Rapid and Visual Detection of Toxoplasma gondii in Blood Samples from Pet Cats and Dogs by Loop-Mediated Isothermal Amplification.

Background: Toxoplasma gondii is an obligate intracellular parasite that invades nearly all nucleated cells of a broad spectrum of vertebrate hosts, and which may cause serious disease in immunocompromised patients, as well as in the immunologically incompetent fetus. This study aimed to establish a loop-mediated isothermal amplification (LAMP) technique to rapidly detect T. gondii in the blood infection by targeting the 529 bp repeat element of T. gondii. Materials and Methods: A turbidity monitoring system, together with visual reagent, was used to test the amplification result of the LAMP assay. In addition, the specificity and sensitivity of the LAMP assay were measured. Results: The results suggest that the successfully established LAMP assay profile can detect the DNA of T. gondii at 67°C within 40 min. The limit of detection of the LAMP assay was 101 copies/µL. No cross reaction occurred with Plasmodium vivax, Toxocara cati, Clonorchis sinensi, Spirometra mansoni or Cryptosporidium parvum. We validated the developed LAMP assay by detecting T. gondii in DNA extracted from 353 blood samples collected from domestic cats and dogs. The percentages of positive results in detecting these blood samples by LAMP and conventional PCR were 5.38% and 2.83%, respectively. Conclusions: Our findings show that the developed LAMP assay offers higher analytical sensitivity than conventional PCR and good analytical specificity, minimizes aerosol contamination, and can be applied to on-site rapid detection of T. gondii.

Glass transitions in frozen systems as influenced by molecular weight of food components.

Freezing is a frequently used way to expand the storage life of foods with high water content. Under suitable cooling rates, frozen systems attain a condition of maximum freeze concentration, which is characterized by the glass transition temperature (Tg '), end point of freezing or onset of melting (Tm '), and concentration of solids (Xs ') in the maximum-freeze-concentrated matrix. The value of Tg ', Tm ', and Xs ' depends on the chemical composition of frozen system. Below Tg ', the rates of deteriorative reactions are significantly reduced. In this article, the data for Tg ', Tm ', and Xs ' of different frozen systems including sugars, starches, proteins, and food are collected and compiled. The trends in Tg ' and Tm ' data of food are investigated using molecular weight (MW) of food components. The Tg ' and Tm ' of most starches (increased by 2.46% to 87.3% and 10.8% to 85.0%) and some protein-rich foods (increased by 5.00% to 53.4% and 25.0% to 52.9%) were higher than the maximum values of sugar-rich foods. Both Tg ' and Tm ' values increased with increasing MW of solids in frozen food, reaching an asymptotic value. Moreover, there were exponential relationships between Tg ' or Tm ' values and MW for sugar and starch-rich foods taken together. Some studies found that frozen storage below Tg ' maintains the higher quality of food that was achieved by fast freezing. However, other studies found that there was no significant difference in the quality of frozen foods between storage temperature below and above Tg '. Therefore, storage below Tg ' is not the only factor for predicting the stability of frozen foods.

Influences of PON1 on airway inflammation and remodeling in bronchial asthma.

