Ticks and tick-borne pathogens infecting livestock and dogs in Tchicala-Tcholoanga, Huambo Province, Angola.

The diversity of ticks and tick-borne pathogens (TBPs) infesting domestic animals in Tchicala-Tcholoanga, Angola, in 2016 was investigated. Seventeen tick species were recorded, Amblyomma pomposum being the most abundant on cattle (40%), goats (38%) and sheep (35%); Rhipicephalus turanicus was the most abundant on dogs (46%). This study presents new records of Haemaphysalis paraleachi, R. compositus, R. kochi and R. sulcatus in Angola, the first georeferenced population of Ha. leachi in southern Africa and the second record of R. microplus in Angola. Using the reverse line blot (RLB) hybridisation assay, fifteen TBP species were detected in blood samples from cattle (n = 88), goats (n = 82), sheep (n = 85) and dogs (n = 85). F The most frequently detected species were Theileria velifera in cattle (78%), Theileria ovis in sheep (80%) and Babesia vogeli in dogs (35%). Species-specific quantitative PCR assays detected Babesia bigemina in 43% (35/80) of blood samples of cattle, while E. ruminantium was detected in 4% (3/70) of blood samples and in 7% of A. pomposum ticks. Anaplasma platys was detected from cattle (18%) and sheep (6%) during RLB analysis. These findings constitute pioneering research in Angola.

Utilizing attached hard ticks as pointers to the risk of infection by Babesia and Theileria species in sika deer (Cervus nippon yesoensis), in Japan.

Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the genera Ixodes and Haemaphysalis. In total 163 samples (31.4%) were positive for Babesia and Theileria spp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes of Theileria sp. Thrivae (27.0% of ticks; 140/519), Theileria capreoli (10.6%; 55/519), Babesia divergens-like (1.7%; 9/519), Babesia sp. (Bab-SD) (0.6%; 3/519), Babesia microti U.S. (0.4%; 2/519), and B. microti Hobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens.

First molecular survey of piroplasm species in cattle from Kyrgyzstan.

Bovine piroplasmosis is a tick-borne disease caused by apicomplexan hemoparasites of the genera Theileria and Babesia. This study was carried out to assess the presence and frequency of piroplasm parasites in apparently healthy cattle in Kyrgyzstan. A total of 454 blood samples were collected from animals of various ages in eight villages located in the Chu valley and around the Lake Issyk Kul. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers specific targeting members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic and species-specific oligonucleotide probes were covalently linked. The results revealed the presence of three piroplasm species (Theileria orientalis, Babesia major, Theileria annulata). Theileria orientalis was the most prevalent species (32.8%; CI 28.5-37.3). Babesia major was the only species of Babesia found in any of the samples (1.3%; CI 0.5-2.8). The co-existence of Theileria annulata and T. orientalis was detected in nine animals (1.9%; CI 0.9-3.7). BLAST search revealed that the Theileria sequences shared 100% identity with the recently reported sequences for T. buffeli and T. annulata. The sequence of B. major was also 100% identical to an existing B. major sequence. This molecular survey provides important epidemiological data for control of bovine piroplasmosis caused by T. orientalis, B. major, and T. annulata in Kyrgyzstan.

Approaches for Reverse Line Blot-Based Detection of Microbial Pathogens in Ixodes ricinus Ticks Collected in Austria and Impact of the Chosen Method.

Ticks transmit a large number of pathogens capable of causing human disease. In this study, the PCR-reverse line blot (RLB) method was used to screen for pathogens in a total of 554 Ixodes ricinus ticks collected from all provinces of Austria. These pathogens belong to the genera Borrelia, Rickettsiae, Anaplasma/Ehrlichia (including "Candidatus Neoehrlichia"), Babesia, and Coxiella The pathogens with the highest detected prevalence were spirochetes of the Borrelia burgdorferisensu lato complex, in 142 ticks (25.6%). Borrelia afzelii (80/142) was the most frequently detected species, followed by Borrelia burgdorferisensu stricto (38/142) and Borrelia valaisiana (36/142). Borrelia garinii/Borrelia bavariensis, Borrelia lusitaniae, and Borrelia spielmanii were found in 28 ticks, 5 ticks, and 1 tick, respectively. Rickettsia spp. were detected in 93 ticks (16.8%): R. helvetica (39/93), R. raoultii (38/93), R. monacensis (2/93), and R. slovaca (1/93). Thirteen Rickettsia samples remain uncharacterized. "Candidatus Neoehrlichia mikurensis," Babesia spp. (B. venatorum, B. divergens, B. microti), and Anaplasma phagocytophilum were found in 4.5%, 2.7%, and 0.7%, respectively. Coxiella burnetii was not detected. Multiple microorganisms were detected in 40 ticks (7.2%), and the cooccurrence of Babesia spp. and "Candidatus Neoehrlichia mikurensis" showed a significant positive correlation. We also compared different PCR-RLBs for detection of Borrelia burgdorferisensu lato and Rickettsia spp. and showed that different detection approaches provide highly diverse results, indicating that analysis of environmental samples remains challenging.IMPORTANCE This study determined the wide spectrum of tick-borne bacterial and protozoal pathogens that can be encountered in Austria. Surveillance of (putative) pathogenic microorganisms occurring in the environment is of medical importance, especially when those agents can be transmitted by ticks and cause disease. The observation of significant coinfections of certain microorganisms in field-collected ticks is an initial step to an improved understanding of microbial interactions in ticks. In addition, we show that variations in molecular detection methods, such as in primer pairs and target genes, can considerably influence the final results. For instance, detection of certain genospecies of borreliae may be better or worse by one method or the other, a fact of great importance for future screening studies.

