The Aedes albopictus (Diptera: Culicidae) microbiome varies spatially and with Ascogregarine infection.

The mosquito microbiome alters the physiological traits of medically important mosquitoes, which can scale to impact how mosquito populations sustain disease transmission. The mosquito microbiome varies significantly within individual mosquitoes and among populations, however the ecological and environmental factors that contribute to this variation are poorly understood. To further understand the factors that influence variation and diversity of the mosquito microbiome, we conducted a survey of the bacterial microbiome in the medically important mosquito, Aedes albopictus, on the high Pacific island of Maui, Hawai'i. We detected three bacterial Phyla and twelve bacterial families: Proteobacteria, Acitinobacteria, and Firmicutes; and Anaplasmataceae, Acetobacteraceae, Enterobacteriaceae, Burkholderiaceae, Xanthobacteraceae, Pseudomonadaceae, Streptomycetaceae, Staphylococcaceae, Xanthomonadaceae, Beijerinckiaceae, Rhizobiaceae, and Sphingomonadaceae. The Ae. albopictus bacterial microbiota varied among geographic locations, but temperature and rainfall were uncorrelated with this spatial variation. Infection status with an ampicomplexan pathosymbiont Ascogregarina taiwanensis was significantly associated with the composition of the Ae. albopictus bacteriome. The bacteriomes of mosquitoes with an A. taiwanensis infection were more likely to include several bacterial symbionts, including the most abundant lineage of Wolbachia sp. Other symbionts like Asaia sp. and several Enterobacteriaceae lineages were less prevalent in A. taiwanensis-infected mosquitoes. This highlights the possibility that inter- and intra-domain interactions may structure the Ae. albopictus microbiome.

The effect of enterococci on feline Tritrichomonas foetus infection in vitro.

Tritrichomonas foetus is a common cause of large bowel diarrhea in cats. Probiotics have been suggested to be effective for many intestinal pathogens; however, there are a lack of studies evaluating the effect of probiotics in T. foetus infection. In vitro studies were performed to evaluate the effect of a probiotic containing Enterococcus faecium (Efm) SF68 and a novel probiotic, Enterococcus hirae, on the inhibition of T. foetus growth, adhesion to, and cytotoxicity towards the intestinal epithelium. The effect of enterococci on T. foetus proliferation during co-culture was evaluated throughout log phase T. foetus growth. The previously validated in vitro co-culture model system using porcine intestinal epithelial cells (IPEC-J2) was used to evaluate the effect of enterococci on T. foetus adhesion and cytotoxicity towards intestinal epithelial cells. Cytotoxicity was assessed using fluorescent microscopy and spectrophotometry. Interactions of T. foetus, enterococci, and intestinal epithelial cells were assessed using scanning electron microscopy and immunofluorescence assays (IFA). Enterococcus-induced inhibition of T. foetus growth was demonstrated at concentrations as low as 104 enterococci colony forming units (CFU)/mL and was dependent, in part, on environmental pH and the presence of viable enterococci organisms. T. foetus adhesion, including with a ronidazole-resistant strain, was reduced with pretreatment of intestinal epithelial cells with enterococci but was not significantly affected when enterococci were introduced simultaneously or following T. foetus infection. Compared to Efm, E. hirae more effectively decreased T. foetus adhesion, suggesting its superior potential as a novel probiotic for T. foetus infection. There was no effect of enterococci treatment on T. foetus-induced intestinal epithelial cell cytotoxicity. Our results support further study into the investigation of a possible benefit of enterococci-containing probiotic treatment for prevention of T. foetus infection in at-risk uninfected cats.

Molecular characterization of Histomonas meleagridis exoproteome with emphasis on protease secretion and parasite-bacteria interaction.

