Blood parasites (Trypanosoma, Leucocytozoon, Haemoproteus) in the Eurasian sparrowhawk (Accipiter nisus): diversity, incidence and persistence of infection at the individual level.

BACKGROUND: A high prevalence of parasites may result from life-long persistence of infection or from high reinfection rates. We have studied blood parasites in a breeding population of the accipitrid raptor, Eurasian sparrowhawk (Accipiter nisus), to determine parasite diversity and turnover. METHODS: During this 7-year study, 210 adult Eurasian sparrowhawks breeding in the city of Prague were checked for parasites using several diagnostic methods. RESULTS: In both female and male raptors, parasites of the genus Leucocytozoon were the most prevalent (92% and 85%, respectively) followed in decreasing order of prevalence by those of genus Trypanosoma (74% and 68%, respectively) and genus Haemoproteus (46% and 16%, respectively). The prevalence of all parasites increased with age in both sexes, with the females at each respective age having the higher prevalence. There was a positive association between Haemoproteus and Leucocytozoon infections. Persistence at the individual level was higher than incidence for Trypanosoma and Haemoproteus. In the case of Leucocytozoon and Trypanosoma, most individuals probably become infected in their first year of life or even before dispersal from the nest. The detected parasites belonged to Trypanosoma avium sensu stricto, Leucocytozoon sp. (haplotypes ACNI1 and ACNI3) and Leucocytozoon mathisi (haplotype ACNI4) and two new lineages of the Haemoproteus elani complex (ACCNIS6 and ACCNIS7). Detailed analysis of parasite lineages in individuals that were repeatedly sampled revealed lineage turnover that would otherwise remain hidden. Phylogenetic analysis revealed that the detected Haemoproteus belongs to a phylogenetically distant group whose taxonomic position requires further analysis. CONCLUSIONS: All three genera of blood parasites persist in infected individuals, thus enabling sustainability of vector transmission cycles. Prevalence increases with age; however, there is a high turnover of Leucocytozoon lineages. No clear evidence of parasite-induced mortality was found, and most of the individuals were infected early in life, particularly in the case of Leucocytozoon.

Preliminary study on investigation of zoonotic visceral leishmaniasis in endemic foci of Ethiopia by detecting Leishmania infections in rodents.

OBJECTIVE: To investigate the zoonotic visceral leishmaniasis (ZVL) by identification of the most probable reservoir hosts using parasite isolation and analysis of a possible transmission dynamics of the disease in extra-domestic agricultural fields and rural villages. METHODS: Rodents were collected from selected study sites in kala-azar endemic areas based on information for localities of kala-azar cases for screening of Leishmania infections using parasitological, serological and polymerase chain reaction (PCR) from March, 2013 to January, 2014. Ketamine (Clorketam Veterinary) was used to anaesthesize the rodents according the prescribed dosage (average 2 mg/kg for intra-venous route). The blood obtained using sterile needle was dropped into sterile filter paper and allowed to air dry before sealing in plastic bags. The tissues from liver, spleen and skin were macerated in Locke's solution before transferring them into NNN medium. Blood and touch smears of liver, spleen, skin and bone marrow were prepared for fixing using methanol and staining by Giemsa stain for microscopy. These tissues were also used for DNA extractions and PCR amplification of Leishmania infection. RESULTS: A total of 335 rodents (13 species) were analyzed by sampling internal organs. The infection rate by PCR was 11.1% (6/54) for Arvicanthis nilothicus compared to 17.6% (3/17) and 12.5% (2/16) for Acomys cahirinus and Tarera (G) robustus respectively. Almost all the infections were found from bone marrow samples (8/48 or 16.7%) compared with 1/91 (1.1%) liver, 2/87 (2.2%) spleen and 0/87 (0%) skin. In all study sites with past human VL cases, rodents and proved vectors shared similar habitats. CONCLUSIONS: Leishmania donovani might circulate among different species of rodents in kala-azar endemic lowlands and valleys of Ethiopia by Phlebotomus orientalis and Phlebotomus martini. Detailed studies to substantiate the preliminary data on the possible role of these rodents are urgently needed.

Natural infection of bats with Leishmania in Ethiopia.

