most fatal as well as debilitating vectored diseases mostly prevalent in Southeast Asia and Pacific regions, where three billion people are at risk of infection, but has also recently invaded the northern Australia [1-2] . The Japanese encephalitis disease is caused due to infection with the JE virus (JEV), a mosquito-borne flavivirus. Approximately 20–30% of JE cases are fatal and 30–50% of survivors have significant neurologic sequelae [3]. JE is primarily a disease of children and most adults in endemic countries have natural immunity after childhood infection, but all age groups are affected. In most temperate areas of Asia, JEV is transmitted mainly during the warm season, when large epidemics can occur. In the tropics and subtropics, transmission can occur yearround but often intensifies during the rainy season. The disease is now considered a serious public health problem since it often tends to damage brain with increased case fatality rate (CFR) [4] . The main JEV transmission cycle involves Culex tritaeniorhynchus mosquitoes and similar species belonging to the ‘vishnui’ subgroup (e.g., Culex vishnui and Culex pseudovishnui) that lay eggs in rice paddies and other open water sources, with pigs and aquatic birds as principal vertebrate amplifying hosts [5]. JEV is maintained in an enzootic cycle between mosquitoes and amplifying vertebrate hosts, primarily pigs and wading ardeid birds. Humans are generally thought to be dead-end JEV hosts, i.e., they seldom develop enough viremia to infect feeding mosquitoes [6]. A fewer than 1% of human JEV infections result in JE. ~ 39 ~ International Journal of Mosquito Research vectorial capacity (VC) for Cx. tritaeniorhynchus necessary cues had been inculcated from different models used for malaria, West Nile Virus (WNV) and Western Equine Encephalomyelitis (WEE) [7-10]. First reported in Japan in 1924, Japanese encephalitis was subsequently reported in other Asian countries including India, where 17 States/Union Territories were affected with periodic outbreaks particularly in years 2005, 2008, 2011 and 2012, respectively. Due to multiplicity of vectors and the changing dynamics of disease transmission in the wake of dramatic shift in agricultural practices, human mobility and climate warming, one of the intriguing questions underlying transmission dynamics being brainstormed globally among public heath entomologists is about the ‘critical vector density’ or ‘vectorial capacity’ of the main vector, Cx. tritaeniorhynchus which has so far never been worked out. In this paper, therefore, based on the massive data available on varied biological parameters of the vector and disease transmission during a three decade old longitudinal study in the endemic district of Cuddalore, a de novo attempt has been made to determine critical density and vectorial capacity of Cx. tritaeniorhynchus. In the derivation of critical density (CD) and Materials and Methods (i) Study area: Japanese encephalitis has been highly endemic in Cuddalore district, (Area 3678 sq. km) situated in South Arcot, Tamil Nadu, India with its geographical coordinates of 11° 45' 0" North, and 79° 45' 0" East (Figure 1). The district (3564 sq. km; population 2.6 million) is located about 250 km south of Chennai and is copiously rich in paddy cultivation, contributing 6.65% of the total production in the State. During 1970s and 1980s, Cuddalore district had reported several outbreaks of Japanese encephalitis exacting many deaths and high morbidity [11-13]. Fig 1: Study area- South India, Cuddalore district, Tamil Nadu. Daily survival rate (P = Proportion of mosquito surviving one day) of Cx. tritaeniorhynchus was calculated by using the formula Parity rate, where ‘gc’ is the length of gonotrophic cycle [16]. (ii) Mosquito collection and estimation of infection rate: Mosquitoes were collected from three endemic study villages namely Kodikkalam, Eraiyur and SS Puram in Cuddalore district (Figure 1). Mosquitoes were collected between April, 2011 and December, 2012 for one hour during dusk hours using mechanical aspirators and torch lights, particularly in and around the cattle-sheds, pig sties and the surrounding bushes. Results from this exercise were compared with those of the previous study [14] in order to understand any change in biological characteristics of the mosquitoes. The virus infection rate in mosquitoes was expressed as minimum infection rate (MIR) per 1000 females tested [15]. (iii) The JE virus transmission: The life cycle of JEV is maintained between mosquitoes and zoonotic hosts like pigs, birds; humans are incidental or dead-end hosts since they do