Isolation, full genomic characterization and neutralization-based human seroprevalence of Medjerda Valley virus, a novel sandfly-borne phlebovirus belonging to the Salehabad virus complex in northern Tunisia.

A new phlebovirus, Medjerda Valley virus (MVV), was isolated from one pool of Phlebotomus sp. (Diptera; Psychodidae) sandflies trapped in the vicinity of the Utique site, northern Tunisia. Genetic analysis based on complete coding of genomic sequences of the three RNA segments indicated that MVV is most closely related to members of the Salehabad virus species, where it is the fourth virus for which the complete sequence is available. A seroprevalence study was performed to search for neutralizing antibodies in human sera in the same region. The results demonstrate that in this area, MVV can readily infect humans despite low seroprevalence rates. Salehabad species viruses have generally been considered to be a group of viruses with little medical or veterinary interest. This view deserves to be revisited according to our human seroprevalence results, together with high animal infection rate of Adana virus and recent evidence of human infection with Adria virus in Greece. Further studies are needed to investigate the capacity of each specific member of the Salehabad virus species to cause human or animal diseases.

The Role of the Leishmania infantum Infected Dogs as a Potential Reservoir Host for Toscana Virus in a Zoonotic Visceral Leishmaniasis Focus of Northern Tunisia.

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of zoonotic visceral leishmaniasis (ZVL) located in Northern Tunisia from June to October 2020. At the end of the exposition period, infected and healthy dogs were examined for TOSV and L. infantum infections by xenodiagnosis using a colony of Phlebotomus perniciosus. Pools of freshly engorged P. perniciosus at days 0 and those at days 7 post-feeding were screened for TOSV and L. infantum by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. In the exposure site, P. pernicious is the most abundant sandfly species. The infection rates of sandflies with TOSV and L. infantum were 0.10 and 0.05%, respectively. Leishmania infantum DNA and TOSV RNA were detected in P. perniciosus females fed on dog B and C, respectively. The isolation of TOSV in Vero cells was achieved from two pools containing P. perniciosus fed on dog C. No pathogens were detected in P. perniciosus females fed on dog A and on control dog. We report for the first time the reservoir competence of dog with ZVL in the transmission of TOSV to sandfly vectors in natural settings, in addition to its role as a main reservoir host of L. infantum.

Transmission Dynamics of Punique Virus in Tunisia.

A novel phlebovirus, Punique virus (PUNV), was discovered and isolated in 2008 from sandflies from Northern Tunisia. PUNV is now classified as a unique member of the Punique phlebovirus species within the Phlebovirus genus in the Phenuiviridae family (order bunyavirales). In this study, we aimed to investigate the transmission dynamics of PUNV in Tunisia. Sandflies were collected during two consecutive years, 2009 and 2010, by CDC light traps. In 2009, a total of 873 sandflies were collected and identified to the species level. Phlebotomus perniciosus was the most abundant species. One pool of P. perniciosus females collected in autumn contained PUNV RNA, yielding an infection rate of 0.11%. The population densities of circulating sandfly species were assessed during May-November 2010 in Northern Tunisia by using sticky traps. Phlebotomus (Larroussius) perniciosus (71.74%) was the most abundant species, followed by Phlebotumus (Larroussius) longicuspis (17.47%), and Phlebotumus (Larroussius) perfiliewi (8.82%). The densities of dominant sandfly species were found to peak in early spring and again in the autumn. In 2010, species identification was not performed, and sandflies were only discriminated on the basis of sex and collection date. Out of 249 pools, three contained PUNV RNA. Each positive pool allowed virus isolation. The three pools of female sandflies containing PUNV RNA were collected in autumn with an infection rate of 0.05%. These findings provide further evidence that P. perniciosus is the main vector of PUNV in Tunisia, and this phlebovirus is endemic in Tunisia. Our findings provided strong evidence of intensive circulation of PUNV in sandflies and hosts through a viral infection buildup process between sandfly vectors and hosts starting at the beginning of the activity of sandflies in spring to reach a maximum during the second main peak in autumn.

