Label-Free Quantitative Proteomic Analysis of Three Strains of Viscerotropic Leishmania Isolated from Patients with Different Epidemiological Types of Visceral Leishmaniasis in China.

BACKGROUND: There are three epidemiological types of visceral leishmaniasis in China, which are caused by Leishmania strains belonging to the L. donovani complex. The mechanisms underlying their differences in the population affected, disease latency, and animal host, etc., remain unclear. We investigated the protein abundance differences among Leishmania strains isolated from three types of visceral leishmaniasis endemic areas in China. METHODS: Promastigotes of the three Leishmania strains were cultured to the log phase and harvested. The protein tryptic digests were analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free quantitative analysis. The MS experiment was performed on a Q Exactive mass spectrometer. Raw spectra were quantitatively analyzed with the MaxQuant software (ver 1.3.0.5) and matched with the reference database. Differentially expressed proteins were analyzed using the bioinformatics method. The MS analysis was repeated three times for each sample. RESULTS: A total of 5012 proteins were identified across the KS-2, JIASHI-5 and SC6 strains in at least 2 of the three samples replicate. Of them, 1758 were identified to be differentially expressed at least between 2 strains, including 349 with known names. These differentially expressed proteins with known names are involved in biological functions such as energy and lipid metabolic process, nucleotide acid metabolic process, amino acid metabolic process, response to stress, cell membrane/cytoskeleton, cell cycle and proliferation, biological adhesion and proteolysis, localization and transport, regulation of the biological process, and signal transduction. CONCLUSION: The differentially expressed proteins and their related biological functions may shed light on the pathogenicity of Leishmania and targets for the development of vaccines and medicines.

Control of eosinopilic meningitis caused by Angiostrongylus cantonensis in China.

Rat lungworm Angiostrongylus cantonensis is the major infective agent of human eosinophilic meningitis (EM) in the world. The parasite was first noted in China in 1933. However, the public health importance was not realized until several EM outbreaks occurred recent years. Such disease is considered as emerging infectious disease in the People's Republic of China (P.R. China) since the major source of infection is invasive snail species, particularly Pomacea spp. National Institute of Parasitic Diseases (NIPD) initiated a systematic implementation research on this disease since 2003. Our researchers in NIPD developed the lung-microscopy for detecting A. cantonensis larvae in Pomacea snails and further accomplished the atlas of larval morphology by this method. We studied the determinants in infection, which helped the field collection of snails and improved the infection procedure in laboratory. Our researches promoted the promulgation of diagnosis criteria of angiostrongyliasis cantonensis by the Ministry of Health. We explored the molecular diversity of rat lungworm and its major snail host for development of source-tracing technique. The transmission modelling could provide the vulnerable area for surveillance. All the studies supported the surveillance system of EM caused by A. cantonensis in P.R. China. Such implementation research will provide a case study for control of emerging infectious diseases.

The complete mitochondrial genome of the rodent intra-arterial nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis.

The two rodent intra-arterial nematodes, Angiostrongylus cantonensis and Angiostrongylus costaricensis, can cause human ill-health. The present study aimed to characterize and compare the mitochondrial (mt) genomes of these two species, and clarify their phylogenetic relationship and the position in the phylum Nematoda. The complete mt genomes of A. cantonensis and A. costaricensis are 13,497 and 13,585 bp in length, respectively. Hence, they are the smallest in the class of Chromadorea characterized thus far. Like many nematode species in the class of Chromadorea, they encode 12 proteins, 22 transfer RNAs, and two ribosomal RNAs. All genes are located on the same strand. Nucleotide identity of the two mt genomes is 81.6%, ranging from 77.7% to 87.1% in individual gene pairs. Our mt genome-wide analysis identified three major gene arrangement patterns (II-1, II-2, and II-3) from 48 nematode mt genomes. Both patterns II-1 and II-2 are distinct from pattern II-3, which covers the Spirurida, supporting a closer relationship between Ascaridida and Strongylida rather than Spirurida. Thymine (T) was highly concentrated on coding strands in Chromadorea, but balanced between the two strands in Enoplea, probably due to the gene arrangement pattern. Interestingly, the gene arrangement pattern of mt genomes and phylogenetic analysis based on concatenated amino acids indicated a closer relationship between the order Ascaridida and Rhabditida rather than Spirurida as indicated in previous studies. These discrepancies call for new research, reassessing the position of the order of Ascaridida in the phylogenetic tree. Once consolidated, the findings are important for population genetic studies and target identification.

[Investigation on Babesia in ticks infested on police dogs in selected areas of China].