The leishmaniases, a group of diseases with a worldwide-distribution, are caused by different species of Leishmania parasites. Both cutaneous and visceral leishmaniasis remain important public health problems in Ethiopia. Epidemiological cycles of these protozoans involve various sand fly (Diptera: Psychodidae) vectors and mammalian hosts, including humans. In recent years, Leishmania infections in bats have been reported in the New World countries endemic to leishmaniasis. The aim of this study was to survey natural Leishmania infection in bats collected from various regions of Ethiopia. Total DNA was isolated from spleens of 163 bats belonging to 23 species and 18 genera. Leishmania infection was detected by real-time (RT) PCR targeting a kinetoplast (k) DNA and internal transcribed spacer one (ITS1) gene of the parasite. Detection was confirmed by sequencing of the PCR products. Leishmania kDNA was detected in eight (4.9%) bats; four of them had been captured in the Aba-Roba and Awash-Methara regions that are endemic for leishmaniasis, while the other four specimens originated from non-endemic localities of Metu, Bedele and Masha. Leishmania isolates from two bats were confirmed by ITS1 PCR to be Leishmania tropica and Leishmania major, isolated from two individual bats, Cardioderma cor and Nycteris hispida, respectively. These results represent the first confirmed observation of natural infection of bats with the Old World Leishmania. Hence, bats should be considered putative hosts of Leishmania spp. affecting humans with a significant role in the transmission.

Exposure to Leishmania spp. and sand flies in domestic animals in northwestern Ethiopia.

BACKGROUND: Human visceral leishmaniasis caused by Leishmania donovani is considered an anthroponosis; however, Leishmania-infected animals have been increasingly reported in L. donovani foci, and the role of these animals as reservoirs for human L. donovani infection remains unclear. METHODS: We conducted a study of domestic animals (goats, sheep, cows, dogs, and donkeys) in three L. donovani foci in northwestern Ethiopia. Domestic animals were screened for Leishmania DNA and for anti-L. donovani IgG. Serum anti-sand fly saliva antibodies were used as a marker of exposure to the vector sand fly, Phlebotomus orientalis. RESULTS: Of 546 animals tested, 32 (5.9%) were positive for Leishmania DNA, with positive animals identified among all species studied. Sequencing indicated that the animals were infected with parasites of the L. donovani complex but could not distinguish between L. infantum and L. donovani. A total of 18.9% of the animals were seropositive for anti-L. donovani IgG, and 23.1% of the animals were seropositive for anti-P. orientalis saliva IgG, with the highest seroprevalence observed in dogs and sheep. A positive correlation was found between anti-P. orientalis saliva and anti-L. donovani IgGs in cows, goats, and sheep. CONCLUSIONS: The detection of L. donovani complex DNA in the blood of domestic animals, the reported seroprevalence to the L. donovani antigen, and the widespread exposure to sand fly saliva among domestic animals indicate that they are frequently exposed to Leishmania infection and are likely to participate in the epidemiology of Leishmania infection, either as potential blood sources for sand flies or possibly as parasite hosts.

Detection of Leishmania donovani and L. tropica in Ethiopian wild rodents.

Human visceral (VL, also known as Kala-azar) and cutaneous (CL) leishmaniasis are important infectious diseases affecting countries in East Africa that remain endemic in several regions of Ethiopia. The transmission and epidemiology of the disease is complicated due to the complex life cycle of the parasites and the involvement of various Leishmania spp., sand fly vectors, and reservoir animals besides human hosts. Particularly in East Africa, the role of animals as reservoirs for human VL remains unclear. Isolation of Leishmania donovani parasites from naturally infected rodents has been reported in several endemic countries; however, the status of rodents as reservoirs in Ethiopia remains unclear. Here, we demonstrated natural Leishmania infections in rodents. Animals were trapped in 41 localities of endemic and non-endemic areas in eight geographical regions of Ethiopia and DNA was isolated from spleens of 586 rodents belonging to 21 genera and 38 species. Leishmania infection was evaluated by real-time PCR of kinetoplast (k)DNA and confirmed by sequencing of the PCR products. Subsequently, parasite species identification was confirmed by PCR and DNA sequencing of the 18S ribosomal RNA internal transcribed spacer one (ITS1) gene. Out of fifty (8.2%) rodent specimens positive for Leishmania kDNA-PCR and sequencing, 10 were subsequently identified by sequencing of the ITS1 showing that five belonged to the L. donovani complex and five to L. tropica. Forty nine kDNA-positive rodents were found in the endemic localities of southern and eastern Ethiopia while only one was identified from northwestern Ethiopia. Moreover, all the ten ITS1-positive rodents were captured in areas where human leishmaniasis cases have been reported and potential sand fly vectors occur. Our findings suggest the eco-epidemiological importance of rodents in these foci of leishmaniasis and indicate that rodents are likely to play a role in the transmission of leishmaniasis in Ethiopia, possibly as reservoir hosts.

