Publications

4053

Abstract Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm), and Candidatus Mycoplasma turicensis (CMt) are the major feline hemotropic Mycoplasma (FHM) species identified in cats worldwide. Data concerning FHM in Egypt is limited; therefore, the current study aimed to gain further insights into disease epidemiology by investigating FHM molecular prevalence, risk factors, and hemogram abnormalities in 246 Egyptian cats (shelter-housed and client-owned) during 2022–2024. Additionally, 16 S rRNA gene-based maximum-likelihood (ML) phylogenetic analysis was performed for all CMt-positive samples for the first time in Egypt. FHM was detected in 16.3% (n = 40) of cats and typed as CMhm, Mhf, and CMt in 15.4% (n = 38), 3.6% (n = 9), and 2.4% (n = 6) of cats, respectively. Single CMhm and Mhf infections were detected in 12.6% (n = 31) and 0.8% (n = 2) of cats, respectively. Dual (Mhf and CMhm) and triple (Mhf, CMhm, and CMt) infections were found in 0.4% (n = 1) and 2.4% (n = 6) of cats, respectively. CMhm was significantly associated with single infections rather than coinfections compared to other FHM species. Male sex and anemia were identified as predictors of FHM PCR positivity. FHM-infected cats had significantly lower hematocrit %, RBC counts, and hemoglobin concentrations than non-infected ones. Egyptian CMt sequences showed 97.6–100% nucleotide identity with each other. Egyptian and reference CMt strains represented nine nucleotide sequence types clustered into three well-supported clades on the ML tree without clear geographic distinction. The data generated in this study, conducted in Egypt, is crucial for enhancing our understanding of disease epidemiology and implementing effective preventive measures.

Huanglongbing (citrus greening disease) is caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) (Alphaproteobacteria) and is one of the most destructive bacterial-vector diseases affecting the citrus industry. The bacterium is transmitted by the Asian citrus psyllid (ACP; Diaphorina citri). Early detection in citrus trees is challenging due to uneven distribution of CLas throughout the tree and a long pre-symptomatic phase of the disease. Due to these limitations, ACP sampling has been suggested as a more reliable early detection strategy. The objective of this study was to develop and optimize approaches for detecting CLas in ACP adults and nymphs collected in citrus groves in California using real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). The goal was to establish the optimal number of ACP adults and nymphal instar life stages (stages 1–2, 3, or 4–5) that yielded the most reliable detection of CLas (Cq values ≤ 38). Results indicated that CLas detection correlated with psyllid developmental stage, with the 4th–5th instar nymphs (sample size of five to ten per tube) or adult ACP (sample size of three to ten per tube) providing the most consistent qPCR detection. While CLas detection rates increased with adult ACP age, nymphs were preferred for field sampling as adult ACP might have dispersed from non-infected trees, potentially misrepresenting the grove’s CLas status. Detection by droplet digital PCR confirmed the presence and genome copies of CLas in a subset of ACP across life stages. In field populations, detection rates in nymphs were consistent or stable throughout the year, whereas CLas detection in adults exhibited seasonal variation, with CLas detection and genome load peaking in January. These targeted ACP sampling strategies and optimized laboratory processing methods will facilitate CLas detection in psyllids for streamlining citrus greening disease management.

Jujube witches’ broom (JWB) disease, caused by Candidatus Phytoplasma ziziphi (Ca. P. ziziphi), severely threatens the production of Chinese jujube (Ziziphus jujuba Mill.). Emerging evidence highlights the critical role of phytoplasma-secreted effectors in pathogenesis, though few have been functionally characterized. Here, we identified a Sec-dependent effector, JWB790, from Ca. P. ziziphi, which was shown to suppress plant immunity. Through transient expression assays in Nicotiana benthamiana, pathogen inoculation assays, the generation of transgenic Arabidopsis thaliana plants, and RNA-seq-based transcriptomic profiling, we systematically investigated the virulence function of JWB790. Our findings revealed that JWB790 is highly expressed in JWB-infected tissues. The transient expression of JWB790 in N. benthamiana suppressed BAX-induced cell death and H2O2 accumulation. Furthermore, the stable overexpression of JWB790 in A. thaliana compromised disease resistance, accompanied by reduced H2O2 accumulation and callose deposition triggered by flg22. Additionally, the RNA-seq analysis of JWB790 transgenic Arabidopsis plants indicated that the overexpression of JWB790 altered the expression of biotic stress-related genes. In summary, JWB790 is a virulence factor that suppresses plant immunity and promotes pathogen proliferation. These results advance our understanding of Ca. P. ziziphi pathogenesis.

