Detection of Bartonella schoenbuchensis (sub)species DNA in different louse fly species in Saxony, Germany: The proof of multiple PCR analysis necessity in case of ruminant-associated bartonellae determination.

BACKGROUND: Hippoboscid flies are bloodsucking arthropods that can transmit pathogenic microorganisms and are therefore potential vectors for pathogens such as Bartonella spp. These Gram-negative bacteria can cause mild-to-severe clinical signs in humans and animals; therefore, monitoring Bartonella spp. prevalence in louse fly populations appears to be a useful prerequisite for zoonotic risk assessment. METHODS: Using convenience sampling, we collected 103 adult louse flies from four ked species (Lipoptena cervi, n = 22; Lipoptena fortisetosa, n = 61; Melophagus ovinus, n = 12; Hippobosca equina, n = 8) and the pupae of M. ovinus (n = 10) in the federal state of Saxony, Germany. All the samples were screened by polymerase chain reaction (PCR) for Bartonella spp. DNA, targeting the citrate synthase gene (gltA). Subsequently, PCRs targeting five more genes (16S, ftsZ, nuoG, ribC and rpoB) were performed for representatives of revealed gltA genotypes, and all the PCR products were sequenced to identify the Bartonella (sub)species accurately. RESULTS AND CONCLUSIONS: The overall detection rates for Bartonella spp. were 100.0%, 59.1%, 24.6% and 75.0% in M. ovinus, L. cervi, L. fortisetosa and H. equina, respectively. All the identified bartonellae belong to the Bartonella schoenbuchensis complex. Our data support the proposed reclassification of the (sub)species status of this group, and thus we conclude that several genotypes of B. schoenbuchensis were detected, including Bartonella schoenbuchensis subsp. melophagi and Bartonella schoenbuchensis subsp. schoenbuchensis, both of which have previously validated zoonotic potential. The extensive PCR analysis revealed the necessity of multiple PCR approach for proper identification of the ruminant-associated bartonellae.

Molecular pathogen screening of louse flies (Diptera: Hippoboscidae) from domestic and wild ruminants in Austria.

BACKGROUND: Hippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria. METHODS: Louse flies were collected from naturally infested cattle (n = 25), sheep (n = 3), and red deer (n = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses. RESULTS: A total of 282 hippoboscid flies corresponding to three species were identified: Hippobosca equina (n = 62) collected from cattle, Melophagus ovinus (n = 100) from sheep and Lipoptena cervi (n = 120) from red deer (Cervus elaphus). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina. Anaplasmataceae DNA (Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida. CONCLUSIONS: Molecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. Bartonella spp.) and the first report of Trypanosoma sp. in H. equina, suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context.

The genome of sheep ked (Melophagus ovinus) reveals potential mechanisms underlying reproduction and narrower ecological niches.

BACKGROUND: Melophagus ovinus is considered to be of great veterinary health significance. However, little is known about the information on genetic mechanisms of the specific biological characteristics and novel methods for controlling M. ovinus. RESULTS: In total, the de novo genome assembly of M. ovinus was 188.421 Mb in size (330 scaffolds, N50 Length: 10.666 Mb), with a mean GC content of 27.74%. A total of 13,372 protein-coding genes were functionally annotated. Phylogenetic analysis indicated that the diversification of M. ovinus and Glossina fuscipes took place 72.76 Mya within the Late Cretaceous. Gene family expansion and contraction analysis revealed that M. ovinus has 65 rapidly-evolving families (26 expansion and 39 contractions) mainly involved DNA metabolic activity, transposases activity, odorant receptor 59a/67d-like, IMD domain-containing protein, and cuticle protein, etc. The universal and tightly conserved list of milk protein orthologues has been assembled from the genome of M. ovinus. Contractions and losses of sensory receptors and vision-associated Rhodopsin genes were significant in M. ovinus, which indicate that the M. ovinus has narrower ecological niches. CONCLUSIONS: We sequenced, assembled, and annotated the whole genome sequence of M. ovinus, and launches into the preliminary genetic mechanisms analysis of the adaptive evolution characteristics of M. ovinus. These resources will provide insights to understand the biological underpinnings of this parasite and the disease control strategies.