Theileria infection in domestic ruminants in northern Ethiopia.

Piroplasmosis caused by different tick-borne hemoprotozoan parasites of the genera Theileria and Babesia is among the most economically important infections of domestic ruminants in sub-Saharan Africa. A survey for piroplasm infection was conducted in three locations in Northern Ethiopia. Of 525 domestic ruminants surveyed, 80% of the cattle, 94% of the sheep and 2% of the goats were positive for different Theileria spp. based on PCR of blood followed by DNA sequencing. Sheep had a significantly higher rate of infection compared with cattle (P<0.0003) and both sheep and cattle had higher rates of infection compared to goats (P<0.0001). Four species of Theileria were detected in cattle: T. velifera, T. mutans, T. orientalis complex and T. annulata with infection rates of 66, 8, 4, and 2%, respectively. This is the first report of T. annulata, the cause of Tropical Theileriosis in Ethiopia. Of the two Theileria spp. detected in small ruminants, T. ovis was highly prevalent (92%) in sheep and rare in goats (1.5%) whereas T. seperata was infrequent in sheep (2%) and rare in goats (0.4%). None of the animals were positive for Babesia spp.; however, Sarcocystis capracanis and S. tenella were detected in one goat and a sheep, respectively. The widespread distribution of Theileria spp. among cattle in northern Ethiopia including the virulent T. annulata and more mildly pathogenic T. mutans and T. orientalis, and the high infection rate in sheep with the usually sub-clinical T. ovis indicate extensive exposure to ticks and transmission of piroplasms with an important economic impact.

Phlebotomus orientalis sand flies from two geographically distant Ethiopian localities: biology, genetic analyses and susceptibility to Leishmania donovani.

BACKGROUND: Phlebotomus orientalis Parrot (Diptera: Psychodidae) is the main vector of visceral leishmaniasis (VL) caused by Leishmania donovani in East Africa. Here we report on life cycle parameters and susceptibility to L. donovani of two P. orientalis colonies originating from different sites in Ethiopia: a non-endemic site in the lowlands - Melka Werer (MW), and an endemic focus of human VL in the highlands - Addis Zemen (AZ). METHODOLOGY/PRINCIPAL FINDINGS: Marked differences in life-cycle parameters between the two colonies included distinct requirements for larval food and humidity during pupation. However, analyses using Random Amplified Polymorphic DNA (RAPD) PCR and DNA sequencing of cytB and COI mitochondrial genes did not reveal any genetic differences. F1 hybrids developed successfully with higher fecundity than the parental colonies. Susceptibility of P. orientalis to L. donovani was studied by experimental infections. Even the lowest infective dose tested (2×10(3) per ml) was sufficient for successful establishment of L. donovani infections in about 50% of the P. orientalis females. Using higher infective doses, the infection rates were around 90% for both colonies. Leishmania development in P. orientalis was fast, the presence of metacyclic promastigotes in the thoracic midgut and the colonization of the stomodeal valve by haptomonads were recorded in most P. orientalis females by day five post-blood feeding. CONCLUSIONS: Both MW and AZ colonies of P. orientalis were highly susceptible to Ethiopian L. donovani strains. As the average volume of blood-meals taken by P. orientalis females are about 0.7 µl, the infective dose at the lowest concentration was one or two L. donovani promastigotes per sand fly blood-meal. The development of L. donovani was similar in both P. orientalis colonies; hence, the absence of visceral leishmaniasis in non-endemic area Melka Werer cannot be attributed to different susceptibility of local P. orientalis populations to L. donovani.

Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia.

BACKGROUND: Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia. METHODS: The efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus. RESULTS: Of 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major. CONCLUSIONS: Although qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.

Polymorphism in the HASPB repeat region of East African Leishmania donovani strains.

