A 43-kDa circulating filarial antigen fraction of Wuchereria bancrofti in immunoprophylaxis against Brugia malayi in jirds

A 43 kiloDaltan (kDa) antigen fraction (CFA₂-6) isolated from microfilaraemic plasma of bancroftian filarial patients showed selective reactivity with sera samples collected from endemic normals. Antibodies raised against this antigen showed a strong reactivity with the surface of Brugia malayi infective larvae as well as microfilariae. Similar antigenic determinants were detected in the parasite extracts, but not in the excretory–secretory products. Further analysis was done on the immunoprophylactic potential of CFA₂-6 in inducing immunity against Brugia malayi in Meriones unguiculatus (jird) in vivo. A strong protective response of approximately 84% was observed against the development of the filarial parasite in the jirds immunized with CFA₂-6. The immunized jirds also showed a significant clearance (87%) of microfilariae inoculated intravenously. Approximately 65% of infective larvae failed to survive in jirds transferred with anti-CFA₂-6 serum compared to the jirds transferred with sera from the control jirds. Passive transfer of anti-CFA₂-6 antibody to the jirds followed by intravenous inoculation of microfilariae resulted in the reduction of 77% of circulating microfilariae. This study suggests that the 43-kDa CFA₂-6 could stimulate a strong protective immune response against infective larvae and microfilariae in experimental animals.     

Pleural cellular reaction to the filarial infection Litomosoides sigmodontis is determined by the moulting process, the worm alteration, and the host strain

The filarial nematode Litomosoides sigmodontis model was used to decipher the complex in vivo relationships between filariae, granulomas and leukocytes in the host's pleural cavity. The study was performed from D5 p.i.: to D47 p.i. in resistant C57BL/6 mice, to D74 p.i. in susceptible BALB/c mice, and to D420 p.i. in permissive jirds. We showed that, during the first month, leukocytes only clustered as granulomas around shed cuticles (exuviae) and with eosinophils as the major constituents. In addition, carbohydrates residues became abundant on exuviae only, suggesting a glycan-dependent mechanism of eosinophil attachment. Neutrophils were absent from the pleural cavity of all rodents and from the murine granulomas, but they made up 25% of the granuloma cell population in jirds. After the first month of infection granulomas formed around developed adult worms and morphological evidence suggested that leukocytes preferentially clustered around altered, but still motile, worms. No carbohydrates were detected on these worms and neutrophils were abundant in those granulomas. Finally, a rare third type of granuloma was observed in the resistant mice only; they contained young newly moulted adult worms; typically these granulomas were attached to the lateral lines of the worm via eosinophils; this feature correlated with the persistence of carbohydrate residues on the worms' lateral lines. Neutrophils were always in low proportion in all granulomas from resistant mice, suggesting difference in their adhesive properties in these mice. In vitro neutrophil recruitment in resistant mice was similar to that observed in susceptible mice although they expressed less cell surface CD11b.     

The effect of cyclosporin A on Angiostrongylus cantonensis infection and eosinophilia in mice

Male BALB/c mice were infected with Angiostrongylus cantonensis and at various times p.i. treated with cyclosporin A (CsA) either for 5 days continuously or intermittently, or for 12–16 days on alternate days. They were monitored for peripheral blood eosinophilia and at necropsy examined for CSF and bone marrow eosinophilia, and worm recovery. CsA treatment provoked a transient inhibition of peripheral blood eosinophilia in all groups examined, followed by rebounding eosinophilia. There was no significant difference in bone marrow and CSF eosinophilia between CsA- and vehicle-treated groups. Mice treated with CsA on alternate days yielded lower intracranial worm recovery with small-sized worms at days 7, 21 and 30 p.i. than vehicle-treated groups did. No significant reduction in worm recovery was noted in mice treated for 5 days either continuously or intermittently, although worms, especially female ones, harvested from groups treated with CsA from days — 1 to 3 or from days 13 to 17 were significantly smaller than those from vehicle-treated groups. CsA-treatment suppressed blastogenic responses of spleen cells against Con A or worm antigen at days 7 and 21 p.i. In vitro treatment of the 3rd stage larvae with CsA did not adversely influence survival of A. cantonensis in mice. These data indicate that CsA exerts antiparasitic effects against A. cantonensis in mice.     

