Metabarcoding data of mitochondrial cytochrome c oxidase subunit 1 gene from bulk community of aquatic organisms collected from Nara Prefecture, Japan.

Riverine metabarcoding data were obtained from the Takamigawa River, a tributary of the Kinokawa River, in Nara Prefecture (Central Honshu, Japan). We extracted DNA from bulk community samples of aquatic organisms, most of which could not be morphologically identified at species level due to their small body size (0.12 - 2 mm length). A partial coding region of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified using PCR, and the amplicon was subjected to high-throughput parallel sequencing (Illumina MiSeq). The 313 bp paired-end sequence reads were classified into operational taxonomic units (OTUs), their species boundaries were delineated using the Generalised Mixed Yule Coalescent (GMYC) method, and taxonomic names of the GMYC species were assigned using basic local alignment search tool (BLAST) against International DNA Databases (INSD: GenBank, ENA, and DDBJ).

Dirofilaria immitis infestation in imported police (K-9) dogs in Iraq: clinicopathological and molecular investigations study / Infecção por Dirofilaria immitis em cães policiais importados no Iraque: estudo clínico-patológico e de investigações moleculares

Dirofilaria immitis, the cause of heartworm infestation (HWI) or dirofilariasis, affects members of the Canidae and remains a worldwide clinical problem. In Iraq, dirofilariasis was believed absent until 2009, when the Karbala Governorate was reported as an endemic area for canine dirofilariasis. Consequently, this study intended to investigate the occurrence of Dirofilaria immitis in police dogs in one police academy in Iraq and to study the gross and histopathological changes in 5 dead dogs, as well as to identify the species of the causative parasite using PCR technique. Thirty-nine police dogs, aged between 6 months and 12 years were included in this study. For the microfilariae investigation, 5 ml blood samples were collected from all dogs in EDTA tubes and examined by Knott's method. The systemic necropsy performed in five dead dogs showed severe clinical signs of dirofilariasis and tissue specimens were sent for routine histopathological processing. For the molecular analysis, adult worms of the detected Dirofilaria spp. were used for DNA extraction and amplification of the cox1 gene. Fifteen of 39 (38.46%) dogs were diagnosed with moderate to severe microfilariasis. The dead dogs revealed typical severe clinical signs of dirofilariasis. Moreover, typical gross and histopathological changes were also seen, accompanied by generalized thromboembolic lesions, suggesting the occurrence of the caval syndrome. The PCR investigation confirmed that D. immitis was the species present in Iraq. In conclusion, this study establishes that Iraq is a newly reported endemic area for dirofilariasis. Moreover, the infestation occurring in these cases most probably happened inside Iraq. The authors recommend doing further epidemiological studies concerning the occurrence of D. immitis in local dogs as well as in the imported dogs in all Iraqi governorates to better understand the epidemiological map of this disease and to introduce an active treatment and preventive program. Awareness and education regarding this disease should be provided to the veterinarians, dog guiders and people in direct contact with dogs, as this disease is one of the important zoonotic diseases.(AU)

A Dirofilaria immitis, causadora da infestação pelo verme do coração (IVC) ou dirofilariose afeta os membros da família Canidae e ainda é um problema clínico mundial. Até o ano de 2009, acreditava-se que o Iraque fosse livre da dirofilariose, porém nessa ocasião a governadoria de Kerbala foi relatada como uma área endêmica de dirofilariose. Assim, o presente trabalho foi realizado para investigar a ocorrência da Dirofilaria immitis em cães policiais em uma academia de polícia do Iraque, estudar as alterações macroscópicas e histopatológicas em cinco cães mortos, bem como, identificar as espécies do parasita causador com o emprego da técnida de PCR. Trinta e nove cães policiais com 6 meses a 12 anos de idade foram incluídos no estudo. Amostras de sangue de cinco mililitros foram colhidas por animal, em tubos com EDTA e foram examinadas pelo método de Knott. A necropsia sistêmica foi realizada em cinco cães que haviam apresentado sinais clínicos severos de dirofilariose e espécimens dos seus tecidos foram enviados para o processamento histopatológico de rotina. Para a análise molecular dos vermes adultos de Dirofilaria spp, foi empregada a extração do DNA e a amplificação do gene cox1. Quinze de 39(38,46%) cães foram diagnosticados com uma microfilariase variável de moderada para severa. As alterações macroscópicas e histopatológicas foram acompanhadas por lesões generalizadas tromboembólicas sugestivas da ocorrência da síndrome da veia cava. A investigação de PCR confirmou que a D.immitisera a espécie presente no Iraque. A conclusão do estudo estabeleceu que o Iraque deve passar a ser considerado como uma nova área endêmica da dirofilariose. Além da infestação registrada nos casos descritos é provável que ela também esteja presente em outras regiões do Iraque. Os autores recomendam a realização de estudos epidemiológicos para investigar a ocorrência de D.immitis tanto nos cães locais bem como em cães importados em todas as governadorias do Iraque, para ser construído o mapa epidemiológico da distribuição da doença e implantadas as ações de tratamento e de um progrma preventivo. Ações de educação em saúde sobre a doença deverão ser dirigidas para os veterinários, tratadores de cães e para pessoas em geral que tenham contato com os cães, pois esta doença é uma importante zoonose.(AU)