The exoproteome of parasitic protists constitutes extracellular proteins that play a fundamental role in host-parasite interactions. Lytic factors, especially secreted proteases, are capable of modulating tissue invasion, thereby aggravating host susceptibility. Despite the important role of exoproteins during infection, the exoproteomic data on Histomonas meleagridis are non-existent. The present study employed traditional 1D-in-gel-zymography (1D-IGZ) and micro-LC-ESI-MS/MS (shotgun proteomics), to investigate H. meleagridis exoproteomes, obtained from a clonal virulent and an attenuated strain. Both strains were maintained as mono-eukaryotic monoxenic cultures with Escherichia coli. We demonstrated active in vitro secretion kinetics of proteases by both parasite strains, with a widespread proteolytic activity ranging from 17 kDa to 120 kDa. Based on protease inhibitor susceptibility assay, the majority of proteases present in both exoproteomes belonged to the family of cysteine proteases and showed stronger activity in the exoproteome of a virulent H. meleagridis. Shotgun proteomics, aided by customized database search, identified 176 proteins including actin, potential moonlighting glycolytic enzymes, lytic molecules such as pore-forming proteins (PFPs) and proteases like cathepsin-L like cysteine protease. To quantify the exoproteomic differences between the virulent and the attenuated H. meleagridis cultures, a sequential window acquisition of all theoretical spectra mass spectrometric (SWATH-MS) approach was applied. Surprisingly, results showed most of the exoproteomic differences to be of bacterial origin, especially targeting metabolism and locomotion. By deciphering such molecular signatures, novel insights into a complex in vitro protozoan- bacteria relationship were elucidated.

Molecular detection of vector borne pathogens in anemic and thrombocytopenic dogs in southern Brazil / Detecção molecular de patógenos transmitidos por vetores em cães anemicos e trombocitopenicos no Sul do Brasil

Abstract Arthropod-borne pathogens are medically important because of their ability to cause diseases in their hosts. The purpose of this study was to detect the occurrence of Ehrlichia spp., piroplasmids and Hepatozoon spp. in dogs with anemia and thrombocytopenia in southern Brazil. EDTA-whole blood was collected from 75 domestic dogs presenting anemia or/and thrombocytopenia from Guarapuava, state of Paraná, Brazil. DNA samples were subjected to conventional PCR assays for Ehrlichia spp. (dsb), piroplasmids (18S rRNA) and Hepatozoon spp. (18S rRNA), followed by sequencing and phylogenetic analyses. Among the 75 dogs, one (1.33%) was positive for Hepatozoon sp. and six (8%) were positive for piroplasmids in 18S rRNA cPCR assays. None of the dogs showed positive results in Ehrlichia spp.-cPCR targeting dsb gene. The phylogenetic analyses revealed that three piroplasm sequences were clustered with Rangellia vitalii, while one sequence was grouped with B. vogeli. The only sequence obtained from Hepatozoon spp.-PCR protocol was pooled with H. canis. Therefore, there is urgent need for differential molecular diagnosis of the two piroplasm species cited as etiological agents in clinical cases of canine hemoparasitic diseases, given the higher pathogenic potential of R. vitalii than of B. vogeli.

Resumo Agentes transmitidos por artrópodes têm grande importância na medicina veterinária devido à sua capacidade de causar doenças graves em seus hospedeiros. O presente estudo objetivou investigar a ocorrência de três patógenos transmitidos por vetores, Ehrlichia canis, Rangelia vitalii e Hepatozoon canis, em cães na região sul do Brasil. Foram coletadas amostras de sangue total de 75 cães domésticos que apresentavam anemia e/ou trombocitopenia, em Guarapuava, Paraná, Brasil. As amostras de DNA foram submetidas à técnica de PCR convencional para E. canis (dsb), piroplasmídeos (18S rRNA) e Hepatozoon spp. (18S rRNA), seguida de sequenciamento e análises filogenéticas. Das 75 amostras, uma (1,33%) foi positiva para Hepatozoon spp. e seis (8%) foram positivas para Babesia spp. Nenhuma amostra mostrou resultados positivos para Ehrlichia spp. utilizando a detecção pelo gene dsb. As análises filogenéticas revelaram que três sequências obtidas foram agrupadas no mesmo clado que R. vitalii , enquanto uma foi agrupada juntamente com B. vogeli. A única sequência obtida pelo protocolo de PCR para Hepatozoon spp. foi agrupada juntamente com H. canis. Assim, é justificada necessidade de diferenciação das espécies de piroplasmas, através do diagnóstico molecular, como agentes etiológicos nos casos clínicos de hemoparasitose canina, considerando o potencial patogênico de R. vitalii quando comparado à B. vogeli.