not develop required concentration for survival and replication of JE virus in their bloodstreams to infect feeding mosquitoes. As far as the mosquito-borne diseases are concerned, the parasite/pathogen transmission model was initially developed for malaria by MacDonald [7] in 1957 and on the basis of this derivation various subsequent models were developed for West Nile Virus Number of mosquito pools positive MIR/1000 = ---------------------------------------- x 1000 Total number of mosquitoes tested ~ 40 ~ International Journal of Mosquito Research (WNV), Western Equine Encephalitis (WEE) etc [8-10, 17]. Since the life cycle of JEV transmission is closely related to WNV/WEE, we too have ventured to bring in appropriate adjustments in different variables to estimate CD and VC for the JE vector. To estimate transmission potential of vector, factors like mosquito density, blood feeding rate, survival rate, gonotrophic cycle, vector competence and incubation period for JEV in the vector were considered by mining into data of nearly three decade-rich endemic Cuddalore district [14]. (h) / Gonotrophic cycle). P = the Proportion of mosquito surviving one day. b = the vector competence of Cx. tritaeniorhynchus. n = the incubation period of the JE virus in the vector. (vi) Data analysis: Mathematical and statistical analyses of data for derivation of JE transmission model and its graphical presentation were carried out by using Microsoft Excel 2007 version and SPSS version 15.0. We have conducted online searches of published literature on mathematical models related to critical density and vectorial capacity of JE vector through various databases without restriction to languages or geography and finally ensured that the transmission potential was yet to be derived. (iv) Critical Density (CD): The term critical density is described as “the average number of mosquitoes required to bite per host daily in order to transmit virus” [10]. The critical density for JEV transmission to human was estimated based on the assumption that the first bite was on amplifying host and second bite was on human. It was calculated as -ln (P) m = --------------a b Pn Where, a = the blood feeding rate (the proportion blood feeding on human/gonotrophic cycle) P = the Proportion of mosquito surviving one day. B = the vector competence of Cx. tritaeniorhynchus (proportion of vector susceptible to infection). n = the incubation period of the JE virus in the vector. Results and Discussion (i) Mosquito abundance: The mosquito vectors of JEV were longitudinally monitored for their abundance and virus infection in the villages of Cuddalore district. The subgroup Culex vishnui mosquitoes comprising Cx. tritaeniorhynchus, Cx. vishnui and Cx. pseudovishnui are proven vectors of JE in south India [18]. A three-year longitudinal study was conducted during 1991-1994 with the objectives of monitoring vector abundance and JEV infection frequency in mosquitoes in the study villages, an area endemic for JE, in Cuddalore district where a total of 422,621 female mosquitoes were collected and found that Cx. tritaeniorhynchus (62.6%) was the predominant species [13]. Again, during April, 2011 to December, 2012, a total of 15,941 female mosquitoes representing 24 culicine species were collected of which about 90.5% were contributed by the JE vectors, in which, Cx. gelidus was 48.6%, followed by Cx. tritaeniorhynchus (40.7%) and Cx. vishnui (1.8%), in principal. A two decadal monitoring and the comparative analysis of the various studies related to vector abundance revealed that Cx. tritaeniorhynchus was the principal vector. A maximum per man hour (PMH= No. of mosquitoes collected/No. of man hours spent) density was observed during October- December (Figure 2). Therefore, the vectorial capacity has been determined for Cx. tritaeniorhynchus among all the vector species influencing the JEV transmission in Cuddalore district. (v) Vectorial Capacity (VC): The vectorial capacity is defined as “a number of new infections produced per day by a vector”9 and is a commonly used term to predict epidemic dynamics of infectious diseases. The vectorial capacity of Cx. tritaeniorhynchus for JE is therefore now expressed as: m a2 b Pn VC = --------------ln (P) Where, m = the average number of female mosquitoes per host. a = the blood feeing rate (the proportion blood feeing on host Fig 2: Cx. tritaeniorhynchus density in Cuddalore district during April 2011 to December 2012. ~ 41 ~ International Journal of Mosquito Research (ii) Blood feeding habit (h): Assessment of blood feeding habit of Cx. vishnui complex was done during the period