This study aims to explore the influences of Paraoxonase-1 (PON1) involved in airway inflammation and remodeling in asthma. Mice were divided into control, asthma, asthma + PON1 and asthma + NC groups, and asthma models were established via aerosol inhalation of ovalbumin (OVA). HE, Masson, and PAS stains were used to observe airway inflammation and remodeling, Giemsa staining to assess inflammatory cells in bronchoalveolar lavage fluid (BALF), qRT-PCR and Western blot to detect PON1 expression, lipid peroxidation and glutathione assays to quantify malondialdehyde (MDA) activity and glutathione peroxidase (GSH) levels, ELISA to determine inflammatory cytokines and immunoglobulin, and colorimetry to detect PON1 activities. Additionally, mice lung macrophages and fibroblasts were transfected with PON1 plasmid in vitro; ELISA and qRT-PCR were performed to understand the effects of PON1 on inflammatory cytokines secreted by lung macrophages, MTT assay for lung fibroblasts proliferation and qRT-PCR and Western blot for the expressions of PON1, COL1A1, and fibronectin. After overexpression of PON1, the asthma mice had decreased inflammatory cell infiltration, fibrosis degree, and airway wall thickness; inflammatory cells and inflammatory cytokines in BALF were also reduced, expressions of OVA-IgE and IgG1, and MDA activity were decreased, but the expressions of OVA-IgG2a and INF-γ and GSH levels were increased. Besides, PON1 significantly inhibited microphage expression of LPS-induced inflammatory cytokines, lung fibroblast proliferation, and COL1A1 and fibronectin expression. Thus, PON1 could relieve airway inflammation and airway remodeling in asthmatic mice and inhibit the secretion of LPS-induced macrophage inflammatory cytokines and the proliferation of lung fibroblasts.

Effect of osmotic dehydration pretreatment and glassy state storage on the quality attributes of frozen mangoes under long-term storage.

Changes in the quality of frozen mango cuboids were investigated during long-term glassy state storage with and without osmotic dehydration pretreatment. The mango cuboids were dehydrated in mixed solutions (sucrose: glucose: fructose in a ratio of 3.6:1:3) of different concentrations (30, 40, and 50% (wt/wt)) prior to freezing and then stored at -55 °C (in the glassy state) for 6 months. The results revealed that compared with the untreated samples, osmotic pretreatment decreased total color difference (reduced by 15.6-62.3%), drip loss (reduced by 8.2-29.5%) and titration acidity (reduced by 1.3-9.4%), while increasing hardness (increased by 48.8-82.3%), vitamin C content (increased by 72.5-120.6%) and total soluble solids (increased by 21.8-53.7%) of frozen mangoes after 6 months. Dehydration with a sugar concentration of 40% was considered as the optimal pretreatment condition. In addition, a storage temperature of -55 °C provided better retention of quality than rubbery state storage at -18 °C. With prolonged storage time, the quality of frozen mangoes continued to change, even in the glassy state. However, the changes in quality of the osmotic-dehydrated samples were less than those of the untreated samples. The current work indicates that osmotic pretreatment and glassy state storage significantly improved the quality of frozen mangoes during long-term storage.

Investigation on the zoonotic trematode species and their natural infection status in Huainan areas of China.

BACKGROUND: To investigate the species of zoonotic trematodes and the endemic infection status in the domestic animals in Huainan areas, north Anhui province of China, we intent to provide evidences for prevention of the parasitic zoonoses. METHODS: The livestock and poultry (definitive hosts) were purchased from the farmers living in the water areas, including South Luohe, Yaohe, Jiaogang and Gaotang Lakes, and dissected the viscera of these collected hosts to obtain the parasitic samples. Then the specimens were microscopically identified, with reference to the descriptions in previous literatures for counting the zoonotic species found in these areas. RESULTS: A total of 41 species were detected in the domestic samples, in which 23 were zoonotic trematodes, and 18 were internal trematodes of animals. Of the 41 species, 38 were novel records in Huainan areas, and 12 were newly detected in Anhui province, including Metorchis anatinus, Echinostoma hortense, E. cinetorchis, E. angustitestis, E. lindoensis, E. nordiana, E. ilocanum, Metagonimus yokogawai, Prosthogonimus gracilis, P. skrjabini, P. anatinusand Trichobilharzia sp. which generally occurred in definitive hosts of chicken, ducks, geese, dogs, cattle, buffaloes, sheep, goats and pigs, respectively. CONCLUSION: A large quantity of livestock and poultry are fed by the local farmers living along the river banks in Huainan area, suggesting that the population in that area are at higher risks of natural focus of zoonotic infections, since these animals are favorable definitive hosts to the zoonotic trematodes.