Molecular detection of vector borne pathogens in anemic and thrombocytopenic dogs in southern Brazil.

Arthropod-borne pathogens are medically important because of their ability to cause diseases in their hosts. The purpose of this study was to detect the occurrence of Ehrlichia spp., piroplasmids and Hepatozoon spp. in dogs with anemia and thrombocytopenia in southern Brazil. EDTA-whole blood was collected from 75 domestic dogs presenting anemia or/and thrombocytopenia from Guarapuava, state of Paraná, Brazil. DNA samples were subjected to conventional PCR assays for Ehrlichia spp. (dsb), piroplasmids (18S rRNA) and Hepatozoon spp. (18S rRNA), followed by sequencing and phylogenetic analyses. Among the 75 dogs, one (1.33%) was positive for Hepatozoon sp. and six (8%) were positive for piroplasmids in 18S rRNA cPCR assays. None of the dogs showed positive results in Ehrlichia spp.-cPCR targeting dsb gene. The phylogenetic analyses revealed that three piroplasm sequences were clustered with Rangellia vitalii, while one sequence was grouped with B. vogeli. The only sequence obtained from Hepatozoon spp.-PCR protocol was pooled with H. canis. Therefore, there is urgent need for differential molecular diagnosis of the two piroplasm species cited as etiological agents in clinical cases of canine hemoparasitic diseases, given the higher pathogenic potential of R. vitalii than of B. vogeli.

Occurrence of blood parasites in seabirds admitted for rehabilitation in the Western Cape, South Africa, 2001-2013.

Blood parasites are generally uncommon in seabirds, and knowledge on their epidemiology is further limited by the fact that they often inhabit remote locations that are logistically difficult or expensive to study. We present a long term data set of blood smear examinations of 1909 seabirds belonging to 27 species that were admitted to a rehabilitation centre in Cape Town (Western Cape, South Africa) between 2001 and 2013. Blood parasites were detected in 59% of species (16/27) and 29% of individuals examined (551/1909). The following blood parasites were recorded: Babesia ugwidiensis, Babesia peircei, Babesia sp., Plasmodium sp., Leucocytozoon ugwidi, Hepatozoon albatrossi, Haemoproteus skuae and Spirochaetales. Several of the records are novel host-parasite associations, demonstrating the potential of rehabilitation centres for parasite and disease surveillance, particularly for species infrequently sampled from which no host-specific parasites have been described.

Pathologic and molecular characterization of histomoniasis in peafowl ( Pavo cristatus).

Histomonas meleagridis is a flagellate protozoan organism that can cause severe necrotizing typhlitis and hepatitis in gallinaceous birds. Peafowl ( Pavo spp.) have been shown to be susceptible to histomoniasis in experimental settings, but there are few reports of natural histomoniasis in this species. A retrospective study of the archived cases at 2 veterinary diagnostic laboratories in the United States yielded 5 cases of peafowl with gross and histologic findings characteristic of histomoniasis. Lesions included bilateral, transmural fibrinonecrotic typhlitis and multifocal necrotizing hepatitis with associated trophozoites morphologically consistent with H. meleagridis. There was no evidence of Heterakis gallinarum infestation in the studied cases. DNA was extracted from formalin-fixed, paraffin-embedded liver and ceca from all 5 cases and was analyzed using multiple sets of primers with subsequent sequencing and genotyping. Four samples were positive for H. meleagridis, and 1 sample was positive for both H. meleagridis and Tetratrichomonas gallinarum. These results confirm that peafowl develop clinical disease similar to that described previously in other gallinaceous birds infected by H. meleagridis. The role of T. gallinarum remains unknown and further research is necessary to elucidate its role, if any, in the pathogenesis of the observed lesions.

Efficacy of a control program for bovine trichomonosis based on testing and culling infected bulls in beef cattle managed under mountain pastoral systems of Northern Spain.

