Whole transcriptome analysis of the poultry red mite Dermanyssus gallinae (De Geer, 1778)

Although the poultry red mite Dermanyssus gallinae (De Geer, 1778) is the major parasitic pest in poultry farming causing substantial economic losses every year, nucleotide data are rare in the public databases. Therefore, de novo sequencing covering the transcriptome of D. gallinae was carried out resulting in a dataset of 232097 singletons and 42130 contiguous sequences (contigs) which were subsequently clustered into 24140 isogroups consisting of 35788 isotigs. After removal of sequences possibly originating from bacteria or the chicken host, 267464 sequences (231657 singletons, 56 contigs and 35751 isotigs) remained, of which 10·3% showed homology to proteins derived from other organisms. The most significant Blast top-hit species was the mite Metaseiulus occidentalis followed by the tick Ixodes scapularis. To gain functional knowledge of D. gallinae transcripts, sequences were mapped to Gene Ontology terms, Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways and parsed to InterProScan. The transcriptome dataset provides new insights in general mite genetics and lays a foundation for future studies on stage-specific transcriptomics as well as genomic, proteomic, and metabolomic explorations and might provide new perspectives to control this parasitic mite by identifying possible drug targets or vaccine candidates. It is also worth noting that in different tested species of the class Arachnida no 28S rRNA was detectable in the rRNA profile, indicating that 28S rRNA might consists of two separate, hydrogen-bonded fragments, whose (heat-induced) disruption may led to co-migration with 18S rRN

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Whole transcriptome analysis of the poultry red mite Dermanyssus gallinae (De Geer, 1778)

SUMMARY Although the poultry red mite Dermanyssus gallinae (De Geer, 1778) is the major parasitic pest in poultry farming causing substantial economic losses every year, nucleotide data are rare in the public databases. Therefore, de novo sequencing covering the transcriptome of D. gallinae was carried out resulting in a dataset of 232 097 singletons and 42 130 contiguous sequences (contigs) which were subsequently clustered into 24 140 isogroups consisting of 35 788 isotigs. After removal of sequences possibly originating from bacteria or the chicken host, 267 464 sequences (231 657 singletons, 56 contigs and 35 751 isotigs) remained, of which 10·3% showed homology to proteins derived from other organisms. The most significant Blast top-hit species was the mite Metaseiulus occidentalis followed by the tick Ixodes scapularis. To gain functional knowledge of D. gallinae transcripts, sequences were mapped to Gene Ontology terms, Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways and parsed to InterProScan. The transcriptome dataset provides new insights in general mite genetics and lays a foundation for future studies on stage-specific transcriptomics as well as genomic, proteomic, and metabolomic explorations and might provide new perspectives to control this parasitic mite by identifying possible drug targets or vaccine candidates. It is also worth noting that in different tested species of the class Arachnida no 28S rRNA was detectable in the rRNA profile, indicating that 28S rRNA might consists of two separate, hydrogen-bonded fragments, whose (heat-induced) disruption may led to co-migration with 18S rRNA

The predicted secretome and transmembranome of the poultry red mite Dermanyssus gallinae

BACKGROUND The worldwide distributed hematophagous poultry red mite Dermanyssus gallinae (De Geer, 1778) is one of the most important pests of poultry. Even though 35 acaricide compounds are available, control of D. gallinae remains difficult due to acaricide resistances as well as food safety regulations. The current study was carried out to identify putative excretory/secretory (pES) proteins of D. gallinae since these proteins play an important role in the host-parasite interaction and therefore represent potential targets for the development of novel intervention strategies. Additionally, putative transmembrane proteins (pTM) of D. gallinae were analyzed as representatives of this protein group also serve as promising targets for new control strategies. METHODS D. gallinae pES and pTM protein prediction was based on putative protein sequences of whole transcriptome data which was parsed to different bioinformatical servers (SignalP, SecretomeP, TMHMM and TargetP). Subsequently, pES and pTM protein sequences were functionally annotated by different computational tools. RESULTS Computational analysis of the D. gallinae proteins identified 3,091 pES (5.6%) and 7,361 pTM proteins (13.4%). A significant proportion of pES proteins are considered to be involved in blood feeding and digestion such as salivary proteins, proteases, lipases and carbohydrases. The cysteine proteases cathepsin D and L as well as legumain, enzymes that cleave hemoglobin during blood digestion of the near related ticks, represented 6 of the top-30 BLASTP matches of the poultry red mite's secretome. Identified pTM proteins may be involved in many important biological processes including cell signaling, transport of membrane-impermeable molecules and cell recognition. Ninjurin-like proteins, whose functions in mites are still unknown, represent the most frequently occurring pTM. CONCLUSION The current study is the first providing a mite's secretome as well as transmembranome and provides valuable insights into D. gallinae pES and pTM proteins operating in different metabolic pathways. Identifying a variety of molecules putatively involved in blood feeding may significantly contribute to the development of new therapeutic targets or vaccines against this poultry pest