Investigation on the zoonotic trematode species and their natural infection status in Huainan areas of China / Investigación sobre las especies de trematodos zoonóticos y su estado natural de infección en las zonas de Huainan en China

Background: To investigate the species of zoonotic trematodes and the endemic infection status in the domestic animals in Huainan areas, north Anhui province of China, we intent to provide evidences for prevention of the parasitic zoonoses. Methods: The livestock and poultry (definitive hosts) were purchased from the farmers living in the water areas, including South Luohe, Yaohe, Jiaogang and Gaotang Lakes, and dissected the viscera of these collected hosts to obtain the parasitic samples. Then the specimens were microscopically identified, with reference to the descriptions in previous literatures for counting the zoonotic species found in these areas. Results: A total of 41 species were detected in the domestic samples, in which 23 were zoonotic trematodes, and 18 were internal trematodes of animals. Of the 41 species, 38 were novel records in Huainan areas, and 12 were newly detected in Anhui province, including Metorchis anatinus, Echinostoma hortense, E. cinetorchis, E. angustitestis, E. lindoensis, E. nordiana, E. ilocanum, Metagonimus yokogawai, Prosthogonimus gracilis, P. skrjabini, P. anatinus and Trichobilharzia sp. which generally occurred in definitive hosts of chicken, ducks, geese, dogs, cattle, buffaloes, sheep, goats and pigs, respectively. Conclusion: A large quantity of livestock and poultry are fed by the local farmers living along the river banks in Huainan area, suggesting that the population in that area are at higher risks of natural focus of zoonotic infections, since these animals are favorable definitive hosts to the zoonotic trematodes (AU)

Introducción: para investigar las especies de trematodos zoonóticos y el estado de infección endémica en los animales domésticos en áreas de Huainan, al norte de la provincia de Anhui, China, tenemos la intención de proporcionar evidencias para la prevención de enfermedades zoonóticas. Métodos: el ganado y las aves (hospedadores definitivos) fueron adquiridos a los campesinos que viven en las zonas con agua, incluyendo el sur de Luohe, Yaohe, Jiaogang y Gaotang Lagos, y se diseccionaron las vísceras de estos anfitriones recogidos para obtener las muestras parasitarias. Las muestras fueron identificadas microscópicamente, con referencia a las descripciones de la literatura revisada para contar las especies zoonóticas que se encuentran en estas áreas. Resultados: se detectaron un total de 41 especies en las muestras nacionales, de las que 23 eran trematodos zoonóticos y 18 eran trematodos internos de los animales. Estas especies representaron 22 géneros en 12 familias de 4 órdenes. De las 41 especies, 38 fueron registros nuevos en las áreas de Huainan y 12 fueron detectadas recientemente en la provincia de Anhui, incluyendo Metorchis anatinus, Echinostoma hortense, E. cinetorchis, E. angustitestis, E. lindoensis, E. nordiana, Euparyphium ilocanum, Metagonimus yokogawai, Prosthogonimus gracilis, P. skrjabini, P. anatinus y Trichobilharzia sp., generalmente producidos en huéspedes definitivos de pollo, patos, gansos, perros, vacas, búfalos, ovejas, cabras y cerdos, respectivamente. Conclusión: los agricultores locales que viven a lo largo de las orillas del río se alimentan de gran cantidad de ganado y aves de corral de la zona de Huainan, lo que sugiere que la población en esa área tiene mayor riesgo ante el foco natural de las infecciones zoonóticas, ya que estos animales son huéspedes definitivos favorables a los trematodos zoonóticos (AU)

[Exploration on the Technology of Counter Staining of Fluke].