Morphological and molecular characteristics of six Sarcocystis spp. from red deer (Cervus elaphus) in Spain, including Sarcocystis cervicanis and three new species.

Samples of muscle tissue from the diaphragm, oesophagus and/or heart of eight adult red deer (Cervus elaphus hispanicus) from the Quintos de Mora Park in Toledo, Central Spain, were screened for sarcocysts by means of the compression method. From positive samples, individual sarcocysts were excised and examined in wet mounts under a light microscope (LM) in order to study their basic morphology before being preserved for molecular studies. In all red deer examined, only microscopic sarcocysts were found. Those in the diaphragm and oesophagus were spindle-shaped and about 1 × 0.1 mm in size, while those in cardiac muscle were sac-like and 500-800 × 80-180 µm. By LM, the sarcocysts either had densely packed, about 8-µm-long, hair-like protrusions (type 1), sparsely distributed indistinct projections (fuzzy outline; type 2) or no visible protrusions (smooth surface; type 3). In cardiac muscle, only sarcocysts without visible protrusions were found. One of the latter sarcocysts was examined by scanning electron microscopy (SEM) and found to possess thin ribbon-like protrusions. Forty-eight sarcocysts isolated from the diaphragm, oesophagus and heart of one red deer, as well as 55 sarcocysts from the heart of three other red deer, 103 sarcocysts in total, were characterized molecularly through PCR amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of the mitochondrial genome, revealing the presence of six major cox1 sequence types. Each type comprised either a single sequence (three types) or a collection of several identical or nearly identical sequences. From selected isolates possessing each of these cox1 sequence types, the complete 18S ribosomal RNA (rRNA) gene was amplified and sequenced directly and/or after cloning of the 5' end half. Supported by the sequence data from the latter gene, as well as the morphology of the sarcocysts from which the sequences originated, the six cox1 sequence types were considered to represent six separate Sarcocystis spp. Two cox1 sequence types were identified as belonging to the previously characterized species Sarcocystis hjorti (one sequence/sarcocyst) and Sarcocystis linearis (38 sequences/sarcocysts), respectively, whereas four sequence types were new. One of the latter types was assigned to the previously named species Sarcocystis cervicanis from red deer, since this sequence type was obtained from 52 sarcocysts from cardiac muscle, which matched the original morphological description (smooth surface) and habitat of this species. The remaining three sequence types were assigned to the three new species Sarcocystis iberica (one sequence/sarcocyst) Sarcocystis venatoria (10 sequences/sarcocysts) and Sarcocystis morae (one sequence/sarcocyst), respectively. The two species S. iberica and S. venatoria shared the same sarcocyst morphology (type 1) and habitat (diaphragm) and had virtually identical 18S rRNA gene sequences, but differed by 4% at cox1, which was considered sufficient to regard them as separate species. The single sarcocyst of S. morae (from the oesophagus) examined by LM had a smooth wall and this species was therefore believed to have the same type of ribbon-like protrusions (ultrastructurally) as sarcocysts of S. cervicanis and S. linearis, which were also the species most closely related to S. morae at cox1. Thus, there seems to be three species with similar ribbon-like cyst wall protrusions in red deer (S. cervicanis, S. linearis, S. morae), as well as three species with similar hair-like protrusions (S. hjorti, S. iberica, S. venatoria). Sarcocysts of S. cervicanis were only identified in cardiac muscle, whereas sarcocysts of S. linearis were found mainly in the diaphragm and oesophagus and rarely in the heart. The relative number of cox1 haplotypes was greater among sequences/isolates of S. linearis (17/38) than among isolates of S. cervicanis (7/52). Four of the species examined (S. cervicanis, S. linearis, S. iberica, S. venatoria) possessed considerable intra-isolate (intra-genomic) sequence variation (insertions/deletions, substitutions) in the 18S rRNA gene.

