Disease Ecology and Epidemiology of Cutaneous Leishmaniasis Caused by Leishmania Tropica in Palestine
Disease Ecology and Epidemiology of Cutaneous Leishmaniasis caused by Leishmania tropica in Palestine
Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY”
by
Ikram A. Salah
Submitted to the Senate of Ben-Gurion University of the Negev
February 28, 2018
Beer-Sheva
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Disease Ecology and Epidemiology of Cutaneous Leishmaniasis caused by Leishmania tropica in Palestine
Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY”
by Ikram A. Salah
Submitted to the Senate of Ben-Gurion University of the Negev
Approved by:
______Burt Kotler Nadav Davidovitch (Advisor) (Advisor)
______Dudy Bar-Zvi (Dean of the Kreitman School of Advanced Graduate Studies)
February 28, 2018 Beer-Sheva
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This work was carries under the supervision of:
Prof. Burt Kotler
Marco and Louise Mitrani Department of Desert Ecology
The Swiss Institute for Dryland Environmental and Energy Research
The Jacob Blaustein Institute for Desert Research
Ben-Gurion University of the Negev
Prof. Nadav Davidovitch
Department of Health System Management
School of Public Health
Faculty of Health Science
Ben-Gurion University of the Negev
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Research-Student's Affidavit when Submitting the Doctoral Thesis for Judgment
I Ikram A. Salah, whose signature appears below, hereby declare that
(Please mark the appropriate statements):
X I have written this Thesis by myself, except for the help and guidance offered by my Thesis Advisors.
X The scientific materials included in this Thesis are products of my own research, culled from the period during which I was a research student.
This Thesis incorporates research materials produced in cooperation with others, excluding the technical help commonly received during experimental work. Therefore, I am attaching another affidavit stating the contributions made by myself and the other participants in this research, which has been approved by them and submitted with their approval.
Date: February 19, 2018 Student's name: Ikram A. Salah Signature:
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ACKNOWLEDGEMENTS
A special and profound thanks to my Mom, my sister Hala Salah, sisters, brothers, nephews, nieces and the rest of my extended family for theirs help, encouragement and support. How can I forget my angel who supports, encourages and stands by me to pass all difficult days.
I would like to express my gratitude to my supervisor Prof. Burt Kotler, for his good advice, encouragement and support, and for his continuous belief in me. I am truly thankful to my supervisor Prof. Nadav Davidovitch for opening me the window to the enthralling field of Eco- health, and for his encouragement and support. I also thank my committee members Yael Lubin, Hadas Hawlena, and Alon Warburg. Many thanks to my lab mates Elsita Kiekebusch, Austin Dixon, Jorge Menezes, and Stuart Summerfield, who provide input, discussion, help, and encouragement all the time.
Thanks to the whole community of Kisan Village, Arab Ar-Rashaiyda village, and Arab Al ‘Azazma in Bethlehem District. Thanks to the whole community of Tubas District. Special thanks to Taleb Qasal, Ezat Dragmeah family, and Mohammad Shriam family who were hosting me during the time of the field work. Many thanks to Taleb Qasal, Saad Dragmeah, Ahmad Othman, and Mahmood Bsharat who were helping me setting the traps and going with me house by house to conduct the interviews during the field work.
I am indebted to Prof. Alon Warburg for supporting, advising and making the research possible by providing most of the equipment for sand fly collection, and allowing me to conduct all molecular analysis for sand flies in his laboratory. Many thanks to Dr. Amer Al-Jawabreh for teaching me the basis of molecular biology, and giving me the courage to learn all the procedures of collecting, analyzing the patient’s samples. Last but not least, I am grateful to Dr. Ibrahim Abbasi for teaching me how to identify blood meal in sand flies, thanks for all support, help, and patience.
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This study was made possible by a generous grant from Ben-Gurion University president’s Prof. Rivka Carmi, Georg Waechter Memorial Foundation, and Science Training Encouraging Peace – Graduate Training Program (STEP-GTP).
And finally, thank you all. With love
I dedicate this work to the person who encouraged me all the time, my Father, May his soul rest in peace.
Also to the women who was dreaming all the time to have an educated daughter, my Mother, May God grant her good health.
"Save the World from Diseases"
(Ibn Sina – Avicenna)
"O men! Here is a parable set forth! Listen to it! Those on whom, besides Allah, ye call, cannot create (even) a fly, if they all met together for the purpose! And if the fly should snatch away anything from them, they would have no power to release it from the fly. Feeble are those who petition and those whom they petition!"
(Chapter 22, Al-Hajj: Verse 73)
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Table of Constants
Research-Student's Affidavit for Submitting the Doctoral Thesis for Judgment IV
Acknowledgements V
Table of Contents VII
List of Figures and Tables XII
List of Abbreviation and Terms XVI
Abstract XVII
CHAPTER ONE: INTRODUCTION 1
1.1.0.0.0. General background 1
1.2.0.0.0. Leishmaniasis 6
1.2.1.0.0. Geographical distribution 6
1.2.2.0.0. The disease 6
1.2.2.1.0. Cutaneous leishmaniasis (CL) 7
1.2.2.2.0. Mucocutaneous leishmaniasis (MCL) 7
1.2.2.3.0. Visceral leishmaniasis (VL) 7
1.2.2.4.0. Post Kala-Azar dermal leishmaniasis (PKDL) 8
1.3.0.0.0. The causative agent 8
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1.3.1.0.0. The parasite 8
1.3.2.0.0. The life cycle: 8
1.3.3.0.0. Transmission 8
1.4.0.0.0. The vector: Phlebotomine Sandflies (Diptera: Psychodidae) 9
1.4.1.0.0. Feeding behavior 9
1.4.2.0.0. Distributions of sand flies 9
1.4.3.0.0. Population dynamic and seasonal changes 10
1.4.4.0.0. Public health importance of sand fly distributions 10
1.4.5.0.0. Control methods of Sand flies 11
1.5.0.0.0. The Reservoir Host: Rock Hyrax Procavia capensis ( Hyracoidea: 12 Procaviidae)
1.5.1.0.0. Rock Hyrax distribution 12
1.5.2.0.0. Rock Hyrax, Reservoir host of Leishmaniasis 12
1.6.0.0.0. Leishmaniasis in Palestine 13
1.7.0.0.0. Eco-epidemiological approach 14
1.8.0.0.0. Overall aim and specific objectives 15
1.8.1.0.0. Overall aim 15
1.8.2.0.0. Specific objectives 15
1.9.0.0.0. General and specific hypothesis 16
1.9.1.0.0. General hypothesis 16
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1.9.2.0.0. Specific hypothesis 16
1.10.0.0.0. Innovation aspects 16
CHAPTER TWO: MATERIALS AND METHODS 17
2.1.0.0.0. Sand fly sampling at the Bethlehem study sites 17
2.1.0.1.0. Study area 17
2.1.0.2.0. Data collection 17
2.1.1.0.0. Comparison of sand fly activity between KIS, AAR, AZA in 2013 17
2.1.1.1.0. Comparison of sand fly densities among the three sites 17
2.1.1.2.0. Comparison in species for males among the three sites 19
2.1.1.3.0. Host blood species identification and Leishmania parasite detection 19
2.1.1.3.1. Sand flies identification 19
2.1.1.3.2. DNA extraction 19
2.1.1.3.3. Blood meal identification and parasite detection 20
2.1.2.0.0. Comparison of Sand fly activity between AAR, AZA in 2014 20
2.1.0.3.0. Statistical analysis 20
2.2.0.0.0. Epidemiological Investigation in the Tubas study sites 21
2.2.0.1.0. Study area 21
2.2.0.2.0. Data collection 21
2.2.0.3.0. Statistical analysis 23
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2.3.0.0.0. Sand fly transects for sampling the Tubas study site 23
2.3.0.1.0. Study area 23
2.3.0.2.0. Data collection 24
2.3.1.0.0. Sand fly density 24
2.3.2.0.0. Sand fly species identification 25
2.3.3.0.0. Leishmania parasite detection 26
2.3.4.0.0. Host blood species identification 26
2.3.0.3.0. Statistical analysis 26
CHAPTER THREE: RESULTS 27
3.1.0.0.0. Sand fly sampling at the Bethlehem study sites 27
3.1.1.0.0. Comparison of sand fly activity between KIS, AAR, AZA in 2013 27
3.1.1.1.0. Comparison of sand fly densities among the three sites 27
3.1.1.2.0. Comparison in species for males among the three sites 31
3.1.1.3.0. Host blood species identification and Leishmania parasite detection 33
3.1.2.0.0. Comparison of Sand fly activity between AAR, AZA in 2014 38
3.2.0.0.0. Epidemiological Investigation in the Tubas study sites 39
3.2.1.0.0. Demographic characteristics 39
3.2.2.0.0. Clinical information 43
3.2.3.0.0. Human behavior 45
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3.2.4.0.0. Personal protection 45
3.2.5.0.0. Topography and house information 48
3.2.6.0.0. Information about vector and reservoir 48
3.3.0.0.0. Sand fly transects for sampling the Tubas study site 51
3.3.1.0.0. Sand fly density 51
3.3.2.0.0. Sand fly species identification 52
3.3.3.0.0. Leishmania parasite detection 55
3.3.4.0.0. Host blood species identification 57
CHAPTER FOUR: DISCUSSION 60
4.1.0.0.0. Sand fly sampling at the Bethlehem study sites 60
4.2.0.0.0. Epidemiological Investigation in the Tubas study sites 65
4.3.0.0.0. Sand fly transects for sampling the Tubas study site 71
4.4.0.0.0. General discussion 73
REFERENCES 76
APPENDIX A: Villages profile 93
APPENDIX B: Cutaneous Leishmaniasis questionnaire 95
XX תקציר
XX הצהרת תלמיד המחקר עם הגשת עבודת הדוקטור לשיפוט
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List of Figures and Tables Figure 1: Epidemiological triangle of CL infection 3
Figure 2: Ross-Macdonald model schematic diagram for Leishmania 5
Figure 3: Sand fly sampling in Bethlehem study sites, green circles represent traps 18 location at the non-endemic site of KIS, purple triangles represent traps location at the endemic site of AAR, and the red rectangular represent traps location at the endemic site of AZA.
Figure 4: Epidemiological investigation study sites; red starts represent city cases, 23 green triangle represent village cases, and blue squares represent cases in Bedouin encampments
Figure 5: Trapping transects at Aleskan neighborhood in Tubas city, the pins 25 represents the traps stations, and the stars represents the hyrax colonies
Figure 6: Phlebotomus sand fly sex ratio distribution. Blue represents males, and 27 red represents females.
Figure 7: The relationship between trap elevation and mean total number of female 28 Phlebotomus sand flies caught in trap. The curve is fit with a quadratic regression showing that peak densities occur at intermediate elevations. Yellow represents AZA village, red represents AAR village, and blue represents KIS village.
Figure 8: The relationship between trap elevation and mean total number of male 29 Phlebotomus sand flies caught in trap. The curve is fit with a quadratic regression showing that peak densities occur at intermediate elevations. Yellow represents AZA village, red represents AAR village, and blue represents KIS village.
Figure 9: The relationship between trap elevation and proportion of blood fed of 30 female Phlebotomus sand flies caught in trap. Yellow represents AZA village, red represents AAR village, and blue represents KIS village.
Figure 10: The relationship between trap elevation and proportion of gravid female 30 Phlebotomus sand flies caught in trap. Yellow represents AZA village, red represents AAR village, and blue represents KIS village.
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Figure 11: Average number of Phlebotomus sand flies captured per month in the three 31 sites. The yellow curve represents AZA village, red curve represents AAR village, and blue curve represents KIS. Figure 12: Phlebotomus sand fly species composition for males in the three sites. 33
Figure 13: Phlebotomus sand fly species at each trap station for males. Red represents 33 P. sergenti. Blue represents P. papatasi. Light green P. alexandri represents, Violet represents P. jacusieli, Gray represents P. perfiliewi, Orange represents P. syriacus, and dark blue represents P. tobbi.
Figure 14: Gel image of cyto b BCR targeting DNA from blood-fed sand flies. M is 34 DNA ladder, from 171 to 183 PCR product of blood-fed sand flies, +ve, positive control (Cow), -ve negative control (pure water).
Figure 15A: Phlebotomus sand fly species at each trap station for females. Red 35 squares represent P. sergenti, blue diamonds represents P. papatasi, brown circles represent P. alexandri, and green line represents P. syriacus.
Figure 15B: The relationship between the numbers of flies with different types of 35 host blood found in each Phlebotomus species. Brown bars represents P. alexandri, blue bars represents P. papatasi, red bars represents P. sergenti, and green bars represents P. syriacus.
Figure 16: Spatial distribution of the species of host blood with which P. sergenti 36 flies were engorged. The blue curve represents human blood, red curve represents avian blood, green curve represents dog blood, and violet curve represents livestock blood.
Figure 17: Temporal distribution of P. sergenti flies engorged with blood of different 37 host species. The blue curve represents human blood, red curve represents avian blood, green curve represents the dog blood host, and violet curve represents livestock blood.
Figure 18: Gel image of ITS-PCR Leishmania targeting DNA from blood-fed sand 37 flies. M is DNA ladder, from 106 to 115 PCR product of blood-fed sand flies, +ve, positive control (L. infantum), and -ve, negative control (pure water).
Figure 19: Average number of Phlebotomus sand flies captured per month. The red 38 curve represents 2013. The blue curve represents 2014.
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Figure 20: Blood fed Phlebotomus sand flies proportion captured per month. The red 39 curve represents 2013. The blue curve represents 2014.
Figure 21: CL cases distribution by age and sex in Tubas district in 2015. Red bars 40 represent the males, and blue bars represent the females. Figure 22: Temporal distribution of CL cases in Tubas district. The red squares 41 represent the city, the green circles represent the villages, and the blue triangles represent the Bedouin encampments.
Figure 23: The location of CL cases in Tubas district. A represents city, B represents 44 Villages, and C represents Bedouin encampment.
Figure 24: The relationship between transects and the number of Phlebotomus sand 51 flies. The red bars represents total sand flies, green bars represents males, brown bars represents females without blood, and blue bars represents blood-fed females.
Figure 25: The relationship between the distance of trap station from hyrax colony 52 and the number of Phlebotomus sand flies. The red circle curve represents total sand flies, green square curve represents males, brown triangle curve represents females without blood, and straight blue curve represents blood-fed females.
Figure 26: Phlebotomus sand fly species composition. The black bars represents the 53 males. The white bars represent the females.
Figure 27: The relationship between the number of male sand flies and the distance 54 of trap station to the nearest hyrax colony for 6 species of sand flies.
Figure 28: The relationship between the number of female sand flies and distance of 54 trap station to the nearest hyrax colony for 5 species of sand flies.
Figure 29: Gel image of ITS-PCR Leishmania targeting DNA from blood-fed sand 55 flies. M is DNA ladder, from 1 to 12 PCR product of blood-fed sand flies, +ve, positive control (L. infantum), and -ve, negative control (pure water).
Figure 30: The relationship between number of pools of female sand flies with at 56 least one infected individual and distance to the nearest hyrax colony. The pink curve represent the infected pools of female without blood, and the brown curve represents the infected pools of blood-fed females.
Figure 31: The left pie represents the distribution of percentage of pools of female 56 sand flies with at least one infected individual across transects. Blue
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represents transect A, red represents transect B, and green represents transect C. The right pies represent the percentage of infected and non- infected pools in each transect. (See figure 3)
Figure 32: Gel image of cyto b BCR targeting DNA from blood-fed sand flies. M is 57 DNA ladder, from 19 to 28 PCR product of blood-fed sand flies, -ve negative control (pure water).
Figure 33: The number of sand flies on each transect engorged with blood for four 58 categories of host species. Blue bars represents human blood, red bars represents hyrax blood, green bars represents dog blood, and violet bars represents livestock blood.
Figure 34: The number of females engorged with blood as a function of distance of 59 the trapping station to the nearest hyrax colony for four categories of host species. Blue curve diamond represents human blood, red square curve represents hyrax blood, green triangle curve represents dog blood, and violet straight curve represents livestock blood.
Table 1: The Shannon-Weiner diversity index (H), evenness (E) and richness(S) for 32 the Phlebotomus sand fly males in different site.
Table 2: The correlation between site and number of male Phlebotomus sand fly 32 species captured per month. Type III SS is Sum of squares for a fixed factor
Table 3: The correlation between altitude of trap station and number of Phlebotomus 32 sand fly species captured per month for males. Type III SS is Sum of squares for a fixed factor
Table 4: Population Characteristics of case and control respondent 42
Table 5: Clinical information about the CL cases in Tubas District. 43
Table 6: Human behavior information of respondents 46
Table 7: Personal protection used by respondents 47
Table 8: Information about houses of respondents 49
Table 9: Information about vector and reservoir 50 Table 10: Host blood species of different sand fly species in July and September 58 identified by Cyto b PCR and RLB.
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List of Abbreviations and Terms
AAR Arab Ar-Rashaiyda AZA Al ‘Azazma ARIJ Applied Research Institute - Jerusalem CDC Centers for Disease Control CL Cutaneous Leishmaniasis DDT Dichloro-Diphenyl-Trichloroethane (insecticide) EID Emerging Infectious Diseases IR Incidence Rate ITS Internal Transcribed Spacer KIS Kisan MCL Mucocutaneous Leishmaniasis PCBS Palestinian Central Bureau of Statistics PCR Polymerase Chain Reaction PKDL Post-Kala azar Dermal Leishmaniasis RLB Reverse Line Blotting VL Visceral Leishmaniasis WHO World Health Organization
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ABSTRACT
Background: Infectious diseases have comprised an increasingly influential role in the morbidity and mortality of humans during the last several decades. Since the 1970s, there has been a resurgence of many vector-borne diseases including malaria, yellow fever, West Nile virus, and Leishmaniasis. The combination of disease ecology and epidemiology can create a better understanding of how humans and their environment affects the manner in which they come into contact and interact with the vector. It also allows better identification and targeting of the weak links in disease transmission for controlling the disease. In the Palestinian West Bank, leishmaniasis is emerging as a serious public health issue as its incidence continues to increase over time, especially in the western and the northern parts. This study was initiated to improve our understanding of the ecological, epidemiological, and transmission dynamics of cutaneous leishmaniasis (CL) caused by Leishmania tropica in the West Bank. It aims to: (I) evaluate the effect of altitude on sand fly density within and between three villages in the Bethlehem District, (II) carry out risk assessment case-control epidemiological studies of Leishmania tropica based on questionnaires administrated in the endemic area of the Tubas District, and (III) to evaluate the effect of distance from hyrax Procavia capensis (the reservoir hosts) colonies on Phlebotomus sergenti (the vector sand fly species) densities within an urban landscape and between the urbanized area and adjacent hyrax colonies.
Methods: First, I conducted a study in the district of Bethlehem (southeastern West Bank) encompassing three villages that differ in elevation and endemicity: Kisan (KIS), Arab Ar- Rashaiyda (AAR), and Al‘Azazma (AZA). The three villages occur along a cline in elevation, ranging from the non-endemic area of KIS (732-782 m ASL), down to the endemic areas of AAR (522-68 m ASL), and AZA (473-510 m ASL). Sand flies were trapped using CDC traps baited with dry ice to quantify sand fly abundance in the three villages. Second, I divided towns and villages in Tubas District in the northeastern West Bank into three different groups according to their urbanization and socio-economic levels: the city of Tubas, six villages, and Bedouin encampments. A matched case-control study of leishmaniasis was conducted in the endemic areas. One to three controls were selected per case and matched for age, sex, and
XVII socioeconomic background. Questionnaires included demographic data, epidemiologic data, characteristics of the house, and information about the vector and the reservoir host. They were administered to each case and control to determine the epidemiological risk factors that are most closely correlated to disease exposure. Third, in Tubas District, sand flies were trapped in July and September of 2016 in a hot spot for Leishmania infections. The effect of distance from hyrax colonies was examined by quantifying sand fly abundance in the Aleskan neighborhood in the city of Tubas along five trapping transects running from a rocky area containing hyrax into an area of inhabited houses. Trapping was conducted each month using CDC light traps. The trapped sand flies were counted, and each fly was sexed and identified to species. Leishmania parasite detection was conducted on the phlebotomus females using PCR amplification of the internal transcribed spacer (ITS1), and the blood from recently consumed meals identified to species using the Cytochrome b PCR and Reverse Line Blotting (RLB) technique. Results: First, the abundance of sand flies differed among the three villages with AAR> AZA>KIS. The sex ratio of captured flies also differed between the three villages, with even sex ratios in AAR and AZA, and female biased sex ratio in KIS. Elevation correlated with female Phlebotomus sand fly abundance, with greatest densities found at intermediate elevations (AAR). Elevation also correlated negatively with the proportion of blood-fed female Phlebotomus sand flies both within and between villages. Species composition measured in male Phlebotomus sand fly species differed among sites, in which all the species were present in AZA, while some species were absent from AAR and KIS. The host blood species engorged by P. sergenti were grouped into four groups: human (45.5%), livestock (25%), avian (19.9%), and dog (9.6%). Sand fly abundances at AAR and AZA differed seasonally and between years. Second, In the case-control study, I found a trend towards differences in the proportion of infected individuals according to gender and a significant difference in the age of those infected with Leishmania between the three levels of urbanization. The city and villages had more male cases while the Bedouin encampments had more female cases. When comparing age classes, children had the highest number of cases, followed by the most elderly in the city, and the middle aged in the villages and Bedouin encampments. The peak of CL cases occurred in December and January. People in the three areas differed in the precautionary measures taken against sand flies. In the city, cases used vaporizing tablets and spraying inside their houses to repel or kill flies more than controls. Proximity to reservoir hosts appears to provide another risk
XVIII factor as the houses of cases were closer to the city’s edge than controls. Other risk factors include: living closer to farms, raising domestic animals, and living near a known hyrax colony. In the villages, risk was dominated by distance to the village edge, while in the Bedouin areas, risk was driven by how close hyrax colonies were to living quarters. Third, 1,051 Phlebotomus sand flies from 9 species were captured, of which 470 (5.2 per trap/night) were captured in July, and 581 (6.5 per trap/night) were captured in September. Distance to hyrax colonies correlated negatively with the total number of Phlebotomus sand flies, as did the number of P. sergenti males and females. All sand flies that were infected with Leishmania carried Leishmania tropica, and all of the infected flies were P. sergenti. Infected female sand flies carrying blood meals were captured closer to hyrax colonies than infected non-fed females. Sand flies carrying blood meals with hyrax blood occurred significantly closer to hyrax colonies than those with other types of blood. A similar trend was observed for those carrying dog blood.
