Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts.

BACKGROUND: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. METHODS: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. RESULTS: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. CONCLUSIONS: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Ecology and seasonality of sandflies and potential reservoirs of cutaneous leishmaniasis in Ochollo, a hotspot in southern Ethiopia.

BACKGROUND: Ochollo is a village in southern Ethiopia burdened with cutaneous leishmaniasis (CL), where Phlebotomus pedifer is the only vector for Leishmania aethiopica and hyraxes are confirmed reservoir hosts. A detailed description of the different players of transmission, and the ecology and seasonality of the vector needs to be established in order to accomplish efficient control programs. METHODS AND FINDINGS: Between March 2017 and February 2018, a monthly sandfly collection was carried out in different habitats and records of temperature and humidity were taken. Rodents and hyraxes were trapped in the dry and wet season. All samples were screened for Leishmania kinetoplast DNA (kDNA). Positive samples were further processed for determination of the Leishmania species and the species of the sandfly/small mammal that was found infected. Additionally, the species of 400 sandfly specimens from different habitats and seasons was identified. 17,190 Sergentomyia and Phlebotomus sandflies were caught and showed an overall kDNA prevalence of 2.6%, all were L. aethiopica infections only found in P. pedifer. The overall sandfly and P. pedifer abundance peaked in the dry season and was negatively correlated with the %RH. The kDNA prevalence varied over the months and was negatively correlated with the temperature. Total sandfly abundance did not differ between the sampled habitats, but P. pedifer was the distinct predominant species only in caves. Moreover, significantly more infected sandflies were found in caves. Only 1/192 rodents were kDNA positive, while 20.0% (5/25) of Heterohyrax brucei were found infected. CONCLUSIONS: This study suggests that caves may be a source of multiplication of the infection. If an outdoor control program would be considered, it would be useful to focus on caves in the wet season, when the sandfly abundance is lowest. The captured rodent species appear not important for transmission and the contribution of hyraxes in transmission should be further investigated.

Leishmania tropica in rock hyraxes (Procavia capensis) in a focus of human cutaneous leishmaniasis.

Cutaneous leishmaniasis, caused by Leishmania tropica, has recently emerged in urban and rural foci of central and northern Israel, and constitutes a major public health concern. Rock hyraxes (Procavia capensis), the suspected natural reservoir, were trapped in the cutaneous leishmaniasis urban focus of Maale Adumim in central Israel and evaluated for L. tropica infection by real-time kinetoplast DNA (kDNA) polymerase chain reaction (PCR) and serology. Real-time PCR on blood and computerized western blot serology analysis was positive for L. tropica in 58% and 80%, respectively, of the hyraxes tested. Phylogenetic analysis of the ribosomal internal transcribed spacer 1 region indicated that similar genotypes were present in humans and hyraxes from the same habitat. The high rates of infection and exposure to L. tropica among hyraxes supports their involvement in the transmission cycle of this parasite, and their potential role as a reservoir for human disease.

Geographic and epidemiologic analysis of the cutaneous Leishmaniasis outbreak in northern Israel, 2000-2003.

BACKGROUND: The incidence of cutaneous leishmaniasis in northern Israel began to rise in 2000, peaking at 41.0 per 100,000 in the Kinneret subdistrict during the first half of 2003. OBJECTIVES: To examine the morbidity rates of CL in northern Israel during the period 1999-2003, which would indicate whether new endemic areas were emerging in this district, and to identify suspicious hosts. METHODS: The demographic and epidemiologic data for the reported cases (n=93) were analyzed using the GIS and SPSS software, including mapping habitats of suspicious hosts and localizing sites of infected sand flies. RESULTS: The maximal incidence rate in the district was found in the city Tiberias in 2003: 62.5/100,000 compared to 0-1.5/100,000 in other towns. The cases in Tiberias were concentrated on the peripheral line of two neighborhoods, close to the habitats of the rock hyraxes. Sand flies infected with Leishmania tropica were captured around the residence of those affected. Results of polymerase chain reaction were positive for Leishmania tropica in 14 of 15 tested patients. CONCLUSIONS: A new endemic CL area has emerged in Tiberias. The most suspicious reservoir of the disease is the rock hyrax.