IntroductionEndosymbiotic bacteria show diverse strategies to manipulate host reproduction for their survival in nature. The diversity of symbionts infecting hematophagous insects and their impact on host ecology could be crucial for developing effective control measures of disease-transmitting vectors. Sand flies are a group of small insects, with some species serving as vectors for leishmaniasis, bartonellosis, and arboviral diseases. Sergentomyia squamirostris is the only known species of sand flies found on the main islands of Japan. Although no medical implications exist for S. squamirostris, we conducted whole-genome sequencing to explore its potential relevance.MethodsWe conducted whole-genome sequencing and de novo assembly of S. squamirostris using genomic DNA isolated from a single field-collected female insect sample. During this attempt, we incidentally obtained closed genomes of two new bacteria, wSSQ and RiSSQ, belonging to Wolbachia and Candidatus Tisiphia, respectively. We then investigated infection rates of both bacteria in two natural populations of S. squamirostris in Japan.ResultsPhylogenetic analysis indicated that wSSQ and RiSSQ belonged to Wolbachia and Ca. Tisiphia, respectively. Ca. Tisiphia is also known as “Torix Rickettsia,” which is considered a large group of endosymbionts of invertebrates with no known pathogenicity to humans and animals. NGS read depths for both wSSQ and RiSSQ genomes were found to be high coverages, indicating that these bacteria are S. squamirostris endosymbionts. The infection rates of wSSQ and RiSSQ in the wild population of S. squamirostris varied in the two different localities in Japan, and co-infection with both bacteria was commonly seen. wSSQ was detected in both sexes of S. squamirostris, whereas RiSSQ was detected only in female sand flies.ConclusionCa. Tisiphia has recently been recognized as an underexplored endosymbiont with a widespread presence in invertebrates, including disease vectors. RiSSQ represents the first complete genomic information resource of Ca. Tisiphia infecting sand flies. Further research is needed to understand potential interactions between its host and other endosymbionts, as well as to explore the potential implications of disease control in the future.

We present a genome assembly of the diplonemid Rhynchopus euleeides (Euglenozoa; Diplonemea; Diplonemea; Diplonemidae). The genome sequence is 199.0 megabases long, with most of the assembly scaffolded into 88 chromosomal pseudomolecules. The multipartite mitochondrial genome and the 2.0 megabase genome of Ca. Syngnamydia salmonis, a bacterial endosymbiont of R. euleeides, were also sequenced and assembled.

Psyllids (Hemiptera: Psylloidea) are the main vectors of various phloem-limited plant pathogens, including ‘Candidatus Liberibacter’ species. ‘Candidatus Liberibacter solanacearum’ (CLso) has been associated with various plant disorders and economic losses in plants from the Solanaceae and Apiaceae families. Recently, it has been reported in Europe, primarily linked to carrots and celery. This situation presents a significant threat, prompting the need for a survey to assess the presence of the bacterium and its potential vectors. Plant and psyllid samples were collected from potato (Solanum tuberosum), carrot (Daucus carota) and other wild weed species in commercial fields and urban areas over two consecutive years (2022 and 2023). DNA was extracted from the samples, followed by conventional PCR and the sequencing of positive samples. The psyllid species Bactericera nigricornis was the dominant species in potato fields, while Bactericera trigonica was the most abundant in carrot fields, followed by Heterotrioza chenopodii, ranking as the second most abundant species in both cases. CLso-positive samples were found in D. carota, B. trigonica, H. chenopodii, B. nigricornis, and Trioza urticae. The sequencing results suggest the detected haplotypes are D and U. These findings raise concerns about the potential spread of CLso and the associated risk of significant economic losses.