Conclusions: This study helps lead to a better understanding of disease transmission to humans and provides a deeper understanding of zoonotic diseases through the ecological study of zoonoses and its link between epidemiology, sociology, and ecology. In regards to the sand fly vector, I show a relationship between elevation and sand fly density, although more data spanning more villages, greater elevational changes, and more time are needed to generalize this conclusion. Also, I provide the first evidence in support P. sergenti being the vector of L. tropica in Bethlehem and Tubas districts. Disturbances caused by urbanization can provide new habitats for the reservoir. Efficient transmission of the Leishmania parasite occurs if the reservoir and the vector live in close proximity and the flies tend to feed upon the reservoir hosts. This research shows that Phlebotomus sand flies occur in higher densities closer to hyrax colonies. Therefore, understanding leishmaniasis risk factors requires knowing, not only sand fly density, but also species composition, feeding success, and distance from the reservoir. In more urbanized sites, the effect of living next to the city or village’s edges, facing the wadies, or being near open green areas increase the exposure to vectors (sand fly) and the proximity to the reservoir (hyrax). In addition, the risk of disease transmission increased with a high density of reservoirs (Hyrax), closer proximity of reservoirs to human houses, high densities of the vector (sand fly), and the absence of personal protection implemented against the vector. Such knowledge can allow us to better identify areas with the greatest risk of infection and to focus our efforts on these areas to control sand flies and encourage behaviors in local residents that reduce the transmission of
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Leishmania. The results of this research must be taken into account when implementing future interventions to reduce CL incidence in Palestine
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CHAPTER ONE: INTRODUCTION
1.1.0.0.0. General background:
Disease ecology is the study of diseases and their dynamics and distributions through an understanding of evolution and the interaction among humans or other hosts, the vectors, the pathogens, and the environment (Harvell, 2004; Mayer, 1996; Wilcox and Gubler, 2005). This approach merges the principles of ecology, epidemiology, microbiology, geography, genetics, medicine, and mathematics to facilitate a better understanding of emergence, re-emergence, and persistence of diseases, especially for infectious diseases (Plowright, et al., 2008).
Infectious diseases caused by pathogenic microorganism such as viruses, bacteria, fungi, or parasites comprise an increasing cause of morbidity and mortality during the last several decades (WHO, 2012). Adaptation and evolution of infectious disease agents are resulting in the emergence of new infectious diseases and the spread of old ones. 60.3% of Emerging Infectious Diseases (EIDs) are zoonotic; 71.8% originate in wildlife (Jones, et al., 2008). EIDs are correlated with climatic, environmental, ecological, and socio- economic factors such as temperature, rainfall, species distribution, species abundance, and human population density. In addition, vector borne diseases are responsible for 22.8% of EIDs, mostly due to changes in the climate that affect vectors through environmental factors such as temperature and rainfall (Jones, et al., 2008). This has created a need for increased surveillance and monitoring, healthcare, vaccine development and distribution, and disease prevention.
A zoonosis is caused by a disease pathogen that naturally spills over across species from a vertebrate animal source to humans (WHO). It also can be caused by eating undercooked or raw `meat' containing infective tissue stages, or by ingesting environmental transmissive stages that contaminate food or water (Slifko, et al., 2000). The emergence of new zoonotic diseases may depend on different factors, including animal movement (migration, range shift or expansion, etc.), the presence of unrecognized pathogens, and ecological disruption factors (Brown, 2004). The transmission of the pathogen can occur when the reservoir and the vector come in close contact. A zoonosis requires temporal and spatial co-existence of vector, reservoir, and pathogen to be established. This can be specified by a combination of favorable environmental factors, climate, vegetation, and soil (Sousa and Grosholtz, 1991). The host’s
1 reservoir competence, which is the ability to transmit a pathogen to a vector, plays an important role in the infection prevalence of the vector. Increasing the abundance of a weakly competent reservoir in a community will increase the dilution effect (a phenomenon that happens when the community has high evenness in species richness of hosts leading to inefficient transmitting of pathogens to a vector), which lowers the disease transmission probability from a vector (Schmidt and Ostfeld., 2001).
Parasitism is an ecological association between individuals of one species (the parasite) and those of another species (the host) on or in which they live. The parasite normally causes harm, but not immediate death to the host and obtains its nutrients from its host individual. The host- parasite interaction is a dynamic process in which the parasites may regulate the host population dynamics (May and Anderson, 1979). Pathogens are divided into two classes that differ in life form and population parameters; microparasites—including viruses, bacteria, protozoa, and prions—and macroparasites—including parasitic helminthes, arthropods, and fungi and higher plants. The transmission of both groups can occur directly from host to host by inhalation, ingestion, sexual transmission, or penetration of the skin, or indirectly through a blood-sucking vector, intermediate host, and penetration by a free-living stage.
Infectious diseases dynamics and the interactions between host, environment, and pathogen, can be described by the epidemiological triangle. The triangle shows the determinants of the disease that either inhibit or facilitate the infection and their interrelationships. A typical example of the epidemiological triangle model is Cutaneous Leishmaniasis (CL) (Fig. 1). The CL dynamics emerge as a result of the interactions between the infectious agent (Leishmania. tropica), the host (hyrax), and the environment that promote the exposure to the agent (Gordis, 1996).
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Figure 1: Epidemiological triangle of CL infection
Ecologists examine infectious disease occurrence from the perspective of evolution and population dynamics and the ecology of host–pathogen interactions. The conceptual basis for understanding such dynamics begins with mathematical models, (usually a system of differential equations) representing populations of hosts and parasites that allow the analysis of the dynamics, spread, and control of infectious diseases (Hethcote, 2000; Smith et al., 2005). The Ross-MacDonald model provides the basis for examining a vector borne disease. It originated in the work of Ross (1911) and MacDonald (1957), and is a mathematical model that includes epidemiological and entomological concepts for measuring malaria transmission and mosquito- transmitted diseases. One parameter of this model is vectorial capacity, C, which measures the efficiency of vector-born disease transmission (Smith, et al., 2012). It is defined as ‘the number of new infections disseminated per case per day by a vector” (equation 1; Macdonald 1952, Garrett-Jones 1964). Parameters that compose C include m, the density of vectors in relation to density of host, a, the proportion of vectors feeding on a host divided by the length of the gonotrophic cycle of the vector in days, V, vector competence, P, the daily survival of vectors, and n, the extrinsic incubation period:
3
C = ma2VPn/-lnP (Eq. 1)
Let us consider a Ross-Macdonald model (Fig.2) that addresses the dynamics of pathogen transmission and persistence of microparasites, which is especially appropriate for vector-borne diseases such as leishmaniasis. The main results of this model can be examined by considering the reproductive number R0, the number of secondary cases resulting from a single case (Anderson and May 1980; Macdonald, 1957). Here, m is the number of mosquitoes (vector) per host, b1 is the infectiousness of reservoir to vector,b2 is the susceptibility of reservoir, μ is the mortality rate of adult vectors, T is the incubation period of parasites within the vector, and r is the rate of recovery of infected hosts (see Equation 2).
In this model, the parasite needs a minimum vector population density of susceptible individuals in order to sustain itself and persist, i.e., the threshold vector density such that the reproductive rate R0 is equal to or exceeds 1. For this to occur, the parasite needs sufficiently high density of vectors, (m), bite frequency, (a), high number of susceptible reservoir hosts (b2), high number of susceptible vectors (b1), low recovery of infected humans (r), and low mortality rate of vectors (μ). By decreasing vector population density, the number of susceptible vectors can be lowered below the threshold; eradication of the disease will be achieved.
2 -μT R0 = ma b1b2e / rμ (Eq. 2)
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Figure 2: Ross-Macdonald model schematic diagram for Leishmania, Ross (1911), Macdonald (1957).
Other models provide the theoretical foundations for understanding disease dynamics, such as SIR (susceptible, infected, recovered) models originated by the work of Kermack and McKendrick, (1927), Ross’s (1916) epidemic model, related models by Anderson and May (1979, 1982), (Brauer, 2005), and SEIR (susceptible, exposed, infected, recovered) models originated by the work of (Mukandavire et al., 2001) which was developed to understand the climate relationship of cholera in Zimbabwe. Also, Anderson and May (1978, 1979) discuss the importance of combining epidemiology, parasitology, and ecology for modeling and better
5 understanding of host-pathogen interactions. They discuss the important role of merging epidemiological parameters such as transmission rate, β, disease-induced mortality rate, α, and recovery rate, υ, with ecological concepts from predator-prey theory of Lotka and Volterra from the 1920s.
Interdisciplinary approaches have important roles in our understanding of disease transmission from vertebrates to humans. Because of the zoonotic nature of leishmaniasis, merging ecological concepts and modeling with epidemiological parameters and human sociological factors can greatly inform us about disease dynamics. Furthermore, it can help us to forecast upcoming disease 'hot spots' and help us to provide evidence for long-term planning.
1.2.0.0.0. Leishmaniasis 1.2.1.0.0. Geographical distribution:
Leishmaniasis is a group of diseases endemic in many tropical and subtropical countries around the world, and is found in more than 98 countries and 3 territories on 5 continents, with an annual incidence rate of 220,000 cases per year (Alvar et al., 2012). On the list of top 10 most neglected tropical diseases, Leishmaniasis is 8th with a burden of nearly 2 million disability- adjusted life years (McDowell et al., 2011; Reddy et al., 2007). One third of the cases of all types of leishmaniasis combined occur in each of three regions: the Americas, the Mediterranean basin, and Western Asia from the Middle East to Central Asia. In regards to Cutaneous Leishmaniasis (CL), five of the 10 countries that together account for 70 to 75% of the estimated global CL cases are in and around the Middle East, including Afghanistan, Algeria, Iran, Syria and Sudan. In the Mediterranean basin, the number of reported CL cases in the period between 2004 and 2008 was 85,555, with an estimated range of 239,500 to 393,600 during the same time period (Alvar et al., 2012).
1.2.2.0.0. The disease:
Leishmaniasis is a vector-borne disease caused by a protozoan parasite of the genus Leishmania (Kinetoplastida: Trypanosomatidae). It is transmitted by phlebotomine sand flies (Diptera: Psychodidae). The pathology of leishmaniasis varies, ranging from non-ulcerative lesions to ulcerative skin lesions to life threatening visceral leishmaniasis (VL). The clinical manifestations of the disease depend on the infecting Leishmania species and the immune
6 response of the host. Leishmaniasis manifests in three clinical syndromes: CL, VL, and Mucocutaneous leishmaniasis (MCL) (Pearson, & de Queiroz Sousa, 1996).
1.2.2.1.0. Cutaneous leishmaniasis (CL):
CL is caused by Leishmania major, Leishmania Tropica, and Leishmania aethiopica in the Old World, and Leishmania amazonensis, Leishmania guyanensis, Leishmania panamensis, Leishmania peruviana, Leishmania mexicana, and Leishmania braziliensis ssp. in the New World (Ashford, 2000). People who recover from CL are mostly immune to re-infection, but the immunity does not extend to all Leishmania species. Secondary infection may occur in up to 10% of patients (Killick-Kendrick, 1985). Two to six weeks after a patient is bitten by an infected sand fly, a small erythematous papule appears on that person. The papule slowly enlarges and frequently ulcerates (‘wet’) or becomes a nodule (‘dry’). The sores normally heal spontaneously after 6 - 12 months, leaving prominent scars. The lesions caused by L. tropica are more difficult to treat and take longer to heal than those of L. major (Al-Jawabreh et al., 2004; Grevelink and Lerner, 1996; Klaus, 1999).
1.2.2.2.0. Mucocutaneous leishmaniasis (MCL):
MCL is caused by L. braziliensis, L. amazonensis, L. panamensis, and L. guyanensis in the New World, and is occasionally caused by L. infantum in Sudan and other places in the Old World. MCL manifests as chronic lesions in the patient’s mouth and nose, where it erodes underlying tissue and cartilage. MCL is more severe than CL, as it produces disfiguring lesions and mutilations of the face, nose, and throat (Ashford, 2000; Choi and Lerner, 2001; Desjeux, 1999).
1.2.2.3.0. Visceral leishmaniasis (VL):
VL is also named Kala-Azar, meaning “black fever.” It is caused by L. d. donovani or L.d. infantum. VL has an incubation period of 2-4 months. It affects the visceral organs such as the liver, spleen, and bone marrow. The symptoms of VL are prolonged fever, splenomegaly, hepatomegaly, substantial weight loss, progressive anemia, pancytopenia, and hypergammaglobulinemia. 95% to 98% of the cases can be successfully treated, and relapses are not common (Ashford, 2000; Choi and Lerner, 2001; Grevelink and Lerner, 1996).
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1.2.2.4.0. Post Kala-Azar dermal leishmaniasis (PKDL):
PKDL is a serious sequel to Kala-Azar following treatment with penta-valent antimonials (the most commonly used group of drugs). Skin nodules with abundant L. donovani amastigotes (see below section 1.3.1.0.) appear within months or years after symptoms from VL have disappeared following treatment. PKDL starts as a rash around the mouth, and then spreads to other body parts. Clinical diagnosis is mainly used with limited sensitivity for parasite detection, but evidence for the parasite appears in 80% of the cases using PCR (Ashford, 2000; Zijlstra et al, 2003).
1.3.0.0.0. The causative agent: 1.3.1.0.0. The parasite:
The genus Leishmania (Kinetoplastida: Trypanosomatidae) includes approximately twenty species that are pathogenic to humans (Cupolillo et al., 2000). Two morphological forms exist for this parasite: the nonflagellated amastigote (3-5 µm in diameter), which lives intra-cellularly in macrophages of the mammalian host, and the flagellated promastigotes (15-30 µm in length, plus the flagella), which lives extra-cellularly in the intestinal tract of the sand fly-vector.
1.3.2.0.0. The life cycle:
The transmission cycle of the parasite occurs through the bite of female Phlebotomus and Lutzomyia sand flies. When a sand fly female bites an infected reservoir host, it ingests Leishmania amastigotes during the blood meal. Flagellated promastigotes then develop from amastigotes in the gut of the sand fly. Later, mature promastigotes can be transmitted to a new mammalian host individual during subsequent blood meals. After arriving in the vertebrate host, the parasites are engulfed by macrophages where they proliferate by binary fission as intracellular amastigotes.
1.3.3.0.0. Transmission:
The transmission of Leishmania is either zoonotic or anthroponotic. The transmission of vector-borne zoonotic diseases requires reservoir host species and vectors. Leishmania species depend on specific vectors and reservoir hosts. Efficient transmission of the Leishmania parasites occurs if the reservoir and the vector live in close proximity and the flies tend to feed upon the
8 reservoir hosts (Ashford, 2000). A variety of ecological and biological factors affect the presence of leishmaniasis, which requires suitable ecological conditions for both the hosts and the vectors.
1.4.0.0.0. The vectors: Phlebotomine Sand flies (Diptera: Psychodidae):
Phlebotomine sand flies are nocturnal insects that rarely exceed 3 mm in length. Two genera out of six are considered medically important: Phlebotomus in the Old World and Lutzomyia in the New World. The species of these genera are the vectors for leishmaniasis, including 31 species that have been implicated as vectors, and another 43 species as probable vectors (Banuls et al., 2007; Killick-Kendrick, 1990, 1999, Ready 2013).
The Phlebotomine life cycle includes four life stages: eggs, needing approximately 7 days to hatch, followed by four larval instars needing 21 days in total, then a pupa needing 7 days, followed by the emergence of adults. Sand flies hide during daytime hours to avoid desiccation and become active during the night.
Sand fly adults are weak fliers. Their flight distance does not exceed two kilometers, with a speed of less than 1 meter per second. Consequently, their resting or breeding places include: animal shelters, houses, rocks, dense vegetation, tree holes, bird nests, rodent burrows, rocky slopes, and inside caves inhabited by rock hyraxes (Alexander, 2000; Feliciangeli, 2004; Killick- Kendrick, 1999; Moncaz, et. al., 2012).
1.4.1.0.0. Feeding behavior:
Sand fly adults of both sexes feed on plant honeydew and nectar, but females also feed on mammalian blood for egg production (Killick-Kendrick, 1999; Schlein and Warburg, 1986). Knowledge about the feeding success of sand flies and blood host identification is essential for the establishment of an efficient control strategy. Their feeding preference depend on the host availability and accessibility in their feeding. P. sergenti feeds on birds and mammals (Svobodova et al., 2003) or on human, ovine, avian, and feline hosts (Maroli, et al., 2009). And L. longipalpis feeds on humans, chickens, dogs, horses, and cats (Gaudêncio et al., 2015).
1.4.2.0.0. Distributions of sand flies:
Phlebotomine sand flies are distributed over large geographical regions, and are present in warm zones of Africa, Asia, Australia, America, and southern Europe (Killick-Kendrick, 1999).
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1.4.3.0.0. Population dynamic and seasonal changes:
The activity period of sand fly adults is seasonal. The appearance or disappearance of sand flies differs between sites, and there is seasonal variation in densities at each site. In the Mediterranean region the available information about seasonal dynamics of sand flies is spotty (Alten et al., 2016). Sand flies start to appear in spring, with peaks of population densities differing between sites (Alten et al., 2016). Previous studies showed that the peak densities of sand flies differ between sites, and may include peaks in April or May (Wasserberg et al., 2003), August or September (Belen & Alten, 2011; Orshan et al., 2010), and September or October (Wasserberg et al., 2003). In addition, previous studies showed that sand fly species have peak densities at different times. P. sergenti peaked on May or June (Doha & Samy, 2010; Faraj et al., 2013), P. tobbi peaked on June (Alten et al., 2016), and P. alexandri peaked in July and August (Doha & Samy, 2010).
Sand fly abundances and development times are extremely sensitive to high temperature and low humidity (Ibrahim, et. al., 2005; Simsek et. al., 2007; Theodore, 1936). Similarly, temperatures below 10o C are unfavorable for sand fly survival, and all sand flies die when exposed to temperature above 40o C for even short time periods. Suitable temperatures range from 24o C to 34o C. Sand fly survivorship increases with relative humidity, with 30% to 40% RH providing suitable conditions (Cross et al., 1996; Ibrahim, et. al., 2005). Other environmental variables also affect the distribution of sand flies. Previous studies showed that soil moisture affects sand fly densities, which in turn is higher in disturbed areas close to human settlement and with more lush vegetation (Wasserberg, et. al., 2002, 2003). Furthermore, the abundance of the sand fly is significantly higher in loessal soil habitat than in sandy habitats (Wasserberg, et. al., 2002, 2003). Other physical factors affect sand fly distributions as well, including geographical barriers, the distribution of vertebrate hosts, and habitat availability (Cross et al., 1996; Ghosh et al., 1999; Sawalha et al., 2003).
1.4.4.0.0. Public health importance of sand fly distributions:
Sand flies are important from both clinical and public health perspectives because they are the vectors of leishmaniasis, as well as two other major diseases, Sand fly fever and Bartonellosis. Sand fly fever, also called papataci fever, is transmitted by P. papatasi. During
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World War II, sand fly fever and leishmaniasis were considered to be important causes of morbidity among military personnel deployed in the Middle East (Cross et al., 1996; Tavana et al., 2001). Bartonellosis is a fatal disease caused by Bartonella bacilliformis. It is also called Carrión's disease, and is limited to a specific geographic area characterized by high, dry valleys in Peru where the vector is the sand fly Lutzomyia verrucarum (Maco et al., 2004; Maguiña et al., 2009). Furthermore, sand fly bites are painful, promote delayed–type hypersensitivity in exposed people, and may cause considerable discomfort for prolonged periods (Barral, 2000; Belkaid, 2000).
