Visceral Leishmaniasis L. Donovani; P. Orientalis Migrant Labourers Rodents Agriculture Fields Thickets of A. Seyal Dense Mixed Forest
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Date
2015-06-09
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Addis Ababa University
Abstract
Kala-azar (visceral leishmaniasis; VL), due to Leishmania donovani, is transmitted by
Phlebotomus orientalis in Metema Humera lowlands where this disease is a major health
problem. The aims of this study were to determine seasonal dynamics, habitat
preferences, nocturnal activities, host preferences, and Leishmania infections of P.
orientalis in addition to study on sero-prevalence of L. donovani infection in migrant
labourers with associated risk factors and the role of rodents as reservoir hosts of VL.
Centers for Disease Control and Prevention miniature light traps (CDC traps) (John W.
Hock, USA) and/or sticky paper traps were used for sand fly collections. The blood meal
sources for P. orientalis were detected by reverse line blot (RLB) of cytochrome b
polymerase chain reaction (PCR) amplification products using 11 probes for domestic
animals. Polymerase chain reaction amplification of internal transcribed spacer 1 (ITS1)
and kinetoplast DNA (kDNA) markers were used to detect Leishmania infections in P.
orientalis. Blood for direct agglutination test (DAT) was sampled, randomly, from
migrant labourers involved in sesame harvest to study sero-prevalence of L. donovani
infection and entomological risk factors. Sherman collapsible rodent traps were used to
capture rodents. The rodents were anaesthetized before sacrificed for tissue biopsies from
liver, spleen, bone marrow and tip of the nose (skin) in addition to blood for screening of
Leishmania infections. The species of the rodents were identified by their morphological
characters. The tissues from liver, spleen and skin were macerated in Locke’s solution
before transferring them into Novy-McNeal Nicolle (NNN) medium for
Leishmaniacultivation. Blood and touch smears of liver, spleen, skin and bone marrow
were made on microscope slide and allowed to air dry before fixing using methanol and
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staining by Giemsa stain for microscopy. Polymerase chain reaction technique was also
used to identify Leishmania infection in the tissues of the rodents. The studies were
conducted from May, 2011 to January, 2014. During study on bionomics of P.orientalis,
a total of 376, 441 sand flies using CDC light trap (n=955) and sticky traps (n=5, 551)
were collected from May 17, 2011 to June 6, 2012 from agriculture fields, thickets of
Acacia seyal and dense mixed forest. Of these sand flies, 313, 055 (80.45%) were
Sergentomyia species. The highest mean monthly density of P. orientalis trapped by
CDC light traps was found in thickets of Acacia seyal in March (64.11 + 75.87). The
corresponding highest mean monthly density of P. orientalis trapped by sicky traps was
found in April (58.69 + 85.20) in agricultural field. No P. orientalis were caught in
September using CDC traps and July - October using sticky traps. The overall mean
monthly density of P. orientalis (female and male) trapped by CDC light traps was 15.78
+ 28.93 (n=320) in agricultural field, 19.57 + 36.42 (n=255) in thickets of A. seyal, and
3.81 + 6.45 (n=380) in dense mixed forest. For sticky traps, the overall mean monthly
density of P. orientalis was 14.76 ± 38.78 (n=2378) in agricultural fields, 11.45 ± 15.56
(n=1500) in the thickets of A. seyal and 0.95 ± 2.16 (n=1168) in dense mixed forest.
Analysis of variance (ANOVA) result has showed statistically significant mean
difference (p=0.000) for different habitats. Phlebotomus orientalis, P. papatasi, P.
