Malaria in the western highlands of Kenya is unpredictable disease with increasing frequency and intensity in transmission. The establishment and spread of the disease in this highland area depends on the presence of and relationship of several epidemiological factors the most important being, the host, the agent and the environment. Man is the vertebrate host and the Anopheles mosquito in the agent of transmission/vector whereas the Plasmodium parasite is the causative agent of malaria.
Malaria vector and non-vector species population structure and density in any
locality is not static (Duffy, 1977). Malaria vector (s)
and non-vectors may periodically extend their range beyond their normal area
of distribution when temporary suitable conditions occur in neighboring areas
(WHO, 2010). Most Anopheles species are known
to change their ecological range, behavior by adapting to new climatic, ecological
and human induced changes (Muriu et al., 2008).
This may not be frequent but it does occur particularly in this era of global
warming and may result in serious public health implications. Human activities
particularly related to land-use evident in the study area could promote changes
in vector and non-vector species diversity and malaria transmission in future
in several ways as reported elsewhere (Walsh et al.,
1993; Patz et al., 2000). For instance man-made
activities such as swamp reclamation and brick making creates more human-made
aquatic habitats for the Anopheles species. Increased cattle grazing
create more open habitats with elevated temperature that favor faster Anopheles
larval development hence increased adult density and possible malaria transmission
(Minakawa et al., 2004). Man-made environmental
changes may also have a bearing on the diversity of the vectorial systems of
an area and subsequently result in eco-epidemiological stratification and invasion
of area by new vectors. Human activities and topography could also have an effect
on the diversity and distribution of disease vector breeding habitats. Therefore,
we hypothesized that human activities, environmental changes and regular travel
to and from the neighboring Lake Victoria lowlands could result in changes in
Anopheles species composition which may have implications on transmission,
epidemiology and control of not only of malaria but also other vector-borne
diseases. The purpose of the study was to assess the Anopheles species
diversity and locate and identify breeding habitats in two sites in a malaria
epidemic-prone area in the western highlands of Kenya.
MATERIALS AND METHODS
Study sites: The study was conducted in North Nandi District [0°21′52 N and 0°16′56 N in longitude and 35°5′20°E and 34°59′7 E in latitude] in the highland areas of Kipsamoite and Kapsisiywa each with 7 and 10 villages, respectively. The study sites were selected because, they were located 1500 m above sea level, an altitude defined as characterizing the highland area and malaria epidemics and outbreaks had been reported within the sites previously.
The topography of the study sites comprises hills, valleys and plateaus. Rivers and streams run along the valley bottoms in the valley ecosystem and reclaimed and natural swamps are a common feature. The study area has two rainy seasons, long rains season from March to May, referred to long rains on the account of duration and high amount of rainfall received in many parts of the highlands. The second season is the short rains from the months of October to December during which period, the area experiences depressed rainfall that is also poorly distributed both in space and time. There are variations in temperature the warmest temperatures are experienced in March and the coldest in July with the mean monthly temperatures ranging from 17-19248°C.
Anopheles species sampling points: Anopheles mosquito samples were collected from January to December 2008 in a total of 17 villages within Kipsamoite and Kapsisiywa study sites. The 40% (n = 656) of the households were randomly selected, coded and used as sampling points distributed as follows: Kipsamoite with a total of 666 households out of which 265 (40%) were sampled; Kapsisiywa with a total of 982 out of which 391 (40%) were sampled.
Anopheles species breeding habitat identification: Mosquitoes are capable of colonizing just about very conceivable type of water body except fast running water in rivers and streams. Systematic ground surveys were conducted at 2 weeks intervals in months of January to December representing the dry and rainy season to determine the possible Anopheles aquatic breeding habitats. This excluded fast running water because mosquitoes breed in calm, slow, moving water and water in containers in houses because it was meant for household use and tree holes because of non accessibility. The following aquatic habitats were found present in the study sites and constituted potential breeding sites: pools and puddles, foot/hoof prints, drains and ditches, streams and river edges, ponds, natural/ disturbed swamps and marshes, bore holes, wells and springs. The occurrence of anopheline larvae in each habitat was determined by using a standard dipper for large habitats (>0.5 m2) for smaller aquatic habitats (<0.5 m2) like foot/hoof prints a small sieve and pipette were used to scoop water.