1.4.5.0.0. Control methods of sand flies:
Control of sand flies is often a public health priority. Yet control of phlebotomine sand flies is notoriously problematic because the breeding sites of their immature stages are usually inaccessible or unknown. Thus, source reduction is usually impractical. There are three main control methods of the sand fly vector; biological control, chemical control, and environmental management (Warburg and Faiman, 2011). Chemical control: Sand flies are highly susceptible to insecticides (Alexander et al., 1995a; Alexander and Maroli 2003; Orshan et al., 2006; Wilamowski and Pener, 2003). In Latin America, spraying insecticides in homes is effective against endophilic species (Alexander et al., 1995a; Vieira and Coelho, 1998). In the Old World and in the Neotropics, formulations of DDT and pyrethroids have been used as a control for sand flies. (Hertig, 1949; Hertig and Fairchild, 1948; Hertig and Fisher, 1945; Marcondes and Nas- cimento, 1993). In recent years, one of the best methods for controlling sand fly adult and larvae is using food baits for rodents or other mammals that contain a feed-through insecticide, such as Fipronil (Ingenloff et al., 2013; Mascari et al., 2013), Imidacloprid (Wasserberg et al., 2010), or Ivermectin (Mascari et al., 2008). Biological control: Some plants are toxic to adult P. papatasi, including Solanum jasminoides, Ricinus communis, and Bougainvillea glabra, so planting these plants can reduce sand fly densities (Schlein et al., 2001). Environmental management: Environmental modification such as removal of all perennial vegetation that reservoir hosts feed upon has been applied in and around permanent settlements (Alexander and Maroli, 2003; Kamhawi et al., 1993), but is extremely deleterious to the environment and often impractical.
1.5.0.0.0. The Reservoir Host: Rock Hyrax Procavia capensis ( Hyracoidea: Procaviidae):
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Rock Hyraxes (Procavia capensis) (Wabr in Arabic) are the only living species of the genus Procavia in Africa and southwest Asia, and belongs to the Procaviidae family (Olds and Shoshani, 1982). It is a medium size mammal, with the average weight of an adult of 4 kg and average weight of newborns between 170-240g. After 6-7 month of gestation, well developed offspring are born, which can jump 2 days after birth. They reach reproductive maturity in 16 months. Hyraxes are generally diurnal, with their activity starting in the morning shortly before sunrise, dropping at midday, and increasing again in the afternoon. They can also be active at night and on warm days in the desert (Mendelssohn, 1965). Hyraxes are principally folivorous, feeding on leaves of perennial plants but may also consuming grasses, forbs, and fruits. They prefer to forage close to their colony place. Rock hyraxes are highly social and can live in colonies of up to dozens of individuals. They are restricted to rocky habitats and kopjes, use rock crevices and boulder mounds for shelter, may move around from den to den, and range out from rocky refuges into the surrounding habitat for food (Kotler et al., 1994).
1.5.1.0.0. Rock Hyrax distribution:
Rock hyraxes range widely from southern Africa, up the African Rift Valley into Northern Israel and Syria, across sub-Sahara Africa, and along the costal Arabian Peninsula (Harrison and Bates, 1991).
1.5.2.0.0. Rock Hyrax, Reservoir host of Leishmaniasis:
Little information is available on the ecology of reservoir hosts of zoonotic diseases (Ashford, 1996; Mills and Childs, 1998). Reservoir host parameters important for understanding their function in zoonotic systems include host distribution, population dynamics, habitat selection, foraging, demography, and dispersal. Improving our understanding of these important ecological factors constitutes an objective of disease ecology. Such an understanding may illuminate the “weak link” in the transmission cycle at which control efforts should be aimed.
The rock hyrax has several attributes that may make it an effective host. Like the fat sand rat, the rock hyrax has short, thick ear pinnae that may remain infectious for several months, therefore increasing the probability of disease transmission. Rock hyraxes live for many years, potentially creating a bridge over winter without transmission. They are diurnal species, so they sleep when sand flies are active, which helps sand flies avoid the defenses during the blood meal.
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Furthermore, its use of communal latrines, the accumulation of organic material in its den, and the moderate den environment under rocks may be favorable to the sand fly larvae, and its aggregated distribution promotes transmission (Ashford, 1996).
Rock hyraxes may move among several den sites and thus leave behind sand flies that may need to seek out new hosts. Thus, the optimal decisions made by individual hyraxes on habitat selection and burrow occupancy based on density-dependent habitat selection (Fretwell and Lucas, 1969) may greatly affect sand fly population dynamics, species interactions among the disease, the vector, and the reservoir host, and even disease transmission to humans. These characteristics may provide opportunities for more effectively disrupting disease transmission.
1.6.0.0.0. Leishmaniasis in Palestine:
Leishmaniasis is endemic in Palestine. There are two forms of the disease: CL caused by L. major (with the vector being the sand fly P. papatasi and the reservoir host the Sand rat Psammomys obesus) and L. tropica (with the vector being the sand fly P. sergenti and the reservoir host the hyrax), and VL caused by L. infantum (Abdeen et al., 2002; Al-Jawabreh et al., 2003 & 2004; Hamarsheh et al., 2012; Jaffe et al., 2004; Nasereddin et al., 2006). Leishmaniasis in general is reported in all Palestinian districts except the Gaza Strip with an official incidence rate of more than 11 per 100,000 in 2010 (Palestinian Ministry of Health, 2011). In the last decade, CL caused by L. tropica has been spreading rapidly in Palestine while L. major infections remain confined to the Jericho region of the Jordan Valley (Al-Jawabreh et al., 2004; Schnur et al., 2004). CL caused by L. tropica was reported from almost all districts and areas including Nablus, Jenin, Salfeet, and Jericho (Al-Jawabreh et al., 2004, 2017; Azmi et al., 2012).
The most common vector for L. tropica in the area is P. sergenti (Sawalha et al., 2003; Schnur et al., 2004), with P. arabicus serving as vector in a small focus near Tiberias (Jacobson et al., 2003). Although L. tropica is known to be mostly anthroponotic elsewhere, strong evidence shows the zoonotic nature of the infection in this region (Jacobson et al., 2003; Svobodova et al., 2006; Talmi-Frank et al., 2010).
In recent years, molecular diagnosis has been carried out on leishmaniasis in parts of the West Bank (Al-Jawabreh et al., 2005; Bader et al., 2005; Hamarsheh et al., 2007; Hamarsheh et
13 al., 2012; Nasereddin et al, 2009), molecular epidemiology by genetic polymorphisms using microsatellites (Al-Jawabreh et al., 2008; Amro et al., 2009; Azmi et al., 2012; Hamarsheh et al., 2006, 2009), identification of vectors and reservoirs (Sawalha et al., 2003), epidemiology of human CL cases (Al-Jawabreh et al., 2003), and comparative diagnosis (Al-Jawabreh et al., 2006; Nasereddin et al, 2006). At the same time, little is known about the distribution, disease ecology, and epidemiology of CL caused by L. tropica and the distribution, population dynamics, and behavior of phlebotomine sand flies in the West Bank.
1.7.0.0.0. Eco-epidemiological approach:
The research presented here can create a better understanding of how the relationships of humans with their environment affect the manner by which they come into contact and interact with the vector, and will allow us to better identify the weak links in the transmission cycle and target them for better disease control. The study of the ecology of the sand fly vector and hyrax reservoir host of this disease together with, human ecology, entomology, molecular biology, and case-control epidemiological studies will provide a more complete understanding of this disease and help offer a basis for successful, more holistic control efforts.
Quantifying the activity density and the distribution in space and time of sand flies is needed in order to control leishmaniasis. Based on Ross-Macdonald models, higher density of the sand fly vector and the hyrax reservoir host will sustain and allow the persistence of the Leishmania parasite. Therefore, the knowledge of sand fly densities and the environmental factors that influence their existence and survival can lead to better and more innovative control strategies. In this study, I monitored populations of the vector sand flies in and around three selected villages in Bethlehem District for the purpose of quantifying the relationship between elevation and sand fly abundance.
Humans often engage in activities that expose them to sand fly bites. Sleeping outdoors during the season of sand fly activity, living next to habitats supporting sand fly populations or close to reservoir colonies, and raising animals next to their houses will increase human-vector- reservoir contact. Therefore, quantifying these risk factors as well as understanding the life cycle and the behavior of the vector and the reservoir can help in creating a well-designed control program. In this study, I also carried out a risk assessment study of CL caused by L. tropica
14 based on the administration of matched case-control epidemiological questionnaires, including questionnaires administrated in endemic areas differing in their urbanization and socio-economic status in the Tubas District.
Understanding the vector-host relationship is crucial for understanding the disease dynamic and transmission. Sand fly females feed on vertebrate blood for egg production (Killick-Kendrick, 1999; Schlein and Warburg, 1986), which affects their fecundity and survival. However, the feeding success of sand flies depend upon the host availability and accessibility (Azizi et al., 2016). In addition, some types of host blood are suitable for leishmania parasite survival, while other types of host may by lethal to Leishmania parasite (Daba et al., 1997). Thus, efficient transmission of the Leishmania parasites occurs when the appropriate host and the vector live in close proximity and the vector tends to feed upon the reservoir hosts (Ashford, 2000). Finally, I conducted a sand fly survey within an urban landscape including adjacent hyrax colonies to evaluate the relationship between distance from hyrax colonies and sand fly density.
1.8.0.0.0. Overall aim and specific objectives:
1.8.1.0.0. Overall aim:
The overall aim is to study Cutaneous Leishmaniasis (CL) caused by Leishmania tropica in the West Bank using an integrative approach comprised of the study of disease ecology and epidemiology at multiple scales. These will allow us to better identify and target risk factors for disease transmission and control.
1.8.2.0.0. Specific objectives: a. To evaluate the effect of altitude on sand fly densities within and between three villages in the Bethlehem District. b. To carry out risk assessment case-control epidemiological studies of Leishmania tropica based on questionnaires administrated in the endemic areas of the Tubas District. c. To evaluate the effect of distance from hyrax Procavia capensis (the reservoir hosts) colonies on Phlebotomus sergenti (the vector sand fly species) densities within an urban landscape.
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1.9.0.0.0. General and specific hypothesis: 1.9.1.0.0. General hypothesis:
This research is intended to contribute ultimately to reducing morbidity from leishmaniasis caused by Leishmania tropica. Ecological factors have an important influence on disease dynamics. For the zoonosis, the ecology of vector species, reservoir host species, and humans all have consequences for disease dynamics in all three species. I hypothesize that L. tropica morbidity is positively correlated with the density of its sand fly vector. If that is correct, I further hypothesize that L. tropica morbidity will be higher in humans with more contact with the vector or with the proximity of reservoir colonies. I also expect morbidity to decrease with elevation, as I expect altitude to decrease sand fly densities.
1.9.2.0.0. Specific hypothesis: a. Endemic sites have lower elevation than the disease-free sites. b. L. tropica related morbidity should peak during the greatest densities of its sand fly vectors. c. Infected people are more exposed to sand fly vector and hyrax reservoir hosts.
1.10.0.0.0. Innovation aspects:
The information about CL caused by Leishmania tropica in the West Bank is largely lacking, with most of the work having been done on CL caused by Leishmania major. The research is innovative in combining disease ecology with an epidemiological approach. This combination creates better understanding of how the relationship of humans to their environment affects the manner in which they come into contact and interact with the reservoir host and vector. The disease ecology approach allows the identification of risk factors that would not be possible simply by tracking human cases alone. Furthermore, the disease ecology approach helps to identify times and locations at which vector populations are most susceptible to control efforts and helps suggest novel factors to target and methodologies for doing so.
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CHAPTER TWO: MATERIALS AND METHODS
2.1.0.0.0. Sand fly sampling at the Bethlehem study sites: 2.1.0.1.0. Study area:
Sand fly sampling was conducted in three villages in the Bethlehem District; including the villages of Al ‘Azazma (AZA, 31°33′37.84′′N, 35°14′28.32′′E, and altitude 480 m above sea level), Arab Ar-Rashaiyda (AAR, 31°56′90.55′′N, 35°22′97.92′′E, and altitude 512 m above sea level), and Kisan (KIS, 31°61′20.69′′N, 35°22′03.37′′E, and altitude 761 m above sea level). The sampling sites were established along an elevational gradient from low (480 m ASL) to high (761 m ASL), and within each site AZA (473-510 m), AAR (522-681 m), and KIS (732-782 m). The three sites differ in their state of endemicity as to whether or not CL can be found in the area, with CL present at AAR and AZA and absent at KIS. See Appendix for details.
2.1.0.2.0. Data collection: 2.1.1.0.0. Comparison of sand fly activity between KIS, AAR, AZA in 2013: 2.1.1.1.0. Comparison of sand fly densities among the three sites:
According to my 2011 work (Salah 2011), sand fly densities at AAR and KIS vary temporally, but are spatially correlated with topography. Notably, sand fly densities are an order of magnitude higher in the endemic area than in the Leishmaniasis-free area. These results agree with hypothesis I, but are limited to a single year. To further test hypothesis I, I monitored sand fly densities for another two years (2013 and 2014) at both sites. In addition, I added another site in the endemic area that has lower altitude. At each site, in 2013, I deployed traps six nights per month from May through October, with three nights centered at new moon and three at full moon. From my pervious study in the West Bank, the activity of sand flies starts in May and ends almost in October. Also sand flies start to hatch on May or late April, and that the total lifespan of sand flies is from 32 to 98 days (Alten et al., 2016). In addition, the human cases in the West Bank start to appear in August end on following April. Taking in consideration the incubation period of the parasite (inside the fly or the human), May through October is the best time to trap sand flies there. I placed six each at KIS and AZA and seven traps at AAR. Trap locations at these sites varied in elevation from 480 m ASL to 761 m ASL (Fig. 3). Traps were located north, south,
17 east, and west of each village. Traps were numbered from 1 to 19, where traps 1 to 6 were deployed in KIS, traps 7 to 13 in AAR, and traps 14 to 19 in AZA. The coordinates for all trap locations in both study sites were recorded. I trapped sand flies for 36 nights all together, accumulating 684 total trap nights. Sand flies were sampled from the three study sites using Center for Disease Control and Prevention (CDC) traps (Model 512, John W. Hock, Gainesville, FL, U.S.A.), each powered by a 6-volt rechargeable battery (Model 3FM12, Amit Industries LTD. Ashdod, Israel). Traps were suspended upside-down in the up-draft position, with the trap entrance ~ 10 cm above the ground. Each trap was baited with 1.5 kg of dry ice placed in a tightly closed insulated container. The container was connected to a rubber tube affixed to the spout with its distal opening placed near the opening of the trap. Traps were deployed overnight starting at ~5 PM and ending ~6 AM the following morning.
Figure 3: Sand fly sampling in Bethlehem study sites, green circles represent traps location at the non-endemic site of KIS, purple triangles represent traps location at the endemic site of AAR, and the red rectangular represent traps location at the endemic site of AZA.
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Trapped sand flies were placed in petri dishes, marked, numbered and stored in a freezer (- 20°C). Petri dishes were transported on ice to Prof. Alon Warburg’s laboratory in The Kuvin Center for the Study of Infectious & Tropical Diseases, Hebrew University, Jerusalem, where they were counted, sexed, and preserved by freezing (-20 °C) for later analysis. The sand fly identification, DNA extraction, blood meal identification, and parasite detection was done by myself.
2.1.1.2.0. Comparison in species for males among the three sites:
Sand flies were taxonomically identified by species based on the morphology of the external genital apparati (Perfil'ev, 1968; Artemiev, 1978; Lewis, 1982) of all the male flies captured at each site.
2.1.1.3.0. Host blood species identification and Leishmania parasite detection:
Of the female flies captured in 2013, 779 had midguts filled with blood from recent meals. Of these, 200 were chosen at random, and their blood meals identified to species and tested for the presence of Leishmania parasites.
2.1.1.3.1. Sand flies identification:
Selected females were dissected; the heads and terminalia were separated, and mounted in
Hoyer’s medium (15g gum Arabic, 100g chloral hydrate, 10g glycerol, 25ml DH2O) on microscope slides for taxonomical identification based on the morphology of the pharynx and spermatheca (Perfil'ev, 1968; Artemiev, 1978; Lewis, 1982).
2.1.1.3.2. DNA extraction:
The thorax and abdomen of each fly were placed in a 1.5 mL micro centrifuge tube. DNA was extracted from each fly by digestion in a total volume of 200 μL of lysis buffer (50 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl pH 7.4, 1% triton X- 100, and 200 μg/mL of proteinase K). This was then followed by extraction with phenol- chloroform and precipitation using cold ethanol. The precipitated DNA was suspended in Tris- EDTA (TE, 10 mM Tris-HCl pH 7.4, 1 mM EDTA) buffer at a concentration of 50 μL and stored at -20 °C until the DNA was amplified by PCR.
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2.1.1.3.3. Blood meal identification and parasite detection:
Blood meal was identified using Cytochrome (cyt) b PCR and Reverse Line Blotting (RLB) (Abbasi et al., 2009). The extracted DNA from Phlebotomus spp. was PCR amplified using biotinylated vertebrate species-specific cytochrome b primers. The PCR products were blotted on species-specific oligonucleotides (Human, donkey, goat, sheep, camel, dog, hyrax, chicken, and avian). Specific hybridization was visualized using streptavidin horseradish peroxidase.
Leishmania parasite DNA was detected using PCR amplification of the internal transcribed spacer (ITS1), the amplicon of all positive samples was sent for DNA sequencing to identify the Leishmania species (El Tai, et al., 2000; Schönian, et al., 2001).
For DNA sequencing the PCR amplicons from sand flies were sequenced by The Center for Genomic Technologies at the Hebrew University of Jerusalem using automated DNA Sequencing, based on BigDye Terminator cycle sequencing chemistry from Applied Biosystems (ABI), ABI PRISM 3730xl DNA Analyzer and the ABI’s Data collection and Sequence Analysis software. The derived sequences were compared against the GenBank database using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and default search parameters (Madden, 2013).
2.1.2.0.0. Comparison of Sand fly activity between AAR, AZA in 2014:
In 2014, I deployed traps three nights per month from May through October at AAR and AZA at the same sites and trap location used in trapping in 2013. Monthly trapping was centered at new moon. Sand flies were trapped for 18 nights all together, accumulating 342 total trap nights.
2.1.0.3.0. Statistical analysis:
Data were analyzed in SYSTAT 13. Generalized linear regression was used to analyze the relationship between sand fly densities and elevation. I used generalized linear regression to test the effects of elevation on the number of female sand flies captured per day per site, the proportion of blood-fed female sand flies captured per day per site, and the number of male sand flies captured per day per site. Similarly, a generalized linear regression was used to analyze the relationship between total sand fly densities and month. A generalized linear regression was also used to compare the male sand fly species composition at the three sites for effects of species,
20 site, and month. For the blood meal analysis, multi-way contingency tables and log linear models were used to analyze the relationship between the number of female sand fly species and the host blood species. Additionally, multi-way contingency tables and log linear models were used to analyze the effect of month and elevation (trap station) on the number of P. sergenti engorged with various host blood species. All statistical tests were carried out at a significance level of 0.05.
2.2.0.0.0. Epidemiological Investigation in the Tubas study sites: 2.2.0.1.0. Study area:
This study was conducted in the Tubas District. The district was divided into three different groups according to their level of urbanization and socio-economic status: urban, rural, and Bedouin encampments. The city of Tubas (31°19′16.23′′N, 35°22′11.83′′E, and altitude 362 m above sea level) comprised the main urban center for the whole district. Rural areas were comprised of the six villages of Tummun (32°16′59.46′′N, 35°23′8.86′′E, and altitude 332 m above sea level), Tayasir (32°20′25.56′′N, 35°23′48.42′′E, and altitude 331 m above sea level), Khirbet Yarza (32°18′20.05′′N, 35°26′0.86′′E, and altitude 257 m above sea level), Wadi al Far’a (32°17′37.40′′N, 35°20′40.06′′E, and altitude 051 m above sea level), Khirbet Atuf (32°15′53.38′′N, 35°26′12.98′′E, and altitude 50 m above sea level), and Bardala (32°23′10.73′′N, 35°28′55.08′′E, and altitude -71 m below sea level). Bedouin areas were composed of several encampments scattered throughout the district, but centered in two localities: Khirbet Al Malih (32°19′38.54′′N, 35°26′10.51′′E, and altitude 32 m above sea level), and Khirbet Tell el Himma (32°22′17′′N, 35°30′50′′E, and altitude -182 m below sea level). See Appendix for details.
2.2.0.2.0. Data collection:
The Palestinian disease surveillance system depends on passive surveillance from health facilities of different health providers (Primary Health Care Centers, Hospitals, and Laboratories), both governmental and nongovernmental (MOH, UNRWA, NGOs and private sector). In case of leishmaniasis, for all suspected patient samples collected from the local primary health care centers that belong to the MOH in each city and village, these samples go to the central epidemiology department in the MOH for molecular tests. Cases used in this study were all patient who were PCR-positive for Leishmania tropica from Tubas District.
