A Study on the Malaria Vector (Anopheles Spp) in a Sudano-Sahelian Savannah Area of Borno State North Eastern Nigeria and the Insect Growth Regulator Pyriproxyfen (S-31183)

A Study on the Malaria Vector (Anopheles Spp) in a Sudano-Sahelian Savannah Area of Borno State North Eastern Nigeria and the Insect Growth Regulator Pyriproxyfen (S-31183)

Chapter One

AIM AND OBJECTIVES OF THE STUDY

This study aims to identify local malaria vectors, determining their vectorial importance/status and the suitability of the insect growth regulator for their control in North-eastern Nigeria.

 Objectives

1. To identify the predominant sibling species of Anopheles gambiae complex in the study area using the polymerase chain reaction assay (PCR).
2. To carry out vector-incrimination study to identify the probable vectors responsible for malaria by determining the circumsporozoite (CSP) rate in the vector using the
3. To determine the seasonal peaks of malaria transmission in the study area through a longitudinal parasitological
4. To evaluate the inhibitory action of the insect growth regulator pyriproxyfen(S 31183) on the 4th stage larvae of  gambiae under laboratory conditions.

CHAPTER TWO 

LITERATURE REVIEW

  THE DISEASE MALARIA

Malaria, a mosquito borne, protozoal disease, is older than recorded history. It is believed that man and malaria evolved together, that populations of human malaria may have had their origin in West Africa (Rugemalila et al., 2006).

Hippocrates was the first to describe the manifestation of the disease, and relate them to the time of the year and to where the patients live before this, the supernatural was blamed. The association with stagnant waters (breeding grounds for Anopheles) led the Romans to begin drainage programs, the first interventions against malaria .The word malaria meaning bad air has its origins there (Rugemalila et al., 2006)

Not until 1889 was the protozoal cause of malaria elucidated by Laveran working in Algeria, and only in 1897 was the Anopheles mosquito demonstrated to be the vector of the disease. Ronald Ross of the Indian Medical Service, working on Manson’s suggestion, succeeded in establishing the Anopheles mosquito-malaria relationship. He found oocysts of malaria parasites, Plasmodium on the stomach of the female Anopheles mosquito previously fed on the blood of a malaria patient. This discovery was made on August 29, 1897, ever since called the “mosquito day” (Jordan and Verma 2005; Rugemalila et al., 2006) .At this point the major features of the epidemiology of malaria seemed clear, and control measures started to be implemented (Rugemalila et al., 2006).

Malaria is caused by single celled protozoan parasites of the genus Plasmodium. According to Bruce-Chwatt (1986) these microorganisms are commonly referred to simply as malaria parasites; this term is usually restricted to the family Plasmodiidae within the order Coccidiida, sub order Haemosporidiidea which comprises various parasites found in the blood of reptiles, birds and mammals. The classification of Haemosporidiidea as a suborder of the coccidiida is complex and controversial, an alternative system has been proposed by Levine. However, Garnham’s classification of Haemosporidiidea into Plasmodiidae, Haemoproteidae and Leucocytozoidae has however been maintained (Bruce-Chwatt, 1986). The zoological family of Plasmodiidae includes the parasites, which undergo two types of multiplication by asexual division (schizogony) in the vertebrate host and a single sexual multiplication (sporogony) in the mosquito host. The genus Plasmodium has been defined on the basis of one type of the asexual multiplication by division occurring in the parenchymal cells of the liver of the vertebrate host (exoeythrocytic schizogony); the other characteristic of this genus is that the mosquito hosts are various Anopheles. There are nearly 120 species of Plasmodia, including at least 22 species found in primate hosts and 19 in rodents, bats or other mammals. About 70 other plasmodial species have been described in birds and reptiles. Plasmodia of the primate hosts are divided into three sub-genera and within the sub genus of Plasmodium there are four groups classified according to the periodicity of their erythrocytic schizogony (Bruce-Chwatt, 1986).