Bovine trichomonosis (BT) is a sexually transmitted disease that is considered a cause of early reproductive failure in cattle under extensive management conditions. Recently, Tritrichomonas foetus was detected in 41.5% of herds from one representative beef cattle breed (Asturiana de la Montaña; AM) reared in traditional mountain systems in Spain. The objective of the present study was to evaluate the effect of BT on reproductive performance and the economic consequences in AM herds. The benefits of a control program based on testing and culling infected bulls were also studied by comparing T. foetus prevalence and reproductive data before and after the implementation of the control measures. In infected herds, T. foetus infection increased calving intervals by 79 days (P<0.0001) and resulted in a higher percentage of cows-not-in calf (36% vs. 19%; P<0.001). An economic analysis showed that BT could reduce income by 68.7% in AM herds. The implementation of the control program decreased calving intervals (P<0.0001) and increased calving percentage (P<0.05). T. foetus prevalence showed a significant decline compared with the prevalence before implementing the control program (P<0.05). Nevertheless, after 2 years, the herd prevalence did not decrease (12.7-13.6%; P>0.05) and the herd incidence was 22.72%. The testing and culling policy was effective in improving reproductive efficiency but the complete elimination of BT without substantial changes in management appears unlikely because putative risk factors associated with the disease are present in the management of this breed.

Cytauxzoon felis and 'Candidatus Mycoplasma haemominutum' coinfection in a Brazilian domestic cat (Felis catus).

This article describes the first detection of Cytauxzoon felis, using molecular techniques, in a naturally infected domestic cat from Brazil, South America. Coinfection with 'Candidatus Mycoplasma haemominutum' was also found. The molecular identification of the piroplasmid species was performed by Polymerase Chain Reaction (PCR) and sequencing analysis. A 284 pb fragment of the gene encoding the 18S ribosomal RNA region was amplified and showed 99% identity with other C. felis strains from North America. In addition, PCR-RFLP (restriction fragment length polymorphism) analysis, which amplifies a 595 bp fragment of the gene encoding 16S ribosomal RNA of some bacterial species, identified the co-infecting species as 'Candidatus M. haemominutum'.

Cytauxzoon felis and 'Candidatus Mycoplasma haemominutum' coinfection in a brazilian domestic cat (Felis catus) / Co-infecção por Cytauxzoon felis e 'Candidatus Mycoplasma haemominutum' em um gato doméstico (Felis catus) no Brasil

This article describes the first detection of Cytauxzoon felis, using molecular techniques, in a naturally infected domestic cat from Brazil, South America. Coinfection with 'Candidatus Mycoplasma haemominutum' was also found. The molecular identification of the piroplasmid species was performed by Polymerase Chain Reaction (PCR) and sequencing analysis. A 284 pb fragment of the gene encoding the 18S ribosomal RNA region was amplified and showed 99% identity with other C. felis strains from North America. In addition, PCR-RFLP (restriction fragment length polymorphism) analysis, which amplifies a 595 bp fragment of the gene encoding 16S ribosomal RNA of some bacterial species, identified the co-infecting species as 'Candidatus M. haemominutum'.

Este artigo descreve a primeira detecção de Cytauxzoon felis em um gato doméstico naturalmente infectado no Brasil, América do Sul, através de técnicas moleculares. Também foi encontrada co-infecção com 'Candidatus Mycoplasma haemominutum'. A detecção molecular da espécie do piroplasmídeo foi realizada através da reação em cadeia pela polimerase (PCR) e sequenciamento. Um fragmento de 284 pb do gene codificador da região 18S do RNA ribossomal do parasito foi sequenciada e mostrou 99% de identidade com outros isolados de C. felis da América do Norte. Ademais, através da análise por meio de PCR-RFLP (Polimorfismo no comprimento de fragmentos de restrição), que amplifica um fragmento de 595 pb do gene codificador da porção 16 do RNA ribossomal de algumas espécies de bactérias, concluiu-se que a espécie com-infectante era 'Candidatus M. haemominutum'.