Distribution and Molecular Epidemiology of Anaplasma ovis in Melophagus ovinus and Small Ruminants in Border Regions of South Xinjiang, China.

Background: Anaplasma ovis are obligate intracellular bacteria that can endanger human and animal health, and they can be transmitted by arthropod vectors, such as Melophagus ovinus and ticks. Materials and Methods: In this study, 433 specimens, including 370 M. ovinus and 63 sheep blood samples, were collected from nine districts of South Xinjiang to investigate the distribution and molecular epidemiology of A. ovis in M. ovinus and small ruminant. Results: DNA of A. ovis was detected in 109 (25.2%, 109/433) of the 433 samples using PCR and sequencing. The analysis of A. ovis msp4 sequences revealed four different genotypes, including genotype III (47.7%; 52/109), GB3 (34.0%; 37/109), AoGOv3 (15.6%; 17/109), and XJ9 (2.8%; 3/109). Conclusions: To the best of our knowledge, A. ovis genotypes GB3, AoGOv3, and XJ9 detected in this study are the first to be reported in M. ovinus, and our data indicate that XJ9 is a novel A. ovis genotype presented herein for the first time. These findings provide important references for the new understanding and prevention of A. ovis in border counties in China.

First determination of DNA virus and some additional bacteria from Melophagus ovinus (sheep ked) in Tibet, China.

Melophagus ovinus (sheep ked) is one of the common ectoparasites in sheep. In addition to causing direct damage to the host through biting and sucking blood, sheep ked is a potential vector of helminths, protozoa, bacteria, and viruses. Sheep M. ovinus samples from three regions in Tibet were selected for DNA extraction. The 16S rDNA V3-V4 hypervariable region was amplified, after genomic DNA fragmentation, Illumina Hiseq libraries were constructed. The 16S rRNA sequencing and viral metagenomics sequencing were separately conducted on the Illumina Novaseq 6000 platform and molecular biology software and platforms were employed to analyze the sequencing data. Illumina PE250 sequencing results demonstrated that the dominant bacteria phylum in M. ovinus from Tibet, China was Proteobacteria, where 29 bacteria genera were annotated. The dominant bacterial genera were Bartonella, Wolbachia, and Arsenophonus; Bartonella chomelii, Wolbachia spp., and Arsenophonus spp. were the dominant bacterial species in M. ovinus from Tibet, China. We also detected Kluyvera intermedia, Corynebacterium maris DSM 45190, Planomicrobium okeanokoites, and Rhodococcus erythropolis, of which the relative abundance of Kluyvera intermedia was high. Illumina Hiseq sequencing results demonstrated that 4 virus orders were detected in M. ovinus from Tibet, China, and 3 samples were annotated into 29 families, 30 families, and 28 families of viruses, respectively. Virus families related to vertebrates and insects mainly included Mimiviridae, Marseilleviridae, Poxviridae, Ascoviridae, Iridoviridae, Baculoviridae, Hytrosaviridae, Nudiviridae, Polydnaviridae, Adomaviridae, Asfarviridae, Hepeviridae, Herpesviridae, and Retroviridae; at the species level, the relative abundance of Tupanvirus_soda_lake, Klosneuvirus_KNV1, and Indivirus_ILV1 was higher. African swine fever virus and many poxviruses from the family Poxviridae were detected, albeit their relative abundance was low. The dominant bacterial phylum of M. ovinus from Tibet, China was Proteobacteria, and the dominant bacterial genera were Bartonella, Wolbachia, and Arsenophonus, where 23 out of 29 annotated bacteria genera were first reported in M. ovinus. Kluyvera intermedia, Corynebacterium maris DSM 45190, Planomicrobium okeanokoites, and Rhodococcus erythropolis were detected for the first time. All DNA viruses detected in this study have been reported in M. ovinus for the first time.

Tandem mass tag labeled quantitative proteomic analysis of differential protein expression on total alkaloid of Aconitum flavum Hand.-Mazz. against melophagus ovinus.