21
This work was approved by Physicians for Human Rights-Israel Ethics Institutional Review Board (IRB) and subject to all national laws of human care.
A survey was conducted in the three different areas (Fig. 4). The cases of CL were referred to individuals who were PCR-positive for Leishmania tropica parasite according to the Palestinian Ministry of Health. The control group was composed of healthy subjects showing no signs of CL and living in different households within the same study area at the time when the corresponding case presented signs of disease. Three controls were selected per each case in the city and villages, while one to three controls were selected per case in the Bedouin encampments. Whenever possible, the controls were chosen from the house located in the same row of cases houses, and then from the second row, and so on. Cases and controls were matched by sex, age, and residency. Cases less than two years of age were matched to controls within two years of age, cases 2–4 years old with cases within three years, cases 5–19 years old with cases within five years, cases 20–59 years old with cases within 10 years, and cases more than 60 years old with cases within 20 years.
Study subjects were interviewed for 30 minutes by myself using a pre-designed questionnaire. The questionnaire was translated into the local language (Arabic). It included: I) demographic parameters such as age, gender, occupation, and education; II) epidemiological data such as history of leishmaniasis, high season of sand fly activity, and the use of personal protection against the vector; III) clinical and laboratory information such as the date of onset of the disease, number of lesions, other family members infected; IV) characteristics of the houses such as the location of the house, and the presence of a garden, and V) information about vector and reservoir (see appendix).
22
Figure 4: Epidemiological investigation study sites; red starts represent city cases, green triangle represent village cases, and blue squares represent cases in Bedouin encampments
2.2.0.3.0. Statistical analysis:
The sample size was 274 cases and controls in total, comprised of 45 cases and 134 controls in the city, 16 cases and 46 controls in the villages, and 15 cases and 18 controls in the Bedouin encampments. Data were analyzed using IBM SPSS Statistics 23. To compare differences in cases between sites, r x c contingency tables and χ² tests for independence were used. The risk factor data were analyzed by χ² as well. All statistical tests were carried out at a significance level of 0.05.
2.3.0.0.0. Sand fly transects for sampling the Tubas study site: 2.3.0.1.0. Study area:
Sand fly transect sampling was conducted in the Aleskan neighborhood of Tubas city. This neighborhood is a hot spot where especially high numbers of human cases were clustered.
23
2.3.0.2.0. Data collection: 2.3.1.0.0. Sand fly density:
A sand fly survey was conducted to evaluate the effect of distance from hyrax colonies on sand fly density within an urban landscape and between the urbanized area and adjacent hyrax colonies. Previously, I identified elevation as a risk factor for CL in the West Bank, Palestine. However, I could not previously partition effects of elevation from those of distance between human habitation and hyrax colonies. Subsequently, I used Aleskan neighborhood in the endemic city of Tubas to examine the role of distance. Aleskan is an ideal location for this study because it is a hot spot where high numbers of human cases recently occurred over an area of similar elevation. It also seems that this site and its immediate surroundings serve as breeding sites for sand flies and as a focus for Leishmania infection within the city.
I tested the effect of distance from hyrax colonies by quantifying sand fly abundance along five east-west 0.6 km long trapping transects running parallel from hyrax colonies across an area of inhabited houses. I placed 3 traps along each transect, with the closest distance of two meters and farthest distance of 400 meters to the nearest hyrax colony (Fig. 5). I conducted two 6-night trapping sessions, one in July and one in September, 2016. Traps were numbered from 1 to 15. The GPS coordinates for all trap locations were taken. I trapped sand flies for 12 nights all together accumulating 180 trap nights.
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Figure 5: Trapping transects at Aleskan neighborhood in Tubas city, the pins represents the traps stations, and the stars represents the hyrax colonies.
Sand flies were sampled using light Center for Disease Control and Prevention (CDC) traps (Model 512, John W. Hock, Gainesville, FL, U.S.A.), powered by a 6-volt rechargeable battery (Model 3FM12, Amit Industries LTD. Ashdod, Israel). Traps were suspended upside-down in the up-draft position, with the trap entrance ~ 10 cm above the ground. The traps were deployed overnight starting at ~5 PM and ending at ~6 AM the following morning. The trapped sand flies were preserved in 70 % ethanol, and then transported to Alon Warburg’s laboratory. The sand fly identification, DNA extraction, blood meal identification and parasite detection was done by myself.
2.3.2.0.0. Sand fly species identification:
Sand flies were dissected and mounted in Hoyer’s medium (15g gum Arabic, 100g chloral hydrate, 10g glycerol, 25ml DH2O) on microscope slides for taxonomical identification. Taxonomical identification was conducted on the males using the morphology of their external
25 male genital apparati, and on the females using the morphology of the pharynx and spermatheca (Perfil'ev, 1968; Artemiev, 1978; Lewis, 1982).
2.3.3.0.0. Leishmania parasite detection:
The thorax and abdomen of sand flies from the same species were pooled (1-5 sand flies) and placed in a 1.5 mL micro centrifuge tube. DNA was extracted from each pool (See section 1.1.3.2.).The presence of Leishmania parasite DNA in pooled samples was detected by PCR amplification of the internal transcribed spacer (ITS1) (El Tai, et al., 2000; Schönian, et al., 2001; See section 1.1.3.3.).
2.3.4.0.0. Host blood species identification:
All blood-fed females were used to test the host blood species, The thorax and abdomen of each fly were placed in a 1.5 mL micro centrifuge tube for blood meal analysis using Cytochrome (cyt) b PCR and Reverse Line Blotting (RLB) techniques (Abbasi et al., 2009; See section 1.1.3.3.).
2.3.0.3.0. Statistical analysis:
Data were analyzed in SYSTAT 13. Generalized linear regression was used to analyze the relationship between sand fly densities and the distance from hyrax colonies. All statistical tests were carried out at a significance level of 0.05. I used generalized linear regression to test the effects of closest distance from hyrax colonies on the number of total Phlebotomus sand flies, the number of non-fed Phlebotomus females, the proportion of blood-fed females, and number of Phlebotomus male captured per day. A generalized linear regression was also used to analyze the relationship between transects and the total number of Phlebotomus sand flies, the number of non-fed females, the proportion of blood-fed females, and the number of males captured per day.
I used multi-way contingency tables and log linear models to analyze the relationship between the distance from hyrax colonies and the number of infected non-fed female pools, and the infected blood-fed female pools. In addition, I also used multi-way contingency tables and log linear models to analyze the relationship between the number of infected non-fed female pools and transects. A final set of multi-way contingency tables and log linear models were used to analyze the relationship between host blood species and transects.
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CHAPTER THREE: RESULTS
3.1.0.0.0. Sand fly sampling at the Bethlehem study sites: 3.1.1.0.0. Comparison of Sand fly activity between KIS, AAR, AZA in 2013: 3.1.1.1.0. Comparison of sand fly densities among the three sites:
The total number of Phlebotomus sand flies collected was 18,416, of which 13,254 (19.4 per trap/night) were captured in AAR, 4,746 (6.94 per trap/night) in AZA, and 416 (0.61 per trap/night) in KIS. The male to female ratio was significantly different among the three villages
(χ² (2) = 79.6, p< 0.00001), with nearly even sex ratios in AAR and AZA, and female biased sex ratio in KIS (Fig. 6).
Figure 6: Phlebotomus sand fly sex ratio distribution. Blue represents males, and red represents females.
There were significant differences in female Phlebotomus sand fly densities among sites
(MS = 43,568, F(2, 658) = 39.5, p< 0.0001). However, when including the elevation of trapping stations as a covariate, the effect of site lost significance, suggesting that the difference between sites was driven by the effect of elevation. Elevation of trapping stations (MS = 13,207, F(18, 642) = 14.75, p< 0.0001), as well as quadratic elevation of trapping stations has a significant association on female Phlebotomus sand fly abundance (MS = 36,919, F(2, 658) = 32.88, p<
27
0.001), where both low and high elevation had lower abundances and the intermediate elevation of 604 m had a greater abundance (Fig. 7).
Figure 7: The relationship between trap elevation and mean total number of female Phlebotomus sand flies caught in trap. The curve is fit with a quadratic regression showing that peak densities occur at intermediate elevations. Yellow represents AZA village, red represents AAR village, and blue represents KIS village.
Males also differed significantly in density among sites (MS = 33,995, F(2, 658) = 32.9, p< 0.0001). However, when including the elevation of trapping stations as a covariate, the effect of site lost significance, suggesting again that the difference between sites was driven by the effect of elevation. Elevation of trapping stations (MS = 9,950, F(18, 642) = 11.23, p< 0.0001), as well as quadratic elevation of trapping stations has a significant association on male Phlebotomus sand fly abundance (MS = 28,599, F(2, 658) = 27.25, p< 0.001), where both low and high elevations had lower abundances and the intermediate elevation of 604 m had greater abundance (Fig. 8).
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Figure 8: The relationship between trap elevation and mean total number of male Phlebotomus sand flies caught in trap. The curve is fit with a quadratic regression showing that peak densities occur at intermediate elevations. Yellow represents AZA village, red represents AAR village, and blue represents KIS village.
Phlebotomus sand flies differed in feeding success among sites. The total number of blood-fed sand flies number was 779, of which 423 (54.3%) were captured in AAR, 343 (44%) in AZA, and 13 (1.7%) in KIS. These differences were significant (MS = 0.503, F(2, 658) = 19.92, p< 0.0001). However, when including the elevation of the trapping stations as a covariate, the effect of site lost significance, suggesting that the difference among sites was driven by the effect of elevation. Elevation of trap stations was negatively correlated with the proportion of blood fed
Phlebotomus sand flies across all sites (MS = 0.094, F(18, 642) = 3.78, p< 0.0001), (Fig. 9).
The total number of gravid females was 451, of which 243 (54%) were captures in AAR, 201 (44.5%) were captured in AZA, and 7 (1.5%) were captured in KIS. The elevation of trapping stations was negatively correlated with the proportion of gravid female Phlebotomus sand flies across all sites (MS = 0.046, F(18, 642) = 3.73, p< 0.0001), (Fig. 10).
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Figure 9: The relationship between trap elevation and proportion of blood fed of female Phlebotomus sand flies caught in trap. Yellow represents AZA village, red represents AAR village, and blue represents KIS village.
Figure 10: The relationship between trap elevation and proportion of gravid female Phlebotomus sand flies caught in trap. Yellow represents AZA village, red represents AAR village, and blue represents KIS village.
30
Phlebotomus sand fly abundance was consistently higher in AAR throughout the study (푋= 53.6 ± 4 (SE)), followed by AZA (푋= 22.5 ± 4.3 (SE)), and KIS (푋= 2.065 ± 4.4 (SE)). These abundances were marginally significantly different in total Phlebotomus sand fly densities between months (F (5, 655) = 2.07, p= 0.067). Also, the seasonal activity pattern did not significantly differ between sites (Fig. 11).
Figure 11: Average number of Phlebotomus sand flies captured per month in the three sites. The yellow curve represents AZA village, red curve represents AAR village and blue curve represents KIS village.
3.1.1.2.0. Comparison in species for males among the three sites:
Of the captured male Phlebotomus sand flies, 75.6% were P. sergenti, 23.4% P. papatasi, 0.95% P. syriacus, 0.34% P. alexandri, 0.1% P. perfiliewi, 0.08% P. jacusieli, and 0.06% P. tobbi. The species diversity of male Phlebotomus sand flies varied between study sites as indicated by the values of Shannon-Wiener index (H), richness (S), and evenness (E) reported in Table 1. Higher diversity and richness were measured in AZA where elevation was lowest (473- 510 m), but it showed low evenness. However, richness at the higher elevation of AAR (522-681 m), and KIS (732-782 m) was equal, but KIS had higher diversity and evenness than AAR.
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Male Phlebotomus sand flies differed by species and site (Table 2). There was a significant difference in male Phlebotomus sand fly species composition between sites (MS =
4,154.8, F(12, 4607) = 28.5, p< 0.001). All the species collected in this study were present in AZA, while P. jacusieli and P. tobbi were absent from AAR and KIS. The most abundant species was P. sergenti followed by P. papatasi, with higher abundance of P. sergenti in AAR and KIS, and P. papatasi in AZA (Fig. 12). Regarding elevation of trapping stations, there was a significant difference between male Phlebotomus sand fly species and the elevation of trapping stations (MS
= 1,072.02, F(108, 4495) = 11.558, p< 0.001), (Table 3). Trapping stations at lower elevations had more species than other stations (Fig. 13).
Table 1: The Shannon-Weiner diversity index (H), evenness (E) and richness(S) for the Phlebotomus sand fly males in different site Site/ Altitude (H) (S) (E) AZA (473-510 m) 0.83 7 0.43 AAR (522-681 m) 0.44 5 0.27 KIS (732-782 m) 0.81 5 0.50
Table 2: The correlation between site and number of male Phlebotomus sand fly species captured per month. Type III SS is Sum of squares for a fixed factor Source Type III SS df Mean Squares F-Ratio P-Value Males Species 46,125.223 6 7,687.537 52.739 <0.001 Site 9,699.121 2 4,849.561 33.270 <0.001 Species* site 49,858.265 12 4,154.855 28.504 <0.001 Error 671,542.779 4,607 145.766
Table 3: The correlation between altitude of trap station and number of Phlebotomus sand fly species captured per month for males. Type III SS is Sum of squares for a fixed factor Source Type III SS df Mean Squares F-Ratio P-Value Males Species 52,390.007 6 2,408.951 71.085 <0.001 Elevation 25,614.633 18 2,329.578 11.585 <0.001 Species* Elevation 153,323.913 108 1,072.023 11.558 <0.001 Error 552,143.424 4,495 122.835
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Figure 12: Phlebotomus sand fly species composition for males in the three sites.
Figure 13: Phlebotomus sand fly species at each trap station for males. Red represents P. sergenti. Blue represents P. papatasi. Light green represents P. alexandri, Violet represents P. jacusieli, Gray represents P. perfiliewi, Orange represents P. syriacus, and dark blue represents P. tobbi.
3.1.1.3.0. Host blood species identification and Leishmania parasite detection:
A total of 200 blood-fed Phlebotomus spp. (56% P. sergenti, 33.5% P. papatasi, 9% P. alexandri, and 1.5% P. syriacus) were analyzed for blood meal identification. Phlebotomus spp.
33 abundance displayed significant spatial variation during the study, in which the number of P. sergenti (χ² (11) = 40.87, p< 0.0001) and P. papatasi (χ² (9) = 17.3, p= 0.044) were significantly different among trapping stations. The spatial distribution of P. alexandri was marginally different among trapping stations (χ² (8) = 13.4, p= 0.062). P. syriacus were only found in two trapping stations, with no difference (Fig 15A). Of these, 181 (90.5%) were positive for cyto b PCR (Fig. 14). All PCR-positive samples were used for blood meal identification using RLB. The host blood type according to species were categorized into four groups: human (45%), avian (23.9%), livestock (23.6%), and dog (7.5%). There was a significant difference among Phlebotomus species in the number of flies that had in their crops human host blood (χ² (3) = 110.2, p< 0.0001), avian host blood (χ² (3) =
47.6, p< 0.0001), dog host blood (χ² (3) = 25.2, p< 0.0001), and livestock host blood (χ² (3) = 53.8, p< 0.0001; Fig. 15B).
Figure 14: Gel image of cyto b BCR targeting DNA from blood-fed sand flies. M is DNA ladder, from 171 to 183 PCR product of blood-fed sand flies, +ve, positive control (Cow), -ve negative control (pure water).
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Fig 15A: Phlebotomus sand fly species at each trap station for females. Red squares represent P. sergenti, blue diamonds represents P. papatasi, brown circles represent P. alexandri, and green line represents P. syriacus.
Figure 15B: The relationship between the numbers of flies with different types of host blood found in each Phlebotomus species. Brown bars represents P. alexandri, blue bars represents P. papatasi, red bars represents P. sergenti and green bars represents P. syriacus.
35
The host blood species engorged by P. sergenti were grouped to four groups: human (45.5%), livestock (25%), avian (19.9%), and dog (9.6%). P. sergenti abundance displayed significant spatial and temporal variations during the study, in which the number of P. sergenti, engorged with human blood were significantly different among trapping stations (χ² (11) = 30.6, p= 0.001). The number of flies engorged with other host blood species did not differ among trapping stations (Fig. 16). The number of flies engorged with human host blood (χ² (5) = 24.6, p<
0.0001) and avian host blood (χ² (5) = 14.6, p= 0.013) were significantly different between months of the year for P. sergenti (Fig. 17).
Figure 16: Spatial distribution of the species of host blood with which P. sergenti flies were engorged. The blue curve represents human blood, red curve represents avian blood, green curve represents dog blood, and violet curve represents livestock blood.
36
Figure 17: Temporal distribution of P. sergenti flies engorged with blood of different host species. The blue curve represents human blood, red curve represents avian blood, green curve represents the dog blood host and violet curve represents livestock blood.
200 females were tested for Leishmania parasite presence. One sample was positive by ITS-PCR Leishmania (0.5% infection rate) (Fig. 18). This sample was positive with Leishmania tropica according to the DNA sequencing.
Figure 18: Gel image of ITS-PCR Leishmania targeting DNA from blood-fed sand flies. M is DNA ladder, from 106 to 115 PCR product of blood-fed sand flies, +ve, positive control (L. infantum), and -ve, negative control (pure water).
37
3.1.2.0.0. Comparison of Sand fly activity between AAR and AZA in 2014:
Sand fly abundances at AAR and AZA differed in seasonal variation in densities between years. Data were available for both 2013 and 2014 only at AAR and AZA. Over both years, sand flies were in greater abundance at AAR (MS = 103,677.8, F (1, 466) = 32.25, p< 0.0001). Seasonally, there were significant differences in sand fly densities between months in 2013 compared with 2014 (interaction of month and year: MS = 11,220.9, F (5, 456) = 3.37, p= 0.005). In 2013, sand flies peaked in abundance in September, while in 2014 they peaked in July (Fig. 19).
Figure 19: Average number of Phlebotomus sand flies captured per month. The red curve represents 2013. The blue curve represents 2014.
Phlebotomus sand flies differed in feeding success between years. There were significant differences in proportion of blood fed Phlebotomus sand flies between months in 2013 compared with 2014 (interaction of month and year: MS = 0.11, F (5, 443) = 7.6, p> 0.001). In 2013, the feeding success was high early in the season and decreased over time, while in 2014 it was low early in the season and get increase over time (Fig. 20).
38
Figure 20: Blood fed Phlebotomus sand flies proportion captured per month. The red curve represents 2013. The blue curve represents 2014.
3.2.0.0.0. Epidemiological Investigation in the Tubas study sites:
Total number of human cases were 76, of which 45 cases were found in the city, 16 in the villages, and 15 in the Bedouin encampments. Controls totaled 198, of which I chose 134 controls to match cases in the city, 46 in the villages, and 18 in the Bedouin encampments. The incidence rate (IR) in the city during 2015 was 21.6 case per 10,000 individuals. IR in the villages ranged between (2.15-14.2) case per 10,000 individuals and IR in Bedouin encampments was 111.4 case per 10,000 individuals.
3.2.1.0.0. Demographic characteristics:
Sites differed in several aspects in regards to cases of CL in humans. There was a marginally significant difference in gender distribution of cases among sites (χ² (8) = 5.16, p= 0.076). In the city and villages (57% and 62% male: 43% and 38% female respectively), the cases tended to be males, while in the Bedouin encampments the cases tended to be females (26% male: 74% female, Fig. 21). There was a significant difference between sites in age distribution of cases (χ² (8) = 14.78, p= 0.022). In the city all age groups were found, but the
39 highest percentage of cases was children less than 17 years old and the second highest percentage were elderly people. In the village, there was a missing age class (31-45 years old age class), but the greatest percent of cases still occurred among children and the second highest percentage were from the 18-30 years old age class. In the Bedouin encampments, the elderly age class was missing, the highest percent of cases were children and the second highest percentage was in the 31-45 years old age class (Fig. 21).
Figure 21: CL cases distribution by age and sex in Tubas district in 2015. Red bars represent the males, and blue bars represent the females.
Cases and controls at each site differed in their demographic aspects. The average age in the city was 25.5 (+ 20.9), in the villages was 25.8 (+ 18.4), and in the Bedouin encampments was 13.9 (+ 12.9). There was no significant difference between case and controls in the city, villages, and Bedouin encampments when it comparing their education level. In the city and the Bedouin encampments the cases and controls did not differ significantly in their occupation, but they differ significantly in the villages where students were underrepresented in the cases and those staying at home were over-represented (Table 4).
The peak of (CL) cases occurred from December 2014 to January 2015. Cases in the city appeared mostly from December 2014 through March 2015, cases in the Bedouin encampments appeared from October 2014 through March 2015 and peaked in December, and cases in the villages appeared from November 2014 through April 2015 and peaked in January. (Fig. 22).
