[Expression of Proteus mirabilis polyphosphate kinase and preparation of its polyclonal antibodies].

OBJECTIVE: To express and purify polyphosphate kinase (PPK) from Proteus mirabilis and prepare the polyclonal antibody against PPK. METHODS: The antigenicity and hydrophobicity of PPK were analyzed using software. The N-terminal conservative sequence containing 309 amino acids was selected as the target peptide, and its corresponding gene sequence with modification based on prokaryotic cells-preferred codon was synthesized and inserted into plasmid pET28b(+). The constructed recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced with IPTG. The expressed fusion protein was purified using Ni-affinity chromatography. The purified protein was injected along with adjuvant in rabbits to prepare the polyclonal antibodies against PPK. RESULTS AND CONCLUSION: PPK fusion protein expressed by E. coli was purified successfully using Ni-affinity chromatography. ELISA result demonstrated that the harvested rabbit anti-sera against PPK had a high titer of 1:512 000, and Western blotting showed a good specificity of the antibody, which can be used further study of the role of PPK in the pathogenesis of Proteus mirabilis infection.

Involvement of polyphosphate kinase in virulence and stress tolerance of uropathogenic Proteus mirabilis.

Proteus mirabilis (P. mirabilis), a gram-negative enteric bacterium, frequently causes urinary tract infections. Many virulence factors of uropathogenic P. mirabilis have been identified, including urease, flagella, hemolysin and fimbriae. However, the functions of polyphosphate kinase (PPK), which are related to the pathogenicity of many bacteria, remain entirely unknown in P. mirabilis. In this study, a ppk gene encoding the PPK insertional mutant in P. mirabilis strain HI4320 was constructed, and its biological functions were examined. The results of survival studies demonstrated that the ppk mutant was deficient in resistance to oxidative, hyperosmotic and heat stress. The swarming and biofilm formation abilities of P. mirabilis were also attenuated after the ppk interruption. In vitro and in vivo experiments suggested that ppk was required for P. mirabilis to invade the bladder. The negative phenotypes of the ppk mutant could be restored by ppk gene complementation. Furthermore, two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry were used to analyze the proteomes of the wild-type strain and the ppk mutant. Compared with the wild-type strain, seven proteins including TonB-dependent receptor, universal stress protein G, major mannose-resistant/Proteus-like fimbrial protein (MR/P fimbriae), heat shock protein, flagellar capping protein, putative membrane protein and multidrug efflux protein were down-regulated, and four proteins including exported peptidase, repressor protein for FtsI, FKBP-type peptidyl-prolyl cis-trans isomerase and phosphotransferase were up-regulated in the ppk mutant. As a whole, these results indicate that PPK is an important regulator and plays a crucial role in stress tolerance and virulence in uropathogenic P. mirabilis.

[Impact of pesticide use on the density of Anopheles anthropophagus and malaria incidence].

OBJECTIVE: To investigate the impact of the application of chemical pesticides on the distribution of Anopheles anthropophagus in rice fields and the malaria incidence. METHOD: Twenty-four villages from 16 counties in the provinces of Zhejiang, Sichuan and Guangxi were chosen for the surveys in the period of 1983-1987. For the survey of An. anthropophagus, indoor human bait trapping until midnight and catching the mosquitoes in all the nets in early morning were carried out to get the density and population ratio of the mosquitoes. Historical data on Anopheles spp., malaria incidence, acreage of the single or double season cropping of rice and on the quantity of chemical pesticide used in rice fields were collected from the study areas. RESULTS: In Hang-jia-hu region of Zhejiang Province, double season rice cropping was performed at that time, the quantity of pesticides used in 1973 was 45 kg/hm2, which was as high as 50 times than that in the 1950s. The density of An. anthropophagus decreased yearly, no An. anthropophagus could be found at 11 survey points in late 1980s. The malaria incidence dropped to less than 1 per 10000. In Leshan and Yibin areas of Sichuan Province, the major cultivation was single cropping, pesticides were applied in paddy fields since 1960s, and the average quantity of pesticides used was 8.6 kg/hm2 during 1970s-1980s. No significant difference on the density of An. anthropophagus was revealed between 1980s (86.2%) and 1960s (82.2%) (chi2=0.63, P>0.05). After mid-1980s, pesticide use gradually increased, and reached to 18.18 kg/hm2 in average in the years after 2000. The density of An. anthropophagus decreased, no An. anthropophagus was found in 2010 in the area surveyed and no malaria cases were reported as well. With double season cropping in Huanjiang County of Guangxi, the pesticide amount consumed was 1.79kg/hm2, 25.13 kg/hm2 and 7.68 kg/hm2 in paddy fields in 1960s, 1970s, and 1980s, respectively. The proportion of An. anthropophagus in anophelines was 52%(1 747/3 392) in the beginning of the 1980s. After the year 2000, the average pesticide use increased to 20.38 kg/hm2 in paddy fields. It was difficult to find An. anthropophagus in human dwellings after 2008. The average annual malaria incidence dropped to 0.14 per 10 000. CONCLUSION: Change of farming activities and especially use of chemical pesticides in high quantity at the rice fields undermine the breeding environments of An. anthropophagus, greatly reduce the mosquito population and therefore the malaria incidence.