AbstractBackgroundRickettsiaare obligate intracellular gram-negative bacteria of the class Alphaproteobacteria and are vector-borne species that cause diseases in humans and animals. NewRickettsiaspecies have been involved as human pathogens; however, the pathogenic potential of other species is unknown.CandidatusRickettsia colombiensis is a new species in the spotted fever group of unknown pathogenicity that is phylogenetically related to other pathogenic species.ObjetiveTo evaluate the pathogenic potential ofCa. R. colombiensis in Syrian hamsters (Mesocricetus auratus) and analyzed its cross-reactivity against other SFG rickettsia species in human and Syrian hamster sera.MethodsCa. R. colombiensis was isolated fromAmblyomma dissimileusing a shell vial. Subsequently, five male hamsters were inoculated intraperitoneally (IP) and five intradermally (ID) with 1 × 106 Vero cells infected withCa. R. colombiensis. One control animal was used in each group of the study. The health status of the animals was assessed daily, and necropsies were performed on days 5, 10, 15, and 16 DPI. Serum samples for indirect immunofluorescence and tissues were processed for qPCR and immunohistochemistry.ResultsAll animals remained healthy during the trial and showed no changes in their physiological parameters. No Rickettsia DNA was detected in any of the tissues. Animals infected withCa. R. colombiensis showed IgG antibody titers ranging from 1:64 to 1:1024. The control animals were negative. Regarding human sera, 56% (84/150) had IgG antibodies against Ca. R. colombiensis antigen. Sera with titers equal to or higher than 1:64 were 100% seroreactive.ConclusionsCa. R. colombiensis caused a subclinical infection in hamsters, suggesting the possibility of infecting other mammals. However, the clinical, pathological, and molecular findings are inconclusive in establishing thatCa.R. colombiensis is a pathogenic species. Regarding cross-reactivity, it is possible to serologically diagnose Rickettsial infection usingCa. R. colombiensis as antigens.Author summaryRickettsiosis is a human and animal disease caused by the bacterial Rickettsia species, which are mainly transmitted by arthropods. The clinical spectrum of rickettsiosis ranges from asymptomatic infection to severe and fatal diseases, such as Rocky Mountain spotted fever caused by Rickettsia rickettsii. In recent years, the number of species belonging to the genus Rickettsia has increased, in part due to the progress in the detection and identification of these agents by the application of modern molecular techniques. Much of this newRickettsiaspecies, known as Candidatus, has been involved as animal and human pathogens; however, for other Candidatus Rickettsia species, the ability to cause diseases is unknown.CandidatusRickettsia colombiensis is a new species phylogenetically related to pathogenic species. Here, we evaluated, for the first time, the pathogenic potential of this microorganism in a hamster animal model and analyzed its cross-reactivity against other members of the Rickettsia genus in human and hamster sera to determine the possibility of usingCandidatusRickettsia colombiensis as antigens to diagnose rickettsiosis in regions where immunologic reagents are scarce and high biosafety level laboratories do not exist. In this report, we demonstrate thatCandidatusRickettsia colombiensis causes an inapparent infection in hamsters and cross-reactivity between other members of the Rickettsia genus.

Abstract The genus “Candidatus Kouleothrix” (Eikelboom type 1851) is the major filamentous bacterium responsible for activated sludge bulking in Japanese activated sludge plants, where it is present in many in unusually high abundances. Global surveys have shown this genus embraces several species, although the contribution each of these makes to bulking is not known. This study followed their population dynamics in a full-scale bulking sludge plant in Japan over a 12-month period, which required the development of species-specific FISH probes, used to measure their relative abundances. Six of the 9 recognised species of “Ca. Kouleothrix” were detected there, two of which (midas_s_3423 and midas_s_35412) were at consistently high abundances and responsible for increases in the sludge volume index (SVI). Their abundances were also statistically correlated with their filament lengths, which showed higher correlations with SVI increases than did species abundances. This study examined the impacts of both operational parameters and other communities on the abundances of these two species over the 12-month period and showed marked interspecies differences in responses to both. Together these findings stress the importance in studies of this kind to identify populations to species level to reveal possible important ecological differences not seen at the genus level.