The zoological classification of Plasmodia is complex, and even today there is some difference of opinion with regard to taxonomic position of the parasite causing falciparum malaria. In this malaria parasite the cresentric shape and lengthy development of sexual erythrocytic forms has been accepted by some authors as a valid argument for recognition of the parasite as belonging to a separate genus,

Laverania falcipara. While this view may be correct in a context of zoological systematics, the rejection of the familiar name Plasmodium falciparum might be confusing and since the use of this well known name is still taxonomically permissible it is retained (Bruce-Chwatt, 1986). There are four generally recognised species of malaria parasites of man: Plasmodium malariae (Laveran) P. vivax (Grassiand Feletti,), P. falciparum (Welch), P. ovale (Stephens) (Bruce-Chwatt, 1986).Their worldwide distribution and characteristics are as follows:

falciparum or malignant tertian malaria (Together with P. vivax) are themost common human malaria parasite and are found in most malarious areas mainlytropical Africa, Asia and latin America including Haiti, Dominican Republic French Guinea, Surinam, parts of Asia and Papua New Guinea. P. falciparum is the most dangerous form of the disease; the high levels of parasites in the blood (parasitaemia) alone can result in death or produce fatal cerebral, renal or pulmonary complications, particularly in non-immune individuals.

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CHAPTER THREE 

MATERIALS AND METHODS

The research was carried out in Borno State; one of the five states belonging to the former northeastern state which now has Maiduguri as the state capital (Figure 3). The two study sites were (a) Maiduguri town and (b) Damboa, 85 kilometres from Maiduguri. The two places are 85 kilometres apart. The choice of the two zones is based on epidemiological and practical considerations such as record of malaria high endemicity and relatively high human population (Molta et al., 2004).

Maiduguri Study Site

The study lasting for a period of one year centred at the Emergency Paediatric Unit (EPU) of the University of Maiduguri Teaching Hospital (UMTH). The UMTH serves as a referral centre catering for the needs of the entire northeast region. Maiduguri is located on latitude 11⁰40’ N and longitude 13⁰05’ E, with mean annual rainfall of 650mm.Maiduguri is considered as the transition zone of the Sudan Sahel Savanna (Sara, 1990). The town is inhabited by about 877, 925 people (1991 census figure). The prevalence of Plasmodium infection here has been put at 35.2% (95/270). The Plasmodium falciparum is the most common cause of malaria and accounting for about 95-98% of infection, while the other species P. malariae and P. ovale account for 2-5% of the infection (Molta et al., 1993) in Maiduguri.

 Damboa Study Site

Damboa is located at the southern edge of the Sahel within the Sudan savannah (Fig 3). 85 kilometres away from Maiduguri, Borno state along Maiduguri-Biu road highway it lies along the bank of one of the seasonal rivers. It has a marked rainy season that spans the period June/July to September/October, and dry season between October/November and May/June. The climate is semi arid, with malaria transmission of the mesoendemic type (Watila et al., 2006). It is essentially a subsistent agricultural community, although considerable animal husbandry and trading are also undertaken. Several man-made ponds created by construction work and a poor drainage system provide favourable breeding sites for mosquitoes that transmit malaria among other diseases.

Damboa, the headquarters of Damboa Local Government Area has a population of 24, 421 (9.9% of the LGA’s Population of 247,904 (Molta et al., 2000) Record also indicated high incidence of malaria in this area. It is endemic for falciparum infection. (Molta et al., 1992, Molta et al., 2004. BOMOH 1986 -1988). The prevalence of Plasmodium infection has been put at 70% (433/617)(Molta et al., 1993). Damboa is a sentinel site of the National Malaria surveillance network.

CHAPTER FOUR 

RESULTS

  ENTOMOLOGICAL INVESTIGATION

 Relative Abundance and Species Composition

A total of 16 houses were sampled across the 6 different wards of Damboa .The mean vector population density was approximately two persons per house in the other places. Overall a total of 1030 adult female Anopheles mosquitoes were collected 979 (95%) were caught indoors. Mosquitos caught outdoors accounted for 51(5%) of the total collected (n=1030) (Table 3). Significant differences was observed between mosquitoes caught indoors and those caught outdoors (P<0.01).