Melophagus ovinus disease is a common ectoparasitosis, which can lead to a decrease in animal production performance, product quality, and even death. Aconitum flavum Hand.-Mazz. has many pharmacological activities including insecticidal, heat-clearing, analgesic, and dehumidifying. However, there are few researches focused on the effects and related mechanism of Aconitum flavum Hand.-Mazz. in killing Melophagus ovinus. In this study, 11 alkaloids of Aconitum flavum Hand.-Mazz. were detected, and its total alkaloid activity was determined. The results showed when the total alkaloid concentration was 64 mg/ml and the treatment time was 16 h, the killing rate of Melophagus ovinus reached 100%. Through the observation of the differences in the surface of Melophagus ovinus in each experimental group, it was found that the morphology of the posterior end of the female Melophagus ovinus in the alkaloid treatment group was significantly different from that of the blank and positive control groups, and most of the epidermal tissue was obsessive and missing. Moreover, the enzyme activity determination results of 64 mg/ml group were significantly different when compared with the normal control group, while there was no significant difference in other groups. Then, the Melophagus ovinus gene library was established by the unreferenced genome transcriptome sequencing, the proteomic comparison was performed using tandem mass tag labeled protein detection technology, and finally, the samples were quantitatively analyzed by liquid chromatography-mass spectrometry tandem and bioinformatics methods. Based on the above experimental results, it was speculated that Aconitum flavum Hand.-Mazz. total alkaloids may cause the imbalance of protein disulfide isomerase expressions by affecting the regulation of Hsp40 cellular protein homeostasis and the oxidation of protein disulfide isomerase and related proteins. This would affect the selective recognition of signal sequence, the targeted transport of Sec 61, and the correct folding of the three-dimensional structure of amino acid chain, weakening the clearance of amino acid chains that cannot be correctly folded and eventually resulting in the killing of Melophagus ovinus. This study preliminarily revealed the mechanism of Aconitum flavum Hand.-Mazz. total alkaloids against Melophagus ovinus and provided a theoretical basis for the screening of Melophagus ovinus action targets and the development of new veterinary drugs.

Possible Arbovirus Found in Virome of Melophagus ovinus.

Members of the Lipopteninae subfamily are blood-sucking ectoparasites of mammals. The sheep ked (Melophagus ovinus) is a widely distributed ectoparasite of sheep. It can be found in most sheep-rearing areas and can cause skin irritation, restlessness, anemia, weight loss and skin injuries. Various bacteria and some viruses have been detected in M. ovinus; however, the virome of this ked has never been studied using modern approaches. Here, we study the virome of M. ovinus collected in the Republic of Tuva, Russia. In our research, we were able to assemble full genomes for five novel viruses, related to the Rhabdoviridae (Sigmavirus), Iflaviridae, Reoviridae and Solemoviridae families. Four viruses were found in all five of the studied pools, while one virus was found in two pools. Phylogenetically, all of the novel viruses clustered together with various recently described arthropod viruses. All the discovered viruses were tested on their ability to replicate in the mammalian porcine embryo kidney (PEK) cell line. Aksy-Durug Melophagus sigmavirus RNA was detected in the PEK cell line cultural supernate after the first, second and third passages. Such data imply that this virus might be able to replicate in mammalian cells, and thus, can be considered as a possible arbovirus.

Vector-Borne Pathogens with Veterinary and Public Health Significance in Melophagus ovinus (Sheep Ked) from the Qinghai-Tibet Plateau.

Melophagus ovinus (sheep ked) is a hematophagous ectoparasite that mainly parasitizes sheep. In addition to causing inflammation, wool loss, and skin damage to the animal hosts, M. ovinus also serves as a vector for a variety of pathogens and is highly likely to participate in the life and transmission cycle of pathogenic organisms. Herein, we investigated the presence and molecular characterization of vector-borne pathogens in M. ovinus from Qinghai-Tibet Plateau, China. A total of 92 M. ovinus pools collected from the Qinghai province of China were screened for the presence of selected vector-borne pathogens. The overall positive rate of A. ovis, A. bovis, A. phagocytophilum, and T. ovis in M. ovinus was 39.1%, 17.4%, 9.8%, and 89.1%, respectively. All of the samples were negative for Border disease virus (BDV), other Anaplasma species, Babesia spp., Rickettsia spp., and Borrelia spp. Co-infection of different Anaplasma species and T. ovis occurred in 51.2% of all samples with T. ovis. The positive rates of A. ovis, A. bovis, and A. phagocytophilum in different regions and altitudes of the sampling sites were significantly different. Sequence and phylogenetic analysis of target genes confirmed their identity with corresponding pathogens. Our results elucidate the occurrence and molecular characterization of Anaplasma spp. and Theileria spp. in M. ovinus, which could act as potential zoonotic reservoirs. To the best of our knowledge, this is the first report of the detection of A. bovis and A. phagocytophilum DNA in M. ovinus. This study gives the first extensive molecular survey of vector-borne pathogens with veterinary and public health significance in M. ovinus from the Qinghai-Tibet Plateau, China.

Keds, the enigmatic flies and their role as vectors of pathogens.

Hippoboscid flies (Diptera: Hippoboscidae), commonly known as keds or louse flies, have been for long time overlooked by the scientific community, and their vector role of infectious agents to humans and domestic animals has been scantly investigated. This is partly due to the fact that the host range for most species is primarily restricted to wildlife, being rarely reported on domestic animals and humans. This led to a scarce scientific knowledge about their biology, ecology, behaviour, epidemiology as well as vector competence. However, the life history of some hippoboscid species, e.g., Melophagus ovinus, Lipoptena cervi and Hippobosca equina, suggests that these ectoparasites are important candidates to vector infectious disease agents (e.g., Rickettsia spp., Borrelia spp., Bartonella spp., Anaplasma phagocytophilum, Theileria ovis). Indeed, the peculiar biological and behavioural traits (i.e., obligatory blood sucking and reproductive physiology) of many ked species make them a suitable pabulum for pathogen's multiplication and for their transmission to receptive hosts. Therefore, studies focusing on the ked bio-ecological aspects as well as on their vector role are advocated along with the control of keds affecting different animal species. This review discusses current information on keds, highlighting their importance as vectors of pathogens of medical and veterinary concern to all animal species, with a special focus on mammals.

Comparative analysis of microbial community in the whole body and midgut from fully engorged and unfed female adult Melophagus ovinus.

Melophagus ovinus is a type of ectoparasite infesting sheep. Data regarding the comprehensive bacterial community associated with the whole body and midgut of M. ovinus under different engorged statuses are required. Melophagus ovinus were collected from the city of Jiuquan, China. Bacterial DNA was extracted from the whole body and midgut of fully engorged female adults, or newly hatched and unfed adult female M. ovinus. The 16S rRNA gene V3-V4 hypervariable regions were sequenced using the IonS5™XL platform (Thermo Fisher Scientific, Waltham, MA, U.S.A.). The whole body bacterial diversity of the newly hatched, unfed adult females was greater compared with that of the other three samples. Proteobacteria was the dominant bacterial phylum in all of the samples. Of the 42 total bacterial genera present in all of the experimental samples, Arsenophonus, Bartonella and Wolbachia were the dominant genera. The relative abundance of Arsenophonus in midgut was greater than that in the whole body. The relative abundance of Bartonella in fully engorged adults was far greater than those in newly hatched, unfed adults. The relative abundance of Wolbachia was highest in the whole body of newly hatched, unfed adults. Seventeen bacterial species were identified in all experimental samples. Bartonella chomelii, Streptococcus hyointestinalis and Escherichia coli were the first species reported in M. ovinus.

Theileria ovis (Piroplasmida: Theileriidae) Detected in Melophagus ovinus (Diptera: Hippoboscoidea) and Ornithodoros lahorensis (Ixodida: Argasidae) Removed From Sheep in Xinjiang, China.

Theileria spp. are tick-transmitted, intracellular apicomplexan protozoan parasites that infect a wide range of animals and, as such, can cause significant economic losses. The aim of the present study was to detect and analyze apicomplexan parasites from two different ectoparasites that were collected from Xinjiang Uygur Autonomous Region, China. The PCR-based detection of 18S rRNA indicated that Ornithodoros lahorensis specimens from Kashgar, Xinjiang, and Aksu were positive for Theileria spp., as were Melophagus ovinus specimens from Aksu. Meanwhile, phylogenetic analysis, based on the 18S rRNA gene sequences, revealed that the four amplified Theileria sequences could be attributed to T. ovis. To the best of our knowledge, this is the first study to report the detection of T. ovis DNA in M. ovinus and the first molecular identification study to confirm the detection of T. ovis in O. lahorensis in China. Accordingly, the present study extends the known distribution of T. ovis.

Molecular evidence of bacteria in Melophagus ovinus sheep keds and Hippobosca equina forest flies collected from sheep and horses in northeastern Algeria.

The sheep ked, Melophagus ovinus, and the forest fly, Hippobosca equina, are parasitic dipteran insects of veterinary importance. As hematophagous insects, they might be considered as potential vectors of diseases which may be transmissible to humans and animals. The purpose of this study was to present initial primary data about these two species in Algeria. To do so, we conducted a molecular survey to detect the presence of bacterial DNA in flies collected in Algeria. A total of 712 flies including, 683 Melophagus ovinus and 29 Hippobosca equina were collected from two regions in northeastern Algeria. Monitoring the monthly kinetics of M. ovinus infestations showed something resembling annual activity, with a high prevalence in January (21.67%) and May (20.94%). Real-time quantitative PCR assays showed that for 311 tested flies, 126 were positive for the Bartonella spp. rRNA intergenic spacer gene and 77 were positive for Anaplasmataceae. A random selection of positive samples was submitted for sequencing. The DNA of Bartonella chomelii and Bartonella melophagi were amplified in, respectively, five and four H. equina. 25 M. ovinus positive samples were infected by Bartonella melophagi. Amplification and sequencing of the Anaplasma spp. 23S rRNA gene revealed that both species were infected by Wolbachia sp. which had previously been detected in Cimex lectularius bed bugs. Overall, this study expanded knowledge about bacteria present in parasitic flies of domestic animals in Algeria.

Molecular Identification of Bartonella melophagi and Wolbachia Supergroup F from Sheep Keds in Xinjiang, China.

To confirm that Bartonella and Wolbachia were carried by sheep keds (Melophagus ovinus) in southern Xinjiang of China, 17 M. ovinus samples, which were collected in Aksu Prefecture, Xinjiang, were randomly selected. In this study, the Bartonella gltA and Wolbachia 16S rRNA gene were amplified through conventional PCR and the sequence of those amplified products, were analyzed. The results demonstrated that Bartonella was carried by all of the 17 sheep keds and Wolbachia was carried by 15 out of them. Bartonella was identified as B. melophagi. Three strains of Wolbachia were supergroup F and 1 strain has not been confirmed yet. It is the first report about Wolbachia supergroup F was found in sheep keds and provided the molecular evidence that B. melophagi and Wolbachia supergroup F were carried by sheep keds in Aksu Prefecture of southern Xinjiang, China. The 2 pathogens were found in sheep keds around Taklimakan Desert for the first time.

Comparative analyses of the mitochondrial genome of the sheep ked Melophagus ovinus (Diptera: Hippoboscidae) from different geographical origins in China.

The sheep ked Melophagus ovinus is mainly found in Europe, Northwestern Africa, and Asia. Although M. ovinus is an important ectoparasite of sheep in many countries, the population genetics, molecular biology, and systematics of this ectoparasite remain poorly understood. Herein, we determined the mitochondrial (mt) genome of M. ovinus from Gansu Province, China (MOG) and compared with that of M. ovinus Xinjiang Uygur Autonomous Region, China (MOX). The mt genome sequence (15,044 bp) of M. ovinus MOG was significantly shorter (529 bp) than M. ovinus MOX. Nucleotide sequence difference in the whole mt genome except for non-coding region was 0.37% between M. ovinus MOG and MOX. For the 13 protein-coding genes, comparison revealed sequence divergences at both the nucleotide (0-1.1%) and amino acid (0-0.59%) levels between M. ovinus MOG and MOX, respectively. Interestingly, the cox1 gene of M. ovinus MOX is predicted to employ unusual mt start codons AAA, which has not been predicted previously for any parasite genome. Phylogenetic analyses showed that M. ovinus (Hippoboscoidea) is related to the superfamilies Oestroidea + Muscoidea. Our results have also indicated the paraphylies of the four families (Anthomyiidae, Calliphoridae, Muscidae, and Oestridae) and two superfamilies (Oestroidea and Muscoidea). This mt genome of M. ovinus provides useful molecular markers for studies into the population genetics, molecular biology, and systematics of this ectoparasite.

First report of Anaplasma ovis in pupal and adult Melophagus ovinus (sheep ked) collected in South Xinjiang, China.

BACKGROUND: Melophagus ovinus (sheep ked) is a blood-feeding ectoparasite that belongs to the family Hippoboscidae (Diptera: Hippoboscoidea) and mainly parasitizes sheep. The life-cycle of M. ovinus consists of three stages: larva, pupa and adult. It has a worldwide distribution and has been found in four provinces of China, especially South Xinjiang. In addition to causing direct damage to animal hosts, M. ovinus serves as a vector for disease transmission. In this study, our aim was to investigate the presence of Anaplasma spp. in pupal and adult M. ovinus. METHODS: A total of 93 specimens (including eight pupal specimens) of M. ovinus collected in South Xinjiang were selected for isolation of genomic DNA, followed by PCR amplification and sequencing of the msp4 gene of Anaplasma spp. The sequences were analyzed in MEGA 7.0 software and via online BLAST. RESULTS: PCR and sequencing results showed that all the specimens collected in 2013 were free of Anaplasma spp., whereas three and 25 specimens (including five pupal specimens) collected in 2016 and 2017, respectively, tested positive for Anaplasma spp. The analysis of 24 msp4 gene sequences (from four pupal specimens) confirmed the presence of A. ovis in M. ovinus specimens collected in South Xinjiang, China. The detected A. ovis isolates belong to Genotypes II and III. CONCLUSIONS: To the best of our knowledge, this is the first report of the detection of A. ovis DNA in pupal M. ovinus, confirming the vertical transmission of A. ovis in M. ovinus and the potential of M. ovinus to serve as a vector for A. ovis.

Molecular Identification of Bartonella melophagi and Wolbachia Supergroup F from Sheep Keds in Xinjiang, China

To confirm that Bartonella and Wolbachia were carried by sheep keds (Melophagus ovinus) in southern Xinjiang of China, 17 M. ovinus samples, which were collected in Aksu Prefecture, Xinjiang, were randomly selected. In this study, the Bartonella gltA and Wolbachia 16S rRNA gene were amplified through conventional PCR and the sequence of those amplified products, were analyzed. The results demonstrated that Bartonella was carried by all of the 17 sheep keds and Wolbachia was carried by 15 out of them. Bartonella was identified as B. melophagi. Three strains of Wolbachia were supergroup F and 1 strain has not been confirmed yet. It is the first report about Wolbachia supergroup F was found in sheep keds and provided the molecular evidence that B. melophagi and Wolbachia supergroup F were carried by sheep keds in Aksu Prefecture of southern Xinjiang, China. The 2 pathogens were found in sheep keds around Taklimakan Desert for the first time.

Microbial population analysis of the midgut of Melophagus ovinus via high-throughput sequencing.

BACKGROUND: Melophagus ovinus, one of the most common haematophagous ectoparasites of sheep, can cause anaemia and reductions in weight gain, wool growth and hide value. However, no information is available about the microfloral structure of the midgut of this ectoparasite. In the present study, we investigated the microbial community structure of the midgut contents of fully engorged female and male M. ovinus using Illumina HiSeq. RESULTS: The phylum showing the highest abundance was Proteobacteria (99.9%). The dominant bacterial genera in females and males were Bartonella, Arsenophonus and Wolbachia. Some less abundant bacterial genera were also detected, including Enterobacter, Acinetobacter, Halomonas, Shewanella, Bacillus and Staphylococcus. CONCLUSIONS: Bartonella, Arsenophonus and Wolbachia were the dominant bacterial genera in the midgut of female and male M. ovinus. Although detected, Enterobacter, Acinetobacter, Halomonas, Shewanella, Bacillus and Staphylococcus showed low abundances. Importantly, this is the first report of the presence of Arsenophonus, Wolbachia, Enterobacter, Halomonas, Shewanella, Bacillus and Staphylococcus in the midgut of M. ovinus.

First report of Rickettsia raoultii and R. slovaca in Melophagus ovinus, the sheep ked.

BACKGROUND: Melophagus ovinus (Diptera: Hippoboscidae), a hematophagous ectoparasite, is mainly found in Europe, Northwestern Africa, and Asia. This wingless fly infests sheep, rabbits, and red foxes, and causes inflammation, wool loss and skin damage. Furthermore, this parasite has been shown to transmit diseases, and plays a role as a vector. Herein, we investigated the presence of various Rickettsia species in M. ovinus. METHODS: In this study, a total of 95 sheep keds were collected in Kuqa County and Alaer City southern region of Xinjiang Uygur Autonomous Region, northwestern China. First, collected sheep keds were identified on the species level using morphological keys and molecular methods based on a fragment of the 18S ribosomal DNA gene (18S rDNA). Thereafter, to assess the presence of rickettsial DNA in sheep keds, the DNA of individual samples was screened by PCR based on six Rickettsia-specific gene fragments originating from six genes: the 17-kilodalton antigen gene (17-kDa), 16S rRNA gene (rrs), surface cell antigen 4 gene (sca4), citrate synthase gene (gltA), and outer membrane protein A and B genes (ompA and ompB). The amplified products were confirmed by sequencing and BLAST analysis ( https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome ). RESULTS: According to its morphology and results of molecular analysis, the species was identified as Melophagus ovinus, with 100% identity to M. ovinus from St. Kilda, Australia (FN666411). DNA of Rickettsia spp. were found in 12 M. ovinus samples (12.63%, 12/95). Rickettsia raoultii and R. slovaca were confirmed based on phylogenetic analysis, although the genetic markers of these two rickettsial agents amplified in this study showed molecular diversity. CONCLUSIONS: This is the first report of R. raoultii and R. slovaca DNA in M. ovinus. Rickettsia slovaca was found for the first time around the Taklimakan Desert located in China. This finding extends the geographical range of spotted fever group rickettsiae.

Molecular survey of arthropod-borne pathogens in sheep keds (Melophagus ovinus), Central Europe.

In the study, we screened a total of 399 adult sheep keds (Melophagus ovinus) for the presence of RNA and DNA specific for arboviral, bacterial, and protozoan vector-borne pathogens. All investigated keds were negative for flaviviruses, phleboviruses, bunyaviruses, Borrelia burgdorferi, Rickettsia spp., Anaplasma phagocytophilum, "Candidatus Neoehrlichia mikurensis," and Babesia spp. All ked pools were positive for Bartonella DNA. The sequencing of the amplified fragments of the gltA and 16S-23S rRNA demonstrated a 100 % homology with Bartonella melophagi previously isolated from a sheep ked and from human blood in the USA. The identification of B. melophagi in sheep keds in Central Europe highlights needs extending a list of hematophagous arthropods beyond ticks and mosquitoes for a search of emerging arthropod-borne pathogens.

Bartonella melophagi in Melophagus ovinus (sheep ked) collected from sheep in northern Oromia, Ethiopia.

Melophagus ovinus (sheep ked) is one of the most common ectoparasites that contributes to enormous economic losses in the productivity of sheep in many countries. The present study was conducted from January 2012 to July 2013 on M. ovinus collected from sheep at three sites in Ethiopia. Of the sheep studied, 65.7% (88/134) were infested with M. ovinus. The prevalence of M. ovinus was 76% (76/100), 47% (8/17) and 23.5% (4/17) at the Kimbibit, Chacha and Shano sites, respectively. An overall number of 229 M. ovinus specimens (138 females, 86 males and five pupae) and 554 M. ovinus specimens (272 females, 282 males) were collected from young and adult sheep, respectively. Bartonella DNA was detected in 89% (694/783) of M. ovinus using a quantitative Bartonella genus-specific PCR assay targeting the 16S/23S rRNA intergenic spacer region. The sequencing of the PCR products of fragments of the gltA and rpoB genes showed 99.6-100% and 100% homology, respectively, with B. melophagi. Statistically significant variation was not noted in the overall prevalence of Bartonella DNA between female and male M. ovinus. All of the sheep infested with M. ovinus 100% (88/88) harbored at least one M. ovinus specimen that contained Bartonella DNA. This study highlights that B. melophagi in M. ovinus from sheep in highlands in Ethiopia possibly has certain zoonotic importance.