Publications

4795

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.

Abstract Background Candidatus Liberibacter asiaticus (CLas) is one the causative agents of greening disease in citrus, an unccurable, devastating disease of citrus worldwide. CLas is vectored by Diaphorina citri, and the understanding of the molecular interplay between vector and pathogen will provide additional basis for the development and implementation of successful management strategies. We focused in the molecular interplay occurring in the gut of the vector, a major barrier for CLas invasion and colonization. Results We investigated the differential expression of vector and CLas genes by analyzing a de novo reference metatranscriptome of the gut of adult psyllids fed of CLas-infected and healthy citrus plants for 1-2, 3-4 and 5-6 days. CLas regulates the immune response of the vector affecting the production of reactive species of oxygen and nitrogen, and the production of antimicrobial peptides. Moreover, CLas overexpressed peroxiredoxin, probably in a protective manner. The major transcript involved in immune expression was related to melanization, a CLIP-domain serine protease we believe participates in the wounding of epithelial cells damaged during infection, which is supported by the down-regulation of pangolin. We also detected that CLas modulates the gut peristalsis of psyllids through the down-regulation of titin, reducing the elimination of CLas with faeces. The up-regulation of the neuromodulator arylalkylamine N-acetyltransferase implies CLas also interferes with the double brain-gut communication circuitry of the vector. CLas colonizes the gut by expressing two Type IVb pilin flp genes and several chaperones that can also function as adhesins. We hypothesized biofilm formation occurs by the expression of the cold shock protein of CLas. Conclusions The thorough detailed analysis of the transcritome of Ca. L. asiaticus and of D. citri at different time points of their interaction in the gut tissues of the host led to the identification of several host genes targeted for regulation by L. asiaticus, but also bacterial genes coding for potential effector proteins. The identified targets and effector proteins are potential targets for the development of new management strategies directed to interfere with the successful utilization of the psyllid vector by this pathogen.

Abstract Members of the verrucomicrobial clade ‘ Candidatus Udaeobacter’ rank among the most dominant bacterial phylotypes in soil. Nevertheless, despite this global prevalence, in‐depth analyses with respect to pH preferences of ‘ Ca . Udaeobacter’ representatives are still lacking. Here, we utilized a recently designed primer pair, specifically targeting ‘ Ca . Udaeobacter’, to investigate links between soil pH and the abundance as well as phylotype composition of this largely unexplored verrucomicrobial clade. Based on 150 forest and 150 grassland soils, comprising a broad pH range, we determined the highest total abundance of ‘ Ca . Udaeobacter’ in strongly acidic soil (pH, ~5.1) and, noteworthy, in ultra‐acidic soil (pH < 3.5) and at a pH ≥ 7, its abundance drastically declined. When we analysed the six most dominant amplicon sequence variants affiliated with ‘ Ca . Udaeobacter’ separately, their abundances peaked within a pH range of approximately 4.7–5.2, and only in one case at slightly acidic soil pH (pH, 6.1). Our study benefits from a combination of quantitative real‐time PCR and high‐throughput amplicon sequencing, enabling for the first time a highly specific abundance analysis of representatives affiliated with ‘ Ca . Udaeobacter’, which revealed that this globally abundant verrucomicrobial clade shows preferences for acidic soil.

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.

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.

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.

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.

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