BACKGROUND/OBJECTIVES: Visceral leishmaniasis (VL) caused by Leishmania donovani is a major health problem in Ethiopia. Parasites in disparate regions are transmitted by different vectors, and cluster in distinctive genotypes. Recently isolated strains from VL and HIV-VL co-infected patients in north and south Ethiopia were characterized as part of a longitudinal study on VL transmission. METHODOLOGY/PRINCIPAL FINDINGS: Sixty-three L. donovani strains were examined by polymerase chain reaction (PCR) targeting three regions: internal transcribed spacer 1 (ITS1), cysteine protease B (cpb), and HASPB (k26). ITS1- and cpb--PCR identified these strains as L. donovani. Interestingly, the k26--PCR amplicon size varied depending on the patient's geographic origin. Most strains from northwestern Ethiopia (36/40) produced a 290 bp product with a minority (4/40) giving a 410 bp amplicon. All of the latter strains were isolated from patients with HIV-VL co-infections, while the former group contained both VL and HIV-VL co-infected patients. Almost all the strains (20/23) from southwestern Ethiopia produced a 450 bp amplicon with smaller products (290 or 360 bp) only observed for three strains. Sudanese strains produced amplicons identical (290 bp) to those found in northwestern Ethiopia; while Kenyan strains gave larger PCR products (500 and 650 bp). High-resolution melt (HRM) analysis distinguished the different PCR products. Sequence analysis showed that the k26 repeat region in L. donovani is comprised of polymorphic 13 and 14 amino acid motifs. The 13 amino acid peptide motifs, prevalent in L. donovani, are rare in L. infantum. The number and order of the repeats in L. donovani varies between geographic regions. CONCLUSIONS/SIGNIFICANCE: HASPB repeat region (k26) shows considerable polymorphism among L. donovani strains from different regions in East Africa. This should be taken into account when designing diagnostic assays and vaccines based on this antigen.

Disseminated cutaneous leishmaniasis resembling post-kala-azar dermal leishmaniasis caused by Leishmania donovani in three patients co-infected with visceral leishmaniasis and human immunodeficiency virus/acquired immunodeficiency syndrome in Ethiopia.

We report paired strains of Leishmania parasites, one from the viscera and the other from skin lesions that were isolated from three patients with visceral leishmaniasis and disseminated cutaneous leishmaniasis that were co-infected with human immunodeficiency virus. The causative parasites were characterized by polymerase chain reaction-restriction length polymorphism of the ribosomal DNA internal transcribed spacer 1 and by a panel of multilocus microsatellite markers. We demonstrated that the causative agent was Leishmania donovani in all cases, irrespective of the phenotype of the disease. The paired strains from viscera and skin lesions of the same patients showed genetic identity across the 14 microsatellite markers investigated. These findings demonstrate that the skin lesions in these human immunodeficiency virus-positive patients with visceral leishmaniasis were caused by dissemination of viscerotropic L. donovani parasites as a consequence of severe immunosuppression. However, in all three patients, rapid clearance of the skin lesions was observed after antimonial therapy.

Inference of population structure of Leishmania donovani strains isolated from different Ethiopian visceral leishmaniasis endemic areas.

BACKGROUND: Parasites' evolution in response to parasite-targeted control strategies, such as vaccines and drugs, is known to be influenced by their population genetic structure. The aim of this study was to describe the population structure of Ethiopian strains of Leishmania donovani derived from different areas endemic for visceral leishmaniasis (VL) as a prerequisite for the design of effective control strategies against the disease. METHODOLOGY/PRINCIPAL FINDINGS: Sixty-three strains of L. donovani newly isolated from VL cases in the two main Ethiopian foci, in the north Ethiopia (NE) and south Ethiopia (SE) of the country were investigated by using 14 highly polymorphic microsatellite markers. The microsatellite profiles of 60 previously analysed L. donovani strains from Sudan, Kenya and India were included for comparison. Multilocus microsatellite typing placed strains from SE and Kenya (n = 30) in one population and strains from NE and Sudan (n = 65) in another. These two East African populations corresponded to the areas of distribution of two different sand fly vectors. In NE and Sudan Phlebotomus orientalis has been implicated to transmit the parasites and in SE and Kenya P. martini. The genetic differences between parasites from NE and SE are also congruent with some phenotypic differences. Each of these populations was further divided into two subpopulations. Interestingly, in one of the subpopulations of the population NE we observed predominance of strains isolated from HIV-VL co-infected patients and of strains with putative hybrid genotypes. Furthermore, high inbreeding irreconcilable from strict clonal reproduction was found for strains from SE and Kenya indicating a mixed-mating system. CONCLUSIONS/SIGNIFICANCE: This study identified a hierarchical population structure of L. donovani in East Africa. The existence of two main, genetically and geographically separated, populations could reflect different parasite-vector associations, different ecologies and varying host backgrounds and should be further investigated.