Identification of a Novel Uncultured Rickettsia Species Strain (Rickettsia Species Strain Tselenti) in Cyprus

In a previous study conducted in Cyprus, various spotted fever group Rickettsia species were detected and identified in ticks by molecular analysis. Among them, a partially characterized Rickettsia species was detected in Hyalomma anatolicum excavatum and Rhipicephalus turanicus ticks. We report characterization of this rickettsial strain by using polymerase chain reaction sequencing analysis of partial citrate synthase A, outer membrane protein A, outer membrane protein B, and 17-kD protein genes. We propose a provisional name Rickettsia sp. strain Tselenti for this strain until it is isolated and further characterized.     

Ticks infesting wild and domestic animals and humans of Sri Lanka with new host records

An island-wide collection of tick species infesting humans, domesticated and wild animals and questing ticks in domestic and peridomestic environments was carried out during 2009–2011. A total of 30,461 ticks were collected from 30 different hosts and free living stages from the ground. The collection consisted of 22 tick species from 30 different hosts recording 12 tick species from humans, 19 from domesticated animals and 21 from wild animals, with a total of 97 new host records. The most common tick species on humans were Dermacentor auratus and Amblyomma testudinairum, while Haemaphysalis intermedia, Rhipicephalus microplus and Rhipicephalus sanguineus were common in domesticated and wild animals sharing 20 host species. Among the questing ticks, immature D. auratus was the most abundant. Humans and domesticated animals were mostly infested by the nymphal stages while adult ticks were found on wild animals. High number of new host records could be due to domestic animals picking tick species from wildlife and vise versa at the human/animal interface. Habitat destruction due to forest fragmentation has lead to wild animals roaming in urban and semi-urban neighbourhoods increasing the interactions of wild animals with domesticated animals. Wild animals play a significant role as a reservoir of many tick borne infections which can easily be spread to domesticated animals and then to humans via tick infestations. Data in this paper are useful for those interested in tick infesting wild and domestic animals and humans in describing the zoonotic potential of tick borne infections.     

Survey of tick species parasiting domestic ruminants in Ghaemshahr county,Mazandaran province,Iran

ObjectiveTo determine the tick species parasitizing domestic ruminants in Ghaemshahr county in Mazandaran, a Caspian province in the north of Iran.MethodsAbout 361 sheep, 54 goats and 10 cattle of 18 herds in several villages in Ghaemshahr were inspected for tick infestation. Separated ticks were preserved in 70% alcohol and identified.ResultsAbout 323 ticks (207 female, 116 male) were collected, the occurrence of ticks on sheep, goats and cattle were 28.3%, 22.2% and 20.0% respectively. The mean number of ticks on each animal was low (3-5 ticks per animal). Rhipicephalus sanguineus, Rhipicephalus bursa, Ixodes ricinus, Boophilus annulatus, Haemaphysalis punctata and Haemaphysalis numidiana were the tick species we found. Rhipicephalus sanguineus were the most abundant species in the study area. The largest number of ticks were generally present from April to July, mostly in animal ears and tails. Ixodes, Boophilus and Haemaphysalis occurred in mountainous areas of Ghaemshahr, whereas Rhipicephalus were present in both mountains and plains of the study area.ConclusionsThe result of this study is a survey of tick species from domestic animals in Iran and implication of possible prevention measures for diseases transmitted by ticks.     

Evasin‐3‐like anti‐chemokine activity in salivary gland extracts of ixodid ticks during blood‐feeding: a new target for tick control

Ticks exploit many evasion mechanisms to circumvent the immune control of their hosts including subversion of the communication language between cells of the immune system provided by chemokines and other cytokines. One subversive molecule secreted in the saliva of Rhipicephalus sanguineus is Evasin‐3, a structurally unique 7 kDa protein that selectively binds the neutrophil chemoattractants, CXCL8 and (with lower affinity) CXCL1. We compared anti‐human CXCL8 and anti‐mouse CXCL1/KC activities in salivary gland extracts prepared from adult Amblyomma variegatum, Rhipicephalus appendiculatus and Dermacentor reticulatus ticks during blood‐feeding. Both anti‐CXCL8 activity and anti‐CXCL1 activity were detected in all species and in both adult females and males, with consistently higher activity levels against CXCL8. These results suggest that Evasin‐3‐like activity is common amongst metastriate ixodid tick species, and provide further evidence of the importance to ticks in controlling neutrophils during blood‐feeding. As such, Evasin‐3 offers a new target for anti‐tick vaccine development.     