A dipper (13 cm in diameter and 6.5 cm deep) with a handle)/sieve/pipette was gently lowered at an angle of 45°C just below the water surface so that water flowed into it or sucked (by pipette) together with any larvae that might be present. During dipping, care was taken not to disturb water too much and make larvae swim downwards. The filled dipper/sieve/pipette was carefully lifted, taking care not to spill the water containing the larvae. The drawn water containing the larvae was poured into white rectangular trays and checked visually and larval samples when present were collected by pipette. Ten dipper/pipette/sieve collections were made per each aquatic habitat. If none had anopheline larvae then the site was declared anopheline negative. The larval samples from each habitat were transferred into labeled (with date, collection site and type of habitat) vials and transported to Kenya Medical Research Institute (KEMRI) for rearing into adults and subsequent identification of adults. The identified anopheline positive breeding sites were noted, location recorded, counted and categorized as Anopheles larval collection points for outdoor collections. The results of the rainy and dry season breeding habitat number are shown in Table 1.
Collection of outdoor larvae and adult Anopheles species: Anopheles larval collection from identified breeding sites was carried out every 2 weeks from 0600-0900 h. The collected larval samples were packet in cool-box and transported to the Kenya Medical Research Institute (KEMRI) laboratory and reared to adults under the following conditions: temperature 27°C, 80% relative humidity and 12:12 light: dark schedule and brewer’s yeast as food. Upon emergence from pupa, the adults were identified by morphological features using identification keys. Out-door adult Anopheles mosquitoes in animal shelters were collected by use of Centers for Disease Control (CDC) miniature light traps (J.W. Hock Ltd, Gainsville, Fl., USA).
|| Rainy and dry season Anopheles identified breeding sites
|| Anopheles diversity and abundance
|*Known human malaria vectors; +Ancestor of all human malaria
vectors (Anthony et al., 1999)
Collection of indoor adult Anopheles species and identification:
Total indoor resting mosquitoes were collected from randomly selected households
by the pyrethrum space spray method also called Pyrethrum Spray Collection/Catches
[PSC] method (WHO, 1975) every fortnight. White sheets
were spread on the floor and all the windows, doors and all other exit points
closed. Pyrethrum extract [0.2% in kerosene] was sprayed on all eaves, doors,
windows and in the entire space of all rooms in the house and the house closed
for 10-15 min. All knocked down Anopheles species were collected carefully
with the forceps and placed in petri dishes lined with moist filter paper. The
collections were transported to KEMRI laboratories preservation on silica gel
in Eppendorf tubes prior to species identification.
All the anopheles mosquito collections were sorted out to separate the females
from males. The females were identified to species level using morphological
features with the aid of identification manuals (Gillies and
De Meillon, 1968; Gillies and Coetzee, 1987) and Anopheles
identification soft ware CD. The results are shown in Table 2.
The females of all morphologically identified female Anopheles gambiae s.l.
mosquitoes collected from houses and those reared to adults from larval mosquitoes
were identified to sibling species using PCR as described by Scott
et al. (1993). The PCR assay involved the following key steps: mosquito
DNA extraction using the potassium acetate precipitation technique making of
PCR master mix [mixture of buffer, ions, primers and enzymes in water]; electrophoresis;
gel visualization and photography.
|| PCR comparative band distribution of An. gambiae s.s
and An. arabiensis. Sample A, B, J, L. O and R were An. arabiensis
controls. Sample S was An. gambiae s.s control; Samples C, D, E,
F, G, H, I, K, M, N, P, Q were An. gambiae confirmed as An. gambiae
s.s. Assay conditions: 3% Agarose, 200 V, 149 mA, 029, 20 min
Samples not identified after 3 PCR reactions were marked as unknown. The distribution
of bands in the gel after electrophoresis was used to identify and determine
Anopheles gambiae siblings as An. gambiae s.s. and An. arabiensis
species whose oligo primers had been included in the master mix as controls.
The results are shown in Fig. 1.
Anopheles larval habitat types: Most of the larval habitats were man-made that is arise from human related activities and confined to valley bottoms. The area has experienced extensive swamp reclamation for crop production and creation of tree nurseries. The drainage channels formed water collection points in both dry and wet season. Bore holes common in tree nurseries and homes had water throughout the year. Hoof/foot prints in drained swamps, pastures were important breeding sites during dry season. Natural springs, undisturbed swamps, marshes were important permanent breeding sites particularly sunlit edges with less vegetation. In rivers and streams, breeding was common on the edges where water flow was slow and calm. During heavy rains when most of the channels become fast flowing streams/rivers, the Anopheles shifted breeding to water collections in cattle hoof prints in the reclaimed swamps. Brick making was a common economic activity that created suitable mosquito breeding grounds. Brick making resulted in innumerable borrow pits (flooded depressions left by soil excavation for brick material) provided prolific breeding sites for vectors. Most of the breeding sites easily dried up in a few days of dry spell, a few become more confined towards the valley bottoms and reappeared after rainfall while others were easily washed out (flush-out effect) following heavy downpour.
A variety of anopheline larval habitats identified in the study area were rivers, streams, boreholes, springs, swamps and animal hoof-prints that were more open to sunlight and plenty of algae. A majority of them were categorized as man-made. During the rainy season, 169 aquatic habitats were identified as potential breeding habitats and anopheline larvae were found in 72 (43%) habitats. In the dry season, the potential breeding habitats identified were 90 and 39% of them were anopheline positive larval habitats (Table 1). Man-made habitats for instance depressions associated with brick-making supported Anopheles mosquitoes in overcoming the dry period in many areas. These and other man-made habitats played a role in maintaining breeding throughout the year. Results of the ground survey showed that the number of potential anopheline aquatic habitats increased 1.9 fold during the rainy season over the dry season (169 vs. 90). However, there was no significant difference (ANOVA, p = 0.436) between the number of anopheline-positive breeding habitats in the rainy and in the dry season. Anopheline larval habitats were clustered at valley bottoms and composed of mainly man-made habitats.
Anopheles species diversity and abundance: A total of 387 female
adult Anopheles belonging to 11 species collected from study area. They were
identified by their morphological features and categorized in non-vectors and
known malaria vectors. The non-vectors belonged to 8 species comprising of An.
christyi Newstead and Carter, the most predominant species and ancestor
of all malaria vectors (Anthony et al., 1999)
followed by An. demeilloni Evans, An. coustani Levaran, An.
squamosus Theobald and An. harperi Evans. Other species found in
low numbers were: An. ziemanni Grunberg, An. leesoni Evans and
An. longipalpis Theobald. The known human malaria vectors were An.
gambiae, An. funestus and An. arabiensis comprising 11% in
3 species. An. gambiae s.l. was the most predominant known malaria vector
species while the other two species were rare. All known malaria vectors were
collected from indoors an indication of their close association with human habitation.
The diversity, relative abundance of vector and non-vector Anopheles mosquitoes
in the study area is shown in Table 2.
PCR assay of anopheles gambiae: The 41 An. gambiae sample specimens collected from the study sites were analyzed by Polymerase Chain Reaction (PCR). All were found to belong to one sibling species An. gambiae sensu stricto (s.s) indicating that it was possibly the only sibling species from the An. gambiae complex circulation in the study area. For quality assurance and comparative purposes, a run containing both An. gambiae and An. arabiensis was done to show how the results would have been in case the other sibling species Anopheles arabiensis would have been present in the samples. In both cases, single bands were visualized at different levels and photographed for An. arabiensis and An. gambiae s.s. The results are presented in form of photographed gel under UV light in (Fig. 1).
The study area is characterized by hill-valley topography. These topographical
features determine the formation of aquatic habitats. Hill-valley topography
facilitates run-off down-hill to settle in the valley bottoms forming aquatic
habitats such as springs, streams, rivers and swamps. The aquatic habitats surveyed
indicated that Anopheles breeding sites were confined at the valley bottoms
mainly in temporary, man-made habitats. It is suggested that probably Anopheles
species prefer open sunlit man-made habitats in which predation on larvae is
less prevalent in temporary habitats than it is in large permanent habitats
and competition for resources is less common in newly created man-made habitats
(Service et al., 1977; Washburn,
1995). In related studies, Minakawa et al. (2004)
reported a similar An. gambiae s.s larval habitat distribution in a highland
site in Kenya. The locations of breeding sites at valley bottoms suggest that
these locations could be associated with higher risk of malaria because they
are more likely to become malaria hot spots (transmission focal points) (Ernst
et al., 2006). Therefore, communities living near valleys have a
higher risk of contracting disease than those living uphill (Lindblade
et al., 2000; Balls et al., 2004).