CHAPTER FIVE 

DISCUSSION

 PCRANALYSIS OF ANOPHELES SPECIES

The present investigation was prompted by the scarcity of information on the precise identity of sibling species of the Anopheles gambiae complex in the semi arid Sudano-sahelian area of Borno State and the need to ascertain the vectorial status of these Anopheles species using high precision biomolecular tools, including the Polymerase Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA) (Gadzama, 1983; Molta et al., 1999).

Polymerase Chain Reaction (PCR) and ELISA showed that An. arabiensis was the predominant member of the Anopheles gambiae complex and the major vector of malaria in Damboa northeastern Nigeria (Samdi et al., 2006).This is agreement with the findings of Hinzoumbe et al.(2009). An. arabiensis is associated with more arid habitats (Coetzee et al., 2000; Davidson 1967; Omer and Cloudsley-Thompson 1970;El Rayah and Abu Groun 1983; Hamad et al., 2002). The density of An. arabiensis resting indoors increased rapidly following the beginning of the rains a situation consistent with the observation of Molta et al.(1995)that annual increase was dependent on rainfall . The direct influence of rainfall on the density of An. arabiensis is in agreement with the study done in Tanzania by Charlwood et al.(1995).

Furthermore findings from this study accords well with Gadzama (1983) and Coetzee et. al., (2000) who found that An. arabiensis was associated more with arid habitats. Earlier Fontenille et al., (1999) had observed that in areas of unstable malaria, An.arabiensis was the predominant species of the An.gambiae complex. Similarly, Himeidan et al. (2004) found that An. arabiensis was the main vector (99.9%) in Semi-arid eastern Sudan and An.pharoensis was only 0.1%. The same results have been observed in areas of low malaria endemicity in Tanzania where entomological monitoring showed that 99% of malaria vectors caught were An. Arabiensis (Mwerinde et al., 2005). From this study it was observed that malaria vector populations of the Sahel, Northeastern Nigeria clearly differs significantly with those of Southern Nigeria, a Polymerase Chain Reaction based test in southern Nigeria identified only 6.3% as An.arabiensis while in this work 95% percent observed were An. Arabiensis (Awolola et al., 20

 CONCLUSION

In conclusion, Polymerase Chain Reaction (PCR) showed that An. arabiensis is the predominant Anopheles in the study area,the vector is anthropophilic (preference for humans)and endophilic(bites indoors). This may help narrow down vector control measures in the sahel to a selective, targeted, site specific, ecologically sound and cost effective (malaria) vector control strategy suited to local environmental and epidemiological conditions of the northeastern Nigeria but the same is not easily applicable to South western Nigeria where PCR based test identified a more diverse mosquito fauna.This may have implications on control measures targeting a single species, which will have little impact on malaria infection associated with either An. funestus or An. arabiensis. In the South, the contribution of the three most important afrotropical malaria vectors may account for the perennial malaria transmission, compared to other parts of Nigeria like the Sahel where one vector species predominates and transmission is seasonal.

The high number of An. arabiensis caught indoors and the positive sporozoite rate indicate their epidemiological impotance in malaria transmission in this part of the Sahel.

Mosquito infection by malaria (Plasmodium) parasites determined using the ELISA method for the detection of Plasmodium falciparum sporozoite protein showed that out of 289 randomly selected Anopheles mosquitoes 7(2.4%) were positive for P. falciparum circumsporozoite antigen. All seven were An. arabiensis indicating susceptibility of An. arabiensis to local strains of the malaria parasite Plasmodium falciparum and its epidemiological importance in malaria transmission in the Sahel.

Mosquito blood feeding preferences was determined by direct ELISA using the Human Blood Index (HBI) which is an indication of the degree of anthropophily showed that the proportion of An.arabiensis with human blood was ninety eight (98%) percent and only two (2%) percent tested positive for bovine blood indicating a tendency to feed on man in preference to other animals (anthropophily as opposed to zoophily). Mosquitoes that prefer human blood to other animals are more dangerous as they more likely to transmit diseases notably malaria. Findings from the longitudinal parasitological study indicated the peak period of malaria transmission as September with the highest Geometric Mean Asexual Parasite Densities (GMPD) of 13,655 asexual parasites per microlitre of blood. This finding is strategic to timely planning and correct application of vector control operations such as Indoor Residual Spraying (IRS) in the study area to prevent seasonal peaks of malaria transmission. The high prevalence of gametocytes amongst the vector age group reservoir (children aged 1-8years). screened during the study indicates the possibility of a continual increase in antimalarial drug resistance in the Sahel.

Though the doses of pyriproxyfen used in this work under field conditions have been shown to produce extended residual activity and much higher inhibition rates but the persistence of pyriproxyfen in water in the absence of organic matter and increased temperature and sunlight declines as was observed in this case. However the important larvicidal potentials of pyriproxyfen at low dose has been demonstrated by this preliminary study. It deserves further consideration as a candidate larvicide for integrated vector management (IVM) in the urban areas of Nigeria. The major interventions for diseases like malaria are indoor residual sprays and the use of long lasting insecticidal treated nets. These do not provide complete control especially in the urban area where recent data indicates that urban m alaria is being associated with the adaptation of An.gambiae s.s to a wide range of polluted water and temporary breeding sites. The addition of an effective larviciding program with S-31183 pyriproxyfen would further increase the impact on vectors and their associated disease agents. This is in view of the several studies conducted in Africa which have shown 80% of Anopheline breeding sites to be man made and close to human habitation (Awolola et al., 2007; Invest and Lucas, 2008).

 RECOMMENDATIONS

1. The Polymerase Chain Reaction (PCR) results showed that 95% of themembers of  gambiae complex in the study area are An.arabiensis. They are endophilic and anthropophilic can help narrow down vector control options.
2. Interruptionof vector-human contact is of priority in breaking the transmission chain of malaria parasites .The human blood index (HBI) of 98 percent shows a very high human-vector contact. Reducing this contact is  This could be through screening of houses in urban quarters and environmental.
3. The timing of residual insecticide spraying is crucial in obtaining maximum Results from the longitudinal parasitological study indicated that the peak period of malaria transmission is September with the highest Geometric Mean asexual Parasite Densities (GMPD) of 13,655 asexual parasites per microlitre of blood. This finding is strategic to timely planning and correct application of vector control operations such as Indoor Residual Spraying (IRS) in the study area to prevent seasonal peaks of malaria transmission.
4. Malaria Early Warning Systems (MEWS) which employs the use of Remotesensing (RS) and Geographic information Sensing (GIS) are important meteorological data based surveillance tools should be put in  Findings from this work showed that malaria transmission intensity varies according to rainfall in the semi arid Sudano Sahel, under such situations Seasonal epidemics (type 11) of malaria are a major threat.

5. The high prevalence of gametocytes amongst the children screened duringthe study indicates the possibility of a continual increase in antimalarial drug resistance in the Sahel. Therefore, this study recommends reduction in the number of gametocyte carriers in the population by the timely use of gametocytocidal anti-malarial drugs such as the dihydroartemisinins in line with World Health Organization recommendation that the use of artemisinin–based combinations (ACTs) as the gold standard antimalarial for managing uncomplicated malaria cases. It has an impact on transmission as
6. Both the longitudinal entomological and parasitological studies clearly showthat vector control activities should target the period starting from June(when the vector buildup begins) up to (December ,covering both the early and late peak transmission for maximum impact.

SUGGESTIONS FOR FUTURE RESEARCH

1. Extensive studies must be carried on Entomological Inoculation Rates (EIR)of malaria vectors of the study area to assess the epidemiological impact of vector control
2. Studies on Man Biting Rate (MBR) rate of the Anopheline fauna in the studyarea is recommended for the future.
3. A baseline determination of Susceptibility of malaria vectors to WHOPES-approved insecticides for key vector control measures such Indoor Residual Spraying (IRS) is recommended for the
4. A baseline study determination of the susceptibility of malaria vectors of thestudy area to all pyrethroids used in insecticide treated nets widely distributed by the Federal Ministry of Health must be done in the
5. Field trials of environmentally friendly larvicides with wide safety marginssuch as pyriproxyfen (s-31183) on discrete malaria vector breeding places and polluted places in urban areas is needed in the future.
6. Acircumsporozoite analysis of  pharoensis a secondary malaria vector of importance in the Sahel is recommended in the future.