Fixed samples of Clonorchis sinensis and Fasciolopsis buski were stained with acetocarmine and malachite green, or stained with acetocarmine only. The samples displayed three different colors after staining with acetocarmine and malachite green. The digestive system, excretory system and the surrounding muscle tissue were stained reddish, the uterus was bright green, and the vitellarium at each side of the worm was tan. Staining with the two dyes resulted in clear structure and moderate degree of staining, and allowed three-dimensional observation, while staining with acetocarmine highlighted the testis tissue. Therefore, combination of the two staining methods is recommended in teaching and research to more effectively facilitate observation.

[Metorchis orientalis founded in basin of Wuhu City, Anhui Province].

OBJECTIVE: To investigate the epidemic situation of Metorchis orientalis in Wuhu City, Anhui Province. METHODS: Three lakes (Fengming, Longwo and Kui lakes) were selected in Wuhu City, and the poultries around the lakes and freshwater fishes in the lakes were captured to test the infection of M. orientalis, and the infection rates were calculated. RESULTS: The adult worms of M. orientalis were found in gall bladder and hepatic duct of domestic ducks with infection rate of 16.7%. The metacercariae of M. orientalis were discovered in the muscle of Pseudorasbora parva and Pseudogobio rivularis with infection rates of 7.13% and 3.38% respectively. CONCLUSION: M. orientalis is endemic in Wuhu City, which should be paid enough attention to the animal husbandry, aquaculture and medicine.

Purification and structural analysis of membrane-bound polyphenol oxidase from Fuji apple.

Membrane-bound polyphenol oxidase (mPPO) in Fuji apple (Malus domestica Borkh. cv. Red Fuji) was purified and analyzed with a nanoelectrospray ionization mass spectrometer. The three-dimensional model and binding site of mPPO to 4-methyl catechol were also studied using molecular docking. mPPO was purified 54.41-fold using temperature-induced phase partitioning technique and ion exchange chromatography. mPPO had a molecular weight of 67.3kDa. Even though a significant level of homology was observed between mPPO and the soluble polyphenol oxidase in the copper binding sequence, there was another region, rich in histidine residues, which differed in 13 amino acids. The three-dimensional structure of mPPO consisted of six α-helices, two short ß-strands, and ten random coils. The putative substrate-binding pocket contained six polar or charged amino acids, His191, His221, Trp224, Trp228, Phe227, and Val190. Trp224 and Trp228 formed hydrogen bonds with 4-methyl-catechol.

[Investigation on schistosomiasis cognitive levels of people from Chaohu area].

OBJECTIVE: To investigate the cognitive levels of people from Chaohu area, Anhui Province about the prevention and control knowledge of schistosomiasis before the water transfer project operation, so as to provide the reference for formulating the health education intervention. METHODS: A schistosomiasis questionnaire survey was conducted among the students, fishermen, boat people and villagers from Chaohu selected by the random cluster sampling from May to October, 2014. RESULTS: A total of 1 140 questionnaires were issued with the actual recovery of 1 096 copies and 1 032 valid questionnaires. The awareness rate of prevention and control knowledge of schistosomiasis was from 11.72% to 71.71%. The highest awareness rate was in the "know schistosomiasis", however the lowest awareness rate was in the "know the intermediate host of schistosomiasis". Catching fish or shrimp in river or lake was the main way of contacting water in production activities (39.05%), and swimming or playing in river or lake was the main way of contacting water in life activities (63.18%). The prevention and control knowledge of schistosomiasis by teaching was at a rate of 41.47% on average, and the main way was anti-schistosomiasis professionals going to countryside to do health education (49.53%). That the feces directly discharged into water accounted for 91.14% in fishman and boatman. CONCLUSION: It is necessary for people from Chaohu to carry out various forms of health education of schistosomiasis prevention and control before the water transfer project operation.

Comparison of membrane-bound and soluble polyphenol oxidase in Fuji apple (Malus domestica Borkh. cv. Red Fuji).