Morphological and molecular characterization of four Sarcocystis spp., including Sarcocystis linearis n. sp., from roe deer (Capreolus capreolus) in Italy.

Fresh (frozen/thawed) muscle samples from four 2-12-year-old roe deer (Capreolus capreolus) from the Sondrio province in north-eastern Italy were examined under a dissecting microscope, and about 180 sarcocysts were isolated and identified to morphological type in wet mounts by light microscopy (LM). Seventy-seven of these sarcocysts were subsequently examined by molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of all isolates, as well as PCR amplification, cloning and sequencing of the complete18S ribosomal RNA (rRNA) gene of two isolates of each species found. By LM, three major sarcocyst types were recognised: spindle-shaped sarcocysts, 0.5-3 mm long, either with no clearly recognisable protrusions (thin-walled) or with finger-like protrusions (thick-walled); and slender, thread-like sarcocysts, 2-3 mm long, with hair-like protrusions. Sequencing of cox1 revealed that the sarcocysts belonged to four different species. Those with no visible protrusions either belonged to Sarcocystis gracilis (n = 24) or to a Sarcocystis taeniata-like species (n = 19), whereas those with finger- and hair-like protrusions belonged to Sarcocystis silva (n = 27) and Sarcocystis capreolicanis (n = 7), respectively. The 19 cox1 sequences of the S. taeniata-like species, comprising five haplotypes, differed from each other at 0-16 of 1038 nucleotide positions (98.5-100% identity). They differed from 25 previous cox1 sequences of S. taeniata from moose and sika deer (with 98.0-100% intraspecific identity), at 33-43 nucleotide positions (95.9-96.8% interspecific identity), and there were 20 fixed nucleotide differences between the two populations. In the phylogenetic analysis based on cox1 sequences, the two populations formed two separate monophyletic clusters. The S. taeniata-like species in roe deer was therefore considered to represent a separate species, which was named Sarcocystis linearis n. sp. At the 18S rRNA gene, however, the two species could not be clearly separated from each other. Thus, there was considerable intraspecific sequence variation in the 18S rRNA gene of S. linearis (98.1-99.9% identity between 24 sequences), which was similar both in magnitude and nature to the variation previously found in this gene of S. taeniata. The new 18S rRNA gene sequences of S. linearis shared an identity of 97.9-99.6% with those of S. taeniata (overlap between intra- and interspecific identity), and in the phylogenetic tree, sequences of the two species were interspersed. By scanning electron microscopy (SEM), the sarcocysts of S. linearis were found to possess regularly spaced, thin and narrow ribbon-like cyst wall protrusions (about 2.8-3.2 µm long, 0.3-0.4 µm wide and about 0.02-0.03 µm thick), terminating in a plate-like structure of the same thickness but with an elliptic outline (about 0.3-0.4 µm wide and 0.7-0.9 µm long). The terminal plates were connected in the middle with the band-like portion of the protrusions like the board of a seesaw (tilting board). The terminal plates of adjacent protrusions were neatly arranged in a hexagonal pattern resembling tiles on a roof. Together, they formed an outer roof-like layer facing the surrounding cytoplasm of the host cell and completely covering the band-like proximal portion of the protrusions, which overlapped and were stacked in three to four layers close to the cyst surface. The sarcocyst morphology of S. linearis was consistent with that of an unnamed Sarcocystis sp. in roe deer previously found by transmission electron microscopy in several countries, including Italy. A few sarcocysts of S. gracilis and S. silva were also examined by SEM, confirming the presence of regularly distributed, short knob-like protrusions in S. gracilis (as seen in previous SEM studies) and revealing tightly packed, erect 6-7-µm-long villus-like protrusions having regularly distributed round depressions on their surface in S. silva. The sequencing of cox1 of 7, 24 and 27 new isolates of S. capreolicanis, S. gracilis and S. silva, respectively, recovered 7, 11 and 10 new haplotypes from each of the three species and expanded our knowledge on the intraspecific sequence variation at this marker. Similarly, the study revealed a more extensive intragenomic sequence variation at the 18S rRNA gene of S. capreolicanis and S. silva than known from previous studies and confirmed a near absence of such variation in the 18S rRNA gene of S. gracilis.