duboscqi, P. bergeroti, P. rodhaini, P. martini and P. alexandri were the Phlebotomus
species found in the area. Phlebotomus orientalis was the dominant species (99%) in the
extra-domestic study sites. During January to May, 2013 collection of P. orientalis at
hourly intervals using 22 CDC light traps, the peak activities of P. orientalis were at 1.00
hr (134.0 ± 7.21) near animal shelters, 3.00 hr (66.33 ± 46.40) in agricultural fields and
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21:00 hr (40.6 ± 30.06) in thickets of A. seyal. This species was not attracted to the
different species of rodents in trials carried out in March and April, 2013. Riverse line
blot PCR identified 7 human (28%), 9 mixed (human and cattle) (36%) and 2 cattle (8%)
blood meals while 7 were unknown (28%). Molecular screening of 30 pools (1 pool = 5
individual P. orientalis) from dense mixed forest in July (rainy season), 2011, for
Leishmania infection, was performed by targeting kDNA in a PCR assay. Five pools
(5/30, 16.7%) were positive for Leishmania kDNA PCR. For March–May, 2013 dry
season collections, 9/15 pools (60%) were ITS1 PCR positive. Of the total 359 labour
migrants screened during October – November (2013), using DAT, 45(12.5%) were
seropositive (≥1:800) for L. donovani infections with risk of VL development in 3 (0.8%)
individuals who had very high titer (1:12800). Leishmania donovani infection in labour
migrants seemed to correlate more with relatively higher density of P. orientalis during
the June – August weeding season than the September – October harvest season. Staying
in the areas both in the weeding and harvesting seasons (p=0.035; odds ratio (OR) = 2.83)
and sleeping in the agricultural fields (p=0.01; OR=15.096) were positively correlated
with L. donovani infection. Night harvest (p=0.028; OR=0.133) and knowledge about
sign or symptoms (p=0.042; OR=0.383) were negatively associated with this infection. A
total of 128 rodents such as Arvicanthis niloticus (n=68), Acomys cahirinus (n=25),
Tatera (Gerbilliscus) robustus (n=21), Mastomys erythroleucus (n=3), Mylomys albipes
(n=2), Rattus rattus (n=5), Paraechimus aethiopicus (Hedgehog) (n=2) and Xerus
erythropus (striped ground squirrel) (n=2) were trapped and screened for Leishmania
infections by parasitological, serological and PCR techniques. Of 91 rodents collected
from extra-domestic habitats of Beaker and Gelanzeraf (Kafta-Humera district) and
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analyzed by ITS1 PCR using skin, spleen, liver and bone marrow samples, 6/54 (11.1%)
of Arvicanthis nilothicus were positive compared to the infection rates in Acomys
cahirinus (3/17 or 17.6%) and Tarera (G) robustus (2/16 or 12.5%). Almost all the PCR
infections were found from bone marrow samples (8/48 or 16.7%) compared with
1/91(1.1%) liver, 2/87(2.2%) spleen and 0/87 (0%) skin. Different organs on the same
rodent were not found infected. These rodents were negative with NNN-medium,
microscopy (Giemsa stains) and direct agglutination tests (DAT) except 2 Arvicanthis
niloticus NNN-medium positives spleen samples from Baeker. The remaining 37
Arvicanthis niloticus,Acomys cahirinus, Tatera (G) robustus, Mastomys erythroleucus, Mylomys
albipes,Paraechimus aethiopicus (Hodge hoge), Rattus rattus and Xerus erythropus (striped
ground squirrel) collected from Baeker, Ademiti, Mayhas and Adijamus (western
Tigray)screening using NNN-medium, Giemsa stain and DAT were negative.
Agricultural fields and thickets of A. seyal habitats are the breeding sites for P. orientalis
in extra-domestic habitats of Kafta Humera lowlands where female P. orientalis can bite
humans at any hour of the night with peak biting after mid night. Sleeping in open
agricultural fields was related to L. donovani infections in labour migrants. Arvicanthis
niloticus, Acomys cahirinus and Tarera (G) robustus might play important role in the
transmission cycle of zoonotic VL in endemic lowlands areas of Ethiopia. Further studies
are required for L. donovani isolation from rodents in the endemic areas in addition to
experimental infection for xenodiagnosis before considering these rodents as reservoir
hosts of L. donovani conclusively. Access and proper use of bed nets, especially during
crop growing season, are required for reducing the incidence of the infection.
Key words:Visceral leishmaniasis; L. donovani; P. orientalis; migrant labourers;
rodents; agriculture fields; thickets of A. seyal; dense mixed forest
Description
Keywords
Visceral Leishmaniasis, L. Donovani; P, Orientalis, Migrant Labourers, Rodents, Agriculture Fields, Thickets of A. Seyal, Dense Mixed Forest