This study compared membrane-bound with soluble polyphenol oxidase (mPPO and sPPO, respectively) from Fuji apple. Purified mPPO and partially purified sPPO were used. mPPO was purified by temperature-induced phase partitioning and ion exchange chromatography. The specific activity of mPPO was 34.12× higher than that of sPPO. mPPO was more stable than sPPO at pH 5.0-8.5. Although mPPO was more easily inactivated at 25-55 °C, it is still more active than sPPO in this temperature range. The optimum substrate of mPPO was 4-methyl catechol, followed by catechol. L-cysteine had the highest inhibitory effects on mPPO followed by ascorbic acid and glutathione. Surprisingly, EDTA increased mPPO activity. The results revealed that purified mPPO is a dimer with a molecular weight of approximately 67 kDa.

Dujardinascaris gigantea sp. n. (Nematoda: Ascaridida) from the critically endangered crocodile Alligator sinensis Fauvel (Reptilia: Crocodylia).

The Chinese alligator Alligator sinensis Fauvel (Reptilia: Crocodylia) is considered as one of the most critically endangered species of the 23 extant crocodiles. However, our knowledge of the helminth parasites of this rare animal is completely lacking. During a helminthological survey of reptiles in China, we found a new ascaridoid nematode, Dujardinascaris gigantea sp. n. from A. sinensis. The morphology of D. gigantea sp. n. was studied using light and scanning electron microscopy. The new species was also characterised using molecular methods by sequencing and analysing the small ribosomal DNA (18S) and the second internal transcribed spacer (ITS-2).

[Zoonotic trematode species identified in domestic animals from Huainan area].

OBJECTIVE: To understand the zoonotic trematode species identified in domestic animals from Huainan area, Anhui Province, so as to supply the evidence for the prevention of zoonoses. METHODS: The livestock/definitive hosts were commercially available on category basis, and sacrificed for obtaining the parasitic samples via dissecting the viscera. The specimens were microscopically identified, with reference to the descriptions in the previous literature. RESULTS: By identification, sorting and classification, 41 species of internal trematodes were found so far in the domestic animals fed in Huainan areas, in which 23 species were zoonotic trematodes and 18 were internally parasitic in livestock, belonging to 4 orders, 12 families and 21 genera, and occurred in 9 definitive hosts including chicken, ducks, geese, pigs, cattle, buffaloes, sheep, goats and dogs, respectively. CONCLUSION: The findings suggest that the zoonotic trematodes belong to many kinds and prevalent in the domestic animals in Huainan areas, and such prevalence should call for high attention since it is urgent risk factors for zoonoses in this geographic area.

[Investigation on species and community ecology of cheyletoid mites breeding in the stored traditional Chinese medicinal materials].

OBJECTIVE: To investigate the species of Cheyletoid mites breeding status in the stored traditional Chinese medicinal materials and the relationship between its community and habitats. METHODS: A total of 60 samples of the traditional Chinese medicinal materials were collected from Huainan, Wuhu and Xuancheng in China. The mites were isolated with shakesieve shock, directicopy, Tullgren funnel, waternacopy and redricopy, and identified and counted under the light microscope. RESULTS: Cheyletoid mites were represented in 48 of the 60 samples, and the breeding rate accounted for as high as 80.0% (48/60). Totally, 6 species of Cheyletoid mites were identified, which belonged to Cheyletus, Acaropsis, Cheletomorpha, Eucheyletic and Pseudocheyles genera under the family of Cheyletidae. In the three investigated areas, the average breeding density of Cheyletoid mites was 109.75 heads/g, and average richness index was 1.54. The index of species diversity was 2.55 and the number of evenness was 0.95. CONCLUSION: This result entails positive prevention and control of the mite breeding in storage and processing of herbal materials.

[Reform and practice on the experiment teaching of medical parasitology].

A new model of education is investigated to meet the new idea of experiment teaching in university. Therefore the establishment of experiment teaching model of medical parasitology needs to be correspondently reformed. A variety of new management measures are taken to raise the efficiency of experiment teaching in training the students in the College.

[Effect of Sry silencing by siRNA on the expression of sex determining genes in mouse embryos].

