Phlebotomine sand flies (Diptera: Psychodidae) of the Maghreb region: A systematic review of distribution, morphology, and role in the transmission of the pathogens

Background Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of various human and animal pathogens such as Bartonella bacilliformis, Phlebovirus, and parasitic protozoa of the genus Leishmania, causative agent of leishmaniases that account among most significant vector-borne diseases. The Maghreb countries Mauritania, Morocco, Algeria, Tunisia, and Libya occupy a vast area of North Africa and belong to most affected regions by these diseases. Locally varying climatic and ecological conditions support diverse sand fly fauna that includes many proven or suspected vectors. The aim of this review is to summarize often fragmented information and to provide an updated list of sand fly species of the Maghreb region with illustration of species-specific morphological features and maps of their reported distribution. Materials and methods The literature search focused on scholar databases to review information on the sand fly species distribution and their role in the disease transmissions in Mauritania, Morocco, Algeria, Tunisia, and Libya, surveying sources from the period between 1900 and 2020. Reported distribution of each species was collated using Google Earth, and distribution maps were drawn using ArcGIS software. Morphological illustrations were compiled from various published sources. Results and conclusions In total, 32 species of the genera Phlebotomus (Ph.) and Sergentomyia (Se.) were reported in the Maghreb region (15 from Libya, 18 from Tunisia, 23 from Morocco, 24 from Algeria, and 9 from Mauritania). Phlebotomus mariae and Se. africana subsp. asiatica were recorded only in Morocco, Ph. mascitti, Se. hirtus, and Se. tiberiadis only in Algeria, whereas Ph. duboscqi, Se. dubia, Se. africana africana, Se. lesleyae, Se. magna, and Se. freetownensis were reported only from Mauritania. Our review has updated and summarized the geographic distribution of 26 species reported so far in Morocco, Algeria, Tunisia, and Libya, excluding Mauritania from a detailed analysis due to the unavailability of accurate distribution data. In addition, morphological differences important for species identification are summarized with particular attention to closely related species such as Ph. papatasi and Ph. bergeroti, Ph. chabaudi, and Ph. riouxi, and Se. christophersi and Se. clydei.

Molecular Detection of Bartonella sp. in Psathyromyia shannoni and Lutzomyia cruciata From Northeastern Mexico

Phlebotomine sand flies are vectors of Leishmania spp., Bartonella bacilliformis, and several arboviruses worldwide. In Mexico, the presence of Bartonella species is associated sporadically with arthropods and little is known on the diversity of insects that could be incriminated with its transmission. The aim of this study was to perform a molecular detection of Bartonella DNA in sand fly species collected in northeastern Mexico. Sand flies were collected at the states of Nuevo Leon and Tamaulipas from June to August 2010, using 16 light traps per night. Sand fly species were morphologically identified, and for Bartonella detection, we amplified ~378 bp of the citrate synthase gene (gltA). DNA sequences were compared in a phylogenetic reconstruction based on maximum likelihood. A total of 532 specimens from seven sand fly species were morphologically identified, where 11 specimens from Tamaulipas tested positive for the presence of a new lineage of Bartonella sp. associated with Psathyromyia shannoni and Lutzomyia cruciata. This work represents the second record of Bartonella-associated with sand flies outside of the endemic area of Carrion’s disease. More studies are necessary to understand their life cycle, transmission dynamics, and their relationship with sand fly species.

Immunogenic Peptides from Pap31 and SCS-α of Bartonella bacilliformis: One Step Closer to a Rapid Diagnostic Tool for Carrion’s Disease