The Limnohabitans Genus Harbors Generalistic and Opportunistic Subtypes: Evidence from Spatiotemporal Succession in a Canyon-Shaped Reservoir.

We studied the diversity of Limnohabitans using reverse line blot hybridization with Limnohabitans lineage-specific probes in the freshwater canyon-shaped Rímov reservoir (Czech Republic). To examine the succession of distinct lineages, we performed (i) a study of an intensive spring sampling program at the lacustrine part of the Rímov reservoir (from ice melt through a phytoplankton peak to the clear-water phase), and (ii) a seasonal study (April to November) when the occurrence of distinct Limnohabitans lineages was related to the inherent longitudinal heterogeneity of the reservoir. Significant spatiotemporal changes in the compositions of distinct Limnohabitans lineages allowed for the identification of "generalists" that were always present throughout the whole season as well as "specialists" that appeared in the reservoir only for limited periods of time or irregularly. Our results indicate that some phytoplankton groups, such as cryptophytes or cyanobacteria, and zooplankton composition were the major factors modulating the distribution and dynamics of distinct Limnohabitans lineages. The highest Limnohabitans diversity was observed during the spring algal bloom, whereas the lowest was during the summer cyanobacterial bloom. The microdiversity also markedly increased upstream in the reservoir, being highest at the inflow, and thus likely reflecting strong influences of the watershed.IMPORTANCE The genus Limnohabitans is a typical freshwater bacterioplankton and is believed to play a significant role in inland freshwater habitats. This work is unique in detecting and tracing different closely related lineages of this bacterial genus in its natural conditions using the semiquantitative reverse line blot hybridization method and in discovering the factors influencing the microdiversity, subtype alternations, and seasonality.

An in-house multilocus SNP genotyping assay for evaluation of complex genetic diseases.

BACKGROUND: With an increase in the discovery of newer genetic loci/polymorphisms in complex multifactorial diseases, there is also an increased need for methods that can simultaneously genotype multiple loci in a cost-effective manner. Using coronary artery disease (CAD) as a model, the study aimed to develop an in-house multilocus assay for simultaneous detection of 17 genetic variants in 11 genes implicated in CAD. METHODS: A multiplex polymerase chain reaction (PCR)-based reverse line blot hybridization (MPCR-RLBH) approach was used, where each DNA sample was amplified using two separate MPCRs, and the alleles were genotyped using covalently immobilized, amino-linked sequence-specific oligonucleotide probes using an enhanced chemiluminescence system. The assay performance was tested on 75 healthy controls and 75 angiographically proven CAD cases. Validation was done by automated Sanger sequencing. RESULTS: The assay could successfully discriminate both the alleles at CETP (I405V), LPL (D9N), NOS3 (T-786G and E298D), LIPC (C-514T), FGB (G-455A), ITGB3 (L33P), AGT (M235T), and MTR (A2756G) loci. Certain mutations included in this assay such as ins242G, ins397G, E387K, L393K in the LDLR; N291S in the LPL; D442G in the CETP; and T833C in the CBS genes were found to be absent. The genotype results obtained using this assay showed 100% concordance with sequencing. CONCLUSION: The study demonstrated development and validation of a multiplex SNP genotyping assay that can be used to assess genetic risk factors in CAD. The assay provides a cost-effective alternative to expensive high throughput genotyping systems in common molecular research laboratories.