 SUMMARY OF RESULTS

1. A total of 1030 female Anopheles mosquitoes were caught consisting of five Namely, 1026 (99.6%) of Anopheles gambiae complex while 2 (0.19%) were An. pharoensis; 1 (0.09%) An. squamosis while 1 (0.09%) was An. rhodesiensis.
2. Two hundred and thirty three (233) mosquitoes randomly taken fromthe one thousand and thirty (1030) morphologically identified gambiae s.l subjected further to PCR analysis which showed that the predominant sibling species were An. arabiensis Patton 95%(n=221) and An. gambiae s.s. 5% (n=12). Three other species of Anopheles mosquitoes collected were morphologically identified to be An. pharoensis, An. squamosus and An. rhodesiense. Results obtained showed that the population of Anopheles arabiensis was significantly higher than that of Anopheles gambiae ss (χ ² = 8.56 df=1, p<0.05).

3. Mosquitoinfection by malaria (Plasmodium) parasites was determined using the ELISA method for the detection of Plasmodium falciparum out of 289 randomly selected Anopheles mosquitoes 7 (2.4%) were positive for P. falciparum circumsporozoite antigen.All seven were An. arabiensis.
4. Mosquito bloodmeal source was determined using DIRECT ELISA and the Human Blood index (HBI) gave a proportion of mosquitoes with human blood and hence an indication of anthropophily. Ninety eight (98%) percent of An. arabiensis mosquitoes tested had fed on human blood. Only two (2%) percent tested positive for bovine blood.

5. Of a total of 692 children consecutively screened over a period of one year,169(24%) were positive for malaria parasite; 114(67.46%) of whom were males and 55 (32.54%) females. Significant difference (p<0.05) in infection rates was observed between the males and the females. The levels of parasitaemia asexual parasite were significantly related to age (p<0.05). The majority of infected children (68.0%) were aged between 12-60 months and their asexual parasite density was between 1000-5000 of whole  The month of September recorded the highest geometric mean parasite density (GMPD) of 13,655 while the lowest parasite densities were observed during the dry season months of March, April, and May with GMPD values of 0.98, 7.17 and 4.69 respectively.

6. Gametocytaemia was not significantly affected by the age of neither thepatients nor the season (χ ² =0.04; df=2 p>0.05).
7. During the period between 2001 and 2004, a total of 13,901 patientsattended the outpatient clinic. Two thousand eight hundred and eighteen (2,818) were admitted and 342 died in Damboa General Hospital. Detailed yearly outpatient clinic attendance, hospital admissions and deaths recorded for 2001, 2002, 2003 and
8. A total of 4,929 children aged 0-4 years attended the Damboa GeneralHospital from 2001 to 2004 .Three hundred and twenty (33%) children aged 0-4 years had malaria, of this number 188(58.75%) were male and 132(41.25%)
9. Themonthly malaria cases,admissions and deaths show seasonal fluctuations with peak incidence of malaria cases in June 2001, October 2002 and 2003 and August 2004.The dry season is characterized by lower numbers of cases compared with those of rainy season.
10. Pyriproxyfen was effective against the larvae of Anopheles gambiae. Theresidual activity and Emergence Inhibition (EI) increased steadily post
11. Average Emergence Inhibition (EI) rates ranged from 86 to 100 percentduring the first seven days of application, though a slight decline to 96 percent was observed in the second week a peak across all doses and the EI declined to 74 percent in the third and finally 34 percent in the fourth week respectively.

12. Asignificant correlation was observed between dose and Emergence Inhibition (r=1, P<0.01), while means of mortality between 0.1mg/L and 5mg/L treatments were not significant different (P> 0.05).
13. Meansof percentage adult emergence inhibition (EI) was found to be statistically significant at all the application rates (χ² = 3.49; df = 3; p < 05.)
14. The means of Emergence Inhibition (EI) between 0.1ppm and 0.5ppmtreatments were not significantly different (P> 0.05). Emergence Inhibition (EI) rates ranged between 86-100 percent during the first seven days of application,   48-96 percent during the second week, 43-74percent during the third and subsequently 18-64 percent by the fourth
15. Meansof percentage adult emergence inhibition (EI) was found to be statistically significant at all the application rates (χ² = 3.49; df = 3; p < 05.).

