Fate of GFP-expressing Escherichia coli in the midgut and response to ingestion in a tick, Ornithodoros moubata (Acari: Argasidae)

Ticks are well-known vectors of various pathogens but migration of the pathogens in the tick midgut is not fully understood. In the present study, the fate of microbes in the midgut of Ornithodoros moubata was observed using green fluorescent protein (GFP)-expressing Escherichia coli. Fluctuations in the percentage of hemocytes in the hemolymph (Hc) and expression of an antimicrobial peptide, defensin, in the midgut was also investigated. Most E. coli gradually disappeared in the midgut after ingestion fluctuations in Hc coincided with the changes. Expression of defensin was also confirmed and slightly up-regulated after E. coli ingestion. Moreover, it was demonstrated that E. coli can not pass through the tick midgut epithelium after ingestion by the hemolymph cultures. It is known that various pathogens and host immunoglobulins ingested with a blood meal can enter into the hemocoel, which suggests the presence of unique and complex passage mechanisms for each molecule and organism. The results obtained here help to clarify that digestion enzymes is an important function of the tick midgut to protect against invading molecules and organisms.     

Recent Situation of Taeniasis in Mongolia (2002-2012)

Epidemiological situation of taeniasis in Mongolia was assessed based on mitochondrial DNA identification of the parasite species. Multiplex PCR was used on a total of 194 proglottid specimens of Taenia species and copro-PCR and loop-mediated isothermal amplification (LAMP) assays were utilized for detection of copro-DNA of 37 fecal samples from taeniasis patients submitted to the Mongolian National Center for Communicable Diseases (NCCD) from 2002 to 2012. In addition, 4 out of 44 calcified cysts in beef kept in formalin since 2003 were evaluated for histopathological confirmation of cattle cysticercosis. All proglottid specimens and stool samples were confirmed to be Taenia saginata by multiplex PCR and by copro-PCR and LAMP, respectively. Cysts collected from cattle were morphologically confirmed to be metacestodes of Taenia species. T. saginata taeniasis was identified from almost all ages from a 2-year-old boy up to a 88-year-old woman and most prominently in 15-29 age group (37%, 74/198) followed by 30-44 age group (34.8%, 69/198 ) from 15 of Mongolia''s 21 provinces, while cattle cysticerci were found from 12 provinces. The highest proportion of taeniasis patients was in Ulaanbaatar, the capital of Mongolia.     

A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity

Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins has been proposed to be the regulation of protein turnover and defense against pathogens as well as the balance of the host-parasite immune relationship. Genes encoding cystatins have been found in several species of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from tick H. longicornis and designated it as Hlcyst-2 (H. longicornis cystatin-2). Its full-length cDNA is 569 bp, and it encodes a putative 133 amino acid protein with an obvious signal peptide. Sequence analysis demonstrated that it has significant homology with the known cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain, cathepsin L, and cathepsin B was identified by fluorogenic substrate analysis. Cystatin was mostly expressed in the tick midgut and hemocyte. Blood feeding induced significantly increased expression in the midgut. Real-time PCR confirmed that LPS-injected adult ticks expressed Hlcyst-2 1.6 more times than the PBS-injected control; Babesia gibsoni-infected larvae ticks expressed Hlcyst-2 1.8 more times than normal larvae ticks. The recombinant protein also showed a significant growth-inhibitory effect on Babesia bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.     

Expression of truncated Babesia gibsoni thrombospondin-related adhesive proteins in Escherichia coli and evaluation of their diagnostic potential by enzyme-linked immunosorbent assay

