Publications

4474

Abstract Little is known about the cell physiology of anammox bacteria growing at extremely low growth rates. Here, “Candidatus Brocadia sinica” and “Candidatus Scalindua sp.” were grown in continuous anaerobic membrane bioreactors (MBRs) with complete biomass retention to determine maintenance energy (i.e., power) requirements at near-zero growth rates. After prolonged retentostat cultivations, the specific growth rates (μ) of “Ca. B. sinica” and “Ca. Scalindua sp.” decreased to 0.000023 h−1 (doubling time of 1255 days) and 0.000157 h−1 (184 days), respectively. Under these near-zero growth conditions, substrate was continuously utilized to meet maintenance energy demands (me) of 6.7 ± 0.7 and 4.3 ± 0.7 kJ mole of biomass-C−1 h−1 for “Ca. B. sinica” and “Ca. Scalindua sp.”, which accorded with the theoretically predicted values of all anaerobic microorganisms (9.7 and 4.4 kJ mole of biomass-C−1 h−1at 37 °C and 28 °C, respectively). These me values correspond to 13.4 × 10−15 and 8.6 × 10−15 watts cell−1 for “Ca. B. sinica” and “Ca. Scalindua sp.”, which were five orders of magnitude higher than the basal power limit for natural settings (1.9 × 10−19 watts cells−1). Furthermore, the minimum substrate concentrations required for growth (Smin) were calculated to be 3.69 ± 0.21 and 0.09 ± 0.05 μM NO2− for “Ca. B. sinica” and “Ca. Scalindua sp.”, respectively. These results match the evidence that “Ca. Scalindua sp.” with lower maintenance power requirement and Smin are better adapted to energy-limited natural environments than “Ca. B. sinica”, suggesting the importance of these parameters on ecological niche differentiation in natural environments.

A protocol to successfully transmit the huanglongbing (HLB) pathogen, ‘Candidatus Liberibacter asiaticus’, between citrus plants by using the Asian citrus psyllid (ACP) and an alternative way to help growers control ACP are proposed. Best results were obtained when pathogen acquisition by adults reared on fully symptomatic ‘Ca. Liberibacter asiaticus’-positive plants, latency, and inoculation occurred at ambient air temperatures ranging from 24 to 28°C and when a single infective adult ACP was confined for 7 days on soft, newly developing vegetative shoots (stages v2 to v4). No infection resulted from confinement of infective ACP adults on mature leaves (stage v6). Under the described conditions, single ACP adults could successfully transmit ‘Ca. Liberibacter asiaticus’ to an average of 56.5% (35 to 83%) of plantlets with v2 to v4 shoots growing in 0.3-liter tubes and to 80.5% (76 to 86%) of plants with v2 to v4 shoots growing in 4.7-liter pots. The use of single insects and plantlets reduces labor, space, and other resources necessary to undertake transmission tests. It also reduces time needed for transmission studies and should help accelerate research on HLB. The results were used to develop an index for favorability to infection (IFI) to determine orchard vulnerabilities to ‘Ca. Liberibacter asiaticus’. The IFI is based on the heterogeneous population of new shoots that occurs on tree canopies and may offer alternative or complementary alternatives to the laborious and costly insect surveys currently used in most instances to determine threshold levels for insecticide applications.

Citrus huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, ‘Candidatus Liberibacter asiaticus’ (CLas), is critical for early diagnosis and successful management of HLB. However, current nucleic acid–based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy nrdB gene specific to CLas, couples isothermal amplification with Cas12a transcleavage of a fluorescent reporter oligonucleotide and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle, and psyllid samples and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.

Abstract Background Rickettsia spp. are human pathogens that cause a number of diseases and are transmitted by arthropods, such as ixodid ticks. Estonia is one of few regions where the distribution area of two medically important tick species, Ixodes persulcatus and I. ricinus, overlaps. The nidicolous rodent-associated Ixodestrianguliceps has also recently been shown to be present in Estonia. Although no data are available on human disease(s) caused by tick-borne Rickettsia spp. in Estonia, the presence of three Rickettsia species in non-nidicolous ticks has been previously reported. The aim of this study was to detect, identify and partially characterize Rickettsia species in nidicolous and non-nidicolous ticks attached to rodents in Estonia. Results Larvae and nymphs of I.ricinus (n = 1004), I. persulcatus (n = 75) and I.trianguliceps (n = 117), all removed from rodents and shrews caught in different parts of Estonia, were studied for the presence of Rickettsia spp. by nested PCR. Ticks were collected from 314 small animals of five species [Myodes glareolus (bank voles), Apodemus flavicollis (yellow necked mice), A.agrarius (striped field mice), Microtus subterranius (pine voles) and Sorex araneus (common shrews)]. Rickettsial DNA was detected in 8.7% (103/1186) of the studied ticks. In addition to identifying R.helvetica, which had been previously found in questing ticks, we report here the first time that the recently described I.trianguliceps-associated Candidatus Rickettsia uralica has been identified west of the Ural Mountains. Graphical Abstract

Abstract Background Candidatus Neoehrlichia mikurensis (CNM) is an emerging tick-born pathogen and usually causes symptomatic infection only in immunocompromised patients. Apart from one described case found in the literature where cultivation was successful, all cases so far were diagnosed by using broad-range 16S rDNA PCR. Case presentation Our patient presented with a prolonged febrile state of unknown origin. Clinical presentation, extensive medical workup and classic microbiologic testing were non-conclusive. Several infectious agents and other causes for the febrile state were excluded. In the end, a broad-range 16S rDNA PCR was to be performed to confirm the diagnosis of CNM infection. Treatment was successful with doxycycline. Conclusions Due to the obscurity of the pathogen, diagnostic workup in CNM is prolonged and challenging. More awareness is need about this emerging infectious disease in countries with high prevalence of tick-borne diseases as standard microbiological methods are not successful in confirming the diagnosis.