Population dynamics of the parasitic stages of Oesophagostomum dentatum in pigs in single and trickle infections

A mathematical model was developed to describe the dynamics of the parasitic stages of Oesophagostomum dentatam in pigs. An immigration-death model with constant establishment, development and death rates was fitted to L3, L4 and adult worm burdens observed in a single-infection experiment. Female worm length was modelled by a function of worm age and total worm burden, while worm egg production (eggs per gram faeces per female worm) was modelled by a function of worm age and worm length. The model was then used to predict worm burdens observed in a trickle-infection experiment. The predicted worm burdens were much higher than those observed, suggesting that worm death rates were higher during the trickle infection. After increusing worm death rates to fit the observed worm burdens, female worm lengths and egg production in the trickle infection were predicted. At the medium- and high-dose rates, predicted worm lengths and, thus, egg preduction were lower than observed, while at the low-dose rate predicted egg production was too high. It appeared that in the trickle infections, total worm burden had less influence on observed female worm length and egg production than in the single infections. The results suggest that the demography of O. dentatum in pigs differs between single and trickle infections.     

Millardia meltada, a new host for Acanthocheilonema viteae and a simple technique for separation of microfilariae from peripheral blood

, and 1992. Millardia meltada, a new host for Acanthocheilonema viteae and a simple technique for separation of microfilariae from peripheral blood. International Journal for Parasitology 22: 1165–1168. Millardia meltada were infected with Acanthocheilonema viteae and examined for their susceptibility. The morbidity of infected M. meltada was low compared with that of jirds. On day 47 post-infection (p.i.), 13 of 14 M. meltada developed microfilaremia. Male M. meltada then showed gradually increasing microfilaremia with a peak level of 7000 per 30 μl blood at week 20 p.i., which was much higher than that (3000) of male jirds. In contrast, microfilarial densities of female M. meltada were markedly low with a peak level of 200 during weeks 10–12 p.i. A simple centrifugation technique with Lympholyte-M was devised for microfilarial separation from the peripheral blood of infected M. meltada and yielded approximately 17 × 10⁵ viable microfilariae from 1 ml of blood. This method also makes it possible to collect microfilariae from the same individuals repeatedly. M. meltada, coupled with this microfilarial separation technique, serves as a useful animal model for microfilarial studies of A. viteae.     

Brugia pahangi: granulomatous lesion development in jirds following single and multiple infections

The development of adult worm burdens and microfilaremias were determined in jirds which received 2, 3, or 4 subcutaneous inoculations of 50 Brugia pahangi infective larvae. Parasite burdens in multiply inoculated jirds were compared to those in four different groups of jirds which received single inoculations of 50 infective larvae. One of each of these singly inoculated groups was infected on the same day that one of the inoculations was given to the multiply infected jirds. Thus, the duration of the infections in the four groups of jirds receiving one inoculation was 54, 118, 189, and 254 days. The development of lymphatic lesions and granulomatous hypersensitivity to B. pahangi antigen was assessed in all jirds at necropsy. The percentage recoveries of adult worms and their locations did not differ in the singly inoculated jirds with infections of different durations. A protective resistance to reinfection, as measured by adult worm recovery in multiply infected jirds, did not occur. The lymphatic lesion scores and numbers of intralymphatic thrombi was greatest in singly inoculated jirds examined 54 days after infection. Pulmonary granuloma areas around adult filarial antigen coated beads embolized in the lungs of jirds 3 days prior to necropsy were also greatest in singly inoculated jirds examined 54 days after infection. Using criteria of lesion scores and lymph thrombi numbers to assess lymphatic lesion severity, a decrease in lesion severity as well as pulmonary granuloma size around antigen coupled beads was seen by 118 days after infection in singly inoculated jirds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice. International journal for Parasitology 3: 773–781. Babesia rodhaini infection was compared in irradiated, splenectomized and control mice. Although irradiation reduced the weight of the spleen by as much as 95 per cent, this reduction in size did not result in parasitaemia levels comparable to those seen in splenectomized mice, which were consistently higher. Parasitaemias were similar in irradiated and control mice, but the mean survival time in control mice was longer than that of irradiated or splenectomized mice, which were comparable. Splenectomy generally resulted in higher parasitaemias than those seen in non-splenectomized mice.