40
Figure 22: Temporal distribution of CL cases in Tubas district. The red squares represent the city, the green circles represent the villages, and the blue triangles represent the Bedouin encampments.
41
Table 4: Population Characteristics of case and control respondent
City Village Bedouin Factor Case Control Case Control Case Control χ² p-value χ² p-value χ² p-value (n= 45) (n= 134) (n= 16) (n= 46) (n= 15) (n= 18) n % n % n % n % n % n %
Age (average) 25.5 ( + 20.9) 25.8 ( + 18.4) 13.9 ( + 12.9) Education (year) <1 7 15.6 19 14.2 2 12.5 4 8.7 6 40 7 38.9 1-6 16 35.6 47 35.1 3 18.8 13 28.3 4 26.7 3 16.7 0.59 0.9 1.1 0.78 0.65 0.72 7-12 11 24.2 40 29.9 6 37.5 19 41.9 5 33.3 8 44.4 >12 11 24.4 28 20.9 5 31.3 10 21.7 n/a n/a Occupation Student 23 51.1 68 50.7 5 31.3 23 50 4 26.7 8 44.4 Housewife 6 13.3 24 17.9 2 12.5 9 19.6 3 20 3 16.7 At home 5 11.1 12 9 3 18.8 0 0 7 46.7 5 27.8 6.3 0.28 10.4 0.035* 4.4 0.35 Farmer 3 6.7 1 0.7 4 25 11 23.9 0 0 2 11.1 Employee 6 13.3 24 17.9 2 12.5 3 6.5 1 6.7 0 0 Business owner 2 4.4 5 3.7 n/a n/a n/a n/a
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3.2.2.0.0. Clinical information:
Infections were clustered (Fig. 23). In the city and Bedouin encampments, cases tended to be clustered, with houses or tents often having more than one case. In contrast, in the villages most of the cases were in different houses (Table 5).
The location and number of lesions per case were different between sites. Most cases had just one lesion regardless of degree of urbanization (Table 5). In contrast, the location of the lesions on the body significantly differed among sites. The lesions of cases in the village were located on the head, limbs, and other body sites, while most lesions of the cases in the city occurred on the limbs. Cases in the Bedouin encampments had the lesions mostly on the face (Table 5).
Table 5: Clinical information about the CL cases in Tubas District. City Village Bedouin χ² p-value Factor (n= 45) (n= 16) (n= 15) n % n % n % Other family member infected Yes 26 57.8 2 12.5 8 53.3 9.97 0.007* No 19 42.2 14 87.5 7 46.7 How many family member infected 1 16 61.5 2 100 2 25 5 0.08* > 1 10 38.5 0 0 6 75 Number of lesions 1 22 48.9 9 56.3 6 40 1.5 0.8 2-3 17 37.8 4 25 6 40 >3 6 13.3 3 18.7 3 20 Lesions location Face 15 33.3 5 31.3 7 46.7 Limbs 24 53.3 5 31.3 5 33.3 27.5 <0.001* Face + Limbs 3 6.7 4 25 1 6.7 Other 3 6.7 2 12.5 2 13.3
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Figure 23: The location of CL cases in Tubas district. A represents city, B represents Villages, and C represents Bedouin encampment
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3.2.3.0.0. Human behavior:
Factors related to human behavior, including the time of the day that individuals were bitten, the time spent outdoors in the garden near their homes and in the neighborhood, and the time of the day during which people were outdoors did not differ between CL cases and controls in the city, villages, or Bedouin encampments (Table 6). Moving to a place recognized as a CL focus increased the chance of getting an infection. In the city and villages, the cases traveled to a CL focus significantly more than the controls during the active sand fly season (Table 6).
3.2.4.0.0. Personal protection:
Using personal protection measures can help inhibit disease transmission in the city. Personal protection measures included the spraying of insecticide outside the house, the use of repellents on skin to prevent bites from adult sand flies, and spraying or using diffusible insecticides inside the house. Controls used these measures significantly more than cases. In the villages and Bedouin encampments, there were no significant differences between cases and controls in the use of personal protection measures when it was most relevant (Table 7).
45
Table 6: Human behavior information of respondents
City Village Bedouin Factor Case Control p- Odd Case Control Odd Case Control p- Odd χ² χ² p-value χ² (n= 45) (n= 134) value ratio (n= 16) (n= 46) Ratio (n= 15) (n= 18) value Ratio
n % n % n % n % n % n %
People moved homes in the
last 6 months
Yes 6 13.3 2 1.5 7 43.8 1 2.2 1 6.7 0 0 11.06 0.004* 10.15 18.3 <0.001* 35 1.2 0.45 N/A No 39 86.7 132 98.5 9 56.3 45 97.8 14 93.3 18 100 Time spent
outdoor Morning 6 13.3 27 20.1 1 6.3 7 15.2 3 20 5 27.8 1.3 0.25 0.61 0.85 0.33 0.37 0.27 0.46 0.65 Evening 39 86.7 107 79.9 15 93.8 39 84.8 12 80 13 72.2 Season when they were mostly bitten Spring 12 26.7 48 35.8 1 6.3 5 10.9 1 6.7 0 0 1.2 0.27 0.65 0.29 0.51 0.55 1.2 0.45 N/A Summer 33 73.3 86 64.2 15 93.8 41 89.1 14 93.3 18 100 Time of day when they were bitten Morning 2 4.4 7 5.2 2 12.5 1 2.2 3 20 1 5.6
Evening 6 13.3 7 5.2 2.12 0.15 N/A 3 18.8 4 8.7 4.3 0.12 N/A 2 13.3 1 5.6 2.5 0.29 N/A
Night 37 82.2 120 89.6 11 68.8 41 89.1 10 66.7 16 88.9
46
Table 7: Personal protection used by respondents
City Village Bedouin Factor Case Control p- Odd Case Control p- Odd Case Control p- Odd χ² χ² χ² (n= 45) (n= 134) value ratio (n= 16) (n= 46) value Ratio (n= 15) (n= 18) value Ratio
n % n % n % n % n % n % People used fans 37 82.2 111 82.8 12 75 36 78.3 0.01 0.9 0.96 0.07 0.53 0.83 N/A No 8 17.8 23 17.2 4 25 10 21.7 People used Insect repellent on skin
(in or out the house) Yes 3 6.7 19 14.2 1 6.3 2 4.3 1.8 0.17 0.43 0.09 0.56 0.68 N/A No 42 93.3 115 85.8 15 93.8 44 95.7 People used vaporizing tablets or liquid Yes 23 51.1 95 70.9 9 56.3 23 50 5.8 0.013* 2.3 1.8 0.45 0.78 N/A No 22 48.9 39 29.1 7 43.7 23 50 People Spraying
inside the house Yes 20 44.4 83 61.9 8 50 27 58.7 4.2 0.03* 2.04 0.67 0.42 0.7 N/A No 25 55.6 51 38.1 8 50 19 41.3 People Spraying outside or around the house/tent Yes 28 62.2 74 55.2 9 56.3 31 67.4 4 26.7 8 44.4 1.1 0.67 0.42 0.79 0.64 0.31 0.6 0.25 0.45 No 17 37.8 60 44.8 7 43.7 15 32.6 11 73.3 10 55.6
47
3.2.5.0.0. Topography and house information:
The location of houses correlated with disease transmission. In the city and villages, case houses were significantly closer than controls to the site edge where people may be exposed to vectors entering there. Similarly, the houses of cases in the city were more likely to have nearby farms (within 200 m) and to raise domestic animals themselves than controls, both of which may increase exposure to sand flies. However, the cases and controls in the villages and Bedouin encampments were both surrounded by farms and domesticated animals. Cases in the villages tended to have domestic livestock more than did controls, although this difference is only marginal, and there was no significant difference in having farms between cases and controls in both sites (Table 8).
Other factors can enhance disease transmission in the houses such as screened windows, cracks or crevices in the wall or roof of the house, and construction near houses which all may serve as breeding or resting sites for sand flies. There was no difference between CL case and control houses in the three sites; both groups had almost no cracks or crevices in the roof and house walls, windows were screened, and construction work was close to their houses (Table 8).
3.2.6.0.0. Information about vector and reservoir:
Cases were more associated with hyraxes, the presumptive reservoir host of L. tropica, than controls. In the city and Bedouin encampments, cases were more likely to have reported seeing hyraxes near their homes than controls, and their houses were located closer to a colony of hyraxes (Table 9). Factors including seeing hyrax, distance to hyrax colonies, proximity of farm, and the use of personal protection did not appear to play a role in the villages, despite its importance in urban settings. There was agreement between cases and controls in their perception that sand flies were more common in the summer of 2014 than previous years (Table 9).
48
Table 8: Information about houses of respondents
City Village Bedouin
Factor Case Control Odd Case Control p- Odd Case Control p- Odd χ² p-value χ² χ² (n= 45) (n= 134) ratio (n= 16) (n= 46) value Ratio (n= 15) (n= 18) value Ratio
n % n % n % n % n % n %
Distance from city/village
edges less than 20m
Yes 38 84.4 63 47 13 81.2 23 50 19.2 <0.001* 6.10 4.8 0.027* 4.33 100% No 7 15.6 71 53 3 18.8 23 50
Homes located on hill tops
Yes 24 53.3 41 30.6 5 31.3 17 37 3 20 6 33.3 7.5 0.006* 2.59 0.17 0.46 0.77 0.73 0.32 0.5 No 21 46.7 93 69.4 11 68.7 29 63 12 80 12 66.7
Farm within 200m
Yes 35 77.8 52 38.8 20.5 <0.001* 5.52 100% 100% No 10 22.2 82 61.2
Having domesticated
animals Yes 16 35.6 24 17.9 12 75 22 47.8 6.04 0.014* 2.53 3.5 0.054 3.30 100% No 29 64.4 110 82.1 4 25 24 52.2
Construction works during summer near your house
Yes 27 60 79 59 11 68.8 26 56.5 2 13.3 0 0 0.15 0.52 0.96 0.73 0.29 0.58 2.5 0.20 N/A No 18 40 55 41 5 31.3 20 43.5 13 86.7 18 100
49
Table 9: Information about vector and reservoir
City Village Bedouin
Case Control Odd Case Control p- Odd Case Control p- Odd χ² p-value χ² χ² (n= 45) (n= 134) Ratio (n= 16) (n= 46) value Ratio (n= 15) (n= 18) value Ratio
n % n % n % n % n % n % Density of sand flies in summer 2014 is more than previous years Yes 30 66.7 82 61.2 8 50 26 56.5 9 60 15 83.3 0.43 0.32 0.79 0.2 0.44 0.77 2.5 0.14 0.3 No 15 33.3 52 38.8 8 50 20 43.5 6 40 3 16.7
The reason behind sand
flies density High temperature & Low 15 33.3 55 41 8 50 20 43.5 9 60 15 83.3 2.5 rainfall
Constructions 7 15.6 3 2.2 n/a n/a
Don't spray insecticides 1 2.2 20 14.9 0.15 0.52 N/A 0 0 5 10.9 0.34 0.39 N/A n/a 0.14 N/A Increase in hyrax 7 15.6 4 3 0 0 1 2.2 n/a population
N/A 15 33.3 52 38.8 8 50 52 38.8 6 40 3 16.7
People who had seen hyraxes in the vicinity of house Yes 32 71.1 46 34.3 9 56.3 22 43.8 10 66.7 13 72.2 18.5 <0.001* 4.7 0.77 0.39 0.71 0.12 0.51 0.77 No 13 28.9 88 65.7 7 43.8 14 52.2 5 33.3 5 27.8
Distance of house from
hyraxes colonies Less than 100m 28 62.2 22 16.4 4 25 6 13 10 66.7 4 22.2 35.1 <0.001* 8.3 1.25 0.23 0.45 6.6 0.013* 7 More than 100m 17 37.8 112 83.6 12 75 40 87 5 33.3 14 77.8
50
3.3.0.0.0. Sand fly transects for sampling the Tubas study site: 3.3.1.0.0. Sand fly density:
The total number of Phlebotomine sand flies was 3771, of which 1050 were Phlebotomus and 2721 Sergentomyia.
There was a significant difference in Phlebotomus sand fly densities among transects, in which transects close to hyrax colonies had the highest sand fly densities. The total number of Phlebotomus sand flies were significantly different among transects (MS = 1,786, F (4, 163) = 14.6, p< 0.0001), as were the numbers of Phlebotomus males (MS = 759, F (4, 163) = 13.7, p< 0.0001), the numbers of Phlebotomus females without blood (MS = 169.5, F (4, 163) = 12.6, p< 0.0001), and the numbers of Phlebotomus blood-fed females (MS = 4, F (4, 163) = 8, p< 0.0001), (Fig. 24).
Figure 24: The relationship between transects and the number of Phlebotomus sand flies. The red bars represents total sand flies, green bars represents males, brown bars represents females without blood, and blue bars represents blood-fed females.
Distances of trap stations to hyrax colonies were negatively correlated with the total number of Phlebotomus sand flies (MS = 5,943, F (1, 166) = 46.63, p< 0.0001), the number of Phlebotomus males (MS = 2,565, F (1, 166) = 44.8, p< 0.0001), the number of Phlebotomus
51 females without blood (MS = 698.6, F (1, 166) = 42.2, p< 0.0001), and the number of Phlebotomus blood-fed females sand fly (MS = 7.5, F (1, 166) = 13.7, p< 0.0001), (Fig. 25).
Figure 25: The relationship between the distance of trap station from hyrax colony and the number of Phlebotomus sand flies. The red circle curve represents total sand flies, green square curve represents males, brown triangle curve represents females without blood, and straight blue curve represents blood-fed females.
3.3.2.0.0. Sand fly species identification:
Of the captured Phlebotomus sand flies, 469 (298 male, and 171 female) were captured in July, and 581(384 male, and 197 female) were captured in September. Phlebotomus males were composed of P. sergenti (95.6%), P. alexandri (1.3%), P. arabicus (1.3%), P. perfiliewi (1.03%), P. syriacus (0.6%), and P. simici (0.14%). Phlebotomus females were composed of P. sergenti (94%), P. perfiliewi (2.5%), P. papatasi (1.9%), P. tobbi (1.1%), and P. kazeruni (0.5%) (Fig 26).
52
Figure 26: Phlebotomus sand fly species composition. The black bars represents the male species. The white bars represent the female species.
The numbers of P. sergenti males were negatively correlated with the distance of trap stations from to the nearest hyrax colony (MS = 3,188, F (1, 166) = 59.6, p< 0.0001). Other male species (P. alexandri, P. arabicus, P. perfiliewi, P. syriacus, and P. simici) did not vary significantly among trap stations. (Fig 27). Similarly, the numbers of P. sergenti females were negatively correlated with the distance of trap stations to the nearest hyrax colony (MS = 829.4,
F (1, 166) = 53.65, p< 0.0001), while other female species (P. perfiliewi, P. papatasi, P. tobbi, and P. kazeruni) did not vary significantly (Fig 28).
53
Figure 27: The relationship between the number of male sand flies and the distance of trap station to the nearest hyrax colony for 6 species of sand flies.
Figure 28: The relationship between the number of female sand flies and distance of trap station to the nearest hyrax colony for 5 species of sand flies.
54
3.3.3.0.0. Leishmania parasite detection:
All captured female Phlebotomus sand flies were tested for the presence of Leishmania parasites. In non-fed females (331 female), out of 173 pools, 30 pools were positive for Leishmania ITS1 (9% minimal infection rate). In blood fed females (37 females), six pools out of 37 were positive for Leishmania parasite (16.2% minimal infection rate; Fig 29). All positive samples were infected with L. tropica according to the DNA sequencing, and all of the infected flies were P. sergenti.
Figure 29: Gel image of ITS-PCR Leishmania targeting DNA from blood-fed sand flies. M is DNA ladder, from 1 to 12 PCR product of blood-fed sand flies, +ve, positive control (L. infantum), and -ve, negative control (pure water).
Most of the infected Phlebotomus females were captured close to the hyrax colonies. Infected sand flies as measured by the number of pools with infected flies occurred in greater numbers at some trapping stations than others. This pattern held both for non-fed female pools
(χ² (3) = 22.7, p< 0.0001), as well as for infected blood-fed female pools (χ² (1) = 3.96, p= 0.047; Fig 30). For both cases, largest numbers occurred at 3 and 4 m from the nearest hyrax colony.
There was a significant difference between transects in infected non-fed females (χ² (2) = 42.6, p> 0.0001), while in blood-fed females all the infections were in transect (A) the closest transect to hyrax colonies (Fig 31).
55
Figure 30: The relationship between number of pools of female sand flies with at least one infected individual and distance to the nearest hyrax colony. The pink curve represents the infected pools of female without blood, and the brown curve represents the infected pools of blood-fed females.
Figure 31: The left pie represents the distribution of percentage of pools of female sand flies with at least one infected individual across transects. Blue represents transect A, red represents transect B, and green represents transect C. The right pies represent the percentage of infected and non-infected pools in each transect. (See figure 3)
56
3.3.4.0.0. Host blood species identification:
A total of 50 blood-fed Phlebotomus spp. (96% P. sergenti, 2% P. perfiliewi, and 2% P. tobbi) were analyzed for blood meal identification. All samples were positive to Cyto b PCR (Fig. 32), however, only in 37 samples (74%) was the host blood species detected using RLB.
Figure 32: Gel image of cyto b BCR targeting DNA from blood-fed sand flies. M is DNA ladder, from 19 to 28 PCR product of blood-fed sand flies, -ve negative control (pure water).
Sand flies fed on different host species. The host species most commonly fed on by sand flies were dogs (39.1%), flowed by hyrax (28.3%), humans (23.9%), and livestock (8.7%). Most females were engorged with the blood of a single host species (73%), and less were engorged with the blood of multiple host species (27%). Six species of hosts were detected in July and September (Table 10).
Host blood species were significantly different among transects. Human blood was significantly different among transects (χ² (2) = 10.96, p= 0.004). However, when accounting for the effect of distance from hyrax colonies, the difference was not significant (χ² (3) = 5.8, p=
0.12). Hyrax blood was significantly different among transects (χ² (2) = 10.7, p= 0.005). Hyrax blood remained significantly different when accounting for the effect of distance from hyrax colonies (χ² (3) = 8.17, p= 0.043). Dog blood was significantly different among transects (χ² (2) = 13.27, p= 0.001). However, when accounting for the effect of distance from hyrax colonies, the difference was marginally significant (χ² (5) = 10.04, p= 0.074). Livestock blood was only found in transect A. In addition: there was no difference when accounting for the effect of distance from hyrax colonies (χ² (1) = 0 p= 1; Fig. 33 and Fig. 34)
57
Table 10: Host blood species of different sand fly species in July and September identified by Cyto b PCR and RLB.
Number of flies Number of Sand fly species Host blood species July September infected flies P. sergenti P. perfiliewi P. tobbi
Human 62.5% (5) 37.5% (3) 12.5% (1) 87.5 % (7) 0 12.5% (1)
Hyrax 20% (1) 80% (4) 20% (1) 87.5% (4) 20% (1) 0
Dog 50% (6) 50% (6) 16.6% (2) 100% (12) 0 0
Cow 50% (1) 50% (1) 0 100% (2) 0 0
Donkey + Sheep 100% (1) 0 0 100% (1) 0 0
Human + Cow 100% (1) 0 100% (1) 100% (1) 0 0
Human + Hyrax 50% (1) 50% (1) 0 100% (2) 0 0
Hyrax + Dog 0 100% (6) 16.6% (1) 100% (6) 0 0
16 21 35 1 1 Total 6 37 37
Figure 33: The number of sand flies on each transect engorged with blood for four categories of host species. The blue bars represents human blood, red bars represents hyrax blood, green bars represents dog blood, and violet bars represents livestock blood.
58
Figure 34: The number of females engorged with blood as a function of distance of the trapping station to the nearest hyrax colony for four categories of host species. The blue curve diamond represents human blood, red square curve represents hyrax blood, green triangle curve represents dog blood, and violet straight curve represents livestock blood.