Effects of multiple feedings on sensitized rabbits on the fitness of Phlebotomus papatasi (Diptera: Psychodidae).

In the present study, we evaluated the effects of antibodies anti-sandfly saliva on the fecundity of Phlebotomus papatasi, vector of zoonotic cutaneous leishmaniasis in the Old World. Rabbits were repeatedly exposed to sandfly bites. Immune sera showed increased levels of anti-sandfly saliva antibody compared to the pre-exposition period. The analysis of biological parameters revealed no decline on the feeding success of females P. papatasi fed on rabbits repeatedly exposed to sandfly bites. Our results showed that anti-sandfly saliva antibodies of rabbits are not detrimental to the fitness of females P. papatasi. Thus, rabbits did not acquire resistance to sandflies following repeated exposures, and that contribute in maintaining a high density of P. papatasi. To control sandfly infestations and Leishmania transmission, more studies are needed for a better understanding of the mechanisms governing the resistance of hosts to bites of sandflies.

Investigation of the Sandfly Fauna of Central Arid Areas and Northern Humid Regions of Tunisia, with Morphological and Molecular Identification of the Recently Established Population of Phlebotomus (Larroussius) perfiliewi.

Based on nucleotide sequences, we re-identified representative samples of Phlebotomus perfiliewi originating from two different biogeographical areas of Tunisia, whose populations had previously been identified based on morphological criteria. A partial region of the mitochondrial DNA cytochrome b gene was targeted, and sandfly species was determined by analogy with DNA sequences available in the GenBank database via a BLAST analysis, taking into account the query coverage and percentage identity. The recognized species presents the most substantial homology with the analyzed sequence. The results of the molecular identification showed complete agreement with the morphological identifications, and Phlebotomus perfiliewi is genetically variable.

Independent Circulation of Leishmania major and Leishmania tropica in Their Respective Sandfly Vectors for Transmission of Zoonotic and Chronic Cutaneous Leishmaniasis Co-Existing in a Mixed Focus of Central Tunisia.

Zoonotic cutaneous leishmaniasis (ZCL) and chronic cutaneous leishmaniasis (CCL) are known to overlap in Central Tunisia. Sandflies were collected using sticky traps and CDC light-traps set in rodent burrows at the ecotones surrounding the village, in houses, and in animal shelters during July-October 2017, 2018, and 2019. A total of 17,175 sandflies were collected during the three sandfly seasons and identified morphologically to species level. Of a total of 18 sandfly species reported in Tunisia, 16 were identified in this mixed focus of ZCL and CCL. Except for the rocky mountainous areas, Phlebotomus papatasi was the most abundant sandfly species in all biotopes. In the mountainous areas, Phlebotomus sergenti is the most abundant sandfly species belonging to the genus Phlebotomus. Female sandflies were tested for the presence of Leishmania species by PCR. The overall infection prevalence of sandflies with Leishmania major and Leishmania tropica was 0.42% and 0.065%, respectively. The sequencing of PCR-amplified ITS1 products showed that L. major is the predominant species in all biotopes and transmitted mostly by P. papaptasi followed by Phlebotomus longicuspis and Sergentomyia species. Leishmania tropica was detected in Phlebotomus sergenti and in Phlebotomus longicuspis collected in bedrooms and in the ecotone of rocky mountainous areas. Our results provided strong evidence that the proximity of human settlements to biotopes of rodent reservoir hosts of L. major and of L. tropica resulted into the cocirculation of both Leishmania species leading to a mixed focus of ZCL and CCL. The epidemiology of leishmaniases in North Africa is highly complex by the high diversity of sandfly vectors and their associated Leishmania species, leading to a mixed form of cutaneous leishmaniasis. It is of major epidemiological importance to point to the risk of spillover from rural to urban areas leading to the anthroponization of cutaneous leishmaniasis. Therefore, efficient control to reduce the indoor abundance of sandfly vectors in order to reduce the incidence of leishmaniases is urgently needed.

Molecular Characterization of Leishmania Species among Patients with Cutaneous Leishmaniasis in Asir Province, Saudi Arabia.