Identification of piroplasm infection in questing ticks by RLB: a broad range extension of tick-borne piroplasm in China?

Sensitive and specific diagnostic method for rapid and simultaneous detection and discrimination of the different species is needed for an effective control of piroplasmosis. Here, a reverse line blot (RLB) assay was developed for piroplasm detection. A general pair of primer based on 18S ribosomal RNA (rRNA) gene was used to amplify V4 region of 18S rRNA gene. General and specific probes for 13 piroplasm species were cited from previous publications or designed according to the alignment of 18S rRNA gene sequences. For sensitivity test of RLB assay, serially diluted plasmids of the different species were used to access the sensitivity of the RLB. Four hundred and fifty tick samples collected from grass from different provinces of China were then detected. The result indicated that the RLB assay is highly specific and sensitive, detecting up to 10(2) copies/µl of recombinant plasmid DNA. Multiple piroplasms were detected as single or mixed infection from tick species. Eight piroplasm species, most of which were Theileria annulata (33/450, 7.3 %) or Babesia sp. Xinjiang (30/450, 6.7 %), were found to infect with 89 tick samples in four tick species; no infections with Babesia major, Babesia ovata, Babesia bigemina, Theileria sergenti, or Theileria equi were detected. The piroplasms species-specific RLB assay may have potential clinical application in the simultaneous detection and differentiation of Babesia and Theileria species.

Molecular and Parasitological Survey of Bovine Piroplasms in the Black Sea Region, Including the First Report of Babesiosis Associated with Babesia divergens in Turkey.

Clinical cases of babesiosis were evaluated, and the frequency of bovine Babesia and Theileria parasites was determined in cattle. Blood samples and thin blood smears were collected from 23 cattle exhibiting clinical signs of babesiosis. In addition, tick and blood samples were collected from 100 apparently healthy cattle cograzing from the same area. Egg masses obtained from fully engorged female ticks were included. DNA isolated from blood and tick samples was screened for Babesia and Theileria by reverse line blot assay. Piroplasms compatible with Babesia spp. were observed microscopically for symptomatic cattle as circular, oval, elongated, or pear-shaped bodies. Parasitemia ranged from 0.08 to 0.9% for Babesia bovis, 2.5 to 15.4% for Babesia bigemina, and 7.4% for Babesia divergens. Reverse line blot showed positivity in 13 (13%) of the sampled clinically normal cattle and revealed the presence of three Babesia species. Babesia bovis was the most prevalent (9/100, 9%), followed by Babesia occultans (3/100, 3%) and B. bigemina (1/100, 1%). One animal infected with B. bigemina was also infected with B. bovis. The single animal infected with B. divergens showed symptoms of babesiosis. Ticks were identified as Rhipicephalus annulatus, Rhipicephalus turanicus, and Ixodes ricinus. One female R. annulatus and its egg mass were infected with B. bigemina. Neither Theileria annulata nor Theileria buffeli/orientalis infections were observed in cattle or ticks. This is the first report of clinical babesiosis caused by B. divergens in cattle from Turkey.

Rifoligotyping assay: an alternative method for rapid detection of rifampicin resistance in Mycobacterium tuberculosis isolates from Morocco.

One of the greatest threats to global tuberculosis (TB) control is the growing prevalence of drug resistant strains. In the past decades, considerable efforts have been made upon the development of new molecular technologies and methodologies for detection of drug resistance in Mycobacterium tuberculosis (MTB). A sensitive, specific reverse line blot assay, called rifoligotyping (RIFO), for the detection of genotypic resistance to rifampicin (RIF), was designed and evaluated. RIFO includes oligonucleotide probes specific for wild-type and mutant sequences, allowing specific and sensitive detection of both genotypes in a single assay. The RIFO was applied on 500 MTB isolates from Morocco. The results of the RIFO showed a good sensitivity (90.9%) and high specificity (100%); the positive and negative predictive values were 100% and 96.1%, respectively. This rapid, simple, economical assay provides a practical alternative for RIF genotyping, especially in low-income countries, to improve TB control and management.

Simultaneous detection of seven bacterial pathogens transmitted by flies using the reverse line blot hybridization assay.

BACKGROUND: Traditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control. METHODS: We aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/µl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China. RESULTS: The established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/µl for S. aureus, 103 copies/µl for S. flexneri, 105 copies/µl for A. caviae, 105 copies/µl for V. vulnificus, 100 copies/µl for S. enterica, 105 copies/µl for P. vulgaris, and 100 copies/µl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species. CONCLUSIONS: Overall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.