[Stabilities of two kinds of Plasmodium antigen substrate slides under different storage temperatures and time].

OBJECTIVE: To observe and compare the inspection effects of antigen substrate slides of Plasmodium cynomolgi (P. c) and Plasmodium falciparum (P. f) on the malaria antibody titer under different storage temperatures and time. METHODS: The densities of Plasmodium of P. c and P. f antigen slides were counted through a microscope, and the average numbers of Plasmodium in each field of vision were calculated. The pooled serum of patients with tertian malaria and falciparum malaria were treated as antibody serum, and the dilutions were from 1:5 to 1:1 280. The two kinds of antibody slides were placed under the temperature of 4-6, 25-27, 33-35 degrees C, and two slides of each kind were selected at Day 3, 5, 7, 10 post-storage to detect the end point antibody titer of malaria by the indirect fluorescent antibody test. Meanwhile, the P. c and P. f antigen slides storing under -20 degrees C for 1 year and 2 years were placed under the aforementioned 3 temperature conditions for 3 d, and the changes of the antibody titers were compared. RESULTS: The densities of Plasmodium in blood cells of P. c and P. f antigen slides were 2.00 x 10(5)/microl and 1.89x 10(5)/microl, respectively, and the average numbers of Plasmodium in each field of vision were 157 +/- 13 and 142 +/- 9, respectively. The end point titers of antibody of P. c and P. f antigen slides placed under the temperatures of 4-6, 25-27, 33-35 degrees C were all on a downward trend after storing for 5 d. The average antibody titers of the two kinds of slides under temperature of 4-6 degrees C and above were 1:440 and 1:80, respectively, and there was a significantly statistic difference between them(t = 1.940, P < 0.05). When P. c and P. f antigen slides storing under -20 degrees C for 1 year were placed under 4-6 degrees C for 3 d, the end point antibody titers were both 1:640, while for those storing under -20 degrees C for 2 years, the end point antibody titers were 1:320 and 1:160, respectively, both the differences were statistically significant (t(P. c) =11.362, P(P. c) < 0.01; t(P. f) = 38.845, P(P. f) < 0.001). CONCLUSION: The end point antibody titers for malaria detection of P. c and P. f antigen slides decrease gradually with the raise of temperature and the prolonging of time for storage.

Vector capacity of Anopheles sinensis in malaria outbreak areas of central China.

BACKGROUND: Both falciparum and vivax malaria were historically prevalent in China with high incidence. With the control efforts, the annual incidence in the whole country has reduced to 0.0001% except in some areas in the southern borders after 2000. Despite this, the re-emergence or outbreak of malaria was unavoidable in central China during 2005-2007. In order to understand the role of the vector in the transmission of malaria during the outbreak period, the vector capacity of An. sinensis in Huanghuai valley of central China was investigated. FINDINGS: The study was undertaken in two sites, namely Huaiyuan county of Anhui province and Yongcheng county of Henan province. In each county, malaria cases were recorded for recent years, and transmission risk factors for each study village including anti-mosquito facilities and total number of livestock were recorded by visiting each household in the study sites. The specimens of mosquitoes were collected in two villages, and population density and species in each study site were recorded after the identification of different species, and the blood-fed mosquitoes were tested by ring precipitation test. Finally, various indicators were calculated to estimate vector capacity or dynamics, including mosquito biting rate (MBR), human blood index (HBI), and the parous rates (M). Finally, the vector capacity, as an important indicator of malaria transmission to predict the potential recurrence of malaria, was estimated and compared in each study site.About 93.0% of 80 households in Huaiyuan and 89.3% of 192 households in Yongcheng had anti-mosquito facilities. No cattle or pigs were found, only less than 10 sheep were found in each study village. A total of 94 and 107 Anopheles spp. mosquitos were captured in two study sites, respectively, and all of An. sinensis were morphologically identified. It was found that mosquito blood-feeding peak was between 9:00 pm and 12:00 pm. Man biting rate of An. sinensis was 6.0957 and 5.8621 (mosquitoes/people/night) estimated by using half-night human bait trap method and full-capture method, respectively. Human blood indexes (HBI) were 0.6667 (6/9) and 0.6429 (18/28), and man-biting habits were 0.2667 and 0.2572 in two sites, respectively. Therefore, the expectation of infective life and vector capacity of An. sinensis was 0.3649-0.4761 and 0.5502-0.7740, respectively, in Huanhuai valley of central China where the outbreak occurred, which is much higher than that in the previous years without malaria outbreak. CONCLUSIONS: This study suggests that vivax malaria outbreak in Huanhuai valley is highly related to the enhancement in vector capacity of An. sinensis for P. vivax, which is attributed to the local residents' habits and the remarkable drop in the number of large livestock leading to disappearance of traditional biological barriers.

Anopheles pseudowillmori is the predominant malaria vector in Motuo County, Tibet Autonomous Region.

BACKGROUND: Malaria is endemic in Linzhi Prefecture in the Tibet Autonomous Region (TAR), but the vector for malaria transmission had never been identified. METHODS: Adult Anopheles spp. were collected in Motuo County, Linzhi Prefecture on the Sino-Indian border in July and August, 2007. Multiplex PCR was adopted for species identification, and a nested PCR approach was used to detect sporozoites in the salivary glands of the mosquitoes. RESULTS: 3,675 mosquitoes of the Anopheles maculatus group were collected and processed for species identification. Among them, 3,602 (98.0%) were Anopheles pseudowillmori and 73 (2.0%) were Anopheles willmori. The Plasmodium vivax SSUrDNA fragment was amplified in two of 360 pooled An. pseudowillmori samples. CONCLUSION: The local An. maculatus group comprises the species An. pseudowillmori and An. willmori. Anopheles pseudowillmori is considered the sole malaria vector in Motuo County in Linzhi Prefecture.

[Species identification of Anopheles maculatus complex in malaria endemic area Motuo County, Tibet].

OBJECTIVE: To identify the species of Anopheles maculatus complex in malaria endemic area Motuo County, Linzhi Prefecture, Tibet Autonomous Region. METHODS: 5190 adult mosquitoes were morphologically identified as An. maculatus complex, and 575 were randomly selected to extract DNA by phenol-chloroform method. According to the rDNA ITS2 variations of An. maculatus s. s., An. willmori, An. pseudowillmori, An. sawadwongporni and An. dravidicus, 5 pairs of specific primers were designed for PCR identification on the species of An. maculatus complex. The PCR products were sequenced in double directions, and homology searches were done over the Web using the program Blast. 22 ITS2 sequence of An. maculatus complex from GenBank was adopted to construct phylogenetic tree with UPGMA method by MEGA (3.1) software. RESULTS: 575 DNA samples were extracted. Among which, 11 were amplified 231 bp An. willlmori fragment (1.9%) and 564 were amplified 119 bp An. pseudowillmori fragment (98.1%). PCR identification, Web homology blast and phylogenetic tree showed same results. CONCLUSION: The predominant anopheline mosquitoes in Motuo County is An. pseudowillmori.

[Investigation on malaria transmission vectors in Motuo County, Tibet Autonomous Region].

OBJECTIVE: To investigate the malaria transmission vectors in Motuo County of Linzhi Prefecture, Tibet. METHODS: Three natural villages with higher malaria incidence rate in Motuo County were selected for the study in July and August, 2007. The anopheline mosquitoes were collected by overnight/semi-overnight trapping indoor and outdoor with human and cattle baits, overnight trapping with ovitrap lights in human dwellings and cowsheds, and by searching in human dwellings in the early morning. The mosquitoes collected were identified morphologically, and the group proportion, density, man-biting rate, blood preference, habits and multiparous ratios were observed. Mosquito larvae breeding place was surveyed, and species of the larvae were identified. RESULTS: A total of 5345 anopheline mosquitoes were captured with 94.71% (5 062/5 345) of An. pseudowillmori, 2.39% (128/5 345) of An. willmori and 2.90% (155/5 345) of An. peditaeniatus. The average density of An. pseudowillmori observed through semi-overnight trapping was 17/per person indoor and 105/per person outdoor. The average man-biting rate of An. pseudowillmori through overnight trapping was 15.80/per person (79/5) indoor and 326.22/per person (1468/4.5) outdoor. The ratio of blood preference to human and cattle through overnight trapping outdoor were 30.51% (714/2340) and 69.49% (1626/2340), and 32.02% (57/178) and 67.98% (121/178) through overnight trapping with ovitrap lights respectively. It suggested that An. pseudowillmori feeding both of the human and cattle blood but preferred to cattle blood. Totally 7 An. pseudowillmori mosquitoes were found in the human dwellings in the early morning, and none of them has digested the engorged blood. The Anopheles larvae were only found in the rice field where 106 larvae were collected, including 62 An. pseudowillmori larvae, An. willmori larvae, and 44 An. peditaeniatus larvae. CONCLUSION: An. pseudowillmori seems qualified as the vector biological perspectives for the local malaria transmission.