Anthroponotic cutaneous leishmaniais (ACL) and zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania tropica and Leishmania major, respectively, are endemic vector-borne diseases in southern Saudi Arabia. In 2021, an outbreak of cutaneous leishmaniasis occurred in the province of Asir. The main objective of our investigation was to analyze the epidemiological features of CL in southern Saudi Arabia. The ministry of health recorded 194 CL patients between January and December 2021 from the Asir province. Our findings showed that the majority of CL patients (87.1%) originated from the governorates of Khamis-Mushait and Abha. Most of the patients were males (62.3%). While CL affected all age groups, those under 13 years old were the most affected (38.1%). For both genders, CL patients were mostly Saudi citizens (90.7%) compared to non-Saudi expatriates. The majority of CL patients (75.2%) suffered from a single lesion, and the majority of lesions (61.3%) were located on the face. The seasonal prevalence of CL showed two peaks, a small one in July-August and a larger one in March. Of a total of 194 Giemsa slides samples, 188 showed positive amplification of Leishmania ITS1 gene. Based on PCR-RFLP and PCR-HMR, 183 patients showed positive amplification of L. tropica and five patients showed positive amplification of L. major. Phylogenetic analysis revealed a clear distinct separation between L. major and L. tropica sequences. Our results provided strong evidence of the pre-domination of L. tropica, the main etiological agent of ACL in Asir province. We reported for the first time the presence of L. major, an etiological agent of ZCL in the study areas. The co-circulation of ACL and ZCL highlighted the complexity of the epidemiology of CL in southern Saudi Arabia, and subsequently, further studies to identify competent vectors and reservoir hosts for the establishment of control strategies are needed.

Tick-borne encephalitis virus in Ixodes ricinus (Acari: Ixodidae) ticks, Tunisia.

Tick-borne encephalitis virus (TBEV) is the etiologic agent of tick-borne encephalitis (TBE) and transmitted by Ixodes ricinus (Linnaeus, 1758) in Europe. The geographical distribution of I. ricinus in the Palearctic region covers also northern Africa, including northwestern Tunisia. While the eco-epidemiology of TBE in Europe is well documented, no data concerning TBEV from northern Africa are available. We investigated whether TBEV is circulating in Tunisia. A total of 877 adult I. ricinus collected from northwestern Tunisia were examined in pools for the presence of TBEV by nRT-PCR. Viral RNA was detected in one pool of three engorged ticks, yielding a minimum infection rate of 0.11 % (1/877). Phylogenetic analysis showed that the Tunisian TBEV strain belongs to the European lineage. We report for the first time the presence of TBEV in I. ricinus from northern Africa. Therefore, more studies are needed to assess the public health importance of TBEV in northern Africa.

Correction: Enhanced attraction of sand fly vectors of Leishmania infantum to dogs infected with zoonotic visceral leishmaniasis.

[This corrects the article DOI: 10.1371/journal.pntd.0009647.].

Risk Assessment of the Role of the Ecotones in the Transmission of Zoonotic Cutaneous Leishmaniasis in Central Tunisia.

Zoonotic cutaneous leishmaniasis (ZCL), endemic in Central and Southern Tunisia, is caused by Leishmania major (Kinetoplastida: Trypanosomatidae), which is transmitted by the sand fly Phlebotomus papatasi. In Tunisia, the fat sand rat Psammomys obesus and the desert jird Meriones shawi are the principal reservoir hosts of L. major. The presence of the P. papatasi vector of the L. major etiologic agent of ZCL was assessed in the vicinity of villages in endemic areas of Central Tunisia. The study was performed from September through October 2019, a period corresponding to the main peak of activity of P. papatasi. Sand flies were collected from rodent burrows located at the ecotone level, which is the transition zone between the natural environment and human settlement. Sand flies were identified to species level and tested for the presence of L. major by PCR. Our entomological survey showed that P. papatasi is the most abundant sand fly species associated with rodent burrows, and this abundance is even higher in ecotones primarily occupied by P. obesus in comparison to ecotones occupied by M. shawi. Infections with Leishmania major were detected only in P. papatasi, with an overall minimum infection rate (MIR) of 2.64%. No significant difference was observed between the MIRs in ecotones of P. obesus and of M. shawi. Incidence of ZCL in the studied areas ranged from 200 to 700 cases per 100,000 inhabitants, with a mean incidence of 385.41 per 100,000. Higher ZCL incidence was identified in ecotones of M. shawi compared to ecotones of P. obesus. ZCL cases are positively correlated with the MIRs. Considering the short flight range of P. papatasi, increases in its densities associated with burrows of P. obesus or M. shawi at the ecotone level expand the overlap of infected vectors with communities and subsequently increase ZCL incidence. Therefore, control measures should target P. papatasi populations at the ecotones.

Enhanced attraction of sand fly vectors of Leishmania infantum to dogs infected with zoonotic visceral leishmaniasis.

BACKGROUND: The sand fly Phlebotomus perniciosus is the main vector of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin. Dogs are the main reservoir host of this disease. The main objective of this study was to determine, under both laboratory and field conditions, if dogs infected with L. infantum, were more attractive to female P. perniciosus than uninfected dogs. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a series of host choice experiments and found that infected dogs were significantly more attractive to P. perniciosus than uninfected dogs in the laboratory as well as in the field. Significantly more P. perniciosus fed on infected dogs than on uninfected dogs. However, the fecundity of P. perniciosus fed on infected dogs was adversely impacted compared to uninfected dogs by lowering the number of laid eggs. Phlebotomus perfiliewi, the second most abundant sand fly species in the field site and a competent vector of L. infantum had similar trends of attractivity as P. perniciosus toward infected dogs under field conditions. CONCLUSIONS: The results strongly suggest that L. infantum causes physiological changes in the reservoir host which lead to the host becoming more attractive to both male and female P. perniciosus. These changes are likely to improve the chance of successful transmission because of increased contact with infected hosts and therefore, infected dogs should be particularly targeted in the control of zoonotic visceral leishmaniasis in North Africa.

Punique virus, a novel phlebovirus, related to sandfly fever Naples virus, isolated from sandflies collected in Tunisia.

Sandflies are widely distributed around the Mediterranean Basin. Therefore, human populations in this area are potentially exposed to sandfly-transmitted diseases, including those caused by phleboviruses. Whilst there are substantial data in countries located in the northern part of the Mediterranean basin, few data are available for North Africa. In this study, a total of 1489 sandflies were collected in 2008 in Tunisia from two sites, bioclimatically distinct, located 235 km apart, and identified morphologically. Sandfly species comprised Phlebotomus perniciosus (52.2%), Phlebotomus longicuspis (30.1%), Phlebotomus papatasi (12.0%), Phlebotomus perfiliewi (4.6%), Phlebotomus langeroni (0.4%) and Sergentomyia minuta (0.5%). PCR screening, using generic primers for the genus Phlebovirus, resulted in the detection of ten positive pools. Sequence analysis revealed that two pools contained viral RNA corresponding to a novel virus closely related to sandfly fever Naples virus. Virus isolation in Vero cells was achieved from one pool. Genetic and phylogenetic characterization based on sequences in the three genomic segments showed that it was a novel virus distinct from other recognized members of the species. This novel virus was provisionally named Punique virus. Viral sequences in the polymerase gene corresponding to another phlebovirus closely related to but distinct from sandfly fever Sicilian virus were obtained from the eight remaining positive pools.

TICKS PARASITIZING THE SPUR-THIGHED TORTOISE (TESTUDO GRAECA) POPULATION OF TUNISIA.

From March to April 2017, a total of 147 free-ranging spur-thighed tortoises (Testudo graeca) was captured in different habitat types of northern Tunisia and examined for tick infestation. A total of 134 was infested, yielding an infestation prevalence of 91.2%. From these tortoises, 1,174 ticks were collected, a subsample (10%, n=120) of which was randomly selected and identified; the remaining ticks were stored at -80 C and examined for zoonotic pathogens. Only adult Hyalomma aegyptium were found among the subsample of ticks collected for spur-thighed tortoise. The prevalence of tick infestation did not vary significantly within the study area but differences in intensity were observed in relation to the size of tortoises and the vegetation coverage. Our results provide strong evidence that adult H. aegyptium is the predominant tick species found on wild spur-thighed tortoises in northern Tunisia, where the species is commonly traded illegally across the Mediterranean Basin. Considering the potential transmission of several zoonotic pathogens by H. aegyptium to humans, we highlight the need to develop a surveillance system to prevent the introduction and the spread of tick-borne pathogens in the Mediterranean Basin.

Ixodes inopinatus and Ixodes ricinus (Acari: Ixodidae) Are Sympatric Ticks in North Africa.

In the present study, we report the sympatric occurrence of Ixodes ricinus (Linnaeus, 1758) and Ixodes inopinatus (Estrada-Peña, Nava, and Petney, 2014) in Tunisia. In total, 173 adult Ixodes ticks were collected from four sites (El Jouza, Tamra, Aïn Soltan, and Jbel Zaghouan) between February and April 2017, a period corresponding to the peak of activity of I. ricinus in North Africa. The morphological characters corresponded to both species; thus, we generated a total of 28 16S rRNA sequences and compared them with previously published data in GenBank. The two species were sympatric in Tamra, Aïn Soltan, and El Jouza, whereas collections in Jbel Zaghouan only yielded I. inopinatus. These results indicate that the two taxa are widespread in the humid area of northern Tunisia. The one tick collected in Jbel Zaghouan suggests that the distribution of at least I. inopinatus might extend to the sub-humid area. More studies are needed to fully comprehend the systematic status of the two taxonomic entities using multiple molecular markers and morphological characters; integrating these two identification methods are a necessary step toward a better understanding of the ecology and epidemiology of tick-borne diseases in Tunisia.

The Impact of Illegal Waste Sites on the Transmission of Zoonotic Cutaneous Leishmaniasis in Central Tunisia.

Illegal waste disposal represents a risk health factor for vector-borne diseases by providing shelter for rodents and their ectoparasites. The presence of the Phlebotomus papatasi vector of Leishmania major, an etiologic agent of zoonotic cutaneous leishmaniasis (ZCL), was assessed at illegal waste sites located at the vicinity of villages in endemic areas of Central Tunisia. The study was performed over a two-year period over three nights from July to September 2017, and over three nights in September 2018. Household waste is deposited illegally forming dumpsites at the vicinity of each village and contains several rodent burrows of Psammomys obesus, the main reservoir host of L. major. Sandflies were collected from rodent burrows in the natural environment and in dumpsites using sticky traps and were identified at species level. Female sandflies were tested for the presence of L. major by PCR. Our entomological survey showed that Phlebotomus papatasi is the most abundant sandfly species associated with rodent burrows in these waste sites. The densities of P. papatasi in dumpsites are significantly higher compared to the natural environment. The minimum infection rate of P. papatasi with L. major in these illegal waste sites is not significantly different compared to the natural environment. Considering the short flight range of P. papatasi, increases in its densities, associated with burrows of P. obesus in illegal waste sites located at the edge of villages, expands the overlap of infected ZCL vectors with communities. Thus, illegal waste sites pose a high risk of spreading ZCL to neighboring home ranges. Waste management is an environmentally friendly method of controlling sandfly populations and should be included in an integrated management program for controlling ZCL in endemic countries.

Establishment, Maintenance of Phlebotomus spp. in the Laboratory, and Infection with Leishmania spp.

Sand fly colonies are of major importance for experimental studies on biology, behavior, vector competence, relationship with Leishmania parasites, and vector control. This chapter is intended to provide methods and techniques used to initiate, establish, and maintain sand fly colonies. Details on collecting sand flies for colonization, colony initiation, maintenance, and experimental infection of Phlebotomus spp. with Leishmania spp. are reported.

Absence of Crimean-Congo haemorrhagic fever virus in the tick Hyalomma aegyptium parasitizing the spur-thighed tortoise (Testudo graeca) in Tunisia.