Detection of Rickettsia africae in Rhipicephalus (Boophilus) decoloratus ticks from the Republic of Botswana, South Africa

A total of 53 engorged adult ticks belonging to the species Rhipicephalus (Boophilus) decoloratus (N = 9), Rhipicephalus evertsi evertsi (N = 27), Rhipicephalus appendiculatus (N = 9), Amblyomma hebraeum (N = 5), and Hyalomma marginatum turanicum (N = 3), were removed from oryx in Botswana (South Africa). They were tested for the presence of spotted fever group (SFG) Rickettsia and Anaplasma phagocytophilum using polymerase chain reaction (PCR). Seventy-seven percent of R. decoloratus as well as twenty percent of A. hebraeum were positive for ompA, gltA and 16S rRNA SFG Rickettsia PCR assays. All nucleotide sequences were homologous to Rickettsia africae, the agent of African tick-bite fever (ATBF). None of the tested ticks was positive for 16S rRNA A. phagocytophilum PCR assays. These results suggest for the first time that R. decoloratus ticks may be reservoirs of R. africae, and support the ATBF risk in this area.     

PARTICIPATION OF TICKS IN THE INFECTIOUS CYCLE OF CANINE VISCERAL LEISHMANIASIS,IN TERESINA,PIAUí, BRAZIL

In this study, we detected Leishmania spp. infection in R. sanguineus collected from dogs that were naturally infected with L. (L.) infantum. We examined 35 dogs of both sexes and unknown ages. The infected dogs were serologically positive by the immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and Quick Test-DPP (Dual Path Platform), as well as parasitological examination of a positive skin biopsy or sternal bone marrow aspiration. Ten negative dogs were included as controls. The ticks that infested these dogs were collected in pools of 10 adult females per animal. The PCR was performed with specific primers for Leishmania spp., which amplified a 720-bp fragment. Of the 35 analyzed samples, a product was observed in eight samples (8/35; 22.9%). We conclude that the presence of parasite DNA suggests that ticks participate in the zoonotic cycle of canine visceral leishmaniasis, in the city of Teresina, Piauí.     

Acariasis on pet Burmese python, Python molurus bivittatus in Malaysia

ObjectiveTo identify the acari present on pet Burmese pythons in Malaysia and to determine whether there is any potential public health risk related to handling of the snakes.MethodsTwo sub-adult Burmese pythons kept as pets for a period of about 6 to 7 months by different owners, were brought to an exotic animal practice for treatment. On a complete medical examination, some ticks and mites (acari) were detected beneath the dorsal and ventral scales along body length of the snakes. Ticks were directly identified and mites were mounted prior to identification.ResultsA total of 12 ticks represented by 3 males, 2 females and 7 nymphal stages of Rhipicephalus sanguineus (R. sanguineus) were extracted from the first python while the other one was with 25 female Ophionyssus natricis (O. natricis) mesostigmatid mites. Only adult female mites were found. These mites are common ectoparasites of Burmese pythons.ConclusionsBoth the acarine species found on the Burmese pythons are known vectors of pathogens. This is the first record that R. sanguineus has been reported from a pet Burmese python in Malaysia.     

First molecular detection of Theileria ovis in Rhipicephalus sanguineus tick in Iran

ObjectiveTo determine tick infestation of domestic ruminants and their infection to ovine theileriosis in northern Iran.MethodsAbout 425 domestic ruminants in Ghaemshahr city in northern Iran were inspected for tick infestations. Twenty tick specimens (13 females and 7 males) of Rhipicephalus sanguineus (R. sanguineus), the most common tick in the study area, were tested by PCR amplification against 18s rRNA genome of Theileria spp using specie specific primers and then the PCR products were sequenced for species identification by comparison with data base available in GenBank.ResultsAbout 323 ticks were collected from 102 animals (88 sheep, 12 goats and 2 cattle). The prevalence of ticks infesting animals was R. sanguineus (82.35%), Rhipicephalus bursa (R. bursa) (0.3%), Ixodes ricinus (I. ricinus) (15.2%), Boophilus annulatus (B. annulatus) (1.2%), Haemaphysalis punctata (H. punctata) (0.3%) and Haemaphysalis numidiana (H. numidiana) (0.6%). Eleven (55%) tick specimens were PCR positive against genome of Theileria ovis (T. ovis). Sequence analysis of the PCR products confirmed presence of T. ovis in one R. sanguinus.ConclusionsThis is the first report of tick infection to T. ovis in Iran. Due to dominant prevalence of R. sanguineus as well as its infection to T. ovis, it is postulated this tick is the main vector of ovine theileriosis in northern Iran.     

