Moneeb A. Qablan

Quest for the piroplasms in camels: Identification of Theileria equi and Babesia caballi in Jordanian dromedaries by PCR

DNA of two species of piroplasmids was detected in dromedaries during a survey of blood protozoans in Jordan between 2007 and 2009. Ten clinically healthy camels (10%) originating from three Jordanian districts were found, using a PCR assay, to harbor Theileria or Babesia species in their blood and no mix infection was determined. Analysis of the partial 18S rRNA gene sequences of these parasites allowed their unambiguous identification as equine piroplasmids Babesia caballi (n=6) and Theileria equi (n=4). In case of latter species, a novel genotype was found in horses. This first molecular-based species determination of piroplasmids from camels further contributes to the growing evidence of low host specificity of piroplasmids.

Stray dogs of northern Jordan as reservoirs of ticks and tick-borne hemopathogens

Dogs are competent reservoir hosts of several hemopathogens including zoonotic agents and can serve as readily available source of nutrition for many blood-feeding arthropods. Three hemopathogens had been detected for the first time in Jordan. The PCR prevalence of Anaplasma phagocytophilum, Hepatozoon canis, and piroplasmid DNA were 39.5%, 28.9%, and 7.9% (n = 38) respectively. Sequencing of amplicons of PCR with universal primers targeting the 18S rRNA gene of piroplasmids shows the highest similarity to equine piroplasmids species Theileria equi from two dogs and Babesia caballi from a single dog. Ticks of two genera Rhipicephalus and Haemaphysalis, were detected in this study (n = 268). The brown dog tick, Rhipicephalus sanguineus was the most abundant species (95.1%, n = 255), followed by Haemaphysalis erinacei (3%, n = 8) and Haemaphysalis parva (1.9%, n = 5). The two Haemaphysalis species were detected for the first time from dogs in Jordan. Regarding its high prevalence, we expect R. sanguineus being a possible vector of detected pathogens.

Genetic diversity of the class II major histocompatibility DRA locus in European, Asiatic and African domestic donkeys.

The major histocompatibility complex (MHC) genes coding for antigen presenting molecules are the most polymorphic genes in vertebrate genome. The MHC class II DRA gene shows only small variation in many mammalian species, but it exhibits relatively high level of polymorphism in Equidae, especially in donkeys. This extraordinary degree of polymorphism together with signatures of selection in specific amino acids sites makes the donkey DRA gene a suitable model for population diversity studies. The objective of this study was to investigate the DRA gene diversity in three different populations of donkeys under infectious pressure of protozoan parasites, Theileria equi and Babesia caballi. Three populations of domestic donkeys from Italy (N = 68), Jordan (N = 43), and Kenya (N = 78) were studied. A method of the donkey MHC DRA genotyping based on PCR-RFLP and sequencing was designed. In addition to the DRA gene, 12 polymorphic microsatellite loci were genotyped. The presence of Theileria equi and Babesia caballi parasites in peripheral blood was investigated by PCR. Allele and genotype frequencies, observed and expected heterozygosities and F(IS) values were computed as parameters of genetic diversity for all loci genotyped. Genetic distances between the three populations were estimated based on F(ST) values. Statistical associations between parasite infection and genetic polymorphisms were sought. Extensive DRA locus variation characteristic for Equids was found. The results showed differences between populations both in terms of numbers of alleles and their frequencies as well as variation in expected heterozygosity values. Based on comparisons with neutral microsatellite loci, population sub-structure characteristics and association analysis, convincing evidence of pathogen-driven selection at the population level was not provided. It seems that genetic diversity observed in the three populations reflects mostly effects of selective breeding and their different genetic origins.

Molecular detection of Anaplasma platys infection in free-roaming dogs and ticks from Kenya and Ivory Coast

BackgroundAnaplasma platys is a bacterium parasitic in the canine platelets, representing the causative agent of canine cyclic thrombocytopenia, with a worldwide distribution, mainly in tropical countries. The agent has zoonotic potential, being reported in several human clinical cases. The suspected vector is the tick Rhipicephalus sanguineus (sensu lato), widely distributed in animals and humans in the tropical regions of South America, Africa, Asia and Australia, but also in southern Europe. Only few scattered data concerning the epidemiology of A. platys in sub-Saharan Africa are available. The aim of our study was to evaluate the epidemiological status of A. platys in dogs and cats from rural communities in eastern and western Africa, where dogs and their ticks live in close contact with humans.MethodsA total of 216 canine and 22 feline blood samples and ticks feeding on them were collected in 2013 and 2014 from eight localities in Ivory Coast and four localities in Kenya. PCR was performed using specific primers targeting a portion of the 16S rRNA gene, followed by sequencing.ResultsThe main results highlight the high prevalence of A. platys infection in dogs in both Eastern and Western Africa and report it for the first time in Eastern Africa and in Rhipicephalus camicasi.ConclusionsThe presence of A. platys DNA in R. camicasi feeding on negative hosts together with the relatively high prevalence of A. platys, correlated with the absence of the probable vector Rhipicephalus sanguineus (s.l.) in Kenyan Island suggest the involvement of other tick species in the transmission of A. platys.