In order to investigate Sry regulation network in the development of male embryo, we inhibited the Sry gene expression by RNAi and then examined expression of other sex-related genes. Six genes (Sox9, Wt1, Sf1, Dax1, Gata4 and Amh), which are suggested to be closely related to Sry regulation were studied. Two siRNA expression vectors pSilencer4.1/Sry217 and pSilencer4.1/Sry565 were constructed and injected into gestated mouse through tail vein at 9.5 day of conception (dpc). The inhibition efficiency of Sry and the expression of other six genes were examined in male embryos at 11.5 dpc by RT-PCR and Western-blot. Expressions of the other six genes were analyzed by fluorescence quantity PCR. The results indicated both the pSilencer4.1/Sry217 and pSilencer4.1/Sry565 could inhibit significantly increased after Sry silencing. In contrast, no significant changes were observed in the expression of Sf1, Amh, Gata4, Dax1 and Sox9 when silencing Sry by siRNA. Our results suggested that the Wt1 transcription was regulated by Sry, whereas the Sox9 expression is not directly regulated by Sry in the development of genital ridge.

Expression of CXC chemokine IP-10 in patients with chronic hepatitis B.

BACKGROUND: Chemokines have strong chemoattractant effects and are involved in a variety of immune and inflammatory reactions, such as attracting activated T lymphocytes, neutrophils, monocytes and natural killer cells via the pathway of G protein-coupled receptors to sites of inflammatory injury and contribute to wound repair. This investigation was designed to assess the levels of chemokine interferon-gamma inducible protein-10 (IP-10) and IP-10 mRNA, and the relationship between IP-10 mRNA and HBV-DNA and alanine aminotransferase (ALT) in patients with chronic hepatitis B. METHODS: The levels of IP-10 mRNA in peripheral blood mononuclear cells (PBMCs) were kinetically detected by real-time polymerase chain reaction (PCR). The rate of chemokine/GAPDH was regarded as the extreme level of chemokine. The level of IP-10 in serum was measured by enzyme linked immunosorbent assay (ELISA), and the expression of IP-10 in hepatic biopsy tissue was detected by streptavidin-peroxidase (SP) immunohistochemistry. RESULTS: The level of IP-10 mRNA in the PBMCs of patients was 0.7387+/-0.0768 (lg cDNA/lg GAPDH); it was significantly higher in patients with chronic hepatitis B than that in normal controls (P<0.001). The level of IP-10 in the serum of patients was 660.9+/-75.5 pg/ml. There was a significant difference between patients with chronic hepatitis B and normal controls (P<0.05). In patients with chronic hepatitis B, the level of IP-10 mRNA in PBMCs was correlated with the IP-10 plasma level (r=0.7312, P<0.001), and the IP-10 plasma level was fairly correlated with the levels of ALT and HBV-DNA plasma (r=0.7235, P<0.001; r=0.7371, P<0.001). IP-10 was found by immunohistochemical analysis to be selectively upregulated on sinusoidal endothelium. CONCLUSIONS: The expression of IP-10 mRNA in PBMCs, IP-10 plasma concentration and the expression of IP-10 in sinusoidal endothelium are all high in patients with chronic hepatitis B. Chemokine IP-10 may play an important role in trafficking inflammatory cells to the local focus in the liver and induce the development of the chronicity of hepatitis B.

[Effect of fertilization condition on mammalian sex determination].

Selection of sex at or before conception is perhaps the most desired biotechnology of all time because many economic traits are related with sex. Sex selection by manipulating the conditions of sperm-oocytes fertilization would be one of the most practicable and widely used methods. This review summarized the progress in the effects of fertilization conditions on mammalian sex. Four main factors which possibly affected sex were described such as arginine concentration in female animal vagina, glucose in the uterus, maturational status of the oocyte at the time they encounter sperm as well as the hormone levels of both parents at the time of conception.