DNA ‘barcoding’ of Schistosoma mansoni across sub-Saharan Africa supports substantial within locality diversity and geographical separation of genotypes

Schistosoma mansoni is a widespread human helminth and causes intestinal schistosomiasis in 54 countries, mainly across Africa but also in Madagascar, the Arabian Peninsula and the neotropics. The geographical range of this parasite relies on the distribution of certain species of freshwater pulmonate snails of the genus Biomphalaria. Whilst S. mansoni is known to exhibit high population diversity the true extent of this diversity is still to be fully elucidated as sampling of this taxon progressively accrues. Here a DNA ‘barcoding’ approach is taken using sequence analysis of a 450 bp region within the mitochondrial cox1 gene to assess the genetic diversity within a large number of S. mansoni larval stages collected from their natural human hosts across sub-Saharan Africa. Five hundred and sixty one individual parasite samples were examined from 22 localities and 14 countries. Considerable within-species diversity was found with 120 unique haplotypes splitting geographically into five discrete lineages. The highest diversity was found in East Africa with samples forming three of the five lineages. Less diversity was found in the Far and Central Western regions of Africa with haplotypes from the New World showing a close affinity to the Far Western African S. mansoni populations supporting the hypothesis of a colonisation of South America via the West African slave trade. The data are discussed in relation to parasite diversity and disease epidemiology.     

DNA barcodes confirm the presence of a single member of the Anopheles maculipennis group in Morocco and Algeria: An. sicaulti is conspecific with An. labranchiae

Anopheles labranchiae Falleroni is the only member of the Maculipennis Group known to occur in northern Africa; however, confusion exists as to the taxonomic status of its junior synonym, An. sicaulti Roubaud (type locality: near Rabat, Morocco). Based on morphological and behavioural distinctions, it has been suggested that Moroccan populations have been isolated from other North African populations by the Atlas Mountains, and that Moroccan populations may represent An. sicaulti, originally described as a variety of An. maculipennis Meigen. DNA barcodes (658 bp of the mitochondrial COI gene) obtained from 89 An. maculipennis s.l. collected in Morocco (n = 79) and Algeria (n = 10) in 2007 and 2008 were used to determine if Moroccan populations are genetically isolated from those east of the Atlas Mountains (Algeria), and whether there is molecular evidence to support the presence of more than one member of the Maculipennis Group in the region. No evidence for speciation was found between Moroccan and Algerian populations, or within populations in northern Morocco. Moreover shared COI haplotypes between Algeria and Morocco indicate ongoing gene flow between populations in these countries, suggesting that the Atlas Mountains are not a boundary to gene flow in An. labranchiae. The synonymy of An. sicaulti with An. labranchiae is confirmed. That An. labranchiae comprises the same species in these North African countries is important for malaria control.     

Association of the variants in AGT gene with modified drug response in Korean aspirin-intolerant asthma patients

The angiotensinogen (AGT) gene enhances the effect of several bronchoconstrictors and produces a peptide that is accumulated in the airways of asthma patients; events that may underpin the pathogenesis of aspirin-intolerant asthma (AIA). To carry out a case-control analysis between AGT and aspirin-induced bronchospasm following treatment with an anti-asthma drug, montelukast (MLK), 38 single nucleotide polymorphisms (SNPs) in AGT were genotyped in 56 AIA cohort. Genotyping was performed with TaqMan assay and haplotypes were inferred using PHASE algorithm ver. 2.0. Statistical analyses of each SNPs and haplotypes were performed using SAS version 9.1. Among 13 variants displaying significant signals, two SNPs (+2401C>G and +2476C>T) in the intronic region of AGT were significantly associated with modification of drug response even after correction for multiple testing (P = 0.0009–0.002; P^corr = 0.02–0.03). Furthermore, the two variants also exhibited associations with MLK response rate (P = 0.0003–0.0006; P^corr = 0.006–0.01). Although our results are preliminary and further replication in a larger-scale group of subjects should be warranted, these observations provide evidence that AGT variants might be one of genetic factors involved in the response of anti-asthma drugs in AIA patients.     

Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T. rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T. rangeli (rangelipain) closest to T. cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T. rangeli supported all the lineages (A–D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T. rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T. rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T. rangeli lineages associated with sympatric species of Rhodnius.     

Lower frequency of the 5/5 homozygous CNDP1 genotype in South Asian Surinamese

We investigated the frequency of the 5/5 homozygous CNDP1 (carnosinase) genotype, which was found to be associated with a reduced risk of developing diabetic nephropathy, in three ethnic groups in The Netherlands. Particularly interesting were the South Asian Surinamese, who have a high prevalence of diabetic nephropathy. Furthermore, we investigated the association between this gene and carnosinase activity in South Asian Surinamese and whether carnosinase was expressed in the kidney.We genotyped 290 South Asian Surinamese, 532 African Surinamese, and 472 White Dutch in a cross-sectional population study. Furthermore, an independent cohort of South Asian Surinamese was genotyped. In this population, carnosinase activity was measured in serum. Immunostaining and in situ hybridization for CNDP1 were performed on kidney tissue.Both South Asian populations had lower frequencies of the 5/5 homozygous genotype than African Surinamese and White Dutch (23.0%, 27.2%, 38.2%, and 41.3%, respectively; chi-square, p < 0.001). This genotype showed a lower carnosinase activity in South Asian Surinamese (Wilcoxon rank-sum, p = 0.03). CNDP1 was expressed in the kidney.South Asian Surinamese have a lower frequency of the 5/5 homozygous genotype, which was associated with lower carnosinase activity. Our study provides an indication that South Asian Surinamese are genetically at risk for developing diabetic nephropathy.     

Fluazuron-induced morphological changes in Rhipicephalus sanguineus Latreille, 1806 (Acari: Ixodidae) nymphs: An ultra-structural evaluation of the cuticle formation and digestive processes

Rhipicephalus sanguineus is a species of tick which is widely distributed in America, Africa and Australia and is probably the most prevalent among all the other ixodid tick species. The present study demonstrated the effects of the arthropod growth regulator fluazuron (Acatak^®), in the formation of the integument and the digestive processes of R. sanguineus nymphs fed on rabbits treated with different doses of this chemical acaricide. For this, three different doses of fluazuron (20 mg/kg, 40 mg/kg and 80 mg/kg) were applied “pour on” to the hosts divided into three different treated-groups (II, III, IV) of three animals each. A fourth group (I) of rabbits (n = 3) was given distilled water as control. On the first day after treatment (24 h), the hosts were artificially infested with R. sanguineus nymphs. After full engorgement (7 days), the nymphs were removed and placed on labeled Petri dishes and kept in BOD incubator for 7 days. The engorged nymphs were then taken to ultra-structural analysis. Results revealed the following main ultra-structural changes in the nymphs integument and midgut of the different treated groups (II, III, IV): cuticle disorganization and the absence of subdivisions, damages in the integument epithelial cells, size of digestive cells, amount of endosomes, autophagic and digestive vacuoles, accumulated digestive residues, lipid droplets and organelles found in the digestive cells’ cytoplasm, as well as the presence of microvilli in their plasma membranes. It is concluded that fluazuron may act on the integument and midgut cells of R. sanguineus engorged nymphs by impairing the synthesis of the new cuticle and the digestive processes (absorption of the blood ingested from the host, digestion – hemolysis, formation of digestive residues and release of nutrients to be converted into lipid, as well as for the synthesis of structural protein), which interfere in the development of nymphs, being able to prevent the emergence of adults after periodical ecdysis. These data indicate the possibility to use this arthropod growth regulator (AGR) in the control of R. sanguineus, at least in the nymphal stage of its biological cycle.     

Immunoglobulin G binding proteins in male Rhipicephalus appendiculatus ticks

Three abundant immunoglobulin G binding proteins (IGBPs) of29kDa (IGBP-MA), 25kDa (IGBP-MB), and 21 kDa (IGBP-MC) were isolated from partly fed male Rhipicephalus appendiculatus ticks using an agar-ose-IgG column. After separating the proteins by SDS-PA GE, each individual IGBP band was cutou tfrom the gel and used to raise antiserum in guinea pigs. Using immuno-blotting, each of the three IGBPs was shown to be antigenically distinct, and specific for feeding male R. appendiculatus ticks. They were detected in the salivary glands of male ticks that had been feeding in either the presence of absence of female ticks, and appeared to be glycoproteins. The smallest protein, IGBP-MC, bound to guinea pig IgG (on which species the ticks were fed) and also bound to human and bovine IgG, whereas IGBP-MA and possibly IGBP-MB only bound to guinea pig IgG. The function of IGBPs in tick salivary glands and their significance in male ticks are discussed.     

First record of larval Pseudoproleptus sp. (Nematoda: Cystidicolidae) in fish host

Infective third-stage larvae of the cystidicolid nematode Pseudoproleptus sp. were found encapsulated in the mesentery of the freshwater fish Satanoperca jurupari Heckel (Cichlidae) from the Guamá River, close to the Amazon River Delta, Pará State, Brazil. The prevalence in fish (total body length 9–24 cm) examined from March 2009 to June 2010 (n = 53) was 37%, with an intensity of 4–45 (mean 14 ± 11) larvae per fish. The nematode larvae (body length 16.2–21.6 mm), characterized by the cephalic end provided with a helmet-like cuticular structure having a thickened free posterior margin, were studied based on light and scanning electron microscopy. Fish play a role of paratenic hosts for this nematode species. This is the first record of a larval nematode of the genus Pseudoproleptus from fish, and the second record of a larval nematode belonging to Cystidicolidae in the Amazon and in the Neotropics. Additional larval specimens were found free in the stomach of only 0.9% Ageneiosus ucayalensis Castelnau (Auchenipteridae) examined (n = 205). This finding can be considered as occasional parasitism.     

Experimental vaccination of sheep and cattle against tick infestation using recombinant 5′‐nucleotidase

Limited prior evidence suggests that 5′‐nucleotidase, an ectoenzyme principally located in the Malpighian tubules of the tick Rhipicephalus (Boophilus) microplus, could be an effective antigen in an anti‐tick vaccine. To assess this, recombinant 5′‐nucleotidase was expressed in Escherichia coli and used in vaccination trials with both sheep and cattle. Vaccinated sheep were challenged with freshly moulted adult ticks. Those with high titres of anti‐nucleotidase antibodies showed significant protection against tick infestation, although protection was less than that found with the previously characterized antigen, Bm86. Cattle were vaccinated, in separate groups, with 5′‐nucleotidase, Bm86 and both antigens combined. Cattle, as the natural host, were challenged with larval ticks. Although Bm86 showed typical efficacy, no significant protection was seen in cattle vaccinated with 5′‐nucleotidase. Cattle receiving a dual antigen formulation were no better protected than those receiving Bm86 alone. One possible reason for the difference between host species, namely antibody titre, was examined and shown to be an unlikely explanation. This demonstrates a limitation of using a model host like sheep in vaccine studies.     

Activity of OZ78 analogues against Fasciola hepatica and Echinostoma caproni

The rapid spread of triclabendazole resistance in veterinary medicine is an important motivation for the discovery and development of novel fasciocidal drugs. The aim of this study was to characterize the fasciocidal properties of 1,2,4,5-tetraoxane (MT04 and MT14) and 1,2,4-trioxane (ST16 and ST28) analogues of the fasciocidal drug candidate OZ78, a 1,2,4-trioxolane. Dose response relationships were determined against juvenile and adult Fasciola hepatica in rats and Echinostoma caproni in mice. The temporal effects of MT04, MT14, ST16, and ST28 compared to OZ78 on the viability of F. hepatica were tested in vitro. The heat flow of OZ78 and MT04 treated flukes was studied with isothermal microcalorimetry. Finally, surface changes to adult flukes were monitored by scanning electron microscopy (SEM) 18, 24, and 48 h post-treatment of rats with 50 mg/kg MT04. Administration of 50–100 mg/kg of the synthetic peroxides resulted in complete elimination of adult F. hepatica from rats. SEM pictures revealed sloughing and blebbing already 18 h post-treatment with MT04. MT04 (100 mg/kg) cured infections with juvenile F. hepatica, whereas MT14, ST16, and ST28 showed only low to moderate worm burden reductions. At 300 mg/kg, MT14 was the only compound to completely eliminate worms from E. caproni infected mice. MT14 showed the highest activity against juvenile F. hepatica in vitro. MT04 was very active against adult F. hepatica in vitro, which was confirmed by heat flow measurements. In conclusion, we have identified MT04 as another lead compound with potential against F. hepatica, hence further preclinical studies are necessary to determine if MT04 can be considered a drug development candidate.