59
CHAPTER FOUR: DISCUSSION
4.1.0.0.0. Sand fly sampling at the Bethlehem sites:
Understanding the activity density of sand fly vectors is essential to understanding the transmission dynamics of leishmaniasis. In this regard, this study documented the sex ratio, species composition, and activity density of Phlebotomus sand flies at three sites along an elevational gradient from low (480 m ASL) to high elevations (761 m ASL) across three sites— AZA, AAR and KIS—in the south western Bethlehem District of Palestine. Comparisons of endemicity between areas differing in elevation and comparisons between an endemic and disease-free area for our region provide new information concerning vector densities. The importance of such research is to provide information about vector density and behavior that influence disease transmission and to monitor risk prone areas. My results show the male: female ratio was significantly different between sites, with females being more common in the non-endemic site KIS (Fig. 6). Because sand fly males disperse much shorter distances than females (Orshan et al., 2016; Yuval et al., 1988), males tend to be closer to their breeding sites than females. This suggests that the breeding sites are closer to human-inhabited areas in the endemic sites of AAR and AZA, rather than in the non-endemic site of KIS. Phlebotomus sand fly densities peaked at an intermediate elevation of 604 m. My results show that sand fly densities differed between the sites (Fig. 7, 8), but this effect disappeared when elevation was included in the analysis. Instead elevation became significant, suggesting that most or all of the site effect is mediated by elevation. The substantial differences in densities between the three sites reflects suitable habitat in the endemic area for the vector of the disease to survive and reproduce, while the disease-free area does not provide the suitable habitat for survival and reproduction of the sand fly vectors. Additional differences between sites may relate to effects of abiotic factors such as temperature, precipitation, moisture, and biotic factors including the presence and distribution of vertebrate hosts, which was previously shown to affect sand fly distributions (Akhoundi et al., 2012; Cross et al., 1996; Ibrahim et al., 2005; Simsek et al., 2007; Theodore, 1936; Wasserberg et al., 2002 and 2003). Age composition of female insects is important in determining the epidemiological danger of that insect (Detinova, 1966). Therefore, age class distributions, which can be determined by
60 examining the parous (females that have deposited eggs) and nulliparous (female that have not deposited eggs) females in the population, can be used to study population dynamics, estimate epidemiological significance of the pathogen’s vector, and evaluate vector control methods (Chaniotis and Anderson, 1960). In regard to sand fly age and state, parous and nulliparous females can be determined using different methods, but samples should be freeze dried or fresh in order to distinguish between them (Anez and Tang, 1997). Sand fly adults of both sexes feed on plant honeydew and nectar, but females also feed on vertebrate blood for egg production (Killick-Kendrick, 1999; Schlein and Warburg, 1986). My results show a negative linear relationship between the elevation of trapping stations and the proportion of blood fed Phlebotomus sand flies (Fig. 9). In contrast, the correlation between the elevation of trapping stations and sand fly (female and male) abundance was quadratic (Fig. 7, 8). This difference can be due to the age difference in sand fly females between sites. Since my samples were too dry, I was not able to test the parousity. Instead, I tested for gravidity of Phlebotomus sand flies as an indicator of sand fly age. The results show a negative linear relationship between the elevation of the trapping stations and the proportion of gravid female Phlebotomus sand flies (Fig. 10), which indicates a difference in age distribution of sand fly females between sites. The greater proportion of gravid females found at lower elevations suggest that females there are older. Older females may be due to greater survivorship, a longer season of activity that starts earlier, or poorer reproduction and recruitment. The first two suggest greater negative epidemiological impact, The seasonal population dynamics of Phlebotomus sand flies were bimodal. In Middle Eastern countries, sand fly population densities are often bimodal across the year, but may differ in when population density peaks occur. Peak density has been shown to occur in August and /or September (Belen and Alten, 2011; Müller et al., 2001; Orshan et al., 2010), from July to September (Doha and Samy, 2010), in April or May, and in September or October (El Sawaf et al., 2016; Wasserberg et al., 2003). My results showed sand fly density was highest earlier in the season at AAR and AZA, while densities at KIS were low. Sand fly abundance was consistently higher at AAR throughout the study, and the fluctuation was mostly between two peaks in June and September. In contrast, the density at AZA decreased each month from May through October (Fig. 11). Sand fly seasonal activity patterns over the years 2013 and 2014 differed, at the two the endemic sites only, with differences in sand fly densities between months in 2013
61 compared with 2014 (interaction of month and year). In 2013, sand flies peaked in abundance in September, while in 2014 they peaked in July (Fig. 19). This resulted in differences in number of blood-fed females, although the numbers of sand flies with human blood peaked in September. The timings of peaks have consequence in disease dynamics; people are more exposed to sand fly bites during peak months. The results further showed also a significant difference in the proportion of blood fed females between 2013 compared with 2014 (interaction of month and year). In 2013, their feeding success was high then gradually decreased over time, while in 2014, it peaked in July then gradually increase in the season (Fig. 20). That can be due to seasonal changes in favorable climate conditions in which sand flies tend to feed at relative humidity of 75%-80% and temperature of at least 20°C (Dinesh et al., 2001). There are also the times when using control methods against adult sand flies will be the most effective for disease control. Distributions of most ectothermic species in time and space, including sand flies, are dependent on ambient temperatures; According to the thermal gradient, temperatures vary with elevation (−0.6◦ C per 100 m), so we can expect sand fly densities to vary with elevation. The distribution of Phlebotomus sand fly males differed significantly from one site to another. P. sergenti, P. papatasi, P. syriacus, P. jacusieli, and P. alexandri were collected from the three sites while other species were restricted to AZA (Table 2, Fig. 12). However, P. sergenti, which is the vector of L. tropica in Palestine (Al-Jawabreh et al., 2004), was more abundant than other species, followed by P. papatasi which is the vector of L. major in Palestine (Al-Jawaberh et al., 2003). This pattern differed among each site with significantly higher abundances of P. sergenti in AAR and KIS, while P. papatasi was higher in AZA. This finding suggests the presence of the vector of L. tropica at high density increases the opportunity for disease transmission. Sand fly species diversity and richness varied among sites/ elevations. Species diversity and richness was highest at AZA with the lowest elevation (473-510 m), where all recorded species were present. The site of KIS, with the highest elevation (732-782 m), also had high diversity and the highest evenness (Table 1). The distribution of sand fly species depends on environmental factors such as temperature, humidity, and precipitation, as well as biotic factors such as the density and distribution of the vertebrate hosts, and physical factors such as habitat availability and geographical barriers. Previous studies showed a direct relation between sand fly species composition and diversity with elevation variation (Doha and Samy, 2010; Guernaoui et al., 2006; Simsek et al., 2007). A study done in southern Turkey found highest species diversity
62 at an elevation range of 400-600 m ASL, which the authors attribute to differences in geographical formations and habitats (Simsek et al., 2007). This concords with my results. Other studies showed the highest diversity and richness at an elevational range of 800-999 m ASL in Morocco, (Guernaoui et al., 2006), or at 800-1200 m ASL in Saudi Arabia (Doha and Samy, 2010). In both examples, the authors attribute the pattern to the transition between lowland and mountains. In addition, species composition differed significantly across trapping stations and elevations. P. sergenti, and P. papatasi were collected at all trapping stations, P. syriacus were found at most trapping stations, P. alexandri and P. perfiliewi in fewer trapping stations, and P. jacusieli and P. tobbi were found only in the traps at the lowest elevations. Lower elevation has larger area, so the highest diversity at the lowest elevational site of AZA can be explained at least in part by the species-area relationship that predicts higher species diversity in larger areas. Many hypotheses have been proposed to explain the relationship, including larger areas receive more solar energy, more resources, larger home range for species, more diversity of environments, and greater potential for having immigrants (Lomolino, 2001) P. sergenti and P. papatasi were collected at all elevations, with their maximum abundance occurring between 522-681 m and 473-510 m, respectively (Fig. 13). Previous studies showed that P. sergenti is largely wide spread while P. papatasi is absent from some elevations. The maximum abundance of P. sergenti and P. papatasi occurred at 0-200 m in Turkey (Simsek et al., 2007), and 800-999 m and 400-599 m in Morocco (Guernaoui et al., 2006), respectively. The authors explain the difference coming from P. papatasi preferring the arid and semiarid areas, while P. sergenti is a “mountain” species. Blood meal identification is essential for determining the feeding success, the host preferences, and the vectorial capacities of hematophagous arthropods. Therefore, studying the feeding behavior of sand flies is important for estimating the efficiency of the Leishmania parasite transmission and to assess the relative human risk for contracting leishmaniasis. In this study, more females of all species of Phlebotomus had abdomens full of human blood, followed by avian, livestock, and dog (Fig. 15). However, P. sergenti showed a slightly different pattern of blood feeding, despite human blood still being the most common (human, livestock, avian, and dog). Previous studies showed that P. sergenti is an opportunistic feeder, feeding on birds and mammals (Svobodova et al., 2003), or on human, ovine, avian, and feline blood (Maroli et al., 2009).
63
Feeding success of sand flies depend upon host availability and accessibility (Azizi et al., 2016). The results showed no difference in the spatial distribution of P. sergenti that engorged themselves with avian, livestock, or dog blood possibly because the farmers in these villages raise these domesticated animals in high numbers. Nonetheless, number of P. sergenti engorged with human blood showed a significant difference among trapping stations. The proportion of P. sergenti engorged with human blood peaked between 522 - 604 m elevations (Fig. 16). At this range of elevations the density of phlebotomus sand flies also peaked (Fig. 5). Furthermore, the density of the human population in the particular village at these elevations is twice that of other villages in the study, and so provides more feeding opportunities for sand flies. Avian blood may affect sand fly fecundity as well as the Leishmania parasite. A pervious study of the new world sand fly Lutzomyia ovallesi showed that females fed on chicken blood had the longest oviposition time, greater egg production, and great fecundity (Noguera et al., 2006). A study showed that avian blood can harm the Leishmania parasite in sand flies (Schlein et al., 1983). My results show that P. sergenti were commonly engorged with avian blood, which was by far the most commonly consumed blood in May, which is the beginning of sand fly season. Thus, P. sergenti may have similar feeding habits to that of Lutzomyia ovallesi, and this may boost sand fly abundances and in this manner contribute to disease dynamics. However, avian blood can harm the parasite, resulting in a reduction in infection rate in sand flies. Human behavior can affect the temporal distribution of sand flies. Dinesh et al. (2001) demonstrated that seasonal variation in the occurrence of sand fly bites could be affected by the seasonal changes in sleeping patterns of humans. Another study referred to changes in climatic factors (Guernaoui et al., 2006). It also can be due to sand flies behavior in which P. sergenti tend to enter houses much more toward the end of the summer (September-October; Orshan et al., 2010, Salah, unpublished). My results show that the numbers of P. sergenti engorged with human blood differed between months, peaking in September (Fig. 17). The villagers from the study sites tend to sleep outside during the summer season and thus are more exposed to sand flies, as would be the case in September when temperatures are higher than other months there. Comparison of endemicity between areas differing in elevation as well as a comparison between an endemic and a disease-free area as presented here is new in our region. The importance of such a study is to provide information about the spatial and temporal distribution, age and species composition, feeding success, and host preference of sand fly vectors that
64 influence disease transmission, and allow us to better target sand flies, to control the disease, and to monitor risk prone areas.
4.2.0.0.0. Epidemiological Investigation in Tubas study sites:
Urbanization influences the epidemiology of infectious diseases, and can increase or decrease disease transmission (Alirol et al., 2011; Neiderud, 2015). In urban areas people often have more access to health services, better sanitation systems, and water, all of which improve health status. However, urbanization increases the risk of emerging and reemerging diseases. Urban environments provide suitable bases for the spread of epidemics because of high human population density (Alirol et al., 2011), the possibility of providing new habitats for the reservoir host species (Orshan et al., 2010), the occasional production of greater contact with wildlife (Neiderud, 2015), and the greater contact of humans with livestock when rural migrants move their domestic animals to urban areas (Desjeux, 2001). All these factors can create ideal condition for disease transmission.
Urbanization may affect the transmission of vector-borne diseases both for better and for worse. For example, during the last four decades, the increase of incidence and the spread of dengue fever was driven by urbanization, globalization, and the lack of mosquito control (Gubler, 2011). In contrast, there is an association between increased urbanization and malaria, in which urbanization decreases disease transmission (Tatem et al., 2013). In regards to CL in particular, Desjeux, (2004) showed that urbanization contributed to increased CL case numbers in Afghanistan and Burkina Faso. Since little information is available about the epidemiology and risk factors of CL caused by L. tropica in the West Bank, we conducted this study to better understand the effect of urbanization and socio-economic status on CL transmission. It serves as a case study of urbanization in a developing country of a region that has not been explored systematically.
To identify risk factors for CL and to examine the consequences of the effect of urbanization on the risk factors, I conducted a matched case-control study based on epidemiological questionnaires filled out by/for CL patients in the District of Tubas. The purpose of the study was to characterize the effect of human exposure on several environmental and behavioral variables.
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In this part of the study, I divided the endemic District of Tubas into three different classes of sites according to their degree of urbanization: city, villages, and Bedouin encampments. The city of Tubas is the most urbanized area with the most spatial complexity. It consists of a central flat area surrounded by mountains and hills, faced by caves, rocky areas, and wadies. The villages comprise a more rural setting with less spatial complexity, forming a smaller area compared to the city and further containing additional flat agricultural areas. The Bedouin encampments are the most rural, with no spatial complexity, and fairly uniform desert habitat.
Comparing individuals with CL (cases) with other individuals who did not have the disease (controls), but live in the same area and under the same conditions, provided a better understanding of the behavioral and environmental risk factors associated with human CL. Most cases of CL occurred in the most urban area, the city of Tubas, compared to the less urban areas, the villages, and Bedouin encampments. The small sample size of CL cases in these areas limits the statistical power and our ability to demonstrate differences that may actually exist.
Population characteristics such as gender, education, and occupation provide insights into the different risk factors associated with CL (Table 4). Urbanization should be understood within the context of gender and division of labor that influence exposure. Gender had a large impact on disease transmission. CL cases in the city and villages were mostly males, but in the Bedouin encampments were mostly females (Fig. 21). This difference may reflect differences between those in the societies that are responsible for working. Females in Bedouin encampments hold responsibility for taking care of family work including tending the livestock near homes, working as shepherdesses on nearby pastures, and doing all the kitchen work at night, nearly all of which occurs outside. In contrast, in the city and villages, the males are responsible for the outside work. A similar study in the nearby city of Jericho (Al-Jawabreh et al., 2003), and in other Middle Eastern countries (Haouas et al., 2015; Khoury et al., 1996) show a higher percent of cases to be males However, a study conducted in the Arab Ar-Rashaiyda Bedouin areas showed that a higher percent of cases were females (Salah, unpublished). These examples highlight the role of urbanization in determining whether males or females are most at risk to CL.
Another important factor in understanding urbanization and the division of labor is age. In all three sites, the highest percent of cases occurred in children less than 17 years old (Fig. 21).
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Other studies in the Jordan Valley area also showed that a large majority of cases were children (Al-Jawabreh et al., 2003; Khoury et al., 1996). These authors suggested that an immature immune system, lack of previous exposure, or the lack of protection against sand fly bites may be the cause for this high incidence. Highest percentage of susceptible individuals occurring in the youngest age categories likely contributes as well. Although there was a significant difference in age distribution of cases among sites, in the city cases occurred in all age classes while, in the Bedouin encampments there were no cases among the oldest age category. This difference may reflect the high incidence of lifetime immunity found in the Bedouin encampments due to likely higher exposure of children and youth in the Bedouin living setting. The urbanization processes and ecological changes found in the villages paradoxically expose more children relative to other age classes than do the other living conditions, thus exposing children also in the non-Bedouin settlements.
Incidence of CL and sand fly population dynamics appear to be coupled. Previous work in the Western Negev Desert has demonstrated that CL cases caused by Leishmania major were positively correlated with sand fly abundances with a time lag of a few months (Wasserberg et al., 2002). Similarly, my work in the Bethlehem District of the West Bank shows strong relationships between sand fly abundances and CL prevalence and endemism (Salah, unpublished). Here, the numbers of new CL cases were also temporally variable. The CL cases peaked during December 2014, and January 2015 (Fig. 22). If CL symptoms appear with the typical time lag as seen elsewhere, this would coincide with peak sand fly abundances that occur in July, August, and September months in the West Bank (Orshan et al., 2010; Section 1). As with other examples, incidence of CL seems to be driven more by vector dynamics than reservoir host dynamics, since the reservoir hosts being long-lived mammals are present close at hand throughout the year. I note, however, that other drivers can also be involved here and in other instances. Recent studies show that disease dynamics can be driven by migration from rural to urban areas, as well as by migration due to crises and wars (Al-Salem et al., 2016; Du et al., 2016).
Distance from reservoir host is a crucial factor in zoonotic disease transmission. The results showed that CL case houses or tents in the city and the Bedouin encampments were closer in distance to hyrax colonies than they were in villages, which increase the risk of disease
67 transmission (Table 9). In part 3, I found that there is a negative correlation between the infection rate in sand flies and the distance from hyrax colonies (rates were higher nearer to hyrax colonies), which increased the exposure of residents to infected sand flies in houses that were closer to these colonies. This helps explain the presence of multiple cases of CL in the same house or tent in the city and in Bedouin encampments. In the villages, proximity of hyrax colonies to human houses was not significant. But another factor, namely the distance to the village edge was correlated to enhanced disease transmission there. Also, for most of village cases they got infected in a place other than their village. This can be found in table 6, where I found a significant difference between cases and controls in moving to a different place in the last six month prior to the infection. Therefore, most of the cases were in different houses.
CL caused by L. tropica normally presents as a single lesion whereas L. major frequently causes multiple lesions (Al-Jawabreh et al., 2017; Bousslimi et al., 2010). Almost half of the cases I studied, had a single lesion with no significant difference between the sites (Table 5). The head and the limbs are the exposed parts of the body to sand fly bites. In the city, most cases had lesion(s) on the limbs; the cases in the Bedouin encampments had lesions mostly on the face, while cases of the villages had a similar proportion of lesions on the face, limbs, and both (Table
5). This can be due to bites on uncovered body parts, and the attraction of sand flies to CO2 exhaled by people as shown in other studies (Al-Jawabreh et al., 2017; Bousslimi et al., 2010; Turan et al., 2015).
Human behavior can increase exposure to infected sand flies. Moving into a place recognized as a Leishmania focus during the sand fly season (May-November) may expose them more to infection. The results showed a significant difference between cases and controls in the city and the villages in moving to a CL focus during the last six months prior to the appearance of the lesion; there was no significant difference in the Bedouin encampments (Table 6). The percentage of cases contracting an infection in an area other than their home town or village were relatively high in the village compared to the other two sites, which can explain the relatively low incidence rate of leishmaniasis in the villages compared to the city and Bedouin encampments. Sand fly activity and biting rates are highest after sunset (Kravchenko et al., 2004; Roberts, 1994), and CL cases in the city, villages, and Bedouin encampments tended to spend more time outdoors in the evenings, than controls did. This is especially true during the summer
68 months when sand flies are active, increasing the risk of getting infected. Respondents suffered from sand fly bites during summer nights especially in July, August, and September, which are considered the peak of sand fly activity in the West Bank (Fig. 19).
Control of sand flies should be a public health priority. The efficacy of using personal protection and other control methods against adult sand flies has been demonstrated in different studies (Alexander et al., 1995; Orshan et al., 2006; Warburg and Faiman, 2011). Awareness of using personal protection to avoid sand fly bites was not found at all sites in our study. Rather, people who live in urban areas were more aware, but people who live in villages and Bedouin encampments were less aware. The city respondents tended to use different personal protection methods to avoid sand fly bites (Table 7). Even in urban settings, there were differences in awareness in ways that affect disease transmission. Controls in the urban settings more often utilized vaporizing tablets or liquid and sprayed insecticides indoors than did cases. These methods were shown to be highly effective (Sirak-Wizeman et al., 2008; Warburg and Faiman, 2011) in controlling sand flies. In contrast, in the villages and Bedouin encampments, almost nobody used these protective methods.
Exposure to vectors as indicated by distance from the edge of the city or villages was associated with disease incidence in both sites. In both instances cases were significantly closer to the edge than the controls within each site. Similarly, most cases’ houses in the city were located on small hill tops facing the slopes of the wadi (Table 8). This likely exposed them to more open areas, caves, rocky areas, and wadies that may serve as breeding habitat for sand flies, and are suitable habitat for the hyrax reservoir host. Whether this is universal is harder to evaluate. In regards to the Bedouin encampments, the scattered nature of the houses means that everyone, including both cases and controls, were effectively living at the edge of the encampments, thus I could not look for consequences of variation in this variable there. Still, this high incidence of CL relative to population size where all people live on edges is consistent with its importance.
Green farms and livestock should provide food resources for adult sand flies. Both sexes of sand flies feed on plant honeydew and nectar, but females also feed on vertebrate blood for egg production (Abbasi et al., 2009; Killick-Kendrick, 1999; Schlein and Warburg, 1986). Therefore, the distance of the house from farms that can provide honey dew, nectar, and animals
69 for blood meals may be crucial for disease epidemiology. Again, in the Bedouin encampments, the cases and the controls or their families were all involved in raising animals next to their tents. Those tents were surrounded by shrubs that can serve as feeding habitat for sand flies that expose people to more bites. There was no variation in this variable, so once more I could not weigh its contribution except in more urban settings (Table 8). A previous study showed that developing CL can be affected by the relationship between human immune system and sand fly saliva and the intensity of exposure to sand fly bites (Mondragon-Shem et al., 2015). In our example, people living in the Bedouin encampments likely are more exposed to sand flies than in the villages or urban settings, and their immunological state may make them more susceptible to infection, although less susceptible to severe effects.