Cytauxzoon Infections in Wild Felids from Carpathian-Danubian-Pontic Space: Further Evidence for a Different Cytauxzoon Species in European Felids

Abstract Parasitic protists of the genus Cytauxzoon are detected in a wide range of wild and domestic felids. Bobcats are a confirmed reservoir of Cytauxzoon felis in North America while domestic cats are susceptible hosts suffering from severe or fatal illness. Cytauxzoon infections are mainly reported from American felids and, recently, several sub-clinical and clinical findings were reported from European, Asian, and African felids. In 2014, the collection of organs of 4 Eurasian lynx and 12 wild cats from 11 Romanian localities was carried out to determine the prevalence and genetic diversity of Cytauxzoon spp. We detected an overall high prevalence of 62.5% in both species of wild felids; 50% in wild cats and 100% in Eurasian lynx. The phylogenetic analysis indicates 2 distinct clades of Cytauxzoon in felids, with all of our sequences clustering with sequences of Cytauxzoon sp./Cytauxzoon manul from Palaearctic felids. Further studies, development of new genetic markers, and experimental transmission studies are required for clarifying the taxonomy and life cycle of feline Cytauxzoon in the Old World.

Ecology of malaria infections in western lowland gorillas inhabiting Dzanga Sangha Protected Areas, Central African Republic

African great apes are susceptible to infections with several species of Plasmodium, including the predecessor of Plasmodium falciparum. Little is known about the ecology of these pathogens in gorillas. A total of 131 gorilla fecal samples were collected from Dzanga-Sangha Protected Areas to study the diversity and prevalence of Plasmodium species. The effects of sex and age as factors influencing levels of infection with Plasmodium in habituated gorilla groups were assessed. Ninety-five human blood samples from the same locality were also analysed to test for cross-transmission between humans and gorillas. According to a cytB PCR assay 32% of gorillas fecal samples and 43·1% human individuals were infected with Plasmodium spp. All Laverania species, Plasmodium vivax, and for the first time Plasmodium ovale were identified from gorilla samples. Plasmodium praefalciparum was present only from habituated individuals and P. falciparum was detected from human samples. Although few P. vivax and P. ovale sequences were obtained from gorillas, the evidence for cross-species transmission between humans and gorillas requires more in depth analysis. No association was found between malaria infection and sex, however, younger individuals aged ≤6 years were more susceptible. Switching between two different Plasmodium spp. was observed in three individuals. Prolonged monitoring of Plasmodium infection during various seasons and recording behavioural data is necessary to draw a precise picture about the infection dynamics.

Questing for the identity of Hepatozoon in foxes

Apicomplexan parasites of genus Hepatozoon invade the blood cells of many mammalian species, being transmitted by range of arthropods. In domestic carnivores, three species of Hepatozoon were described to date: H. canis and H. americanum in dogs and H. felis in cats. The classification of Hepatozoon in wild carnivores is still not complete due to lack of field and experimental data as well as phylogenetic studies. The aim of this study is to carry out a survey on the prevalence and diversity of Hepatozoon sp. in red foxes Vulpes vulpes. Samples of tissues were collected from dead foxes in 11 counties of Romania; 91 samples of liver tissue were examined in total. DNA extraction was performed with commercial kit according to the manufactures protocol. Hepatozoon sp. DNA was detected by PCR using primers amplifying 400-600 bp long part of 18S rRNA gene. These primers are commonly used for diagnostic purposes in dogs and in some studies also for detection of Hepatozoon sp. in wild carnivores. PCR products were sequenced to validate positive results of reaction. DNA of parasite was confirmed in 55% of examined samples. Recent findings classified Hepatozoon sp. in foxes and other wild canids in Europe as H. canis. However, Rhipicephalus sanguineus, the only known vector of H. canis in Europe, is absent in most of our sampling sites. Moreover, this tick is typical for the dogs but rare or even absent in foxes. In order to clarify the identity of the parasite, the next step of our study is to focus on amplification of longer or full segment of 18s rRNA gene to allow more accurate phylogenetic analyses and comparison with H. canis sequences from dogs and Hepatozoon isolates from other carnivores. This study was supported by IGA UVPS Brno, project 115/2013/FVL.

A comparative molecular survey of malaria prevalence among Eastern chimpanzee populations in Issa Valley (Tanzania) and Kalinzu (Uganda).