December 1988 - December 1990 when a relatively high proportion of recognized vectors occurred, and Cx. tritaeniorhynchus and Cx. vishnui had fed (h) mainly on cattle (84.6-88%), followed by pigs (4.4-5.4%) and humans (2.4-6.2%). The proportion of blood feeding habit on pig and human was estimated to the maximum of 0.1. Ironically, there were no confirmation about feeding on ardeid birds [19] and as such these, despite being referred to as one of the important zoonotic hosts for the JEV, could not be considered in deriving the present formula. (iii) Gonotrophic cycle (Gc): The frequency of blood meals taken and the survival rates of vector mosquitoes are important parameters influencing transmitting capacity of pathogens [20]. Mosquito gonotrophic cycle (Blood-feeding → egg maturation → oviposition) is repeated many times by a female mosquito [21]. Mori et al. [22] and Samboon et al. [23] estimated the daily survival rate of JE vectors by using Davidson’s method [16] and found that the duration of gonotrophic cycle was around 3-4 days. In our study, the female mosquitoes of the Cx. vishnui subgroup became infective in 9 days, i.e., after completing 3 gonotrophic cycles, during the transmission season September-November [13] . Assuming that the gonotrophic cycle remains the same throughout the year, the VC was estimated for both transmission and non-transmission seasons. In Cuddalore district, JE cases mainly occurred during September-November every year. During these months, the gonotrophic cycle in females of the Cx. vishnui subgroup reached 3 days. (iv)Vector competence (b): The virus transmitting capacity of a mosquito is influenced by various factors such as the ability of an ingested virus to survive and its development in the mosquito tissues and potential to penetrate the salivary glands in order to become eligible to be inoculated into a new host. Vector competence is estimated as the proportion of mosquitoes with a disseminated infection to the total number of exposed mosquitoes, often expressed as dissemination rates within a vector population [24]. Philip Samuel et al [25]. have developed a system for assessing vector competence of mosquitoes in three different areas, namely, Cuddalore, Madurai and Alleppey, and reported that the estimated vector competence of Cx. tritaeniorhynchus (i.e., transmission rate) actually ranged from 32-74%, with Cuddalore managing a range of about 32%. (v) Survival probability (P): The parous rate (Rate of parous mosquitoes) is one of the useful parameters to describe the age structure and net reproductive rate of the mosquito population. It is not only used to ascertain the daily survival rate of adult mosquitoes but also used to determine the recruitment rate of adults, the adult longevity and the length of a gonotrophic cycle. Therefore, any change in the parous rate will reflect changes in the population dynamics [26]. In Cuddalore district, human cases were mainly affected during the months of September-November every year. It was estimated that the parity rate (PR= the proportion of parous from the total number of ovaries dissected [27]) was 0.37, 0.39, 0.26, respectively, during September, October and November in 2011 and 0.33, 0.42 and 0.32, respectively, for the months in 2012, implying that the probability of the vector surviving one day (survival probability (P)) was 0.72, 0.73 and 0.64 in 2011, while 0.69, 0.75 and 0.69 in 2012 during the transmission season (Table 1). The average survival rate of Cx. tritaeniorhynchus (P) was 0.8 during this transmission season [28] . Table 1: Parity Rate (PR) and survival probability (P) of Cx. tritaeniorhynchus in Cuddalore district during transmission seasons. Year 2011 2012 Month September October November September October November Dissected 106 102 88 27 90 50 (vi) Extrinsic incubation period (EIP) in vector (n): The prolonged development period of mosquito larval and the longer extrinsic incubation period of JE virus at cooler temperature will reduce the virus transmission rate [29]. Due to prolonged viraemia, mosquitoes get the opportunity to pick up infection from pigs easily [30]. After an extrinsic incubation period of 9-12 days, infected female mosquito transmits the virus to other vertebrate hosts [31]. It was estimated that extrinsic incubation period of JEV in Cx. tritaeniorhynchus was 9-10 days (n = 9-10 days) in Cuddalore district. Female Parous 39 40 23 9 29 21 PR 0.37 0.39 0.26 0.33 0.42 0.32 P 0.72 0.73 0.64 0.69 0.75 0.69 Age (days) 3.03 3.24 2.26 2.73 3.49 2.66 infective mosquitoes taking a viremic blood meal would become infective 9 days later (i.e., after completing 3 gonotrophic cycles). Thus, the earlier study observed that the proportion of infective female mosquitoes among those infected was about Pn = 0.13 (P = 0.8, n = 9, Pn = 0.13) [13]. While estimating this it was found that the Pn=9, in 2011, falls within range of 0.019-0.051 and, in 2012, to 0.034-0.076, alluding towards a predictable proportion of viraemic vectors surviving for an extended period of 12 days, as summarized in Table 2. Table 2: The proportion of Cx. tritaeniorhynchus surviving the virus - during in the transmission seasons Year 2011 2012 Month September October November September October November P 0.72 0.73 0.64 0.69 0.75 0.69 P9 0.051 0.062 0.019 0.037 0.076 0.034 ~ 42 ~ P10 0.037 0.046 0.012 0.026 0.057 0.023 P11 0.027 0.033 0.008 0.018 0.043 0.016 P12 0.019 0.025 0.005 0.012 0.032 0.011 International Journal of Mosquito Research and 2.66-3.49 during 2012. These figures are attributable to the transmission season (Table 3) which may vary depending on the survival probability (P = 0.60-0.80) of the mosquito. Therefore, based on the survival probability, the life expectancy (age) and the expected infective life (days) of Cx. tritaeniorhynchus were estimated (Table 4, Figures 3 & 4). (vii) Mosquito life expectancy: Two factors, viz., gonotrophic cycle (gc) and parity rate (PR), are imperative to estimate the life expectancy and infective life of a mosquito. The PR, proposed by Davidson (1954) [16] was used to arrive this derivation. Since the gonotrophic cycle (gc) value was found to be 3 days, the life expectancy (age) of Cx. tritaeniorhynchus was estimated to be 2.26-3.03 during 2011 Table 3: Life expectancy and expectation of infective life (days) of Cx. tritaeniorhynchus in Cuddalore district during the transmission seasons. Year 2011 2012 Month P Life Expectation September October November September October November 0.72 0.73 0.64 0.69 0.75 0.69 3.03 3.24 2.26 2.73 3.49 2.66 n=9 0.156 0.201 0.042 0.102 0.266 0.090 Expectation of infective life (days) n = 10 n = 11 n = 12 0.112 0.080 0.058 0.147 0.108 0.079 0.027 0.017 0.011 0.070 0.049 0.034 0.199 0.150 0.113 0.062 0.043 0.029 P = probbility of Cx. tritaeniorhynchus survival through one day, n = Extrinsic Incubation period (JE virus in the vector). Life expectation is expressed as (1/-ln(p)) and expectation of infective life as (pn/ -ln(p). Table 4: Expectancy of life or age and infective life of Cx. tritaeniorhynchus in Cuddalore district Survival probability (P) 0.60 0.65 0.70 0.75 0.80 Life Expectation (1/-ln(p)) 1.96 2.32 2.80 3.48 4.48 Expectation of infective life (days) n=9 n = 10 n = 11 n = 12 0.020 0.012 0.007 0.004 0.048 0.031 0.020 0.013 0.113 0.079 0.055 0.039 0.261 0.196 0.147 0.110 0.601 0.481 0.385 0.308 Fig 3: Expectancy of life days (age) and infective life (days) of Cx. tritaeniorhynchus in Cuddalore district during April 2011 to December 2012 ~ 43 ~ International Journal of Mosquito Research Fig 4: Survival probability and expectancy of infective life (days) of Cx. tritaeniorhynchus in Cuddalore district. the year 2012. (viii) Critical Density (CD): The lifespan of vector (survival rate) is the vital variable in directly influencing the incubation period (n), gonotrophic cycle and also decides the requirement amount of mosquitoes to transmit virus. In this study, it was observed that during the transmission season in the study period, the survival rate was high in October (Table 3). Therefore, a minimum density of mosquitoes required for virus transmission was estimated for the month of October. It was found that a minimum of an average of 430 and >700 mosquito bites per human/day are necessary during October and the entire transmission season, respectively (Table 5), whereas in the non-transmission season, >900 mosquito bites were required in order to transmit virus. The required number of mosquito bites will be high for the remaining months in the transmission season due to shortened lifespan of the vector. Table 6: Vectorial Capacity (VC) of Cx. tritaeniorhynchus that were estimated for transmission season in Cuddalore district Year 2011 2012 P 0.72 0.73 0.64 0.69 0.75 0.69 n 9 9 9 9 9 9 VC= ma2bpn/-ln(P) 0.001 0.01 0.001 0.001 0.002 0.002 Discussion MacDonald [7] was among the first vector-borne disease experts to propose a potential pathogen transmission model for malaria in 1957. Taking cues from this basic model several different models were developed in due course pertaining to lymphatic filariasis, West Nile Virus etc. As far as culicine-mediated infections were concerned, it was Ciota [8, 9] who had for the first time estimated vectorial capacity (VC) for Culex