Bartonella bacilliformis is the causal agent of Carrion’s disease, an overlooked illness endemic in the Andean Mountains with Peru being the most affected country. The diagnostic of this illness is a challenge due to the limited resources and the common symptomatology with other infectious diseases. The goal of this study was to identify immunogenic peptides from Pap31 and succinyl-CoA synthetase α (SCS-α) of B. bacilliformis that might be suitable for developing a serologic tool. The immunodominant character of Pap31 and SCS-α was determined by Western blotting and in-silico analysis. Subsequently, 35 peptides were selected for epitope mapping and their immunoreactivity was tested by enzyme-linked immunosorbent assay (ELISA). A total of 30 sera were tested including pre-exposed people with high IgM levels for Pap31/SCS-α (23 sera), patients (2 sera) as well as 5 sera with no reactivity to Pap31/SCS-α. The results indicate that Pap31-8 (187QAIGSAILKGTKDTGT202) and SCS-α-12 (59IFASVAEGKEKTGANA74) are the most immunogenic peptides, with Pap31-8 showing potential to discriminate between B. bacilliformis and the remaining Bartonella spp., and SCS-α-12 differentiating Bartonella spp. from other microorganisms.

Comparison of sand fly trapping approaches for vector surveillance of Leishmania and Bartonella species in ecologically distinct, endemic regions of Peru

Background In Peru, the information regarding sand fly vectors of leishmaniasis and bartonellosis in the Amazon region is limited. In this study, we carried out sand fly collections in Peruvian lowland and highland jungle areas using different trap type configurations and screened them for Leishmania and Bartonella DNA. Methodology/Principal findings Phlebotomine sand flies were collected in Peruvian Amazon jungle and inter Andean regions using CDC light trap, UV and color LED traps, Mosquito Magnet trap, BG Sentinel trap, and a Shannon trap placed outside the houses. Leishmania spp. screening was performed by kDNA PCR and confirmed by a nested cytochrome B gene (cytB) PCR. Bartonella spp. screening was performed by ITS PCR and confirmed by citrate synthase gene (gltA). The PCR amplicons were sequenced to identify Leishmania and Bartonella species. UV and Blue LED traps collected the highest average number of sand flies per hour in low jungle; UV, Mosquito Magnet and Shannon traps in high jungle; and Mosquito Magnet in inter Andean region. Leishmania guyanensis in Lutzomyia carrerai carrerai and L. naiffi in Lu. hirsuta hirsuta were identified based on cytB sequencing. Bartonella spp. related to Bartonella bacilliformis in Lu. whitmani, Lu. nevesi, Lu. hirsuta hirsuta and Lu. sherlocki, and a Bartonella sp. related to Candidatus B. rondoniensis in Lu. nevesi and Lu. maranonensis were identified based on gltA gene sequencing. Conclusions/Significance UV, Blue LED, Mosquito Magnet and Shannon traps were more efficient than the BG-Sentinel, Green, and Red LED traps. This is the first report of L. naiffi and of two genotypes of Bartonella spp. related to B. bacilliformis and Candidatus B. rondoniensis infecting sand fly species from the Amazon region in Peru.

Molecular Characterization of Fluoroquinolone-Resistant Bartonella bacilliformis

The presence of amino acid changes in GyrA, GyrB, ParC, ParE, and in a proposed chromosomal chloramphenicol acetyl transferase (CAT), as well as mutations at 23S rRNA, were established by PCR and sequencing in 38 B. bacilliformis clinical isolates from four different areas in Peru. Eighteen out of 24 (75%) isolates showing ciprofloxacin resistance for both disk-diffusion and e-test presented amino acid substitutions in GyrA (G89C, six isolates, A91V, 1 isolate) GyrB (S474F, 10 isolates) or both (GyrA D95N and GyrB S474F, one isolate). Two out of 14 susceptible isolates presented amino acid substitutions at GyrB (S474F) or a double substitution GyrA D95N and GyrB S474F. Of note, ciprofloxacin-resistant isolates were recovered in the four areas studied. No amino acid change was observed at ParC or ParE. Only one isolate showed chloramphenicol resistance, but no alteration was present in either 23S rRNA or CAT. B. bacilliformis resistant to quinolones are extended throughout Peru, with amino acid substitutions at GyrA or GyrB as the main, albeit not exclusive, cause. B. bacilliformis seems to have an apparent facility to develop mutations on GyrB outside the classical positions 91, 95 of GyrA and 85, 88 of ParC.