Disturbances caused by urbanization can provide new habitats for the hyraxes. Respondents in the city and the villages agreed that there was construction work near their houses during the summer of 2014 (Table 8), as well as the building of new neighborhoods in the city, which create suitable habitat for hyrax colonization. Different studies showed that anthropogenic disturbance increases vector and reservoir host densities by providing breeding sites for the reservoir that is also suitable for sand flies to rest and breed (Orshan et al., 2010; Wasserberg et al., 2003).
Efficient transmission of Leishmania parasites occur when the reservoir hosts and the vectors live in close proximity and the vectors tend to feed upon the reservoir hosts (Ashford, 2000). In the city and the villages, cases were significantly closer to the hyrax colonies than were the controls within each site. In regard to vector sand flies, more than half of the respondents from the three sites agreed that the density of sand flies in 2014 was more than in previous years, which likely was due to high temperature, and low rainfall, construction, the municipality not spraying insecticide, and increases in hyrax population close to houses (Table 9). Thus, the presence of high densities of both sand fly vector and reservoir hosts close to houses increase the disease burden.
An understanding of urbanization processes, as they take place in various local contexts— ecological and epidemiological—should be taken into consideration when planning public health interventions to reduce leishmaniasis morbidity.
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4.3.0.0.0. Sand fly transects sampling Tubas site:
Aleskan is a new neighborhood in the city of Tubas, established in 2002. It consists of homes and cultivated gardens located on a small hill facing south west towards a wadi full of rocks and caves that provide suitable shelters for hyraxes. More than half of the cases in the entire city (51%) in 2015 were located in this neighborhood. Therefore, I chose this neighborhood to quantify the effect of distance from hyrax colonies on the density for L. tropica, i.e., Phlebotomus sandflies, the infection rate of Phlebotomus sandflies, and the feeding success of Phlebotomus sandflies.
The results show that the largest number of sandflies were captured along the transect closest to the hyrax colonies, which was also the closest to the edges of the neighborhood (Fig. 24). Other studies show that disturbances can provide new habitats for the reservoir (Orshan et al., 2010; Wasserberg et al., 2002, 2003), which may increase the sand fly density. In addition: Orshan et al. (2010) showed that sand flies tend to be more numerous on the edges of villages rather than in the centers.
Trapping stations that were closer to the hyrax colonies and to the edges had higher densities of sand flies. There was a negative correlation between the sand fly density and the distance of trapping stations from hyrax colonies (Fig. 25). The hyrax colonies appear to serve as a resting place and breeding sites for adult sand flies and may provide the organic materials for larvae development. Thus, sand fly foraging behavior may follows the central place foraging theory (Kacelnik et al., 1986) in which they emerge from the hyrax caves, travel to feed on plants and to seek blood meals, and then return to their breeding sites to lay eggs. If this is true, then the density of sand flies should be higher close to hyrax colonies, and gradually decrease with distance from the colonies.
CL caused by L. tropica incidence increases as P. Sergenti abundances increases. The results show that the most common Phlebotomus sand fly species in the study area was P. sergenti (Fig. 26). Both male and female P. sergenti showed negative correlations between their densities and the distance to hyrax colonies (Fig. 27, 28). Studies show that there is a relationship between sand fly abundances and CL prevalence and endemism (Salah, unpublished; Wasserberg et al., 2002). Furthermore, the presence of the specific vector of L. tropica at high densities
71 increases the opportunity for disease transmission. My results suggest that human infections will be more frequent closer to the hyrax colonies and closer to the city edges.
This study is the first one showing that P. sergenti is the vector of L. tropica and is found in Tubas District. However, P. sergenti had already been known to be the vector of L. tropica in the Jenin District of the West Bank (Samer Sawalha, personal communication). I found high infection rates in the non-blood fed P. sergenti (9%). However, there were higher infection rates in blood fed P. sergenti females (16.2%). This high IR indicates a higher infection among susceptible human populations that are exposed to infected flies. In addition, the results show a significant difference between the infected flies for both non-blood fed and the blood fed Phlebotomus, and the distance to hyrax colonies (Fig. 30). Low infection rate was found in P. sergenti females captured at the trap station 2 m from a hyrax colony, suggesting that the hyraxes close to that particular station were uninfected. But higher infection rates were found in flies captured at the 3 and 4 m distances from an adjacent colony, suggesting that those nearby hyraxes were infected. The distance between the two hyrax colonies was relatively short (~ 100 m), but one appears to have infected animals and the other probably not. That suggests little movement between adjacent groups of hyrax. These results further suggest that the infection in a hyrax population can be heterogeneous. Thus, people who live close to the hyrax colonies are at a higher risk of contracting CL because they are exposed to higher densities of infected sand flies.
Host preference is a crucial parameter in estimating vectorial capacity of hematophagous arthropods since a competent vector will necessarily feed on both reservoir hosts and humans. Therefore, studying the feeding behavior of sand flies is important for estimating the efficiency of Leishmania transmission. In this part of the study, the female P. sergenti females were shown most frequently to rely on dog blood meals, followed by hyrax, humans, and livestock (Table 10). Previous studies show that P. sergenti is an opportunistic feeder, feeding on birds and mammals (Svobodova et al., 2003), or on human, ovine, avian, and felines bloods (Maroli et al., 2009). Feeding success of sand flies depends upon the host availability and accessibility (Azizi et al., 2016). Almost one third of the blood-fed Phlebotomus females had engorged on multiple host species. This can be due to the host’s defensive movement, reduced skin exposure, or
72 difficulty in finding skin blood capillaries, all of which may prevent a sand fly from engorging enough blood in a single meal (Bongiorno et al., 2003).
In the previous section, I showed that the people who lived close to hyrax colonies were at higher risk of contracting the disease. My results in this section show that the number of sand flies engorged with hyrax or dog blood is negatively correlated with the distance to hyrax colonies (Fig. 33, 34). This suggests higher transmission will occur closer to these colonies, resulting in higher infection rates of humans living close to hyrax colonies.
4.4.0.0.0. General discussion:
Leishmaniasis remains one of the world's most devastating neglected tropical diseases, causing substantial morbidity and mortality and contributing to the loss of nearly 2 million disability-adjusted life years (McDowell et al., 2011). Vector-borne disease control can be achieved by better understanding of the ecology of vectors and reservoir hosts as well as other risk factors associated with disease transmission and its epidemiology.
Manipulations of risk factors relevant to the vector and to humans will confirm the importance of those factors in disease transmission and open the way for better control and for better public health practices. Comparison of endemicity between areas differing in elevation as well as a comparison between an endemic and a disease-free area as presented here is new in this region. The importance of such research is to provide information about vector density and behavior that influences disease transmission, and to monitor risk prone areas. Applying real data from the field to the Ross-Macdonald model can promote our understanding of pathogen dynamics and transmission and provide a target for personal protection of susceptible humans and reduction of vector population densities that reduces transmission below the threshold necessary to eradicate the disease.
Understanding the ecological considerations regarding the population dynamics of vector populations as well as human sociological factors and epidemiological approaches can have large impact on zoonotic disease control. This study is important in combining approaches of disease ecology and epidemiology. This combination can provide better understanding of human-vector interaction. Such understanding will allow us to better identify the weak links in disease transmission and target them for controlling the disease. Studying the ecology of the sand fly
73 vector of this disease together with human ecology, epidemiological studies, and sociological methods will provide a more complete understanding of zoonotic CL caused by L. tropica and offer hopes for more holistic and more effective control strategies.
Urbanization influences the epidemiology of infectious diseases, and can increase or decrease disease transmission. Better understanding of urbanization processes, as they take place in various ecological and epidemiological contexts, in my case the Tubas District in the West Bank in Palestine, should be taken into consideration when planning public health interventions to reduce leishmaniasis morbidity.
The construction methods for building infrastructure in a new neighborhood created the rock piles that attracted the hyraxes. Areas in which houses are built along natural contours do not appear to provide valued habitat and refuges for the hyrax and so do not serve foci for the disease. In this manner, we can now talk about the concept of disease –preventive landscape architecture– that combines epidemiology, ecology and urban design when planning and building the places where we live, work, and play.
In regards to the Ross-Macdonald model of Fig.2, in this research I showed how areas differing in endemicity have different densities of sand fly vectors (m), and feeding success (bite frequency (a)), in which the endemic sites of AAR and AZA are higher in vector density and vector biting rate than the non-endemic site of KIS that provide the threshold necessarily to sustain transmission of L. tropica (Fig. 2). Additionally, in the third section I showed the effect of reservoir presence on sand fly population and infection rate by quantifying sand fly densities (m) in relation to the distance from the hyrax colony. Although, I could not estimate the density of reservoir host (b2), I could show that the sand flies trapped closer to hyrax colonies were more frequently infected with L. tropica than the flies trapped farther away, which again confirms the prediction of the Ross-Macdonald model that higher densities of vectors and reservoirs promote transmission.
Based on Ross-Macdonald model higher density of the sand fly vector and the hyrax reservoir host will sustain the disease. I confirmed in this research many of the predictions of the Ross-Macdonald model. Based on my findings, the possibility of the disease spreading to different districts in Palestine appears high because of the increase in the hyrax densities, their movement from place to place, and their establishment in areas where they were never
74 previously observed. As well as unplanned urbanization create suitable habitat for both the hyrax colonization and the sand fly breeding sites. Another factor is the effect of climate change that creates a suitable climate for sand fly development, potentially leading to an increase the densities of sand flies. Thus, having the vector sand fly and the hyrax reservoir in high densities, as well as having infected host individuals in areas previously free of disease will create leishmaniasis outbreaks. In order to control and contain the disease, we need to decrease the vector and reservoir densities, by removing the hyraxes from the periphery of human habitation to a minimum a range of 250-500 m away, the incidence of CL can be reduced by 90%. Another promising methods for decreasing the density and controlling sand fly adult and larvae being the use of food baits for mammals that contain a feed-through insecticides. The intent is to feed individuals of the reservoir with food laced with a systematic insecticide that the reservoir hosts themselves find non-harmful, but that renders their blood and their feces toxic to adults and larvae of the vector.
With CL caused by L. tropica rapidly spreading in the West Bank, research such as that presented here is of crucial importance. My study provided some information about the ecology and epidemiology of CL caused by L. tropica in the Bethlehem and Tubas Districts. The limitations of the study include its short duration, the lack of equipment, manpower, and limited funds. These factors limited my ability to collect more data, and perform experiments on the vector and the reservoir host at these sites and at other sites. Another limitation is the small sample size of cases in the villages and Bedouin encampments, which limits statistical power and my ability to demonstrate differences that may actually exist. In this research I was not able to test for the infection in humans and in the hyrax reservoir host, as well as measure reservoir host density, which are both important parameters to understand leishmaniasis transmission and dynamics. The results of this thesis should be used to monitor areas under risk, as well to increase people’s awareness about the risk of exposure in the peak season of sand flies, and promote the use of personal protection methods to avoid disease transmission. In addition, when planning and building the places where we live, these results should be taken in consideration. Further studies are needed in these districts and in the rest of the West Bank. These studies should include the movement and flight patterns of the sand fly vector and use of behavioral indicators and foraging theory to better understand the distribution, abundance, density dependent habitat selection, optimal patch use, and movement of reservoir host hyraxes.
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APPENDIX A: Villages profile The village of Arab Ar-Rashaiyda is endemic for the disease. Arab Ar-Rashaiyda is divided into two localities, Ar-Rashaiyda (AAR, 31°56′90.55′′N, 35°22′97.92′′E, and altitude 512 m above the sea level), and Al ‘Azazma (AZA, 31°33′37.84′′N, 35°14′28.32′′E, and altitude 480 m above the sea level). These two localities are separated by 1 km. Arab Ar-Rashaiyda is located 18.7 Km south-ease of Bethlehem city. The village comprises a total area of about 4,784 ha; 48 ha are built up area, and 4,700 ha are arable land. The village has an average annual temperature of 19.2 C0, with mean annual rainfall of 246 mm, and average annual humidity of 58%. The village lacks health facilities although a mobile government physician is available. Emergency care for residents is available at the Tuqu’ health centers about 8 Km away. (ARIJ, Arab Ar- Rashaiyda Village Profile, 2010). The total population of the village in 2007 was 1,453 (PCBS, 2007).
The village of Kisan is located 11 Km south of Bethlehem city (KIS, 31°61′20.69′′N, 35°22′03.37′′E, and altitude 761 m above sea level). The village is spread out and covers a total area of about13,333 ha; 7.9 ha are built up, and 13,229 ha are considered arable land. The average annual temperature is 17 C0, with mean annual rainfall of 365 mm, and average annual humidity of 60.3%. The village has a small health clinic housed in a small rented building, so in case of emergency village residents use Tuqu’ health centers located 3 Km from the village. (ARIJ, Kisan Village Profile, 2010). The total population of the village in 2007 was 454 (PCBS, 2007). This village is a non-endemic area for leishmaniasis.
The city of Tubas (31°19′16.23′′N, 35°22′11.83′′E, and altitude 362 m above sea level) is located to the western part of Tubas District. The city comprises a total area of about 29,512.3ha; 300 ha are built up, and 15,000 ha are agricultural. The total population of the city in 2015 was 20,801 (ARIJ, Tubas city Profile, 2006; PCBS, 2015).
The village of Tummun (32°16′59.46′′N, 35°23′8.86′′E, and altitude 332 m above sea level) is located 5 Km south of Tubas city. The village comprises a total area of about 8,100ha; 500 ha are build up area, and 2,000 ha are forests. The total population of the village in 2015 was 13,900 (ARIJ, Tummun village Profile, 2006; PCBS, 2015).
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The village of Tayasir (32°20′25.56′′N, 35°23′48.42′′E, and altitude 331 m above sea level) is located 3 Km north of Tubas city. The village comprises a total area of about 2,600ha; 50ha are build up area, and 500ha are agriculture area. The total population of the village in 2015 was 3,205 (ARIJ, Tayasir village Profile, 2006; PCBS, 2015).
The village of Khirbet Atuf (32°15′53.38′′N, 35°26′12.98′′E, and altitude 50 m above sea level)is located 25 Km southeast of Tubas city. The village comprises a total area of about 2,361.4ha. The total population of the village in 2015 was 220 (ARIJ, Al Bikai’a village Profile, 2006; PCBS, 2015).
The village of Bardala (32°23′10.73′′N, 35°28′55.08′′E, and altitude -71 m below sea level) is located 13 Km northeast of Tubas city. The village comprises a total area of about 2,000ha; 48ha are build up area, and 1000ha are agriculture area. The total population of the village in 2015 was 2,108 (ARIJ, Bardala village Profile, 2006; PCBS, 2015).
The village of Khirbet Yarza (32°18′20.05′′N, 35°26′0.86′′E, and altitude 257 m above sea level) is located 6 Km east of Tubas city. The village comprises a total area of about 2,000ha; 30ha are build up area, and 150ha are agriculture area. The total population of the village in 2015 was 80 (ARIJ, Bardala village Profile, 2006; PCBS, 2015).
The village of Wadi al Far’a (32°17′37.40′′N, 35°20′40.06′′E, and altitude 051 m above sea level) is located 5 Km southwest of Tubas city. The village comprises a total area of about 1,200ha; 33.7ha are build up area, and 1,050ha are agriculture area. The total population of the village in 2015 was 3,515 (ARIJ, Wadi al Far’a village Profile, 2006; PCBS, 2015).
The area of Khirbet Al Malih (32°19′38.54′′N, 35°26′10.51′′E, and altitude 32 m above sea level) is located 10 Km east of Tubas city. The total area of about 1,900ha; 50ha are build up area, 1,200ha are agriculture area, and 40 ha are forests. The total population of the village in 2015 was 476 (ARIJ, Khirbet Al Malih Profile, 2006; PCBS, 2015).
The area of Khirbet Tell el Himma (32°22′17′′N, 35°30′50′′E, and altitude -182 m below sea level) is located 15 Km east of Tubas city. The total area of about 100ha; 5ha are build up area, 40ha are agriculture area, and 55 ha are forests. The total population of the village in 2015 was 100 (ARIJ, Khirbet Al Malih Profile, 2006; PCBS, 2015).
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APPENDIX B: Cutaneous Leishmaniasis questionnaire Patient personal details: 1. Date of Sampling ____/____/______2. Given name ______3. D.O.B. ___/____/______4. Age ______5. Sex: [1] Male [2] Female 6. Occupation (children – record daytime placement): ______7. Education: 1. (<1) 2. (1-6) 3. (7-12) 4. (>12) Current address: 8. District ______9. City/village ______10. Phone no: ______
11. Length of residence in the area ______Epidemiologic data 12. Have you moved house in the 12 months preceding the diagnosis of the illness? [1] Yes [2] No [3] Don’t know 13. If yes: Previous address ______13. a) Date of move ___/____/______13.b) Period spent at that place ______14. Were you bitten by an insect? [1] Yes [2] No [3] Don’t know 14.a) If yes, it was (show the pictures): 1. Mosquito 2. Sand-fly 3. Other Season when bitten How do you know Time of day when Were other people in you were bitten? you bitten the same place bitten at the same time? 14.b 14.c 14.d 14.e
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1 Spring 1 Pricking feeling 1 Evening 1 No 2 Summer 2 Itch 2 Night 2 Only a few 3 Fall (Autumn) 3 Mark on skin 3 Morning 3 Most of them 4 Unknown 4 Other 4 Unknown 4 Unknown
Do you use one of the following as personal protective measures against mosquito, sand fly, or other insect bites? Fans Insect Insect Vaporizin Repellent Spraying Spraying Other repellent repellent g tablets candles or within the outside the on skin on skin or liquid coils house house (in the (outside home) the home) 15 15.a 15.b 15.c 15.d 15.e 15.f 15.j 1 Yes 1 Yes 1 Yes 1 Yes 1 Yes 1 Yes 1 Yes 1 Yes 2 No 2 No 2 No 2 No 2 No 2 No 2 No 2 No 3 3 3 3 3 3 3 3 Sometime Sometime Sometime Sometime Sometime Sometime Sometime Sometime
Clinical and laboratory information: 16. Have been diagnosed with CL before? [1] Yes [2] No [3] Don’t know 16.a) If yes, when______17. Date of appearance of first signs of the disease: __/__/____ 18. Date of definitive diagnosis: __/__/____ 19. Other family member(s) infected? [1] Yes [2] No [3] Don’t know Site of lesion Nature of lesion 20. Face [1] Yes [2] No 23 Ulcer [1] Yes [2] No 21. Limbs [1] Yes [2] No 24. Nodule [1] Yes [2] No 22. Other [1] Yes [2] No 25. Other [1] Yes [2] No 22.a) If yes, specify ______25.a) If yes, specify ______
26. No. lesions ______27. Was a laboratory diagnosis made? [1] Yes [2] No [3] Do not know
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If yes, what were the results of the following tests? Please record as follows: [1] Positive result [2] Negative result [3] Not carried out Test Result 27.a) Direct smear [ ] 27.b) Histologic examination [ ] 27.c) Culture [ ] 27.d) Serology [ ] 27.e) Leishmania skin test [ ] 27.f) PCR [ ] 28. Type of Leishmania (if identified): 1. Tropica 2. Major 3. Infantum 4. Other ______Information about the house: 29. The house is placed on: [1] The top of a hill [2] Inside a Wadi [3] In between (not hill nor wadi) 30. Type of house: [1] Newly built [2] Old built 31. Are there screens on the windows and doors in your home? [1] Yes [2] Partial [3] No 31.a) If yes, are they intact and in good condition? [1] Yes [2] No 32. Does your home have a private garden? [1] Yes [2] No 33. During what hours of the day you tend to spend time outdoors: in the garden, near your home, in the neighborhood? [1] Morning [2] Afternoon [3] Evening [4] After sunset or at night 34. Do you have a farm within 200sqm of your residence? [1] Yes [2] No [3] Don’t know 34.a) What type of farm is it?