Free-ranging spur-thighed tortoises Testudo graeca, captured in different habitat types of Northern Tunisia from March to April 2017, were examined for tick infestation: 134/147 (91%) were infested. The overall infestation intensity and abundance was 8.5 and 7.8, respectively. From these tortoises, 1174 ticks were collected, of which 10% (n = 120) taken from 18 randomly-selected tortoises were identified at the species level; the remaining ticks were examined for the presence of Crimean-Congo haemorrhagic fever virus (CCHFv) by real time RT-PCR. Only adult Hyalomma aegyptium were found, suggesting a high degree of host specificity to tortoises. No CCHFv was detected in ticks. Considering the absence of CCHFv in Hyalomma aegyptium infesting its main host, the spur-thighed tortoise, this tick species is unlikely to play a major role in the epidemiology of CCHF. Therefore, more studies are needed to investigate the circulation of this arbovirus between livestock and other tick species from North Africa.

Modeling and comparative study of the spread of zoonotic visceral leishmaniasis from Northern to Central Tunisia.

During the last twenty years, zoonotic visceral leishmaniasis (ZVL) spread from the north characterized by humid, sub-humid, and semi-arid bio-climates to the arid areas located in Central Tunisia. In order to understand the eco-epidemiological factors involved in the distribution of ZVL, both a sero-epidemiological and an entomological investigation were performed in two governorates situated in two different bio-geographical areas: Bizerte in the north and Kairouan in the center. A mathematical model for transmission of ZVL was built, describing the disease dynamic in these areas. Among 108 sera collected from clinically healthy owned dogs used for guarding houses in the governorate of Bizerte and tested for anti-L. infantum antibodies by indirect immune-fluorescence antibody test (IFAT), 8.3% were positive. From a total of 191 serum samples collected from clinically healthy owned dogs used for guarding houses in the governorate of Kairouan and tested for anti-L. infantum antibodies by IFAT, 26.7% were sero-positive. The cumulative incidences of ZVL in humans in the governorates of Bizerte and Kairouan were 39.17/100,000 and 0.2/100,000 inhabitants, respectively. The infection rates of sand flies with Leishmania DNA in the governorate of Bizerte and Kairouan were 3.8% and 9.4%, respectively. It is important to point out that ZVL epidemic in humans is preceded or concomitant to a high prevalence of Leishmania infantum in dogs and in sand flies. The model showed also a non-linear relationship between dog infection and ZVL in humans. These results suggest an important force of infection (R0) in emerging foci such as the governorate of Kairouan leading to an increased incidence of ZCL in humans. A high prevalence of L. infantum in dogs will result in high prevalence in the vector and obviously high probability to infect human; that it is a key factor for triggering transmission to humans, and subsequently it is an important parameter in the control of ZVL transmission.

Risk factors for mortality of children with zoonotic visceral leishmaniasis in Central Tunisia.

BACKGROUND: Zoonotic visceral leishmaniasis (ZVL) caused by Leishmania infantum is endemic with an epidemiological profile of a paediatric disease in Tunisia. In the context of a high fatality rate, identifying risk factors for in-hospital mortality in children treated for ZVL is of major epidemiological importance. DESIGN: A retrospective (case-control) study included 230 immuno-competent children diagnosed and confirmed with primary ZVL in the paediatric department of the University Hospital of Kairouan between 2004 and 2014. Forty-seven per cent (47%) were children under 18 months of age, and with a male / female ratio of 1.01:1. RESULTS: The overall case-fatality was 6% (n = 14). The risk factors for in-hospital death identified by a multivariate analysis were: bleeding at admission (OR = 25.5, 95% CI: 2.26-287.4; p = 0.009), white cell count less than 4000/mm3 (OR = 5.66, 95% CI: 1.16-27.6; p = 0.032), cytolysis (OR = 28.13, 95% CI: 4.55-173.6; p < 0.001), and delay between onset of symptoms and admission ≥ 15 days (OR = 11, 95% CI: 1.68-72; p = 0.012). CONCLUSION: The results strongly suggest that paediatric patients admitted 15 days after onset of symptoms, with bleeding, white cell counts below 4,000/mm3, and cytolysis at admission should be considered severe cases and subsequently, they are at high risk of mortality. A better understanding of factors associated with death of children from ZVL may contribute to decrease mortality.