399 tick specimens were collected from the body surface of police dogs in Chongqing municipality, provinces of Fujian, Guangdong, Hainan, Guangxi, Hebei, Henan, Shanxi, Jiangsu and Zhejiang. Nested-PCR and sequence testing were taken to investigate the prevalence of Babesia sp. in ticks. The results showed that Babesia vogeli was found in ticks infested on the body surface of police dogs, with a positive rate of 5.3%. The prevalence in Chongqing, Guangdong, Guangxi, Hainan and Zhejiang was 4/16, 3.6% (1/28), 12.5%(11/88), 3.3% (4/121) and 1/15, respectively. It suggested that there was a certain rate of infected ticks infested on the body suriface of police dogs, which contributed to the potential threat to staff. The prevention and control measures should be strengthened.

[Investigation of ticks parasitizing on bodies of dogs in Shanghai].

OBJECTIVE: To investigate the ticks which parasitize on the bodies of dogs in Shanghai. METHODS: The tick samples were collected from the dogs in 18 districts and counties of Shanghai from March to December in 2011, and the ticks were identified for the species through the morphological analysis by the dissecting microscope in laboratory. RESULTS: Totally 1 950 dogs were investigated for ticks parasitizing on the bodies of districts including Jiading, Minhang, Pudong, Songjiang, Huangpu and Jinshan. These ticks were belong to 2 species of 2 genera by morphological analysis in laboratory. Rhipicephalus sanguineus and Haemaphysalis longicornis were the ticks parasitizing on the bodies of pet dogs. R. sanguineus was the main ticks parasitizing on the bodies of police dogs and laboratory dogs. CONCLUSION: R. sanguineus is the domain tick parasitizing on dogs in Shanghai, and H. longicornis is a newly found hard tick species parasitizing on dogs in Shanghai. We should strengthen the prevention and control of ticks.

[Investigation on transmitting vectors of visceral leishmaniasis in Jiashi County, Xinjiang].

OBJECTIVE: To investigate sandfly vectors transmitting visceral leishmaniasis, including species and seasonal distribution in Jiashi county of Xinjiang Uygur Autonomous Region. METHODS: Sandflies were collected in the field, counted and identified. The specimens were dissected to analyze the gonotrophic cycle and to find infection of promastigotes. The resting places were observed by using oil-paper and sandfly-capturing trap. RESULTS: 4540 sandflies were collected with 99.9% of Phlebotomus wui and only 0.1% Sergentomyia minutus sinkiangensis. On the seasonal distribution, the first peak appeared by the end of May and the first ten-day of June, and the second peak was in the middle of August. Observation showed that the activity of sandflies occurred mainly from 22:00 to 4:00, reaching to the maximum in the midnight. Analysis on the gonotrophic cycle revealed that Ph. wui was an exophilic species and appeared nocturnally for feeding with preference to human blood. Natural infection with promastigotes was found in 4 sandflies, more in the field than the residential area. Resting places included the aperture on the wall of livestock sheds and in the caves. CONCLUSION: Ph. wui is the predominant species in Jiashi, with higher infection rate of natural promastigotes in the field and with two life generations annually.

[Multiplex PCR assay for the detection of Angiostrongylus cantonensis larvae in Pomacea canaliculata].

OBJECTIVE: To establish a multiplex PCR assay for detecting Angiostrongylus cantonensis larvae in Pomacea canaliculata. METHODS: A pair of specific primers was designed based on the sequences of the small subunit rDNA of A. cantonensis (GenBank jAY295804), in combination with 16s rDNA specific primers of P. canaliculata, a multiplex PCR was developed. The PCR was performed on positive and negative snails, and the amplified products were analyzed by agarose gel electrophoresis and DNA sequencing. DNA template of 200 III stage larvae of A. cantonensis was diluted by negative snail DNA (1200 ng/microl, 120 ng/microl, 12 ng/microl, 1200 pg/microl, 120 pg/microl and 12 pg/microl), to find the minimum detectable level. Single blind method was used to evaluate the accuracy. After being detected by lung microscopy, 172 snails from field were tested by the multiplex PCR to assess the sensitivity and specificity. RESULTS: Agarose gel electrophoresis and DNA sequencing analysis indicated that the target sequences were efficiently amplified by the PCR assay (550 bp for P. canaliculata, 405 bp for A. cantonensis). The minimum detectable level was 120 pg/microl. The coincidence between the two methods stood for 84.3% (145/172), including 45 positives and 100 negatives. 24 snails were PCR positive and microscopy negative, 3 snails were PCR negative and microscopy positive. The sensitivity and specificity of multiplex PCR was 93.8% and 80.6%, respectively. Its positive rate (40.1%, 69/172) was significant higher than that of lung-microscopy (27.9%, 48/172)(chi2 = 14.8, P < 0.01). CONCLUSION: A multiplex PCR method has been developed for the detection of A. cantonensis larvae in P. canaliculata.