Publications

4855

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 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.

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.

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.

Abstract Background Mandarin ‘Shatangju’ is susceptible to Huanglongbing (HLB) and the HLB-infected fruits are small, off-flavor, and stay-green at the maturity period. To understand the relationship between pericarp color and HLB pathogen and the effect mechanism of HLB on fruit pericarp coloration, quantitative analyses of HLB bacterial pathogens and carotenoids and also the integrative analysis of metabolome and transcriptome profiles were performed in the mandarin ‘Shatangju’ variety with four different color fruits, whole green fruits (WGF), top-yellow and base-green fruits (TYBGF), whole light-yellow fruits (WLYF), and whole dark-yellow fruits (WDYF) that were infected with HLB. Results the HLB bacterial population followed the order WGF > TYBGF > WLYF > WDYF. And there were significant differences between each group of samples. Regarding the accumulation of chlorophyll and carotenoid, the chlorophyll-a content in WGF was the highest and in WDYF was the lowest. The content of chlorophyll-b in WGF was significantly higher than that in other three pericarps. There were significant differences in the total content of carotenoid between each group. WGF and TYBGF pericarps were low in phytoene, γ-carotene, β-cryptoxanthin and apocarotenal, while other kinds of carotenoids were significantly higher than those in WDYF. And WLYF was only short of apocarotenal. We comprehensively compared the transcriptome and metabolite profiles of abnormal (WGF, TYBGF and WLYF) and normal (WDYF, control) pericarps. In total, 2,880, 2,782 and 1,053 differentially expressed genes (DEGs), including 121, 117 and 43 transcription factors were identified in the three comparisons, respectively. The qRT-PCR confirmed the expression levels of genes selected from transcriptome. Additionally, a total of 77 flavonoids and other phenylpropanoid-derived metabolites were identified in the three comparisons. Most (76.65 %) showed markedly lower abundances in the three comparisons. The phenylpropanoid biosynthesis pathway was the major enrichment pathway in the integrative analysis of metabolome and transcriptome profiles. Conclusions Synthesizing the above analytical results, this study indicated that different color pericarps were associated with the reduced levels of some carotenoids and phenylpropanoids derivatives products and the down-regulation of proteins in flavonoids, phenylpropanoids derivatives biosynthesis pathway and the photosynthesis-antenna proteins.

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.