Since B. rodhaini has a predeliction for invading reticulocytes, the apparent failure of irradiated mice to develop parasitaemias comparable to those of splenectomized mice, may have been due to the selective destruction of these immature red cells by irradiation.     

The Mongolian gerbil (Meriones unguiculatus) as a laboratory host for the cystic stage of Echinococcus granulosus of British horse origin

The Mongolian gerbil (Meriones unguiculatus) has been found to be susceptible to secondary equine hydatidosis and fertile cysts can be routinely produced with the subsequent serial passage through further generations. Mice, rats, rabbits, guinea-pigs, hamsters, cotton rats and voles proved to be refractory to infection. Since some of these hosts have been infected experimentally with horse material in other countries, by other workers, these results suggest that strain differences may exist between Echinococcus granulosus from the same host species subjected to geographical separation.     

Brugia pahangi: effects of duration of infection and parasite burden on lymphatic lesion severity, granulomatous hypersensitivity, and immune responses in jirds (Meriones unguiculatus)

The effects of Brugia pahangi infection duration and parasite burden on parasite-associated inflammatory and immune responses were determined over a 181-day period in jirds receiving from one to eight inoculations of infective larvae. Multiple infections did not produce a protective resistance to reinfection as determined by adult worm recovery at necropsy. Intralymphatic granulomatous lesions, lymph thrombi, were first seen at 48 days post initial inoculation (DPI). The numbers of lymph thrombi reached peak levels in singly inoculated jirds at 90 DPI and significantly decreased to low levels by 160 DPI. The ratio of lymph thrombi to adult worms recovered from the spermatic cord lymphatics followed a similar pattern. Sizes of renal lymph nodes, which drain lymphatics containing parasites, followed a temporal pattern of increase and decrease similar to that of lymph thrombi numbers. Peak granuloma areas around antigen-coated beads embolized in lungs were seen at 27 DPI. Granuloma areas around antigen-coated beads began to decrease after 69 DPI and reached sizes not significantly different from uninfected controls by 118 DPI. Multiple inoculations of infective larvae and increasing worm burdens did not affect the pattern of granulomatous response to antigen-coated beads. Eosinophilia of singly and multiply infected jirds peaked at 26 DPI. Eosinophilia of singly infected jirds returned to normal levels by 103 DPI but those of multiply infected jirds remained elevated until 160 DPI. Lymph node cell blastogenic responses to antigen were greater than those of splenocytes at all time intervals measured. However, significant differences in stimulation indexes between groups with different infection durations were not seen with either cell type. Antibody responses to somatic adult worm antigen as measured by ELISA reached near peak levels by 48 DPI and remained elevated for the course of the study in all infected jirds. The decrease in lymphatic lesion severity seen in chronically infected jirds temporally corresponds to the decrease in granulomatous reactivity measured around antigen-coated beads embolized in the lungs. This observation suggests that host and/or parasite factors associated with these two phenomena may be similar. Although these decreases may be the result of down-regulated immune responses, corresponding decreases in antibody levels and blastogenesis of lymphocytes stimulated by crude worm extracts were not observed in chronic infections.     