Among the previously established enzyme-linked immunosorbent assays (ELISAs), an ELISA using the full length of a recombinant thrombospondin-related adhesive protein of Babesia gibsoni (rBgTRAPf) is considered as the most sensitive diagnostic method for the detection of an antibody to B. gibsoni in dogs. However, the expression of rBgTRAPf in high concentration is poor and, thus, limits its usefulness as a diagnostic antigen. To improve its expression level, we have truncated BgTRAPf into two fragments having either an N- or a C-terminus (BgTRAPn or BgTRAPc, respectively). The expression of BgTRAPc protein in Escherichia coli yielded adequate recombinant protein. The specificity and sensitivity of ELISAs with the truncated proteins were determined using dog sera experimentally infected with B. gibsoni and specific pathogen-free (SPF) dog sera. A total of 254 field dog sera were examined by the ELISA with rBgTRAPn, rBgTRAPc, and rBgTRAPf as well as by an indirect fluorescent antibody test (IFAT). The specificity of rBgTRAPc was the highest (97.15%), and its kappa value was more (0.8003) than rBgTRAPn (0.7083). With a sufficient level of expression as well as higher specificity and reliable sensitivity, rBgTRAPc appears to be a potential candidate antigen for the serodiagnosis of B. gibsoni infection in dogs.     

Zoonotic enteric parasites in Mongolian people,animals, and the environment: Using One Health to address shared pathogens

BackgroundCryptosporidium spp. and Giardia duodenalis are important zoonotic enteric pathogens of One Health concern for humans, animals, and the environment. For this study, we investigated parasite prevalence and risk factors among rural, peri-urban, and urban households and environments of Mongolia.MethodsThis cross-sectional study implemented a household risk factor survey at 250 home sites along with sample collection from humans, animals, flies, and drinking water. Multiplex real-time PCR analysis was conducted to look for Cryptosporidium spp. and/or Giardia duodenalis within household samples.ResultsLab analysis found one or both zoonotic parasites at 20% of the participating households (51/250). Human samples had a parasite prevalence of 6.4% (27/419), domestic animals at 3.3% (19/570), pooled filth flies at 14.8% (17/115), and drinking water samples at 2% (5/250). Parasite presence at the household was significantly associated with a household’s use of an improved drinking water source (OR 0.27; CI 0.12–0.61; p = < 0.01), having an indoor handwashing site (OR 0.41; CI 0.19–0.92; p = 0.03), domestic animal ownership (OR 2.40; CI 1.02–5.65; p = 0.05), and rural location (OR 0.50; CI 0.25–0.98; p = 0.04). Household use of an improved drinking water source remained significant in the multivariate model (OR 0.16; CI 0.04–0.68; p = 0.01).ConclusionIn Mongolia, public and veterinary health are intertwined, particularly for rural herding households. Increased access to safe water, sanitation and hygiene infrastructure could help prevent further transmission of zoonotic enteric parasites. Public health interventions, policy and messaging should utilize a One Health framework employing joint leadership from local human and animal health sectors.     

A cysteine protease is critical for Babesia spp. transmission in Haemaphysalis ticks

Vector ticks possess a unique system that enables them to digest large amounts of host blood and to transmit various animal and human pathogens, suggesting the existence of evolutionally acquired proteolytic mechanisms. We report here the molecular and reverse genetic characterization of a multifunctional cysteine protease, longipain, from the babesial parasite vector tick Haemaphysalis longicornis. Longipain shares structural similarity with papain-family cysteine proteases obtained from invertebrates and vertebrates. Endogenous longipain was mainly expressed in the midgut epithelium and was specifically localized at lysosomal vacuoles and possibly released into the lumen. Its expression was up-regulated by host blood feeding. Enzymatic functional assays using in vitro and in vivo substrates revealed that longipain hydrolysis occurs over a broad range of pH and temperature. Haemoparasiticidal assays showed that longipain dose-dependently killed tick-borne Babesia parasites, and its babesiacidal effect occurred via specific adherence to the parasite membranes. Disruption of endogenous longipain by RNA interference revealed that longipain is involved in the digestion of the host blood meal. In addition, the knockdown ticks contained an increased number of parasites, suggesting that longipain exerts a killing effect against the midgut-stage Babesia parasites in ticks. Our results suggest that longipain is essential for tick survival, and may have a role in controlling the transmission of tick-transmittable Babesia parasites.