Specific scopeThis Standard describes a diagnostic protocol for ‘Candidatus Phytoplasma phoenicium’.This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. 1Specific approval and amendmentApproved in 2021–06.

AbstractIn recent years, empty or poor grain bearing corn plants with leaf reddening symptoms have been observed in some commercial maize production areas in the Adana province of Turkey. The disease is increasing and causing economic losses. To verify the possible presence of phytoplasmas, leaf samples were tested with P1/Tint and R16F2n/R2 phytoplasma‐specific primer pairs after DNA extraction. The 16S ribosomal gene sequence analysis followed by sequence in silico enzyme digestions and phylogenetic analyses allowed the identification of ‘Candidatus Phytoplasma solani’, subgroup of 16SrXII‐A related‐strain in all symptomatic samples. The positive samples were characterized using tuf and vmp1 genes. In silico restriction fragment length polymorphism and sequence analyses allowed the inclusion of all the positive samples into tuf‐type b and V15 molecular types. The sequence analysis indicated the presence of a single genotype in all the tested samples (n = 12). This is the first molecular characterization of a ‘Ca. P. solani’ strain infecting maize plants in Turkey. The identified pathogen is listed in the EPPO A2 list.

In sweet cherry (Prunus avium), infection by ‘Candidatus Phytoplasma pruni’ results in small fruit with poor color and taste, rendering the fruit unmarketable. Yet the disease pathology is poorly understood, particularly at the cultivar level. Therefore, in this study we examined the physiological effects of Ca. P. pruni infection across a range of cultivars and locations in eastern Washington. We found that infection could be separated into early and established stages based on pathogen titer, which correlated with disease severity, including fruit size, color, and sugar and metabolite content. Furthermore, we observed that the effects of early-stage infections were largely indistinguishable from healthy, uninfected plants. Cultivar- and location-specific disease outcomes were observed with regard to size, color, sugar content, and citric acid content. This study presents the first in-depth assessment of X-disease symptoms and biochemical content of fruit from commercially grown sweet cherry cultivars known to be infected with Ca. P. pruni.

AbstractBackgroundSerious disease outbreaks in cattle are usually associated with blood pathogens. This study aims to detect blood pathogens namelyTheileriaspecies,Anaplasmaspecies,Candidatus Mycoplasma haemobosandTrypanosoma evansi, and determine their phylogenetic relationships and haemato-biochemical abnormalities in naturally infected cattle.MethodsMolecular analysis was achieved by PCR amplification and sequencing of PCR amplicons of 18SrRNA gene of Theileria species, 16SrRNA genes ofAnaplasmaandMycoplasmaspecies, MPSP genes ofT. orientalisandT. sinensis, MSP4 gene ofA. marginale, 16SrRNA gene ofCandidatus Mycoplasma haemobos, and RoTat1.2 VSG gene ofTrypanosoma evansi, in sixty-one (61) clinically ill Kedah-Kelantan x Brahman cattle in Pahang, Malaysia.ResultsA total of 44 (72.13%) cattle were infected with more than one blood pathogen.Theileriaspecies was the blood pathogen with the highest molecular detection rate (72.13, 95% CI 59.83–81.81%). Nucleotide blast analyses of all sequences demonstrated high degree of molecular similarity (98–100%) in comparison with their respective reference sequences. Analysis of 18SrRNA gene sequences ofTheileriaspecies and 16SrRNA gene sequences ofAnaplasmaspecies revealedTheileria sinensisandAnaplasma platysrespectively as additional species detected in these cattle. MPSP-PCR analysis was conducted for further confirmation ofT. sinensis.The blood picture of eight infected cattle groups revealed poikilocytosis, anisocytosis, rouleaux formation and degenerative left shift. High mean erythrocyte fragility values were common in infected cattle groups. Anaemia of the macrocytic normochromic type and spherocytes were observed in theT. evansi and Anaplasma platys + Theileria sinensisdouble species co-infected cattle group. Normocytic normochromic anaemia was observed in theT. sinensisinfected cattle group. Significant (p < 0.05) increases in serum liver and kidney parameters, total protein, globulin, total and unconjugated bilirubin and decreased albumin values were observed in theT. evansiinfected cattle when compared to clinically healthy cattle.ConclusionWe present the first evidence ofTheileria sinensis-associated bovine anaemia (TSABA) in Malaysian cattle. Because of the high occurrence of bovine theileriosis and detection ofA. platys, there is an urgent need for appropriate preventive and control measures against these blood pathogens.