Brugia pahangi: comparative susceptibility of the Mongolian jird, Meriones unguiculatus, and the PD4 inbred hamster, Mesocricetus auratus

The susceptibility of Mongolian jirds, Meriones unguiculatus, and PD4 hamsters, Mesocricetus auratus, to Brugia pahangi was compared based on the percentage adult worm recoveries, mean microfilaremia levels, and adult worm lengths. Fourteen male jirds and seventeen male PD4 hamsters were each inoculated subcutaneously in the left inguinal region with 90-100 L3 of B. pahangi and necropsied 130-150 days after inoculation. There were no significant differences between jirds and hamsters in mean adult worm recoveries (24.7 vs 25.4%) and prepatent periods (69.9 vs 77 days after inoculation). In hamsters, 85% of recovered worms were found in the heart and lungs and 15% were found in genital lymphatic vessels. In jirds, distribution of recovered worms was 66% in genital lymphatics, 23% in the heart and lungs, 8% in the peritoneal cavity, and 3% in lymphatic vessels in other sites. The mean microfilaremia level in jirds (16.5/20 microliter) was significantly higher than in hamsters (8.7/20 microliter. Female worms in the genital lymphatics of jirds were significantly longer than female worms in the genital lymphatics of PD4 hamsters (33.5 vs 27.3 mm). Lengths of worms in other locations were similar between the two species.     

Apodemus sylvaticus, a new host for Acanthocheilonema viteae (Nematoda: filarioidea)

Susceptibility of Apodemus sylvaticus and A. agrarius to infection with Acanthocheilonema viteae was compared with that of hamsters and jirds. Microfilaremia in A. sylvaticus was first noted on day 52 post-infection (p.i.) and lasted during the course of the study (up to day 150 p.i.). Maximum microfilaremic levels (female worm basis) of A. sylvaticus [mean +/- S.D. (n) = 690 +/- 1288(6)] were considerably higher than those of hamsters [16 +/- 18(6)] and jirds [51 +/- 25(5)]. Adult worm recovery in A. sylvaticus ranged from 2 to 40% of the number of infective larvae inoculated. Worm development in A. sylvaticus resembled that in hamsters and jirds. In contrast, microfilaremia was not detected in, nor adult worms recovered from A. agrarius throughout the study.     

去泛素化酶OTUB1肝脏特异性基因敲除小鼠模型的构建与表型分析

目的:建立OTUB1肝脏特异性基因敲除小鼠模型,初步分析其表型并研究OTUB1基因与肝脏代谢的关系。方法:利用Cre/Loxp系统构建条件性基因敲除小鼠模型,即将OTUB1~(fl/fl)转基因小鼠与Alb-Cre小鼠杂交,子代自交,得到OTUB1肝特异性基因敲除小鼠并进行鉴定。取同窝对照小鼠(control,NC)和肝特异型基因敲除(hepatic-specific OTUB1 knockout,HCKO)小鼠,通过PCR和免疫印迹(Western blot),确证OTUB1肝脏特异性基因敲除小鼠模型是否成功构建。通过组织病理学方法,分析主要组织器官的形态以及是否存在自发的病变;通过血清生化指标检测肝脏脂代谢水平;通过血糖耐受实验(GTT)分析HCKO小鼠对血糖的控制。结果:基因组测序和Western blot检测结果显示HCKO小鼠肝脏中OTUB1被敲除,其他组织中OTUB1表达水平无变化,证明OTUB1肝脏特异性基因敲除小鼠模型构建成功。HCKO小鼠出生正常,各组织器官无异常,生化指标中总胆固醇水平明显降低,表明OTUB1影响肝脏脂代谢水平。糖耐受实验中HCKO小鼠血糖回落迅速,表明敲除OTUB1影响肝脏血糖调节稳态。结论:应用Cre/Loxp技术成功建立OTUB1肝脏特异性基因敲除小鼠模型,为研究OTUB1在肝脏的生理功能和调控机制提供了重要的动物模型。     

Molecular cloning and expression of a Schistosoma japonicum tegumental membrane-associated antigen from Japanese strain