Experimental transmission of Leishmania tropica to hyraxes (Procavia capensis) by the bite of Phlebotomus arabicus.

The ability of the sand fly Phlebotomus (Adlerius) arabicus to transmit Leishmania tropica was studied experimentally using hyraxes (Procavia capensis), natural reservoir hosts of the parasite. Sand flies became infected with L. tropica after feeding on a lesion of needle-inoculated hyrax. Moreover, P. arabicus fed with L. tropica promastigotes transmitted the parasite to hyraxes by bite during a second bloodmeal. Although the animals remained asymptomatic after infective sand fly bite, they were PCR positive and infectious for naive sand flies. We have thus demonstrated cyclical transmission of L. tropica by P. arabicus in hyraxes. This confirms experimentally the vectorial competence of P. (Adlerius) arabicus, and demonstrates that asymptomatic reservoir hosts are infectious to appropriate vectors.

Distinct transmission cycles of Leishmania tropica in 2 adjacent foci, Northern Israel.

Transmission of Leishmania tropica was studied in 2 adjacent foci in Israel where vector populations differ. Only Phlebotomus sergenti was found infected with L. tropica in the southern focus; P. arabicus was the main vector in the northern focus. Rock hyraxes (Procavia capensis) were incriminated as reservoir hosts in both foci. L. tropica strains from the northern focus isolated from sand flies, cutaneous leishmaniasis cases, and rock hyraxes were antigenically similar to L. major, and strains from the southern focus were typically L. tropica. Laboratory studies showed that P. arabicus is a competent vector of L. tropica, and P. sergenti is essentially refractory to L. tropica from the northern focus. Susceptibility of P. arabicus may be mediated by O glycoproteins on the luminal surface of its midgut. The 2 foci differ with respect to parasites and vectors, but increasing peridomestic rock hyrax populations are probably responsible for emergence of cutaneous leishmaniasis in both foci.

Outbreak of cutaneous leishmaniasis in northern Israel.

This study describes a new focus of cutaneous leishmaniasis (CL) due to Leishmania tropica, in the Galilee region of northern Israel. Thirty-three cases from 4 villages (northern part) and from the city of Tiberias (southern part) have been clinically diagnosed since 1996. Parasites from 13 patients and from 6 sand flies were characterized by isoenzyme electrophoresis, 2 immunological methods, and 3 polymerase chain reaction (PCR)-based methods. Isolates from the northern part were antigenically similar to Leishmania major and were different from other L. tropica isolates, including those from the southern part of the focus. They belonged to a newly reported zymodeme and were separable from all known Israeli L. tropica isolates, by use of 2 different PCR-based methods. Five (5.2%) of 97 Phlebotomus (Adlerius) arabicus and 2 (1.2%) of 162 Phlebotomus (Paraphlebotomus) sergenti females from the northern part of the focus were found to be infected with L. tropica. Three of 29 hyraxes (Procavia capensis) were positive for Leishmania ribosomal DNA. Thus, the northern part of this emerging focus of CL in Israel is distinct from all known L. tropica foci. P. arabicus is the main vector, and it transmits parasites that are different from other L. tropica isolates, with respect to antigenic, molecular, and biochemical parameters.

[The genus Ctenocephalides Stiles and Collins, 1930 (Siphonaptera, Pulicidae)]. / Le genre Ctenocephalides Stiles et Collins, 1930 (Siphonaptera, Pulicidae).

After a short historical review of this genus and an outline of biogeographical and paleontological origin of this flea, the 14 taxa are studied (synonymy, repartition, specificity and morphology). A new dichotomic key is given on criteria selected by the authors.

Characterization of Leishmania aethiopica from rock hyrax, Procavia capensis trapped in Najran, Saudi Arabia.