Seroprevalence and current infections of canine vector-borne diseases in Nicaragua

Abstract Background Vector-borne diseases constitute a major problem for veterinary and public health, especially in tropical regions like Central America. Domestic dogs may be infected with several vector-borne pathogens of zoonotic relevance, which may also severely compromise canine health. Methods To assess the prevalence of canine vector-borne diseases in Nicaragua, 329 dogs from seven cities, which were presented to the veterinarian for various reasons, were included in this study. Dogs were examined clinically and diagnostic blood samples were taken for analysis of packed cell volume (PCV) and presence of microfilariae as well as antigen of Dirofilaria immitis and antibodies to Ehrlichia spp., Anaplasma spp. and Borrelia burgdorferi (sensu lato) by use of a commercially available rapid ELISA. To detect current infections, specific PCRs for the detection of E. canis, A. platys and A. phagocytophilum were carried out on blood samples of the respective seropositive dogs. Microfilaremic blood samples, as well as D. immitis antigen positive samples were further subjected to PCR and subsequent sequencing for filarial species identification. Results Antibodies against Ehrlichia spp. were present in 62.9% of dogs, while Anaplasma spp. seroprevalence was 28.6%. Antibodies against species of both genera were detected in 24.9% of dogs. Borrelia burgdorferi (s.l.) antibodies were not detected. Dirofilaria immitis antigen was present in six animals (1.8%), two of which also showed D. immitis microfilariae in buffy coat. In addition to D. immitis, Acanthocheilonema reconditum was identified by PCR and sequencing in two of four additional microfilaremic blood samples, which were tested negative for D. immitis antigen. Current E. canis infections as defined by DNA detection were present in 58.5% of Ehrlichia-seropositive dogs, while 5.3% of Anaplasma-seropositive dogs were PCR-positive for A. platys, 2.2% for A. phagocytophilum and 16.0% for both Anaplasma species. Current E. canis infection had a statistically significant negative impact on PCV, whereas no relationship between infection status and clinical signs of disease could be observed. Conclusions These results indicate that canine vector-borne diseases are widespread in Nicaragua and that dogs may constitute a reservoir for human infection with E. canis, A. phagocytophilum and D. immitis. Thus, the use of repellents or acaricides to protect dogs from vector-borne diseases is strongly recommended

Detection of Rickettsia monacensis and Rickettsia amblyommatis in ticks collected from dogs in Costa Rica and Nicaragua