Diagnosis and vaccine development form the major focus in creating strategies for the control of schistosomiasis. In this study, we established an IgG1 mouse monoclonal antibody (MoAb), SJA111, which strongly reacted with 23–25-kDa Schistosoma japonicum tegumental-associated membrane proteins, but not with eight other parasitic antigens. A λgt 11 cDNA library from the Japanese strain of the Schistosoma japonicum adult worm was screened with SJA111 as a probe. A single positive clone was isolated and the nucleotide sequence of the isolated cDNA was determined. The cDNA clone consisted of 844 bp, and the coding region contained 576 bp which was translated to a 22.6-kDa protein. This region showed 99.0% and 99.3% significant homology with those of the Chinese and Philippine strains of Schistosoma japonicum, respectively. The deduced amino acid sequence of the protein was identical to that of the Philippine strain and only one residue differed from that of the Chinese strain. The recombinant form of the tegumental protein was expressed in Escherichia coli and purified by a combination of ion exchange and affinity chromatography, and the purified protein was found to react with the sera of patients infected with Schistosoma japonicum. This result suggests that this antigen may be useful in the immunodiagnosis of schistosomiasis as well as in the development of an effective vaccine.     

Toll样受体4(TLR4)基因剔除小鼠构建及初步表型分析

目的 利用CRISPR/Cas9技术构建Toll样受体4(TLR4)基因敲除小鼠模型,并观察突变小鼠对革兰氏阴性细菌脂多糖(LPS)刺激响应的变化。方法 针对TLR4基因外显子2设计并合成1对sgRNA片段,与编码Cas9的mRNA混合后通过受精卵显微注射方法,建立TLR4基因敲除小鼠,通过繁育获得基因敲除纯合子小鼠(TLR4^-/-小鼠);通过LPS刺激,分析TLR4^-/-小鼠对炎症应激的反应情况,并在分子和病理水平上和野生型对照(WT)进行比较。结果 PCR及测序检测表明TLR4基因外显子2在小鼠基因中被成功敲除;给予LPS刺激后,IL1β、IL6、MyD88、iNOS及TNFa等炎症因子的表达在野生型小鼠的心、肝和肺组织中显著上调,而在TLR4^-/-小鼠中则几乎没有变化;血生化指标显示LPS刺激后WT小鼠血清中的尿素(Urea)和肌酐(Cre)水平显著升高,而TLR4^-/-小鼠刺激前后无显著变化,病理分析同样发现TLR4^-/-小鼠能够抵抗LPS对肾组织的损伤。结论 利用CRISPR/Cas9技术成功构建了TLR4基因剔除小鼠模型,TLR4的缺失能够降低IL1β、IL6、MyD88、iNOS及TNFa炎症因子对LPS刺激的响应,抑制LPS引起的炎症反应及对组织的损伤。     

Some effects of mermithid infection on metabolic reserves and flight in Locusta

Low infections of Mermis nigrescens reduce the flight performance of adult male Locusta migratoria, and bring about changes in the levels of blood carbohydrate and protein but not lipid. The quantities of all three metabolites in the fat body are reduced, as is the amount of tissue present. Lipid content only, is markedly decreased in the flight muscle, but the water content is increased.

Blood volume and malpighian tubule activity are unaffected by mermithid infection, but the rates of lipid mobilisation in response to injections of extracts of corpora cardiaca or flight are greatly reduced compared with those in uninfected locusts. Additional carbohydrate does not correct flight performance in parasitised locusts as it does in starved animals. Poor flight performance is not due to an effect of the worm on the host endocrine system.     

Growth and development of Haemonchus contortus in jirds, Meriones unguiculatus.

Growth and development of Haemonchus contortus were examined in jirds and were compared to these processes in lambs. Number, sex, size, and stage of development were determined for worms recovered at necropsy at various times postinoculation (PI) from immunosuppressed jirds inoculated with approximately 1,000 exsheathed infective larvae (L3) of H. contortus. In addition, gastric tissue samples from jirds were examined histologically. Parallel studies were done in lambs inoculated with approximately 7,500 L3. Typically, 5-30% of the inoculum established and survived in jirds at reasonably stable numbers to day 14 PI. By day 21 PI, worm numbers in jirds decreased dramatically. Although the parasite was similar in size and development on day 4 PI in jirds and lambs, from day 7 PI on, worms were significantly smaller and less developed in jirds. On histological examination, the parasite was found only in the glandular portion of the stomach of jirds (anatomically similar to its predilection site in the abomasum of lambs), and histological changes were consistent for both host species. Although growth and development of H. contortus are slower and incomplete in jirds, the parasite establishes, grows, and develops (at anatomically comparable sites in both hosts) in this model. Thus, the model appears to provide a useful laboratory host to study H. contortus.     

过表达兴安落叶松TPP基因增强拟南芥对盐胁迫的耐受能力*

兴安落叶松(Larix gmelinii)是极为重要的造林针叶树种,具有早期速生、抗逆性强、生态效益好等特点。海藻糖参与调控干旱、寒冷、盐害等多种逆境胁迫,海藻糖-6-磷酸磷酸酶(TPP)是海藻糖合成通路的重要酶。从兴安落叶松逆境胁迫转录组中筛选到LgTPPI.1基因全长序列,克隆了其编码区(CDS),构建了重组载体并获得过表达LgTPPI.1拟南芥纯合株系。结果表明,LgTPPI.1 CDS全长1 236 bp,共编码411个氨基酸;LgTPPI.1基因的mRNA在根和茎中表达水平较低,在针叶中表达水平较高;过表达LgTPPI.1基因拟南芥在盐胁迫下海藻糖含量显著提高、脯氨酸和抗氧化酶类活性增加、胁迫响应标记基因表达上调、对盐胁迫的耐受性增强。这些结果表明,裸子植物利用与被子植物类似的海藻糖通路来耐受非生物胁迫。为后续进一步解析落叶松中海藻糖合成相关基因的功能,揭示裸子植物中针叶树对逆境的响应机制奠定基础。     

长爪沙鼠与黑线仓鼠的温度选择比较

本文使用自制的陆生动物温度选择仪,对长爪沙鼠和黑线仓鼠的温度选择进行了比较,结果表明,长爪沙鼠的选择温度(29.06±0.62℃)较黑线仓鼠(26.65±0.64℃)高。这种差异可能与两种鼠的栖息环境及昼夜节律的不同有关。     

Growth and development of Gymnophalloides seoi in immunocompetent and immunosuppressed C3H/HeN mice

The growth and development of Gymnophalloides seoi were studied in C3H/HeN mice and effects of immunosuppression of the host on the worm development were observed. Two hundred metacercariae of G. seoi were orally administered to each mouse, and worms were recovered on days 1, 3, 5, 7, 14 and 21 post-infection (PI). The worm recovery rate was significantly higher in immunosuppressed (ImSP) mice than in immunocompetent (ImCT) mice except on days 1 and 3 PI. The worms attained sexual maturity by day 3 PI with eggs in the uterus, and worm dimensions and the number of uterine eggs continuously increased until day 14 PI in ImSP mice. Worms recovered from ImSP mice were significantly larger in size than those from ImCT mice on days 1 and 3 PI, and the number of uterine eggs was significantly larger in ImSP mice on days 5 and 7 PI. Genital organs such as the ovary, testes, and vitellaria, that were already developed in the metacercarial stage, grew a little in size until day 14 PI. The results show that the C3H/HeN mouse is, though not excellent, a suitable laboratory host for G. seoi.     

Litomosoides sigmodontis: A simple method to infect mice with L3 larvae obtained from the pleural space of recently infected jirds (Meriones unguiculatus)

Litomosoides sigmodontis is a filarial nematode that is used as a mouse model for human filarial infections. The life cycle of L. sigmodontis comprises rodents as definitive hosts and tropical rat mites as alternate hosts. Here, we describe a method of infecting mice with third stage larvae (L3) extracted from the pleural space of recently infected jirds (Meriones unguiculatus). This method enables infection of mice with a known number of L3 larvae without the time-consuming dissection of L3 larvae from mites and results in higher worm recovery and patency rates than conventional methods. Additionally, this method allows for geographical separation of the facility maintaining the L. sigmodontis life cycle from the institution at which mice are infected.