Development and application of a novel histological multichrome technique for clam histopathology.

A novel and expedient histological tetrachrome technique was developed and applied to whole-body sections of the clam Ruditapes decussatus (L. 1758). The technique involves fixation in Carnoy's fluid followed by immediate embedding in paraffin with staining with a combination of Alcian Blue, Periodic Acid-Schiff's, Haematoxylin and Picric Acid. Fixation and staining was perfect for all tissues and resolved good identification of Perkinsus sp. infection and high structural detail. Among the surveyed fixatives, Bouin-Hollande's fluid also provided good results, however, fixation is potentially longer, polysaccharide staining was less intense and fibres appeared to be better preserved by Carnoy's.

Parasites and pathogens of the endosymbiotic pea crab (Pinnotheres pisum) from blue mussels (Mytilus edulis) in England.

A histopathological survey of the commensal pea crab (Pinnotheres pisum) from the mantle cavities of blue mussels (Mytilus edulis) has been conducted. A total of 266 pea crabs from eight sites around the English coastline were examined. Of these, 82 were negative for any visible infections by histology. The remaining pea crabs were infected with an intranuclear bacilliform virus designated as P. pisum bacilliform virus (PpBV) in the hepatopancreatic epithelial cells, peritrichous ciliates on the gills, an intracytoplasmic microsporidian infection of the hepatopancreatocytes, a myophilic microsporidian infection, the gregarine Cephaloidophora fossor in the hepatopancreas, the entoniscid isopod Pinnotherion vermiforme, a low level nematode infection and an acanthocephalan cystacanth. Host reactions to infections were generally subdued. Results are discussed in relation to the endocommensal habitat of the pea crabs.

Epidemiology of parasitic protozoan infections in Soay sheep (Ovis aries L.) on St Kilda.

The feral Soay sheep (Ovis aries L.) population on Hirta, St Kilda, is host to a diverse component parasite community, but previous parasitological studies of the population have only focussed on the metazoan species. This paper reports the first epidemiological study of the protozoan species comprising Cryptosporidium parvum, Giardia duodenalis and 11 species of Eimeria in Soay sheep across 3 years of varying host population density. Prevalence and intensity of almost all species of protozoa significantly decreased with host age, with the exception of E. granulosa, which increased in prevalence with host age. The prevalence of C. parvum appeared to vary positively with host population density but that of G. duodenalis did not vary significantly with density. Most species of Eimeria showed a distinct lag in infection level following the host population crash of 2002, taking up to 2 years to decrease. Mixed Eimeria species intensity and diversity were highest in 2002, a year of low host density. Parasite diversity decreased with host age and was higher in males. There were 5 positive pair-wise associations between protozoa species in terms of prevalence. The results of this study highlight the potential for protozoal infection to shape the evolution of parasite resistance in wild host populations harbouring diverse parasite species.

Parasitism by the protozoan Perkinsus atlanticus favours the development of opportunistic infections.

It has been suggested that opportunistic pathogens could contribute to the mortality of Perkinsus atlanticus-infected clams. Examination of Tapes semidecussatus clams from the northern Mediterranean coast of Spain revealed that while 86% of the clams heavily infected with P. atlanticus were co-infected by bacteria and/or viruses, neither non-infected nor lightly P. atlanticus-infected specimens had bacterial or viral infections. The bacteria, which had a Gram-negative cell wall, were always located in the apical pole of gill epithelial cells and enclosed within membranous compartments. Bacteria-containing cells were hypertrophied and showed dysplasia with loss of cilia and microvilli. The viruses shared ultrastructural, morphologic and cytopathic characteristics of a polyomavirus. Viral particles with icosahedral symmetry were found in both the cytoplasm and the nucleus of numerous cell types. Virus-infected cells showed severe alterations, including hypertrophy, reduction of the intracellular compartments and extrusion of the nuclear envelope. Moreover, gill epithelial cells showed disorganization and swelling of the apical region, which affected the ciliary structure. Our findings show that P. atlanticus parasitism favours the development of opportunistic infections which have detrimental effects in this clam population.