BackgroundHabitat types can affect vector and pathogen distribution and transmission dynamics. The prevalence and genetic diversity of Plasmodium spp. in two eastern chimpanzee populations—Kalinzu Forest Reserve, Uganda and Issa Valley, Tanzania—inhabiting different habitat types was investigated. As a follow up study the effect of host sex and age on infections patterns in Kalinzu Forest Reserve chimpanzees was determined.MethodsMolecular methods were employed to detect Plasmodium DNA from faecal samples collected from savanna-woodland (Issa Valley) and forest (Kalinzu Forest Reserve) chimpanzee populations.ResultsBased on a Cytochrome-b PCR assay, 32 out of 160 Kalinzu chimpanzee faecal samples were positive for Plasmodium DNA, whilst no positive sample was detected in 171 Issa Valley chimpanzee faecal samples. Sequence analysis revealed that previously known Laverania species (Plasmodium reichenowi, Plasmodium billbrayi and Plasmodium billcollinsi) are circulating in the Kalinzu chimpanzees. A significantly higher proportion of young individuals were tested positive for infections, and switching of Plasmodium spp. was reported in one individual. Amongst the positive individuals sampled more than once, the success of amplification of Plasmodium DNA from faeces varied over sampling time.ConclusionThe study showed marked differences in the prevalence of malaria parasites among free ranging chimpanzee populations living in different habitats. In addition, a clear pattern of Plasmodium infections with respect to host age was found. The results presented in this study contribute to understanding the ecological aspects underlying the malaria infections in the wild. Nevertheless, integrative long-term studies on vector abundance, Plasmodium diversity during different seasons between sites would provide more insight on the occurrence, distribution and ecology of these pathogens.

A molecular survey on host specificity of feline and canine Hepatozoon in model site of northern Kenya

Species of the genus Hepatozoon are apicomplexan parasites transmitted by variety of hematophagous arthropods to a wide range of intermediate hosts. Hepatozoon infections are broadly distributed around the world and the presence of this parasite is confirmed both in cats and dogs. Until now, two species from canines (H. canis, H. americanum) and a single species from felines (H. felis) have been reported. While the canine Hepatozoon spp. are transmitted by ticks, the vector for H. felis remains unknown. The aim of presented study was to determine the prevalence and the diversity of Hepatozoon in population of domestic cats and dogs living in close contact in rural communities of Samburu pastoralists in northern Kenya and to evaluate the existence of possible cross-transmissions between both hosts. Between years 2007-2012 in total, 135 and 258 blood samples from cats and dogs from the area of Mt. Kulal (5 localities) was collected, respectively. The DNA from blood (preserved in ethanol) was extracted using phenol-chlorophorm method and followed by conventional PCR screening. First round of PCR was done by using Piroplasmid-F and Piroplasmid-R primers, amplifying 18S rRNA gene of Hepatozoon spp. (400 bp). A second PCR assay was performed in samples positive by Piroplasmid primers to amplify a larger fragment (1400 bp) of the 18S rRNA gene. All PCR amplicons comming from positive samples were sequenced and determined according to the BLAST match. In cats, this revealed 110 out of 135 (81.5%) positive samples, of which we obtained 104 sequences with an identity of 98% -100% to an existing GenBank accession. In total, 77 sequences were confirmed as Hepatozoon felis, 26 samples was proven to be Hepatozoon sp. and a single sample was determined as Hepatozoon canis with an identity of 96%. Regarding dogs, 121 out of 258 (47%) were positive and we gained 107 sequences of which 105 showed similarity to Hepatozoon canis and the remaining two proved to be Hepatozoon sp. Despite a close contact of hosts, obviously cats and dogs do not share the same parasite, which demonstrates a great majority of clearly identified Hepatozoon canis/Hepatozoon felis. Mentioned facts could be explained either by strict host specificity or by presence of different vector or by both statements.

Next generation sequencing as a novel tool for diagnostics of apicomplexan pathogen in ticks and mammalian hosts

Among apicomplexan parasites, ticks are known vector of several species belongs to three protozoan genera (Babesia, Theileria and Hepatozoon). During their life cycle, tick-transmitted apicomplexan parasites alternate between asexual (in vertebrate host) and sexual (in ticks) developmental stages. The major constraint for the proper diagnostics of those pathogens is the high possibility of mix infection, both in ticks and vertebrate hosts, with several species or genotypes. The aim of this study was to apply the Next Generation Sequencing (NGS) as a method of choice for simultaneous determination of the full spectrum of apicomplexan pathogens in ticks and the mammalian hosts. Therefore, A pair of universal primers were designed to flank a 167 bp barcode region of the 18s rRNA gene of all Babesia, Theileria and Hepatozoon species. The new protocol was evaluated on DNA samples isolated from 195 dogs and 144 ticks (Rhipicephalus armatus and R. pulchellus) collected from Northern Kenya. In total 301 sample (89%) were positive for apicomplexan infections; ranging from single to multiple infection with one species or several species and/or genotypes in a single sample. The most abundant apicomplexan pathogens were Hepatozoon followed by Babesia and Theileria, respectively. Further, the result shows that the barcode region entails enough variability that allows identifying the pathogens up to the subspecies and genotypes level. The exact methodological and results detailed will be presented later. This work was supported by the project OP VK CZ.1.07/2.3.00/30.0014.