Publications

4373

Background: Tick-borne diseases are frequent in the Southeastern section of Brazil. The most prevalent canine parasites diagnosed are Ehrlichia canis, Babesia gibsoni, Babesia canis and Anaplasma platys, although Mycoplasma haemocanis and candidatus Mycoplasma haematoparvum have been registered in the country. Methods: When two clinically healthy dogs from a suburban area of the state of Rio de Janeiro, Brazil with history of being heavily infested with ticks were examined at Idexx Reference Laboratories, California for tick panel check. Results: One dog harbored DNA of candidatus Mycoplasma haematoparvum and the other DNA of Mycoplasma haemocanis. Conclusions: These results suggest that monitoring for tick infestations and tick-borne parasites must be permanent in southeastern Brazil, especially due to global climate changes which may contribute to spread and increase the number of infections in the Country.

AbstractPlacozoa is an enigmatic phylum of simple, microscopic, marine metazoans1,2. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host3–6. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax sp. H2 lives in symbiosis with two intracellular bacteria. One symbiont forms an undescribed genus in the Midichloriaceae (Rickettsiales)7,8 and has a genomic repertoire similar to that of rickettsial parasites9,10, but does not seem to express key genes for energy parasitism. Correlative image analyses and three-dimensional electron tomography revealed that this symbiont resides in the rough endoplasmic reticulum of its host’s internal fibre cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations11–13. This symbiont lives in the ventral epithelial cells of Trichoplax, probably metabolizes algal lipids digested by its host and has the capacity to supplement the placozoan’s nutrition. Our study shows that one of the simplest animals has evolved highly specific and intimate associations with symbiotic, intracellular bacteria and highlights that symbioses can provide access to otherwise elusive microbial dark matter.

AbstractHuanglongbing (HLB; yellow shoot disease) is a severe worldwide infectious disease for citrus family plants. The pathogenCandidatus Liberibacter asiaticus(CLas) is an alphapro-teobacterium of theRhizobiaceaefamily that has been identified as the cause. The virulence of CLas has been attributed, in part, to prophage encoded genes. Prophage and prophage like elements have been identified in 12 of the 15 CLas available genomes, and are classified into three prophage types. Here, we re-examined all 15 CLas genomes using ade novoprediction approach and expanded the number of prophage like elements from 16 to 33. Further, we find that all CLas contain at least one prophage-like sequence. Comparative analysis reveals a prevalent, albeit previously unknown, prophage-like sequence type that is a remnant of an integrated prophage. Notably, this remnant prophage is found in the Ishi-1 CLas strain that had previously been reported as lacking prophages. Our findings provide both a resource and new insights into the evolutionary relationship between phage and CLas pathogenicity.

Western X (WX) disease, caused by ‘Candidatus Phytoplasma pruni’, is a devastating disease of sweet cherry resulting in the production of small, bitter-flavored fruits that are unmarketable. Escalation of WX disease in Washington State prompted the development of a rapid detection assay based on recombinase polymerase amplification (RPA) to facilitate timely removal and replacement of diseased trees. Here, we report on a reliable RPA assay targeting putative immunodominant protein coding regions that showed comparable sensitivity to polymerase chain reaction (PCR) in detecting ‘Ca. Phytoplasma pruni’ from crude sap of sweet cherry tissues. Apart from the predominant strain of ‘Ca. Phytoplasma pruni’, the RPA assay also detected a novel strain of phytoplasma from several WX-affected trees. Multilocus sequence analyses using the immunodominant protein A (idpA), imp, rpoE, secY, and 16S ribosomal RNA regions from several ‘Ca. Phytoplasma pruni’ isolates from WX-affected trees showed that this novel phytoplasma strain represents a new subgroup within the 16SrIII group. Examination of high-throughput sequencing data from total RNA of WX-affected trees revealed that the imp coding region is highly expressed, and as supported by quantitative reverse transcription PCR data, it showed higher RNA transcript levels than the previously proposed idpA coding region of ‘Ca. Phytoplasma pruni’.

Phytoplasmas are cell-wall-less bacteria that cause diseases in approximately 1,000 plant species. ‘Candidatus Phytoplasma pyri’, the causal agent of pear decline, induces various symptoms on its hosts, leading to weakening and dieback of the plants, reduced fruit size and yield, and, consequently, considerable financial losses in all pear-growing areas. Fighting this disease requires a reliable and inexpensive method for pathogen detection in propagation material as well as plant stocks in orchards and breeding facilities. Here, we present a field-suitable detection protocol for ‘Ca. P. pyri’ based on loop-mediated isothermal amplification (LAMP) targeting the phytoplasmal 16S ribosomal DNA sequence. The combination of a simplified sample preparation method based on sodium hydroxide and colorimetric visualization of LAMP results enables a laboratory-independent pathogen detection. The detection limit is comparable with analysis by polymerase chain reaction; however, the pear decline LAMP detection method is superior in terms of ease of use, cost, and time effectiveness for obtaining results.