This is in contrast to the lowland area of Lake Victoria Basin which is generally
a flat terrain and Anopheles breeding habitats are many and widely dispersed
(Gimnig et al., 2001; Minakawa
et al., 2002a, b). These generate high malaria
vector densities throughout the year hence perennial malaria transmission in
the lowlands (Bodker et al., 2003) compared to
seasonal transmission in the highlands.
The finding that larval habitat distribution was aggregated and mostly man-made
suggested that larval control in the study area could be targeted to the aquatic
habitats at the valley bottoms. Githeko et al. (2006)
suggested that in such situation, effective vector control could target the
confined breeding sites at specific sites and could also involve community participation.
Targeted larval control in the study area would be even more effective if undertaken
during the dry season just before the onset of rainy season when breeding habitat
distribution is limited by high evaporation rate leading to drying of most of
the aquatic habitats. And the few that remained were more confined towards valley
bottoms. It is suggested that source reduction (larval control) is the method
of choice for mosquito control when mosquito species targeted are concentrated
in small number of discrete habitats as is the case in the study sites. However,
in severe epidemic situations as is common in the western highlands of Kenya,
it would be prudent to apply Integrated Vector Management (IVM). In this regard,
source reduction (Githeko et al., 2006) and targeted
IRS (Chrispinus et al., 2011) would be appropriate
control strategies. Source reduction has been one of the important malaria measures
used to suppress malaria in Brazil (Soper and Wilson, 1943),
Tanzania (Bang et al., 1975, 1977)
and United States, Israel, Italy (Kitron and Spielman, 1989).
Human activities particularly related to land-use evident in the study area
are likely to promote malaria transmission in future in several ways as reported
elsewhere (Walsh et al., 1993; Patz
et al., 2000). For instance swamp reclamation creates more man-made
aquatic habitats for the local An. gambiae s.s, the predominant malaria
vector in the area. Increased cattle grazing creates more open habitats with
elevated temperature that favor faster vector larval development hence increased
adult vector density and malaria transmission (Minakawa
et al., 1999, 2004). Higher adult densities
of Anopheles gambiae complex have been reported in houses near established
malaria vector breeding habits in some highland areas (John
et al., 2004) and people living near valley bottoms (near to breeding
habitats) are likely have more vector-human contact than those up-hill and therefore
more malaria cases.
Entomological results indicate the presence of both known malaria vectors and
non-vector species in the study area. The significance of Anopheles species
not known to act as vectors is not clear. However, it is possible that some
of the species present a nuisance of mosquito-bites rather than transmit malaria
(Koenraadt, 2003). The study sites were characterized
with large herds of livestock (cattle, sheep and goats) that were often kept
near/around human habitations. For the zoophilic/ antropophilic species in this
group (including An. christy, An. demillon, An. harperi,
An. leesoni and An. longipalpis), the initial attraction emanating
from cattle/goats/sheep kept inside or around human houses may influence their
feeding behavior (Oyewole et al., 2007). As such
a possible change in behavior in host seeking Anopheles may increase the risk
of man becoming a regular source of blood meal by both zoophilic and anthropophilic
species. This could create a close link between man-animal-mosquito favorable
for the transmission of other mosquito-borne diseases in animals and man. For
instance, An. coustani laveran widespread and abundant over much of the
African continent also encountered in the study area readily feeds on humans
outdoors (Coetzee, 1983) and play a role in the transmission
of disease arboviruses (Logan et al., 1991; Gordon
et al., 1992; Coetzee, 1994). Other non malaria
vector species of the gambiae and funestus complexes are known
to transmit O’nyong nyong virus in East Africa (Williams
et al., 1965). It is possible that some of the non-vector anopheles
species may be of local importance in disease transmission as incidental vectors
as reported elsewhere (Gillies and De Meillon, 1968; Gillies
and Coetzee, 1987). In this regard the non-malaria vectors should not be
ignored. Efforts to eliminate them could turn out to be a community motivation
for adopting malaria control and prevention methods in the present study area
where adoption of malaria control measures is low because of the sporadic nature
of the disease.
