ABSTRACT
Objective: To understand the relationship between chloroquine resistance and the virulence of Plasmodium berghei. Met hods: Dynamic changes of histopathologic features of livers, spleens, brains, hearts, lungs and kidneys of mice infected with the chloroquine-sensitive (N) and the chloroquine-resistant (RC) strains of P. berghei were compared. Results: In mice infected with the N strain, deposition of heavy hemoz oin in livers and spleens, congestive edema in lungs, and congestion and embolis m in the brain capillaries were observed. The histopathologic features revealed ac ute inflammatory reaction. In mice infected with the RC strain, histopathologic variations of livers and spleens were associated with changes of parasitemia. In terstitial pneumonia was displayed in lungs. There were chronic histopathologic changes of the organs in the mice infected with RC strain. Conclusion: The mice infected by the N strain with potent virulence die due to adher ence of the erythrocytes to microvascular endothelia and embolism of the microva scula, especially in their brain. Immune responses of the mice infected by the R C strain with poor virulence may be a delayed-type hypersensitive inflammation a ssociated with CD4+Th1 at an early stage of the infection, but may become anti body-dependent immune response assisted with CD4+Th2, which play a key role in elimination of the malaria parasites at later stage of the infection.
ABSTRACT
To insight into genetic differences between chloroquine-resistant line (RC) and chloroquine-sensitive strain (N) of Plasmodium berghei. MethodsAfter continous cbloroquine-pressure upon RC line at higher dosage (50mg/kg. d) ,total RNAs from the RC line and the N strain of P. berghei were isolated for simplified differential display reverse transcript polymerase chain reaction (sDDRT-PCR ). The generated differential fragments had been repetitively rescued and identified by PCRs before one pair of suspected differential fragments (N25 and R25 )were cloned and sequenced. Then, their sequences were analyzed through PC-gene program and BLAST search. ResultsThough the identity of two nucleotide sequences of N252 and R251 ,cloned separately from the N25 and R25 fragments, were up to 99.8％ ,their predicted amino acid secondary structures were quite different due to multiple mutations. Compared with the published sequences in GeneBank,EMBL,DDBJ and PDB database ,no similar gene was found ,using BLAST search. However their partial nucleotide sequences (62nt from query 128nt to189nt bore highly homology to part sequence(from 1053nt to1114nt)of rattus norvegicus mRNA for phospholipase B,up to 93.5％ in N251 and 91.9％ in N252 respectively. ConclusionIt is feasible to isolate chloroquine-resistant related genes by using simplified DDRT-PCR combined repetitively rescuing and PCR identifying the interest differential DNAs together with sequence analyses.
ABSTRACT
Objective To isolate and identify genes related to malaria parasite infection in vector mosquito, and to explore the mechanisms. Methods Anopheles stephensi infected with Plasmodium yoelii was used as tester (T) group, while uninfected but normal blood fed as driver (D) one. Engorged female mosquitoes of two groups were collected separately at 24 hours after biting. An enriched subtractive cDNA pool was generated through the course of suppression subtractive hybridization (SSH) and selective PCR amplification. The subtracted library was screened by hybridization using T and D cDNA mixture as probes, respectively. The positive clones, which produced stronger signal when probed with T than with D, were sequenced and their sequence homologues in GenBank database were searched with BLAST by internet. Results The analysis of subtraction efficiency showed that the differentially expressed genes in T comparing to in D were enriched significantly. In dot blot screening, 24 of 58 randomly selected clones (41.4%) were shown up regulation in malaria infected mosquitoes. The BLAST search of 23 genes revealed that 12 were homologous to functionally known genes, 4 were homologous to functionally unknown entries, and 7 were novel without any relatives. Nine of the 23 genes (39.1%) also hit homologous sequences in the An. gambiae EST database generated from an immune competent cell line treated with lipopolysaccharide (LPS). Conclusion An enriched cDNA pool of the mosquito genes which up regulated responsively at the early stage of malaria parasite infection was obtained. Expression screening against the pool indicated that various biochemical processes and mechanisms might be involved in the response of mosquito to parasite infection, especially those related with the innate immune system and energy metabolism.
ABSTRACT
Objective To determine sequence of sporogony stage-specific (S type) 18S ribosomal RNA gene of Plasmodium yoelii (P.y) By265 strain, and by using it to detect the malaria parasites within vector mosquito. Methods A pair of conserved DNA primers, universe primer (Pu) and reverse transcription one (Pr), was designed and synthesized according to sequence of the 18S rRNA gene of Plasmodium berghei (P.b). The segment of the S type 18S rDNA of P.y was amplified by reverse transcript-polymerase chain reaction (RT-PCR) from dissected midguts of Anopheles stephensi infected with P.y on the 7th day after infective blood-meal, and its sequence was then determined. One P.y sporogony stage-specific primer (Pys) was selected according to the sequence. Using this primer and Pr, the parasites within mosquitoes were semi-quantitatively detected through RT-PCR between 1-7 d post-infection. Results The length of the amplified segment was 920 bp. Alignment in match region of the 18S rDNA among S type of P.y (PyS), S type of P.b (PbS) and asexual blood stage-specific one of P.y (PyA) revealed that the similarity between the former and the latter two reached 95^3% and 94^0% respectively. The density of amplified band was significantly concordance with the intensity of oocyst in the midgut. Sensitivity of RT-PCR method was higher than that of the traditional dissection and oocyst observation also. The assay could detect the 18S rRNA molecule of the parasites on the third day post-infection while their oocysts were difficult to be recognized under an optical microscope at that time. Conclusion This S type 18S rDNA sequence in P.y species was first reported (AF266261). As a molecular marker, it could be applied to monitoring the parasite development in its vector at an earlier stage semi-quantitatively with an adequate sensitivity and specificity.
ABSTRACT
Objective: To investigate virulence differences of the malaria parasites and responses of their hosts to the malaria infection between chloroquine sensitive (N) and resistant (RC) strains of Plasmodium berghei . Methods: Ultrastructural pathological alterations of liver, kidney and lungs of ICR mice infected with the N or the RC strains were investigated. Results: Congestion and more parasite infected red blood cells in hepatic sinusoids, renal interstitium, capillaries of the liver, kidney and lungs in late period of the infection with the N strain were observed. Necrosis of hepatic cells in liver, juxtaglomerular cells and epithelial cells of renal tubules in kidney, type Ⅰ and type Ⅱ alveolar cells in lungs occurred. Membranes of these cells were broken, resulting in cytoplasm missing and mitochondria swelling infected with the N strain. While degeneration of some hepatic cells of liver, epithelial cells of renal tubules, alveoli cells of lungs appeared during late period of the infection with the RC strain of P. berghei . It was observed that some hepatic cells recoved, the glomeruli basement membrane and mesangial cells contained electron dense deposits,alveoli septa were undergoing the proliferation and infiltration of lymphocytes, macrophages and neutrophilic granulocytes. Conclusion: The N strain is more virulent than the RC strain of P. berghei . Ultrastructures of the liver, kidney and lungs of the mice infected with the N strain show cellular necrosis, while those infected with the RC strain mainly reveal cellular degeneration.
ABSTRACT
Objective: To study the mechanism that erythrocytic stages Plasmodium berghei chloroquine resistant strain (RC strain) were eliminated in their hosts. Methods: Interactions of leukocytes with the parasites in mice livers infected with the RC strain or chloroquine sensitive strain (N strain) of P. berghei were studied by electron transmission microscopy. Results: None of leukocytes proliferated and infiltrated in mice livers infected with the N strain. Whereas in mice livers infected with the RC strain, proliferation and infiltration of monocyte macrophages, lymphocytes and nuetrophils occurred within portal areas and hepatic sinusoids with sequestration of numerous parasitized erythrocytes. The activated monocyte macrophages adhered to the parasitized red blood cells with their surface membranes. A lot of the parasites became crisis form within the parasitized erythrocytes which directly contacted with the phagocytes or not. Phagocytosis of free merozoites by macrophages was rarely revealed, but of the whole parasitized erythrocytes was not found. Conclusion: The crisis form of the parasites induced by the activated monocyte macrophages, rather than the direct phagocytolysis, is mainly responsible for the elimination of the P. berghei RC strain in their hosts. [
ABSTRACT
Objective: To investigate the feature of crisis at asexual stages of chloroqine resistant strain (RC) of Plasmodium berghei . Methods: By means of optical microscope,transmission electron microscope and agarose electrophoresis, microscopic and ultramicroscopic architectures, and genomic DNA electrophoresis at asexual stages of the RC and chloroquine sensitive (N) strains of P. berghei with rising parasitemia and the RC strain of the parasite with declining parasitemia were observed in ICR mice. Results: Under microscope, different developmental stages asexual parasites with normal forms were found in blood smears from the mice infected by the N and RC strains with rising parasitemia, whereas there were a few of the asexual parasites most of which were contracted and vacuolated from the mice recovered from infection by the RC strain. Using electron microscope, intact cytoplasm membranes of the parasites paralleling with parasitophrous membrane of erythrocytes, nuclear laterad, obvious metabilic window and mitochondrions, ribosomes, endoplasmic reticulum in cytoplasm were recognized in erythrocytic stages of the RC and N strains with rising parasitemia. There were rhoptries in merozoites and food vacuoles in trophozoites of the N strain. Erythrocytic parasites of the RC strain from recovered mice with declining parasitemia were spherical or elliptical, with intact cytoplasm membrane, contracted nuclei and concentrated cytoplasm, without mitochondrion membranaceous structure and metabolic windows. The genomic DNA electrophoretogram of the RC strain parasites in the recovered mice showed a ladder pattern, which differed evidently from the parasites of the RC strain with rising parasitemia and the N strain which showed a single band. Conclusion: It is confirmed that crisis form of asexual stages of the RC strain is apoptosis.
ABSTRACT
The nocturnal activities of, biting midges, mainly Culicoides nipponensis Toku-naga and C. schultzei (Enderlein) of Shanghai district were observed. Catches were hourly made with an ultraviolet light trap over nights. Using electronic computer and stepwise regression technique, the relationship between the caught number of the midges and meteorological factors was analysed. The primary comparison of 3 types of the mathematical models with a general regression analysis was made. The results obtained are as follows:1.Wind is the most important weather factor which affects the activities of both the midges. And the temperature is the second. The suitable weather conditions for their activities are windspeed of
ABSTRACT
Objective: To understand the morphologic foundation for differences in drug-resistance, virulence and immunity between chloroquine-sensitive (N) and chloroquine-resistant (RC) strains of Plasmodium berghei. Methods: The RC strain and the N strain were compared concerning the formation and morphologic features of digestive vacuoles and haemozoin with transmission electron microscope. Results: There was a single large digestive vacuole and multiple micro-single-mem-braned vacuole-like structures in the trophozoites of the N strain, and haemozoins centralized and fused during their schizo-gony were situated under the plasma membrane. Whereas there were few digestive vacuoles in the trophozoites of the RC strain, but with multiple micro-single-membraned vacuole-like structures instead. The RC strain formed obviously less hemo-zoins than that of the N strain and the hemozoins were not centralized and fused during the schizogony. Conclusion: The RC strain forms multiple single-membraned food vacuole-like structures in the trophozoites, and has different mechanism for detoxifying free heme with N strain and the features may be the foundation for the difference in drug-resistance, virulence, immunity between RC strain and N strain of Plasmodium berghei.