The neotropical climate of Central America provides ideal conditions for ticks, which may transmit several human pathogens, including spotted-fever group Rickettsia. Dogs may act as sentinels or reservoirs for human tick-borne diseases due to shared tick species. Here, ticks were collected from 680 client-owned dogs in Nicaragua and Costa Rica, and a total of 316 tick pools were investigated for Rickettsia infection by quantitative real-time PCR (qPCR) targeting the gltA gene. Subsequently, up to six further genomic targets (16S rDNA, gltA, sca4, ompA, ompB and the 23S-5S intergenic spacer) were investigated for Rickettsia species determination. The predominant tick species was Rhipicephalus sanguineus sensu lato (s.l.) (19.9% of dogs infested in Costa Rica, 48.0% in Nicaragua), followed by Ixodes boliviensis (3.1% in Costa Rica / none in Nicaragua) and Amblyomma ovale (4.8% in Costa Rica, 0.9% in Nicaragua). In total, 22 of 316 tick pools containing 60 of 1023 individual ticks were Rickettsia-positive as determined by qPCR, resulting in a minimum infection rate (MIR) of 2.2%. In detail, MIR in Rh. sanguineus s.l. was 0.7% (7/281 pools), in I. boliviensis 33.3% (12/13 pools) and in A. ovale 9.7% (3/22 pools). For 11 of 12 positive I. boliviensis pools and one of six positive Rh. sanguineus s.l. pools, the species could be determined as R. monacensis. R. amblyommatis was identified in one Rh. sanguineus s.l. pool from Costa Rica and one A. ovale pool from Nicaragua. Nine of 12 R. monacensis-positive tick pools were collected in San Rafael de Heredia, Costa Rica, indicating a high local occurrence in this area. This study supports recent evidence that R. monacensis is present on the American continent. Its high local occurrence among dog-asso ciated I. boliviensis, which may also parasitize humans, in Costa Rica gives cause for concern, as R. monacensis is also pathogenic to humansEl clima neotropical de Centroamérica ofrece condiciones ideales para las garrapatas, que pueden transmitir varios patógenos humanos, entre ellos la Rickettsia del grupo de la fiebre manchada. Los perros pueden actuar como centinelas o reservorios de enfermedades humanas transmitidas por garrapatas debido a las especies de garrapatas que comparten. En este caso, se recogieron garrapatas de 680 perros propiedad de clientes en Nicaragua y Costa Rica, y se investigaron 316 grupos de garrapatas para detectar la infección por Rickettsia mediante PCR cuantitativa en tiempo real (qPCR) dirigida al gen gltA. Posteriormente, se investigaron hasta seis objetivos genómicos más (16S rDNA, gltA, sca4, ompA, ompB y el espaciador intergénico 23S-5S) para determinar la especie de Rickettsia. La especie de garrapata predominante fue Rhipicephalus sanguineus sensu lato (s.l.) (19,9% de los perros infestados en Costa Rica, 48,0% en Nicaragua), seguida de Ixodes boliviensis (3,1% en Costa Rica / ninguna en Nicaragua) y Amblyomma ovale (4,8% en Costa Rica, 0,9% en Nicaragua). En total, 22 de 316 grupos de garrapatas que contenían 60 de 1023 garrapatas individuales fueron positivas a Rickettsia según la qPCR, lo que dio lugar a una tasa de infección mínima (MIR) del 2,2%. En detalle, la TMI en Rh. sanguineus s.l. fue del 0,7% (7/281 pools), en I. boliviensis del 33,3% (12/13 pools) y en A. ovale del 9,7% (3/22 pools). En 11 de los 12 pozos positivos de I. boliviensis y en uno de los seis pozos positivos de Rh. sanguineus s.l., la especie pudo determinarse como R. monacensis. R. amblyommatis se identificó en un grupo de Rh. sanguineus s.l. de Costa Rica y en un grupo de A. ovale de Nicaragua. Nueve de los 12 charcos de garrapatas positivos para R. monacensis se recogieron en San Rafael de Heredia, Costa Rica, lo que indica una elevada presencia local en esta zona. Este estudio respalda las pruebas recientes de que R. monacensis está presente en el continente americano. Su elevada presencia local entre las garrapatas I. boliviensis asociadas a los perros, que también pueden parasitar a los humanos, en Costa Rica es motivo de preocupación, ya que R. monacensis también es patógena para los humanos.Universidad Nacional, Costa Rica.Escuela de Medicina Veterinari

Seroprevalence and current infections of canine vector-borne diseases in Costa Rica

Domestic dogs may carry several vector-borne pathogens, including zoonotic agents, especially in tropical regions like Central America. The epidemiology of these pathogens is prone to change due to urbanization, trade and travel as well as climate change, necessitating repeated monitoring. This study aims to present a comprehensive picture of canine vector-borne diseases in Costa Rica, combining data on seroprevalence with molecular species identification of the causative pathogens. In this survey, 294 dogs from all seven provinces of Costa Rica were included. After a clinical examination, diagnostic blood samples were analyzed with regard to packed cell volume (PCV) and presence of microfilaria. Serum samples were tested for antibodies against Ehrlichia spp., Anaplasma spp., Babesia spp., Borrelia burgdorferi sensu lato (s.l.) as well as antigen of Dirofilaria immitis. Seropositive and microfilaremic blood samples were analyzed by PCR to detect current infections and identify the pathogen species. Overall, 45.24% (133/294, 95% CI: 39.45–51.11%) of dogs were seropositive for at least one of the tested pathogens. Seroprevalence was highest for Ehrlichia spp. (39.46%, 116/294, 95% CI: 33.83–45.29%), followed by Babesia spp. (23.13%, 68/294, 95% CI: 18.43–28.38%), Anaplasma spp. (13.27%, 39/294, 95% CI: 9.61–17.69%), and B. burgdorferi s.l. (0.34%, 1/294, 95% CI: 0.01–1.88%). Generalized linear mixed models indicated a significant association of Ehrlichia-, Anaplasma- and Babesiaseropositivity, as well as a significant effect of age and breed on Ehrlichia-seropositivity. Furthermore, a statistically significant negative effect of Ehrlichia-, Anaplasma-, and Babesia-seropositivity on PCV was found. Regarding current infections, Ehrlichia canis DNA was detected in 51.72% (60/116, 95% CI: 42.26–61.10%) of Ehrlichia-seropositive dogs, while Ehrlichia ewingii and Ehrlichia chaffeensis were not detected. Furthermore, 10.26% (4/39, 95% CI: 2.87–24.22%) of Anaplasma-seropositive dogs were coinfected with Anaplasma phagocytophilum and Anaplasma platys, while one animal (2.56%, 95% CI: 0.65–13.48%) was infected with A. phagocytophilum only. Among Babesiaseropositive dogs, Babesia vogeli and Hepatozoon canis were detected in one animal each (1.47%, 1/68, 95% CI: 0.04–7.92%). Dirofilaria immitis antigen was detected in 4.42% (13/294, 95% CI: 2.38–7.44%) of dogs. In microfilaremic animals, D. immitis as well as Acanthocheilonema reconditum infections were identified. This survey demonstrates that canine vector-borne pathogens, including zoonotic agents like A. phagocytophilum and D. immitis, are widespread in Costa Rica. Thus, protection of dogs from disease-transmitting vectors is recommended from an animal welfare as well as public health perspective.Los perros domésticos pueden ser portadores de varios patógenos transmitidos por vectores, incluidos agentes zoonóticos, especialmente en regiones tropicales como América Central. La epidemiología de estos patógenos es propensa a cambiar debido a la urbanización, el comercio y los viajes, así como al cambio climático, lo que hace necesario un seguimiento repetido. Este estudio pretende presentar un panorama completo de las enfermedades caninas transmitidas por vectores en Costa Rica, combinando datos de seroprevalencia con la identificación molecular de especies de los patógenos causantes. En este estudio se incluyeron 294 perros de las siete provincias de Costa Rica. Tras un examen clínico, se analizaron muestras de sangre para el diagnóstico en relación con el volumen celular empaquetado (PCV) y la presencia de microfilarias. Las muestras de suero se analizaron para detectar anticuerpos contra Ehrlichia spp., Anaplasma spp., Babesia spp. y Borrelia burgdorferi sensu lato (s.l.), así como el antígeno de Dirofilaria immitis. Las muestras de sangre seropositivas y microfilarémicas se analizaron mediante PCR para detectar las infecciones actuales e identificar la especie patógena. En general, el 45,24% (133/294, IC 95%: 39,45-51,11%) de los perros eran seropositivos para al menos uno de los patógenos analizados. La seroprevalencia fue mayor para Ehrlichia spp. (39,46%, 116/294, IC 95%: 33,83-45,29%), seguido de Babesia spp. (23,13%, 68/294, IC 95%: 18,43-28,38%), Anaplasma spp. (13,27%, 39/294, IC 95%: 9,61-17,69%), y B. burgdorferi s.l. (0,34%, 1/294, IC 95%: 0,01-1,88%). Los modelos lineales mixtos generalizados indicaron una asociación significativa de Ehrlichia-, Anaplasma- y Babesiaseropositividad, así como un efecto significativo de la edad y la raza sobre la Ehrlichia-seropositividad. Además, se encontró un efecto negativo estadísticamente significativo de la seropositividad a Ehrlichia, Anaplasma y Babesia sobre el PCV. En cuanto a las infecciones actuales, se detectó ADN de Ehrlichia canis en el 51,72% (60/116, IC del 95%: 42,26-61,10%) de los perros seropositivos a Ehrlichia, mientras que no se detectaron Ehrlichia ewingii y Ehrlichia chaffeensis. Además, el 10,26% (4/39, IC 95%: 2,87-24,22%) de los perros seropositivos a Anaplasma estaban coinfectados con Anaplasma phagocytophilum y Anaplasma platys, mientras que un animal (2,56%, IC 95%: 0,65-13,48%) estaba infectado sólo con A. phagocytophilum. Entre los perros positivos a Babesias, se detectaron Babesia vogeli y Hepatozoon canis en un animal cada uno (1,47%, 1/68, IC 95%: 0,04-7,92%). El antígeno de Dirofilaria immitis se detectó en el 4,42% (13/294, IC 95%: 2,38-7,44%) de los perros. En los animales microfilarémicos se identificaron infecciones por D. immitis así como por Acanthocheilonema reconditum. Este estudio demuestra que los patógenos caninos transmitidos por vectores, incluidos los agentes zoonóticos como A. phagocytophilum y D. immitis, están muy extendidos en Costa Rica. Así pues, se recomienda proteger a los perros de los vectores transmisores de enfermedades, tanto desde el punto de vista del bienestar animal como de la salud pública.University of Veterinary Medicine Hannover, AlemaniaUniversidad Nacional, Costa RicaIDEXX Laboratories, AlemaniaEscuela de Medicina Veterinari

Mammals Preferred: Reassortment of Batai and Bunyamwera orthobunyavirus Occurs in Mammalian but Not Insect Cells

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the Bunyamwera orthobunyavirus and the Batai orthobunyavirus, can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of Bunyamwera orthobunyavirus and the Batai orthobunyavirus during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (Mesocricetus auratus). In contrast, no reassortment was observed in the examined insect cells from Aedes aegypti (Aag2) and Aedes albopictus (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines