栗绛蚧形态生物指标与发生量关系研究

栗绛蚧是长江流域板栗主要害虫之一 ,常引起板栗大幅度减产。该害虫自然种群内雌虫个体间发育差异很大 ,并与其生殖力、死亡率及后代的发生量有密切关系。以雌虫体重、体积及虫体直径为形态指标 ,将雌虫发育分为 、 、 三个等级 ,各级体重 ( x1 )、体积 ( x2 )、虫体直径 ( x3 )分别为 级 ( 0 .1 0 1± 0 .0 0 4 )g,( 0 .1 6 3± 0 .0 1 2 ) m L,( 6 .6 2± 0 .1 7) mm; 级 ( 0 .0 6 52± 0 .0 0 6 ) g,( 0 .1 4 1± 0 .0 0 9) m L,( 6 .1 4± 0 .1 4 ) mm; 级 ( 0 .0 2 9± 0 .0 0 2 ) g,( 0 .0 6± 0 .0 0 4 ) m L,( 5.0 9± 0 .1 3) mm。发生量 ( Y)分别与体重 ( x1 )、体积 ( x2 )、虫体直径 ( x3 )成正相关关系 ,其线性回归方程分别为 :Y=-6 72 .7+ 381 78.7x1 ,Y=-1 1 4 1 .6 + 2 2 86 0 x2 ,Y=-8588+ 1 7359.3x3 ;相关系数 r1 =0 .9939,r2 =0 .8896 ,r3 =0 .96 39。

Construction and biological function of Toxoplasma gondii rop41 gene knockout strain

Objective:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii(T.gondii),which can lead to complications such as encephalitis and ocular toxoplasmosis.The disease becomes more severe when the host’s immune system is compromised.Rhoptry proteins are major virulence factors that enable T.gondii to invade host cells.This study aims to construct a T.gondii rhoptry protein 41(rop41/ROP41)gene knockout strain and preliminarily investigate the biological function of rop41.Methods:Using CRISPR/Cas9 technology,a specific single-guide RNA(sgRNA)for the target gene was designed and linked to a recombinant plasmid.Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes.The recombinant plasmid and the homologous fragments were electroporated into T.gondii,and PCR identification was performed after drug selection and monoclonal screening.Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T.gondii in host cells.Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity.The STRING database was utilized to construct a protein-protein interaction(PPI)network,and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.Results:The T.gondii rop41 gene knockout strain(RHΔku80Δrop41)was successfully constructed and stably inherited.Plaque assays showed that compared with the parental strain,the number of plaques formed by the rop41 gene knockout strain did not significantly decrease,but the reduction in plaque size was statistically significant(P<0.05).After the rop41 gene was knocked out,the invasion ability of T.gondii was reduced,but there was no statistically significant difference in its proliferation ability(P>0.05).The PPI network revealed that ROP41 was associated with other protein kinases and autophagy related proteins.Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction,biosynthesis,metabolism,and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.Conclusion:The T.gondii RHΔku80Δrop41 strain has been successfully constructed.ROP41 primarily affects the ability of T.gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T.gondii and the host.

Biological role of surface Toxoplasma gondii antigen in development of vaccine

AIM： To analyze the biological role of the surface antigen of Toxoplasma gondii（Tgondii） in development of vaccine. METHODS： The surface antigen of Tgondii （SAG1） was expressed in vitro. The immune response of the host to the antigen was investigated by detection of specific antibody reaction to SAG1 and production of cytokines. Mice were immunized with recombinant SAG1 and challenged with lethal strain of Tgondii RH. The monoclonal antibody to r-SAG1 was prepared and used to study the effects of SAG1 on Tgondii tachyzoites under electromicroscope. RESULTS： The mice immunized with recombinant SAG1 delayed death for 60 h compared to the control group. The recombinant SAG1 induced specific high titer of IgG and IgM antibodies as well as IFN-y, IL-2 and IL-4 cytokines in mice. In contrast, IL-12, IL-6 and TNF-α were undetectable. When T gondii tachyzoites were treated with the monoclonal antibody to r-SAG1, the parasites were gathered together, destroyed, deformed, swollen, and holes and gaps formed on the surface. CONCLUSION： SAG1 may be an excellent vaccine candidate against T gondii. The immune protection induced by SAG1 against Tgondii may be regulated by both hormone- and cell-mediated immune response.

Seasonal fluctuation of Myxobolus gibelioi (Myxosporea) plasmodia in the gills of the farmed allogynogenetic gibel carp in China

The seasonal fluctuation of the plasmodia ofMyxobolus gibelioiWu et Wang, 1982 in the gill filaments of the allogynogenetic gibel carpCarassius auratus gibelio(Bloch) in a fish pond in Hubei Province of China was investigated from August 1999 to July 2000. A total of 445 fish was examined; the overall prevalence of the plasmodium infection in the fish was 64.94% and mean abundance of plasmodia was 11.65 ± 27.85 (mean ± SD). Significant seasonal changes in prevalence and mean abundance, with higher levels of the plasmodia infection from late spring to autumn, were observed.

CD2 deficiency partially prevents small bowel inflammation and improves parasite control in murine Toxoplasma gondii infection

To investigate whether bowel inflammation and/or parasite control is altered in the absence of the T cell adhesion molecule CD2.

Low Sero-Prevalence of Toxoplasma gondii IgG and IgM Antibodies in HIV Sero-Positive Patients Attending National Hospital, Abuja, Nigeria

Several researchers have investigated the association of numerous opportunistic pathogens with HIV, little is documented on its association with T. gondii in our environment. We investigated the prevalence of T. gondii immunoglobulins G and M （IgG and IgM） in HIV positive individuals in relation to their cluster of differentiation 4 （CD4） cells count. IgG, IgM and CD4 were assayed using enzyme immunoassay （EIA） and flowcytometry respectively. 341 HIV positive individuals were studied in the present research, 30 （8.7%） of them had T. gondii IgG and IgM, 297 subjects had CD4 cells count a range of 200-400 cells/μL, 27 （9.7%） and 2 （0.6%） of which had T. gondii IgG and IgM respectively. Of the 44 HIV positive subjects with CD4 〉 400 cells/μL, one （2.2%） was positive for T. gondii IgG. In the control group, all the 177 had CD4 〉 400 cells/μL of which, one （0.5%） had T. gondii IgG. The prevalence of T. gondii infection was significantly higher in HIV positive individuals than in controls （P 〈 0.05）. Male subjects in the age bracket 18-30 years had significantly higher prevalence when compared to other groups （P 〈 0.05）. Although the present findings revealed a low prevalence of T. gondii antibodies in HIV infection, this suggests that a differential toxoplasmosis diagnosis is also necessary in cases of encephalitis in HIV infection.

CORAL AS A CARRIER FOR RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2

By combining coral with recombinant human bone morphogenetic protein-2 (rhBMP-2), rhBMP-2/coral composite was obtained in this study. Following implantation of the composite into the muscle pouches of mice, cartilage growth was induced in the pores or on the surface of the implants at one week, woven bone at three week and lamellar bone with bone marrow at six week, and coral was absorbed partially. The induced formation of endochondral bone was time-related and rhBMP-2 dose-related. The results of this study indicate that the composite possesses a superior ability of osteogenesis, and coral acts as one of the most suitable rhBMP-2 slowrelease carriers currently available. The composite will be a new type of bone substitute to be used in orthopaedics and maxillofacial surgery.

Area-wide Detection of Anti-Toxoplasma gondii Antibodies in Dairy Animals from the Khartoum State, Sudan

Toxoplasmosis is an important cause of reproductive failure in man and farm animals resulting in significant socio-economic losses worldwide. The aim of this study was to avail large scale data-regarding the sero-prevalence of T. gondii infection in dairy animals from the Khartoum State, Sudan using the commercially available Latex Agglutination Diagnostic kits. The study revealed that the overall seroprevalence of T. gondii infection at herd level of dairy animals in the State was 92.7%. The within herd seroprevalence was ranging from 8% up to 100% with mean of 51.3% in different herds of different dairy animals species in the State. Only 6 herds, 3 herds, 3 herds and 1 herd of cattle, camel sheep and goat respectively were found to be clean from anti-T, gondii antibody in the present study. Interestingly, most of the clean herds （12 herds） are located in the Khartoum district. The differences between the three districts, the seven localities and the four animal species were statistically highly significant （P 〈 0.01）. The overall sero-prevalence of T. gondii infection in dairy animals--at individual level--was 45.3%. Sheep scored the highest seroprevalence rate （75.0%） followed by goats （64.0%）, camels （54.1%） and cattle （40.9%） with high statistically significant differences （P 〈 0.01）. The highest level of antibody titration was reported in sheep and goats. This is the first area-wide and large scale report on seroprevalence of T. gondii infection in dairy animals from the Sudan. It was concluded that dairy animals in the Sudan are widely exposed to T. gondii and people in the Sudan should be aware of the possibility of hyper-prevalence of human toxoplasmosis through these food animals. Research on clinical toxoplasmosis in dairy animals and human in the Sudan is recommended to evaluate the role T. gondii infection in economical losses in dairy farm industry and for building strategy of sustainable toxoplasmosis control.

Modus operandi of transnational transfer pricing for window dressing

A goal of transfer pricing may be to maximize after tax revenue by setting transfer prices that reduce the total tax paid. ＂Transfer pricing＂ is the pricing of products or services provided by one division to other division of the same corporate entity. Most of the corporate entities are using the method of ＂Window dressing＂, which is a technique used in preparation of financial statements of corporate entities. A transnational corporation is any enterprise that undertakes Foreign Direct Investment （FDI）, owns or controls income gathering assets in more than one country, produces goods or services outsides its country of origin, or engages in international production. Profitability of the transnational corporate entities is being manipulated by the technique of transfer pricing. Abuse of transfer prices is a key tool used by the corporate entities to think that they have virtually no profit; hence, they shouldn＇t pay any taxes. India needs to realize the fundamental need for co-operation among tax administrations in order to remove the obstacles that international double taxation presents to the free movement of goods, services and capital between various countries. In this context, one needs to consider that transactions among associated enterprises may take place under different conditions from those taking place among independent enterprises, while enforcing the act of transfer price mechanism. This paper focuses on transfer pricing and its implications in transnational transactions.

A New Species of Parodontophora(Nematoda:Axonolaimidae) from the Intertidal Zone of the East China Sea

This study described a new species of free-living nematode discovered in the intertidal mudflat of Ximen Island,East China Sea.The new species,designated Parodontophora longiamphidata sp.nov.,was characterized by a cylindrical body with tapering extremeties;cuticle smooth without somatic setae;four short cephalic setae;cylindrical buccal cavity with six clawlike teeth at the top of stoma;pharynx cylindrical with widened base;amphidial fovea crook-shaped with elongated scalariform branch extending past level of base of pharynx and ventral gland;ventral gland cell long-oval shaped located posterior to pharyngo-intestinal junction;excretory pore at level of middle of buccal cavity;tail conico-cylindrical with enlarged tip;three caudal gland cells,male spicules arched with cephalic proximal end and tapered distal end;gubernaculum with dorso-caudal apophysis;female with two opposed outstretched ovaries;and vulva at slightly post-midpoint of body length.This new species was close to P.wuleidaowanensis Zhang,2005 and P.polita Gerlach,1955 in terms of long amphidial fovea branch.The newly found species was easily distinguishable from the two documented;its amphidial fovea branch(255–290 μm versus 72–106 and 125–150 μm) was obviously longer.Key to the Parodontophora species with a longer amphidial fovea branch was given.

Immunity induced by DNA vaccine of plasmid encoding the rhoptry protein 1 gene combined with the genetic adjuvant of pcIFN-γ against Toxoplasma gondii in mice

Objective To construct the eukaryotic expression recombinant plasmid, pcIFN γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3 rhoptry protein 1 (pc ROP1) combined with pcIFN γ against Toxoplasma gondii (T gondii) infection in mice Methods A fragment of the IFN γ gene was directly inserted into the pcDNA3 plasmid and identified by two restriction endonucleases digestion pcIFN and pcROP1 DNA was injected into the left leg muscle of mice at a dosage of 100?μg, and a booster vaccination was given at the same dosage after two weeks Control groups were injected with pcDNA3 blank plasmid or normal saline At 30, 50 and 70 days after booster injection, kinetic tests were carried out: MTT assay for the proliferation response of T lymphocyte cells and the activity of NK cells, sandwich ABC ELISA for the determination of IFN γ, IL 2 and IL 10; a serum enzymetic aassay for nitric oxide (NO) in sera and ELISA for the titer of IgG antibody in sera Results The recombinant plasmid, pcIFN γ was constructed The proliferation response of spleen T lymph cells, NK cell killing activity, and serum levels of IFN γ, IL 2 and NO in mice injected with pcROP1 and pcIFN γ were higher than in those injected with pcROP1 alone There was no difference in IgG antibody levels between the two groups Conclusion The genetic adjuvant, pcIFN γ, could enhance the cellular immune response induced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection

Construction of a recombinant plasmid harbouring the rhoptry protein 1 gene of Toxoplasma gondii and preliminary observations on DNA immunity

Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3. Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection and boosted after 2 weeks. pcDNA3 and normal saline were used as control. 30, 50 and 70 days after the second immunization, NK cell activity, T lymphocyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The number of CD4+ T cells exhibited no obvious increase compared with that of the control, but CD8+ T cells were obviously increased (P<0.05). At 90 days after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100). Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune responses in immunized mice.

Evolution of discocephalid ciliates:Molecular,morphological and ontogenetic data support a sister group of discocephalids and pseudoamphisiellids (Protozoa,Ciliophora) with establishment of a new suborder Pseudoamphisiellina subord.n.

Discocephalids and pseudoamphisiellids are possibly two of the most confused groups among hypotrichous/euplotid ciliates regarding their systematic position and phylogenetic relationships.The former were often regarded as related to euplotids while the latter,in the absence of molecular data,were mostly assigned to the urostylid-like hypotrichs.In the present work,the small subunit rRNA genes of several rarely observed discocephalid and pseudoamphisiellid genera were analyzed to obtain insights into the phylogenetic relationships of these highly ambiguous Spirotrichea.Four different tree reconstruction algorithms yielded nearly identical topologies,which indicated both groups belong to the same assemblage.This assemblage is clearly isolated as a deep-branching clade and invariably positioned between Euplotida and Hypotricha.The sister group relationship of the Pseudoamphisiellidae and Discocephalidae supports the previous suggestion that they might represent an ordinal taxon,the Discocephalida.Both morphological and morphogenetic features indicate that the pseudoamphisiellids should be placed in the order Discocephalida but as a sister group to other typical discocephalids.Thus we propose establishing a new suborder,Pseudoamphisiellina subord.n.The new taxon is diagnosed by the following characteristics:(i) two distantly separated midventral rows that are morphogenetically formed with an urostylid mode;(ii) absence of the "frontoterminal row",which is formed from the posterior-most frontoventral-transverse cirral anlage in all other typical urostylids;(iii) numerous caudal cirri that derive from each of the dorsal kinety anlagen;(iv) right marginal row that has a unique de novo origin;and (v) inhabiting periphytic communities.The validity of the suborder Pseudoamphisiellina is firmly supported by molecular data.

Protective effect of DNA-mediated immunization with liposome-encapsulated GRA4 against infection of Toxoplasma gondii

The dense granule protein 4 （GRA4） is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3, 1-GRA4 （pGRA4）, encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine （PC）, 8 mmol/L dioleoyl phosphatidylethanolamine （DOPE）, and 4 mmol/L 1,2-diodeoyl-3-（trimethylammonium） propane （DOTAP）. C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay （ELISA） of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon （IFN）-7 and interleukin （IL）-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th 1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 103 tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii.