ABSTRACT
Studies of Anopheles mosquitoes transmitting malaria in some villages of Jigawa State were conducted to establish morpho-species and highliting the members transmitting malaria. A total of 1,863 adult Anopheles were collected, 591 (31.72%) by indoor residual spray collection while 1,272 (68.28%) were reared Anopheles larvae/pupae. Morphological characters and Polymerase Chain Reaction (PCR) were employed in the identification of Anopheles species while Enzyme-linked Immunosorbent Assay (ELISA) was employed to determine their infection by Plasmodium. Morphologically, two species, An. gambiae s.l. and An. funestus were identified, these comprised of 1,362 (73.11%) and 501 (26.89%) respectively. Molecular studies by Polymerase Chain Reaction (PCR) separated An. gambiae s.l. into 1149 (84.36%) An. gambiae s.s and 213 (15.64%) An. arabiensis. Molecular study of An. gambiae s.s. yielded 222 (19.32%) M-form and 927 (80.68%) S-form. The amplification of the 16s rDNA has revealed occurrence of a M/S hybrid in Auyo indoor anopheline population collected. There were 1,373 (73.70%) Anopheles during the rainy season and 490(26.30%) in dry season. The highest population of mosquitoes caught was in September 311 (16.70%). The distribution of Anopheles according to study locations showed that Kirikasamma had the highest 162 (8.70%) mosquito population while Jahun had the lowest 16 (0.85%). Of 591 Anopheles examined by ELISA, 43 (7.28%) were infected with Plasmodium. Of the 43 infected mosquitoes, 10 (23.25%) were An. arabiensis, 5 (11.63%) M-form and 28 (65.12%) S-form. Plasmodium falciparum 32 (74.42%) was more prevalent than P. malariae 11 (25.58%). In conclusion, Anopheles funestus, An. Arabiensis, M-and S-form of Anopheles gambae s.s were the abundant species in the study area. Anopheles mosquitoes were more abundant in rainy season than in dry season. Transmission was more in rainy season than in dry season. Anopheles species M-and S-forms and An. Arabiensis transmitting Plasmodium malariae and P. falciparum.
CHAPTER ONE
INTRODUCTION
Mosquito is a common insect in the family Culicidae. It has mouthparts that are adapted for piercing the skin of animals. While males typically feed on nectar and plant juices, the female needs to obtain nutrients from a blood meal (Jordan and Verma, 2011).
There are about 3,500 species of mosquitoes found throughout the world. In some species, the females feed on human blood, and are therefore vectors of a number of infectious diseases affecting millions of people per year. Species of Anopheles, Culex, Culiseta and Mansonia are common in warm climate and species of Anopheles are responsible for transmission of malaria (Jordan and Verma, 2011).
In a general way, the life cycles of all mosquitoes are much alike, but they differ in the details. The eggs of mosquitoes are usually oval with various surface markings and in Anopheles, with peculiar “floats” of air cells. Anopheles lays eggs singly in loose clusters on water; the larva lies horizontally in water and has no trumpet shaped siphon or breathing tube (Toply, 2008).
Members of the An. gambiae complex are the most important vectors of malaria in sub-Saharan Africa. The complex consists of about seven species that vary in their ability to transmit malaria. Two species of the complex, An. gambiae s.s and An. arabiensis are both the most broadly distributed and the most efficient vectors of malaria (Coetzee et al., 2000).
Singh et al. (1996) observed that the same species of Anopheles can behave differently in different ecological settings. They observed that An. culicifacies in India was mainly
exophilic in forest villages, whereas in villages away from the forest, An. culicifacies was predominantly endophilic.
Due to their variable breeding habits, choice of breeding places, taste in blood, extent of travels and willingness to come indoors to bite or to rest, different species of Anopheles relate differently with humans (Chandler and Read,1961). A few domestic species that feed in or near houses readily enter them and feed on human blood and are regarded as house pests include An. quadrimaculatus in the United States, An. maculipennis in Europe, An. darling in South America and An. gambiae in Africa.
Species that regularly come in contact with humans outdoors may come into homes to feed but not to rest. They breed in rice fields, seepage, overflow, in swamps, temporary rain pools, in tree holes, axile of banana leaves, coconut shells, near villages or cultivated areas. Important species in this group are An. scutedaris in the Pacific Islands and many species of Anopheles in various parts of the world (Jordan and Verma, 2011).
Gilles et al. (1968) described morphological characters of Anopheles and this had aided their classification. Gilles and Coetzee (1987) used the same identification features to report on the distribution of various anopheline species in Nigeria. Anopheles gambiae s.l. was considered a single species (Coluzzi, 1978) although Davidson (1964) distinguished the following species based on chromosomal studies: An. arabiensis, An. bwambe, An. gambiae s.s, An. melas, An. merus, and An. quadriannulatus (Hunt et al., 1998). Scott et al. (1993) reported two sibling species, namely M-and S-forms from An. gambiae s. s. (the M-form is presently known as An. coluzzi using molecular polymerase chain reaction technique).
Service (1980) described An. funestus, An. moucheti, An. nilli, An. hancocki and An. hagreavesi. Anopheles funestus belongs to a group of five sibling species, An. funestus, An. leesoni, An. parensis, An. rivulorum and An. vaneedenip. These species have been identified in North Africa, Western Africa and Central Africa (Frederic et al., 2003). Molineaux and Grammicia (1980) reported eleven species of Anopheles in the Sudan Savannah of West Africa, namely An. gambiae sensu stricto, An. arabiensis, An. funestus, An. rufipes, An. pharoensis, An. wellcomei, An. squamosus, An. coustani, An. maculipalpis, An. nilli and An. pretoriensis.
Onyabe et al. (2003) reported broad distribution pattern of the M- and S-molecular forms in Nigeria and, also the occurrence of An. arabiensis in the Southern Guinea savanna of Nigeria. Ndams (2004) reported eight taxa in Northern Guinea Savanna of Nigeria, namely An. funestus, An. rufipes, An. maculipalpis, An. gambiae s.l, An. gambiae s.s, An. arabiensis, M-and S-forms. The abundance of An. gambiae s.l, An. arabiensis, An. gambiae s.s, M-and S-forms from Guinea, Sahel and Sudan Savanna of northern Nigeria were reported (Tukur, 2010).
There are five species of Plasmodium responsible for human malaria these are Plasmodium falciparum, P. knowlesi, P. vivax, P. ovale and P. malariae. Plasmodium has a complex life-cycle, which includes stages in the female Anopheles mosquito. The infection starts with the bite of an infected mosquito, the sporozoite rapidly makes its way to the liver, where it invades a hepatocyte (Toply, 2008). Different species of Anopheles have been found to inoculate Plasmodium into humans. For instance, An. gambiae s.l and An. funestus transmit malaria in the Sudan savanna of West Africa, An. gambiae M-form is the most important and
widespread malaria vector in Angola and the sporozoite rate of Anopheles, determined by ELISA, was 1.9% for An. gambiae (n=580) and 0.7% for An. funestus (n=140) (Molineuax and Gramiccia, 1980; Martinez – Torres et al, 2002). Ndams (2004) reported the prevalence of Plasmodium circumsporozoite, protein antigens in An. gambiae s.s molecular forms in which 19 (2.84%) (n=668) were positive.
Malaria is a major public health problem and the cause of much suffering and premature death in tropical Africa, Asia and Latin America. In many endemic areas, it is becoming increasingly difficult to control the disease because of the resistance of the parasite to anti-malarial drugs and lack of adequate vector control measures (Cheesbrough, 2005).
In Garki, Jigawa State, there was a WHO intervention programme in 1969, which sought to establish a link between entomological and parasitological variables, especially vectoral capacity and the prevalence of Plasmodium falciparum. The project also measured the effect of specified interventions, such as spraying with residual insecticides, (propoxur alone and in combination) with mass drug administration, using a combination of sulfaline and pyremethamine (Molineaux and Grammicia, 1980). Two species of Anopheles gambiae s.l, namely An.arabiensis and An. gambiae s.s were found transmitting the malaria parasite in Garki (Molineaux and Grammicia, 1980).
Statistics on malaria in Jigawa State shows that it kills more people than HIV/AIDS, tuberculosis, measles and whooping cough (Taiwo, 2010). One out of every three children in Jigawa State that dies before his or her fifth birthday is killed by malaria and 11% of maternal mortality is also due to malaria (Taiwo, 2010). Entomological studies are an essential foundation for malaria control; Anopheles species and Plasmodium transmission
status in Jigawa should therefore contribute to a more accurate design of vector control, and by extension, malaria.
The Federal Ministry of Health (FMH) is currently undertaking malaria control activities in Jigawa State, which involve distribution of insecticide treated bed nets (ITNs), indoor spray of pyrethroid and free distribution of antimalaria drug (coartem). With all these, malaria still remains a problem in the state and this makes the study of Anopheles mosquitoes in Jigawa State important.
The older techniques that were used for the identification of Anopheles and presence of Plasmodium were light microscopy and dissection. Dissection involved first examining the ovaries of each mosquito for parity. Midguts and salivary glands of parous mosquitoes were then examined for oocysts and sporozoites respectively. Oocysts were counted, graded according to relative size, and differentiated oocysts and free sporozoites were noted. Salivary gland sporozoite infections were graded either 1+ (1 – 10 sporozoites), 2+ (11 – 100 sporozoites), 3+ (101 – 1,000 sporozoites), or 4+ (> 1,000 sporozoites) (Bangs et al., 2002). However, availability of skilled microscopists, for processing large number of samples to detect the sporozoites and identification of sporozoite species are the main limitations of this method (Mahapatra et al., 2006). Hence a method with greater sensitivity and specificity has been looked for. The polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA) are highly sensitive and specific (Mahapatra et al., 2006).
Polymerase is an enzyme that participates in making a copy of each Deoxyribonucleic acid (DNA) strand using the original template. Polymerase Chain Reaction (Scott et al., 1993) is a quick technique for determining Anopheles species complex. It involves denaturation in
which the original DNA is heated to make individual strands to separate; annealing in which oligonucleotide primers bind to the separated DNA strands and polymerization in which the polymerase copies the DNA rapidly. Enzyme linked immunosorbent assay also provides immediate result for detection of Plasmodium in Anopheles mosquito. Molineaux and Grammicia (1980) employed the dissection of Anopheles salivary glands technique which is difficult and time consuming.
Therefore, there is need to update the abundance of Anopheles and transmission status in Jigawa State using PCR and ELISA techniques which are more reliable than the techniques initially employed. The outcome of this study will be an essential tool for fighting malaria in the study area.
1.1 Statement of the Problem
Malaria remains one of the greatest human health burdens in Jigawa State. Jigawa has a long history in the research of malaria such as „‟the Garki project‟‟ and recently W.H.O malaria control interventions such as free distribution of antimalaria drugs, insecticide treated bed nets and vector control in some Local Government Areas such as Auyo, Ringim, among others. There are also significant gaps in the existing knowledge on the transmission of malaria. These compound the difficulty of developing targeted and effective control measures that would be evaluated. The sheer complexity and number of malaria vectors in the state are daunting and such receive detailed studies. The difficult task of summarizing the available information for each species and/or species complex is compounded by patchiness of the data: while relatively plentiful in one area or region, it can also be completely lacking in others. Compared to many other states in Nigeria, only scant information on vector
bionomics and response to chemical measures is available in Jigawa State. It is to the light of the complexities highlighted above that this was designed.
1.2 Justification
Published work on malaria vectors in Jigawa State is patchy or scanty. Determination of risk of malaria transmission requires quick, frequent and accurate methods of assessing transmission intensity, which is the product of human-biting rates and infectivity of vectors (WHO, 1975).
There is need to update information on Anopheles species and malaria transmission to assist in developing effective intervention in the study area. The behaviour of Anopheles species largely determines their vector status, and insights into their distribution are essential to evaluate the appropriateness of vector control measures. Therefore, the study will shed lights in greater detail, the species, composition and abundance, the species that are actively involved in transmission of falciparum malaria during the period of the study.
1.3 Aim of the Research
The aim of this research is to evaluate the species composition of malaria vectors and their invovlement in the transmission falciparum malaria in Jigawa State.
1.4 Objectives of the Research
i. To determine the species composition and distribution of Anopheles mosquitoes
ii. To determine the seasonal variation within the Anopheles population and the relationship between seasons and transmission.
iii. To determine Anopheles infection with Plasmodium and Plasmodium falciparum species causing malaria in the study area.
1.5 Research Hypotheses
i. Anopheles species are not abundant and not well distributed in the study area.
ii. Anopheles species are not infected with Plasmodium and only Plasmodium falciparum is responsible for malaria in the study area.
iii. There is no seasonal variation among the Anopheles population and Plasmodium transmission is independent of season.
iv. Plasmodium transmission is not associated with Anopheles species.
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