Cutaneous leishmaniasis caused by L. aethiopica is endemic in Ethiopia, Kenya and South-west Africa. In a previous paper, the present authors reported natural Leishmania species infection in two hyraxes, P. capensis. In this paper, an isolate from a rock hyrax. P. capensis (Pallas, 1766) was enzymatically characterized by demonstrating the mobilities of seven enzymes, to be identical with the reference strain of L. aethiopica.

Natural Leishmania infection in rock hyrax, Procavia capensis (Pallas, 1766) order: Hyracoidea, trapped in Najran, Saudi Arabia.

Cutaneous leishmaniasis caused by Leishmania major (zoonotic, ZCL) and L. tropica (anthroponotic, ACL) is found in most countries of the Eastern Mediterranean Region. Phlebotomus papatasii is the proven vector of L. major and rodents Rhombomys opimus, Psammomys obesus, Meriones spp. and Gerbillus spp. serve as animal reservoirs. Ph. sergenti is the vector of L. tropica in the majority of endemic foci. On the other hand; in the Eastern African highlands (mainly Ethiopia and Kenya), stable foci of L. aethiopica are maintanined by hyraxes and transmitted by Ph. longipes and Ph. pedifer. In this paper, natural Leishmania sp. infection was demonstrated serologically (IHAT) and parasitologically (smear examination) in two out of four rock hyraxes trapped in the highlands of Najran, southern part of the Kingdom. It is concluded that the identity of the Leishmania parasite(s)in such a focus is essential since it has implication in control and treatment. Also, passive case-detection and isolates from man and sandfly in the vicinity of Najran for typing is a must.

Inermicapsifer beveridgei n. sp. (Cestoidea: Anoplocephalidae) from Procavia capensis (Hyracoidea) in Israel, with notes on two species of Hymenolepis.

Inermicapsifer beveridgei n. sp. from Procavia capensis differs from all other species in the genus in having 150-174 testes per segment. The closest species is I. hyracis (Rudolphi, 1810) Janicki, 1919, with 75-120 testes. Hymenolepis vogeae Singh, 1956 and H. horrida (Linstow, 1901) Lühe, 1910, are briefly discussed.

Parasites of domestic and wild animals in South Africa. XIX. Ixodid ticks and fleas on rock dassies (Procavia capensis) in the Mountain-Zebra National Park.

Five or 6 rock dassies (Procavia capensis) were shot at monthly intervals for 13 consecutive months in the Mountain Zebra National Park and processed for arthropod parasite recovery. Ten species of ixodid ticks and 2 flea species were recovered. The seasonal prevalence of the rock dassie ticks, Haemaphysalis hyracophila and Rhipicephalus distinctus, and of the immature stages of Rhipicephalus arnoldi, a tick that usually infests the red rock rabbit (Pronolagus rupestris), was determined. Only 10 of the approximately 10 000 ticks recovered from the rock dassies belonged to species that may infest domestic livestock.

The zoonotic potential of reservoirs of leishmaniasis in the Old World.

A 'good' reservoir of leishmaniasis in the Old World is judged according to five criteria: contact with man, chronic susceptibility, good presentation of the disease organism, intimate contact with the phlebotomine sandfly vector and major source of blood meals for the vector. The endemics of cutaneous leishmaniasis in Soviet Asia and of visceral leishmaniasis in the South of France are examined in the light of these criteria. Other special examples of the criteria are highlighted and the exceptions cited. A table lists the present knowledge of the existence of reservoirs of Old World leishmaniasis.

Notes on African Haemaphysalis ticks, XIII. identity of H. (Rhipistoma) cooleyi, a parasite of the rock hyrax in South Africa (Acarina: Ixodidae).

The lectotype male, allotype female, and paratype nymph of Haemaphysalis (Rhipistoma) cooleyi Bedford, 1929, a parasite of the rock hyrax, Procavia capensis, in the Transvaal, are redescribed and illustrated to provide reliable criteria for differentiating between haemaphysaline parasites of hyraxes. Keys are included for identifying adults of these Haemaphysalis ssp. (orientalis Nuttall & Warburton, 1915; bequaerti Hoogstraal, 1956; cooleyi Bedford, 1929; hyracophila Hoogstraal, Walker & Neitz, 1971).