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[1] Green farm [2] Livestock [3] Other ______35. Is there a spring or a pond in your residence area? [1] Yes [2] No [3] Don’t know 36. Does the house have cracks on the walls and /or crevices on the floor? [1] Yes [2] No 36.a) If yes, where: [1] On the walls [2] On the floor [2] 1+2 37. Are the walls painted? [1] Yes [2] No
37.a) If yes: mention the color of the paint inside ______38. Do you have Air conditioning? [1] Yes [2] No 38.a) If yes, what Type? [1] Fan [2] Water [3] Gas
39. What is the distance of the house to the edge of the village? [1] Less than 20 m [2] More than 20 m
Information about Hyrax host and sand fly vector: 40. Have you seen the in the hyrax vicinity (show the picture)? [1] Yes [2] No 41. How far is the distance of the house to the hyrax colony? [1] Less than 100 m [2] More than 100 m [3] Don't know
42. Does the family have domestic animals next to the house? [1] Yes [2] No
42.a) if yes what kind of domestic animals? ______43. Do you think that the density of sand flies in summer 2010 is more than other years? [1] Yes [2] No [3] Don't know
43.a) If yes, what do you think the reason behind? ______44. Were there any construction works during summer 2014 (tractors moving earth etc.) [1] Yes [2] No
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إستمارة اللشمانيا الجلدية )حبة أريحا( معلومات شخصية: 0.تاريخ تعبئة االستمارة ــــــــ/ ــــــــــ / ــــــــــــــــــ 2. االسم الكامل ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ 3. تاريخ الميالد ــــــــ/ ــــــــــ / ــــــــــــــــــ 4. العمر ـــــــــــــــــــ 5. الجنس: 0. ذكر 2. انثى 6. الوظيفة )لالطفال- سجل مكان تواجدهم خالل اليوم( ______7. التعليم: )1>( .0 )6-0( .2 )02-7( .3 (12<( .4 مكان االقامة الحالي: 8. المحافظة ــــــــــــــــــــــــــــــــ 9. المدينة/ القرية ـــــــــــــــــــــــــــــــ 01. رقم الهاتف ــــــــــــــــــــــــــــــــــ 00. المدة الزمنية القامتك الدائمة في هذه المنطقة ______02. خالل ال 02 شهر السابقة لتشخيص االصابة هل قمت بتغير مكان السكن؟ 0. نعم 2. ال 3. ال اعلم 03. اذا كانت االجابة نعم: اذكر مكان االقامة ـــــــــــــــــــــــــــــــــــــــــــــ 03.ا. تاريخ االنتقال ــــــــ/ ــــــــــ / ــــــــــــــــــ 04. هل تعاني من لدغ الحشرات؟ 0. نعم 2. ال 3. ال اعلم 04.ا. اذا كانت االجابة نعم: الحشرة كانت )انظر للصور( 0. بعوضة 2. ذبابة الرمل 3. اخرى 04.ب. الفصل الذي 04. ج. كيف تشعر عندما 04.د. الوقت الذي تتعرض 04.ه. هل السكان الذين يسكنون تعرضت فيه للقرص تتعرض للقرص فيه للقرص نفس المكان يعانون من القرص 0. الربيع 0. الشعور بالوخ 0. المساء 0. ال 2. الصيف 2. الحكة 2. الليل 2. القليل منهم فقط 3. الخريف 3. عالمات على الجلد 3. الصباح 3. معظم السكان 4. ال اعلم 4. اخرى 4. ال اعلم 4. ال اعلم
هل تستخدم اي من االتية للحماية من التعرض لقرص االبعوض, ذبابة الرمل, او اي حشرات اخرى؟
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05. 05.ا. 05.ب. 05.ج. 05. د. 05.ه. 05.و. 05.ي. المروحة طارد للبعوض طارد للبعوض اقراص او شمع او رش البيت رش اخرى على الجلد)داخل على الجلد)خارج سائل زيت طارد من الداخل البيت من البيت ( البيت ( الخارج 0.نعم 0.نعم 0.نعم 0.نعم 0.نعم 0.نعم 0.نعم 0.نعم 2.ال 2.ال 2.ال 2.ال 2.ال 2.ال 2.ال 2.ال 3.احيانا 3.احيانا 3.احيانا 3.احيانا 3.احيانا 3.احيانا 3.احيانا 3.احيانا
المعلومات السريرة والمخبرية: 06. هل اصبت بداء اللشمانيا )حبة اريحا( سابقا؟ 0. نعم 2. ال 3. ال اعلم 06.ا. اذا كانت االجابة نعم, متى ــــــــــــــــــــــــــــــــــــــــــــــــــــــــ 07. تاريخ ظهور اول عالمة للمرض: ــــــــ/ ــــــــــ / ــــــــــــــــــ 08. تاريخ التشخيص النهائي: ــــــــ/ ــــــــــ / ــــــــــــــــــ 09. هل يوجد لدى العائلة اي افراد مصابين؟ 0. نعم 2. ال 3. ال اعلم موقع االصابة طبيعة االصابة 21. الوجه 0. نعم 2. ال 23. تقرح 0. نعم 2. ال 20. االطراف 0. نعم .2 ال 24. عجيرة 0. نعم 2. ال 22. اخرى 0. نعم 2. ال 25. اخرى 0. نعم .2 ال 22.ا. اذا كانت االجابة نعم , حدد ـــــــــــــــــــــــــــ 25.ا. اذا كانت االجابة نعم , حدد ــــــــــــــــــــــــ 26. ما عدد مواقع االصابة : ـــــــــــــــــــــــــــــــــــــــــــــــــــــ 27. هل تم التشخيص المخبري؟ 0. نعم 2. ال .3 ال اعلم اذا كانت االجابة نعم, ما هي نتيجة الفحوصات التالية؟ الرجاء ان تتم التعبئة كالتالي: الفحص النتيجة 27. ا( Direct smear [ ] 27. ب( Histologic examination [ ] 27.ج( Culture [ ] 27.د( Serology [ ] 27.ه( Leishmania skin test [ ] 27.و( PCR [ ]
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28. ما هو نوع اللشمانيا )اذا تم تصنيفه(: Tropica .0 Major .2 Infantum .3 4. اخرى معلومات خاصة بالمنزل: 29. يقع المنزل على: 0. قمة التلة 2. داخل وادي 3. بينهما )ليست تلة وال وادي( 31. نوع المنزل: 0. بناء جديد 2. بناء قديم 30. هل يوجد شبك لحماية الشبابيك واالبواب من الحشرات في بيتك؟ 0. نعم 2. جزئيا .3 ال 30.ا( اذا كات االجابة نعم, هل الشبك بحالة جيدة؟ 0. نعم 2. ال 32. هل يوجد لديك حديقة خاصة بالمنزل؟ 0. نعم 2. ال 33. في اي وقت من ساعات النهار تقضي معظم وقتك خارج البيت, في الحديقة, بجانب البيت او في الجوار؟ 0. فترة الصباح 2. فترة بعد الظهيرة 3. فترة المساء 4. قبل غروب الشمس 34. هل يوجد مزرعة في حدود 211 متر مربع من مكان سكنك ؟ 0. نعم 2. ال 3. ال اعلم 34.ا( اذا كانت االجابة نعم, ما هو نوع المزرعة؟ 0. مزرعة نباتية 2. مزرعة حيوانات 3. اخرى ـــــــــــــــــــــــــــــــــــــــــــ 35. هل يوجد ينابيع او برك في محيط سكنك؟ 0. نعم 2. ال .3 ال اعلم 36. هل يوجد شقوق في جدران المنزل او جحور في ارضيته؟ 0. نعم 2. ال 36.ا( اذا كانت االجابة نعم, اين: 0. في الجدران 2. في االرضية 3.)2+0( 37. هل جدران المنزل مدهونة؟ 0. نعم 2. ال 37.ا( اذا كانت االجابة نعم, اذكر لون الدهان من الداخل ــــــــــــــــــــــــــــــــــــــــــــــــ 38. هل لديك مكيف تبريد ؟ 0. نعم 2. ال 38.ا( اذا كانت االجابة نعم, ما نوعه: 0. مروحة 2. مكيف ماء 3. مكيف غاز
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39. ما هي المسافة بين منزلك واطراف القرية/المدينة؟ 0. اقل من 21 متر 2. اكثر من 21 متر
معلومات خاصة بالمضيف الوبر الصخري والناقل ذبابة الرمل: 41. هل سبق وان رأيت الوبر الصخري )انظر للصورة(؟ 0. نعم 2. ال 40. ما هي المسافة بين المنازل ومستعمرات الوبر الصخري؟ 0. اقل من 011 متر 2. اكثر من 011 متر 3. ال اعلم 42. هل يوجد لدى العائلة حيوانات اليفة تعيش بالقرب من المنزل؟ 0. نعم 2. ال 42.ا( اذا كانت االجابة نعم, اذكر انواع الحيوانات ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ 43. هل تعتقد ان كثافة ذبابة الرمل في صيف 2101 اكثر من السنوات االخرى؟ 0. نعم 2. ال .3 ال اعلم 43.ا( اذا كانت االجابة نعم, ما هو السبب وراء ذلك حسب رأيك؟ ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ 44. هل كان هاك اي اعمال بناء او انشاء خالل صيف 2101 )شق طرق بالجرافات, بناء منازل, واخرى( 0. نعم 2. ال
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תקציר
רקע: מחלות זיהומיות משפיעות משמעותית על תחלואה ותמותה בבני אדם במהלך העשורים האחרונים. מאז שנות ה07-' של המאה הקודמת, חלה התגברות בשכיחותן של מחלות רבות המועברות על ידי וקטור, כגון מלריה, קדחת צהובה, קדחת מערב הנילוס ולישמניאזיס. שילוב התחומים של אקולוגיית-המחלות ) Disease Ecology( ואפידמיולוגיה הינו בעל פוטנציאל להעמיק את ההבנה של המגע וההשפעה ההדדית של האדם וסביבתו על הוקטור. שילוב זה מאפשר זיהוי מוצלח יותר של "הקשרים החלשים" )weak links( בהעברת מחלות, לטובת שליטה בהן. בגדה המערבית שברשות הפלסטינית, לישמניאזיס מגיחה כנושא מרכזי לבריאות הציבור, ושעור ההיארעות שלה ממשיך לעלות במיוחד בדרום ובצפון הגדה.
מחקר זה נועד להעמיק את הבנתנו לגבי הדינמיקה של מאפייני האקולוגיה, האפידמיולוגיה וההעברה של לישמניאזיס עורית )שושנת יריחו, CL( הנגרמת על ידי הפתוגן לישמניה טרופיקה בגדה המערבית. מטרותיו הינן )1( להעריך את השפעת הגובה על צפיפות זבוב החול בתוך ובין שלושה כפרים במחוז בית לחם ; )2( להעריך את הסיכונים להדבקה בלישמניה טרופיקה באמצעות מחקרי מקרה-בקרה אפידמיולוגיים המבוססים על שאלונים הניתנים במחוז טובאס שהינו אנדמי למחלה ; ו-)3( להעריך את השפעת המרחק ממושבות שפני הסלע Procavia capensis, בעל החיים המהווה את המאגר, על צפיפותם של Phlebotomus sergenti, מין הוקטור, בתוך תבנית נוף עירונית, ובין אזור עירוני למושבת שפני סלע סמוכה.
שיטות: ראשית, נערך מחקר במחוז בית לחם )דרום-מזרח הגדה המערבית( המקיף שלושה כפרים הנבדלים בגובה וברמת האנדמיות: (Kisan (KIS), Arab Ar-Rashaiyda (AAR ו-(Al‘Azazma (AZA . שלושת הכפרים ממוקמים במעלה של מדרון, החל מאזור KIS הבלתי-אנדמי (m ASL 732-782) , ומטה לעבר האזורים האנדמיים m ASL( AAR 522-68) ו-m ASL( AZA 473-510(. זבובי חול נלכדו באמצעות מלכודות CDC המכילות פיתיון קרח-יבש, על מנת לכמת את שפע זבובי החול בשלושת הכפרים.
שנית, עיירות וכפרים במחוז טובאס בצפון-מזרח הגדה המערבית חולקו לשלוש תת-קבוצות בהתאם לרמות העיור והדירוג החברתי-כלכלי - העיר טובאס, שישה כפרים, וישובים בדואיים. נערך מחקר מקרה-בקרה מותאם של לישמניאזיס באזורים אנדמיים. שלוש בקרות נבחרו עבור כל מקרה, תוך התאמה של גיל, מין ורקע חברתי-כלכלי. השאלונים כללו שאלות בנושאים דמוגרפיים, אפידמיולוגיים, מאפייני הבית ומידע על הוקטור ובעל החיים המהווה את המאגר של התחלואה. הם ניתנו לכל מקרה ובקרה על מנת לקבוע את גורמי הסיכון האפידמיולוגיים הנמצאים במתאם הגבוה ביותר לחשיפה למחלה.
שלישית, במחוז טובאס, זבובי חול נלכדו במהלך החודשים יולי וספטמבר 2712 ב"מוקד חם" לזיהומי לישמניה. השפעת המרחק ממושבות שפני הסלע נבחנה תוך כימות שפע זבובי החול בשכונת Aleskan בעיר טובאס, לאורך 5 חתכי רוחב של מלכודות, הפרוסות החל מאזור סלעי המכיל שפני סלע ועד לאזור של בתים מאוכלסים. לכידה התבצעה פעם בחודש תוך שימוש במלכודות אור מסוג CDC. זבובי החול הלכודים נספרו, ובנוסף זוהו המין והמגדר של כל זבוב. זיהוי טפיל הלישמניה בוצע על נקבות phlebotomus באמצעות אמפליפיקציית PCR של ה-(internal transcribed spacer (ITS1 , ומנת הדם מארוחות שנאכלו לאחרונה הותאמו למין באמצעות PCR של ציטוכרום b וטכניקת (Reverse Line Blotting (RLB.
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תוצאות: ראשית, נמצא הבדל משמעותי בשפע זבובי החול בין שלושת הכפרים כאשר AAR>AZA>KIS. כמו כן, נמצא הבדל ביחס המגדרי של זבובים לכודים בין שלושת הכפרים, כאשר נמצא יחס זהה בין זכרים לנקבות ב-AAR וב-AZA, ויחס המוטה לטובת נקבות ב-KIS. נמצא מתאם בין הגובה לבין צפיפות זבובות חול Phlebotomus נקבות, כאשר הצפיפויות הגבוהות ביותר נמצאו בגבהים אמצעיים )AAR(. בנוסף, הגובה נמצא במתאם שלילי ליחס לזבובות חול Phlebotomus מוזנות-דם, גם בתוך ובין כפרים. הרכב המינים הנמדד בזכרים של זבובי חול מסוג Phlebotomus נבדל בין האתרים. בכפר AZA נמצאו כל המינים, בעוד שחלקם של המינים נעדר מ-AAR ומ-KIS. דם המארח, אשר נצרך על ידי P. sergenti נמצא בארבעה מינים: בני אדם )55.5%(, מקנה )25%(, בעלי כנף )11.1%( וכלבים )1.2%(. כמות זבובי החול אשר נמצאו AAR וב-AZA היתה שונה על פי עונות ועל פי שנים.
שנית, במחקר המקרה-בקרה, נמצאו הבדלים בשעורי התחלואה על פי מגדר וגיל, בין שלוש רמות העיור. שעורי התחלואה בעיר היו גבוהים יותר בקרב זכרים מאשר בכפר, בעוד ששעורי התחלואה בישובים בדואיים היו גבוהים יותר בקרב נקבות. כאשר משווים בין קבוצות גיל, בקבוצת הילדים היה שעור המקרים הגבוה ביותר, לאחריה בקרב קשישים בעיר וגילאי הביניים בכפרים ובישובים הבדואיים. לגבי עונתיות שיא מקרי ה-CL ארע בחודשים דצמבר וינואר. אנשים בשלושת האזורים נבדלו במידת השימוש באמצעי זהירות מפני זבובי חול. בעיר, השימוש בטבליות אידוי ובריסוס בתוך הבתים לדחיית או הריגת זבובים היה גבוה יותר בקרב קבוצת המקרים מאשר בקרב הבקרות. גורמי סיכון נוספים לתחלואה שנמצאו הינם: מגורים בקרבת חוות, גידול של חיות בית, ומגורים בקרבת מושבות שפני סלע. בכפרים, גורם הסיכון העיקרי היה מרחק משולי הכפר, ואילו באזורי ישובים בדואיים, הסיכון נמצא כתלוי במרחק מושבות שפני סלע מאזורי המגורים.
שלישית, נלכדו 1,751 זבובי חול מסוג Phlebotomus מתשעה מינים שונים, כאשר 507 מתוכם )ממוצע של 5.2 בכל מלכודות, בכל לילה( נלכדו ביולי, ו581- )ממוצע של 2.5 בכל מלכודת, בכל לילה( נלכדו בספטמבר. המרחק ממושבות שפני הסלע נמצא במתאם שלילי לכמות הכוללת של זבובי החול Phlebotomus, כמו גם כמות זכרי ונקבות P. sergenti. כל זבובי החול שהיו נגועים בלישמניה נשאו את טפיל הלישמניה טרופיקה, וכל הזבובים הנגועים היו מסוג P. sergenti. נקבות זבוב חול נגועות הנושאות ארוחת-דם נלכדו סמוך יותר למושבות שפני סלע, ביחס לנקבות נגועות שלא נשאו ארוחת-דם. זבובי חול הנושאים ארוחות-דם שנלקח משפני סלע נמצאו משמעותית קרובים יותר למושבות שפני סלע, ביחס לזבובי חול הנושאים סוגי דם אחרים. מגמה דומה נצפתה בעבור אלו הנושאים דם כלבים.
מסקנות: מחקר זה מעמיק את ההבנה לגבי העברת מחלות לבני אדם ובמיוחד במחלות זואונוטיות, באמצעות חקר הקשרים בין ההיבטים האפידמיולוגיים, החברתיים והאקולוגיים שלהן. בכל הנוגע לוקטור זבוב החול, המחקר מצא מתאם בין גובה לבין צפיפות זבוב החול, על אף שהכללה של מסקנה זו דורשת איסוף נתונים למשך זמן ארוך יותר ובכפרים ורמות גובה נוספות. בנוסף, המחקר מספק ראיות ראשוניות התומכות בהיות P. sergenti הוקטור של לישמניה טרופיקה במחוזות בית לחם וטובאס. שינויים סביבתיים שיוצר העיור עשויים לייצר בתי גידול חדשים עבור המארח. העברה יעילה של טפיל הלישמניה מתרחשת כאשר המארח והוקטור חיים בקרבה, והזבובים נוטים לאכול מחיות המארחים. המחקר מראה כי זבובי החול מסוג Phlebotomus מצויים בצפיפות גבוהה יותר בקרבת מושבות שפני סלע. בשל כך, חקר גורמי הסיכון ללישמניאזיס דורש הבנה, לא רק של צפיפות זבובי החול, אלא גם של הרכב המינים, הצלחת ההזנה והמרחק
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מהמארחים. באזורים יותר עירוניים, המחייה בקרבת עיר או שולי כפר הגובלים בוואדיות או הסמוכים לאזורים ירוקים פתוחים מגבירה את החשיפה לוקטורים )זבובי חול( ואת הקרבה למארחים )שפני סלע(. בנוסף, הסיכון להעברת המחלה גובר ככל שגוברת צפיפות המארחים, הקרבה של המארחים לבתי בני אדם, צפיפות הוקטור, ואי-הקפדה על אמצעי מיגון מפני הוקטור. ידע זה מסייע לזיהוי של אזורים בסיכון גבוה לזיהום, ולמיקוד מאמצי השליטה באזורים אלה לטובת הסברה מניעתית לתושבים המקומיים בכדי להפחית את העברת הלישמניה. התחשבות בתוצאות מחקר זה תסייע לתכנן ולהטמיע התערבויות עתידיות לטובת הפחתת שושנת יריחו בפלסטין ובאזורים נוספים.
XVIII
הצהרת תלמיד המחקר עם הגשת עבודת הדוקטור לשיפוט
אני אכראם א. צלאח החתום מטה מצהיר/ה בזאת: (אנא סמן):
X חיברתי את חיבורי בעצמי, להוציא עזרת ההדרכה שקיבלתי מאת מנחה/ים.
X החומר המדעי הנכלל בעבודה זו הינו פרי מחקרי מתקופת היותי תלמיד/ת מחקר.
בעבודה נכלל חומר מחקרי שהוא פרי שיתוף עם אחרים, למעט עזרה טכנית הנהוגה בעבודה ניסיונית. לפי כך מצורפת בזאת הצהרה על תרומתי ותרומת שותפי למחקר, שאושרה על ידם ומוגשת בהסכמתם.
שם התלמיד/ה אכראם צלאח תאריך 28 בפברואר 2718 חתימה
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העבודה נעשתה בהדרכת
פרופ' ברט פ. קוטלר
המחלקה לאקולוגיה על שם מיטרני המכונים לחקר המדבר ע"ש יעקב בלאושטיין אוניברסיטת בן-גוריון בנגב
פרופ' נדב דוידוביץ'
המחלקה לניהול מערכות בריאות הפקולטה למדעי הבריאות אוניברסיטת בן גוריון בנגב
XX
אקולוגיה ואפידמיולוגיה של תחלואה בלישמניה עורית
הנגרמת על ידי Leishmania tropica בפלסטין
מחקר לשם מילוי חלקי של הדרישות לקבלת תואר "דוקטור לפילוסופיה"
מאת אכראם א. צלאח
הוגש לסנאט אוניברסיטת בן גוריון בנגב
אושר ע"י:
______ברט קוטלר נדב דוידוביץ' )מנחה( )מנחה( ______דודי בר-צבי )דיקן בית הספר ללימודי מחקר מתקדמים ע"ש קרייטמן(
י"ג באדר תשע"ח 28 בפברואר 2718 באר שבע
XXI
אקולוגיה ואפידמיולוגיה של תחלואה בלישמניה עורית
הנגרמת על ידי Leishmania tropica בפלסטין
מחקר לשם מילוי חלקי של הדרישות לקבלת תואר "דוקטור לפילוסופיה"
מאת
אכראם א. צלאח
הוגש לסנאט אוניברסיטת בן גוריון בנגב
י"ג באדר תשע"ח 28 בפברואר 2718
באר שבע
XXII