The known malaria vectors present in study area were Anopheles gambiae,
An. funestus and An. Arabiensis. The three Anopheles species
are known to be the most efficient malaria vectors in the world (Besansky,
1999). This is because of their marked preference for human environments
and for humans as hosts and due to their rapid adaptation to changes in the
environment induced by human habitation and agricultural development (Collins
and Besansky, 1994; Powell et al., 1999).
An. gambiae was abundant malaria vector and the most prevalent of the
three species in the present study. These findings were consistence with related
studies in the same or neighboring sites as well as other highland regions in
Africa (Collins et al., 1988; Petrarca
et al., 1991). Previous vector studies in Nandi indicated malaria
vector composition of 98% An. gambiae complex and 2% An. funestus
(Roberts, 1964; Ernst et al.,
2006). In related studies in many parts of Africa, An. gambiae is
found together with the equally important vector An. funestus (Charlwood
et al., 2003; Nkuo-Akenji et al., 2006).
An. funestus and An. arabiensis were rarely encountered with only
one specimen of each collected translating into 2.4% of the total malaria vectors.
This could be because the two species have difficulties/apparent failure to
colonize high altitudes successfully. However, there is need for extensive studies
on larval and adult surveys and dispersal experiments targeting these species
to come up with a clear picture on diversity in the study area.
Malaria vector (s) and non-vectors may periodically extend their range beyond
their normal area of distribution when temporarily suitable conditions occur
in neighboring areas (WHO, 1998). This may explain the
presence of An. ziemanni in study area which is widely encountered in
West Africa extending to Ethiopia with scanty, localized distribution in East
Africa (Gillies and De Meillon, 1968). In Kenya, it has
been previously reported mainly in the low lands of Lake Victoria basin (Khamala,
1971; Kamau et al., 2006). In the present
study, the species was encountered but its vectorial importance is not clear
although, it is known to be susceptible to and can maintain malaria parasites
(Gillies and De Meillon, 1968), feeds on both man and
animals (Kamau et al., 2006). The species may
be undergoing a phase of adaptation to live in highland areas in proximity to
the normal habitat, the lowlands. There is need for further field and experimental
studies on An. ziemanni as regards possible role in malaria transmission
in both low and high altitude areas. The presence of a species of well known
malaria vector, An. arabiensis, a predominant vector in lowlands at high
altitude raises curiosity. If determined and confirmed in larger long-term studies,
the presence of An. arabiensis at the present high altitude area would
support scanty reports that the species is capable of breeding and transmitting
malaria in a highland area (Chen et al., 2006).
It is possible that regular travel between Lake Victoria lowlands and the western
highlands could introduce the vector into highlands. Both An. gambiae s.s
and An. arabiensis have similar requirements for their larval environment
(Service et al., 1977; Gimnig
et al., 2001). Therefore, there is a possibility that An. arabiensis
imported into the highlands could become established and become an important
malaria vector together with An. gambiae s.s in future. Whenever these
two species occur together, populations of An. arabiensis are known to
survive the dry season better while populations of An. gambiae s.s. peak
shortly after onset of rainy season (Koenraadt, 2003).
If this scenario is established, then malaria transmission in the western highlands
of Kenya could become perennial as opposed to the current seasonal transmission.
Malaria transmission in the western highlands of Kenya is seasonal, focal in nature, sporadic with more cases reported in communities living at valley bottoms where highest concentration of breeding habitats are located. It is concluded that intervention strategies should therefore be appropriately focused at these points when identified for cost effectiveness and better results.
The researchers acknowledge technical and field assistance from the Division of Vector-Borne Diseases [DVBD], Kapsabet Station and the Ministry of Health, Kenya and Kenya Medical Research Institute [KEMRI], Kisumu, Kenya.