mosquitoes for the West Nile Virus (WNV), whereas Smith [10] estimated anew critical density (CD) for West Equine Encephalitis (WEE). In our study, we have attempted to estimate CD and VC for the main vector of Japanese encephalitis, Cx. tritaeniorhynchus, on the basis of analysis of data mined over two and a half decades with respect to its biology, ecology, physiology and genetics in JE endemic Cuddalore district, Tamil Nadu State, India. The JE vectors feed on amplifying and reservoir hosts (the main target) during the first blood meal and humans (accidental and opportunistic target) during the second blood meal. If the amplifying animal host was earlier infected for a reasonable quantum of days then the vector female mosquitoes become infected, and after passing the full extrinsic period of the virus’s presence in its body becomes infective with viruses in her salivary glands ready to be inoculated to the first human being on whose blood she tends to feed opportunistically. m= -ln(P)/a b pn 501 359 430 2011 9 829 2012 9 700 Average 765 *Month taken where highest survival probability recorded (October). ^ Average survival rate for the transmission season (Oct-Nov). a= blood feeding rate (0.033) b = Vector competence (0.32) P 0.73* 0.75* Average 0.70^ 0.71^ m 20.0 78.0 65.0 17.0 23.0 54.0 a= blood feeding rate (0.033) b = Vector competence (0.32) Table 5: Critical density (CD) of Cx. tritaeniorhynchus that were estimated for transmission season in Cuddalore district Year 2011 2012 Month September October November September October November n 9 9 (ix) Vectorial capacity (VC): Vectorial Capacity (VC), i.e., the JEV transmission potential of Cx. tritaeniorhynchus was calculated from out of the analysis of results for various long-term studies incorporating different parameters necessitated to calculate VC. The vectorial capacity during transmission seasons for the study area ranged between 0.01 and 0.001 for the year 2011, while 0.001 and 0.002 for 2012 (Table 6); in the non-transmission season the VC ranged between 0.001 and 0.003 during 2011, while 0.00 and 0.002 for ~ 44 ~ International Journal of Mosquito Research 3. Infection to human is accidental since the vector is predominantly zoophilic. Generally, because viraemia in human peripheral blood is of a very low titer, therefore, possibility of the vector getting back load of virus is literally obscured [32]. Culex tritaeniorhynchus is the most abundant mosquito in Cuddalore district, accounting for 41-63% of the total mosquito species encountered. It is well known that the mosquito has a predilection to breed in areas with vast paddy cultivation and pig rearing. The vector showed a strong inclination for feeding on cattle (88%), followed by humans (6%) and pigs (5%). The main reason for this result may be due to close approximation of human households and animal sheds, and poor socioeconomic conditions of the rural poor [14]. Interestingly no feeding on ardeid birds could be substantiated in Cuddalore district [19], although pigs offered positive blood meals [33-34]. The EIP in Cx. tritaeniorhynchus was found to be a minimum of 9 days, allowing inter alia 3 gonotrophic cycles each for a period of 3 days. [13] The high parous rates imply that the probability of daily survival of the vectors for efficient transmission of infection is high [35]. High parity values would be an evidence for high egg development and high blood intake. The interaction between the survival probability (P) and EIP influences the infective life days, and it was found that infective life of the vector increased when the survival probability (P) also increased, and vice versa (Figure 4). The average vectorial capacity for the study area was found to be high during transmission season compared to nontransmission season due to higher values of density and feeding related parameters. The inferior VC of the vector and maximum representation of CD indicate that the JEV transmission in human is low. When compared to the previous studies [14] conducted during 2003-2013, it was observed that the mosquito density and survival rate of the vector had substantially reduced, thereby the value of VC being on the lower side. JE cases started reporting during the months of April – May, reaching the peak during late August to early September, and again sliding in October [33]. In the study area, the probability of child receiving an infective bite during September and December was 0.53 which is reasonably close to the estimate of 0.50-0.75 obtained from seroconversion rates in children in the same area [13]. 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