The data generated from all entomological and parasitological studies will provide a useful basis for the purpose of designing a suitable malaria control strategy in line with integrated vector management (IVM) now advocated by the Federal Ministry of Health.

CONTRIBUTIONS TO KNOWLEDGE

This study has made 6 major contributions to knowledge by providing key baseline data for malaria vector control activities the Federal Ministry of Health desires to scale up. This is in line with the principles that, the control of malaria requires a break in the epidemiological chain: either by acting on the human parasite reservoir or by reducing man- vector contact.

1. Thisis one of the works selected and cited by the World Health Organization (WHO) for the document entitled: Malaria Entomological profile for Nigeria (1919-2007) available online at http://www.afro.who.int/vbc/reports/entomological_nigeria.pdf the report represents an analysis of 86 selected baseline research works on malaria vectors in Nigeria over the last nine decades(1919-2007).
2. Thisis the first documented attempt in this part of the Sudan Savanna at the southern edge of Sahelian part of Borno state Northeastern Nigeria at the use of high precision biomolecular tools technique of Polymerase Chain Reaction (PCR) for the identification of the Anopheles gambiae complex and  arabiensis.
3. This is the first documented attempt at the use of ELISA tests to show that arabiensis is the vector of Plasmodium falciparum in the study area and to determine sporozoite rate by a quantification of the number of the sporozoites present based on the detection on a specific antigen on the sporozoite surface,the circumsporozoite protein(or CSP1) and give a precise identification of the sporozoites as those of Plasmodium falciparum.

4. This is the first documented attempt at the use of molecular methodsemploying direct ELISA to show that arabiensis which is known to predominantly feed on animals does not feed on animals in the study area but on mainly on human beings (human blood) thus conclusively incriminating An.arabiensis as the predominant malaria vector in the study area
5. This work determined the degree of man-vector contactbetween ( arabiensis and man (human blood index of 0.98) (98% human biter) This is critical figure for the evaluation of the outcome of the success of the major malaria vector aimed at decreasing Human – Vector Contact. interventions (ITNS and IRS )  currently being scaled up in Nigeria.
6. This work documented proof to show that the resting preference of arabiensis at the study area is not exophilic but endophilic.This is crucial to the recommendation and success of Indoor Residual Spray (IRS) and the promotion of Insecticide treated nets (ITNS) as a malaria vector control strategy in the study area.
7. Where malaria transmission is seasonal, optimal timing of vector controlactivities is crucial to its success.This is the first documented attempt at a longitudinal human parasitological investigation to provide baseline information to further show the period within a season for timing vector control activities in the Sudano-Sahel.

REFERENCES

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• Amajoh, C.N. (1997). A review of Malaria Vector behaviour in Nigeria. Abstracts of two days National symposium on malaria in Nigeria held at Nigerian Institute of Medical Research Yaba, Lagos. 4^th to 5^th November 1997.
• Anderson, D. (1932). Notes on mosquitoes-borne diseases in Southern Nigeria III.Differences in the periodicity of various mosquitoes. Journal of Tropical Medicine and Hygiene 35:305-308.
• Ansari, M.A., Sharman, V.P., Mittal, P.K., Razdan, R.K. (1991). Evaluation of juvenile hormone analogue JHM /5-383 against immature stage of mosquitoes in natural habitats. Indian Journal of Malariology 28: 9-43.
• Anyanwu, G. I. and Iwuala, M.O.E. (1999). Mosquito breeding sites: Distribution and relative abundance. Journal of Medical Entomology and Zoology 50 (3): 234-249.
• A M ZKHCuticular hydrocarbon discrimination between Anopheles gambiae ss and An. arabiensis larval karyotypes. Annals of Tropical Medicine and Parasitology 95(8): 843-852.

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