A human factor H-binding protein of Bartonella bacilliformis and potential role in serum resistance

Bartonella bacilliformis is a Gram-negative bacterium and etiologic agent of Carrión’s disease; a potentially life-threatening illness endemic to South America. B. bacilliformis is a facultative parasite that infects human erythrocytes (hemotrophism) and the circulatory system, culminating in a variety of symptoms, including a precipitous drop in hematocrit, angiomatous lesions of the skin (verruga peruana) and persistent bacteremia. Because of its specialized niche, serum complement imposes a continual selective pressure on the pathogen. In this study, we demonstrated the marked serum-resistance phenotype of B. bacilliformis, the role of factor H in serum complement resistance, and binding of host factor H to four membrane-associated polypeptides of ∼131, 119, 60 and 43 kDa by far-western (FW) blots. The ∼119-kDa protein was identified as ABM44634.1 by mass spectrometry; a protein annotated as a 116.5-kDa outer membrane autotransporter (encoded by the BARBAKC583_1133 locus). We designated the protein as factor H-binding protein A (FhbpA). FhbpA possesses three structural motifs common to all autotransporter proteins (i.e., a signal peptide, autotransporter β-barrel domain and passenger domain). Recombinant FhbpA passenger domain, but not the recombinant autotransporter domain, was able to bind human factor H when analyzed by FW blots. Phylogenetic analyses of the passenger domain suggest that it is well-conserved among Bartonella autotransporters, with closest matches from Bartonella schoenbuchensis. Transcriptomic analyses of B. bacilliformis subjected to conditions mimicking the sand fly vector or human host, and infection of human blood or vascular endothelial cells showed maximal expression of fhbpA under human-like conditions and during infection of blood and endothelial cells. Expression during HUVEC infection was significantly higher compared to all other conditions by DESeq2. Surface binding of serum factor H by FhbpA is hypothesized to play a protective role against the alternative pathway of complement fixation during B. bacilliformis infection of the human host.Author SummaryB. bacilliformis is a bacterial pathogen that colonizes the circulatory system of humans, where it can cause a life-threatening illness unless treated. Serum complement is a major effector of innate humoral immunity and a significant obstacle that must be evaded for successful survival and colonization by pathogens, especially those residing in the vasculature. In this study, we examined the serum complement resistance phenotype of B. bacilliformis and identified four membrane-associated proteins that bind serum factor H; a protein used by the host to protect its own tissues from complement activation. One of the proteins was identified by mass spectrometry, characterized, and designated factor H-binding protein A (FhbpA). FhbpA is a predicted autotransporter, and we determined that the translocated “ passenger” domain of the protein is responsible for binding factor H. We also determined that expression of the fhbpA gene was highest during infection of human blood and especially vascular endothelial cells or under conditions that simulate the human host. The results suggest that FhbpA binding of host serum factor H protects the bacterium against complement activation during infection.

Bartonella henselae Detected in Malignant Melanoma, a Preliminary Study

Bartonella bacilliformis (B. bacilliformis), Bartonella henselae (B. henselae), and Bartonella quintana (B. quintana) are bacteria known to cause verruga peruana or bacillary angiomatosis, vascular endothelial growth factor (VEGF)-dependent cutaneous lesions in humans. Given the bacteria’s association with the dermal niche and clinical suspicion of occult infection by a dermatologist, we determined if patients with melanoma had evidence of Bartonella spp. infection. Within a one-month period, eight patients previously diagnosed with melanoma volunteered to be tested for evidence of Bartonella spp. exposure/infection. Subsequently, confocal immunohistochemistry and PCR for Bartonella spp. were used to study melanoma tissues from two patients. Blood from seven of the eight patients was either seroreactive, PCR positive, or positive by both modalities for Bartonella spp. exposure. Subsequently, Bartonella organisms that co-localized with VEGFC immunoreactivity were visualized using multi-immunostaining confocal microscopy of thick skin sections from two patients. Using a co-culture model, B. henselae was observed to enter melanoma cell cytoplasm and resulted in increased vascular endothelial growth factor C (VEGFC) and interleukin 8 (IL-8) production. Findings from this small number of patients support the need for future investigations to determine the extent to which Bartonella spp. are a component of the melanoma pathobiome.

Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

Bartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs αr45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonella bacilliformis small RNA 9 (BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.