The pathogenesis of street rabies virus in rats

Investigations were made on the spread of street rabies virus after its inoculation into the left hind foot-pads of rats. The virus isolate used was selected because disease was produced after 2 to 3 weeks of incubation. The presence of rabies virus in the central nervous system was first detected in the lubar segment of the spinal cord on the sixth day after inoculation, yet a minimal amount of virus was detected in the pooled sciatic nerves from the inoculated side at 96 hours. Before this time, virus could not be detected in any organ except in the foot-pad immediately after inoculation. Removal of the sciatic nerve or of its fasciculus prior to foot-pad inoculation was a complete saving procedure in all animals, thus giving evidence for the neural spread of the infection; neither the perineural structures nor the axons appeared to be involved.     

Kala-azar: a comparative study of parasitological methods and the direct agglutination test in diagnosis.

In a comparative study 88 patients were diagnosed as suffering from kala-azar (visceral leishmaniasis) using 3 parasitological methods simultaneously. Splenomegaly was absent in 4 cases. In 84 patients with splenomegaly, splenic aspiration appeared to be the most sensitive method (96.4%), followed by bone marrow aspiration (70.2%) and lymph node aspiration (58.3%). There was no relation between titres in the direct agglutination test and parasite load as determined by the number of parasitological methods which were positive or parasite density in splenic aspirates. Splenic aspiration and bone marrow aspiration were compared as an assessment of cure in kala-azar. In 6 (13%) of 46 patients tested, parasites were found, all by splenic aspiration. Bone marrow showed parasites in one of these. The literature with regard to parasitological investigations before and after treatment is reviewed.     

Cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis in Amazonian Brazil, and the significance of a negative Montenegro skin-test in human infections.

The clinical and epidemiological features of 62 cases of cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis, from Pará State, Amazonian Brazil, are discussed. The parasite, isolated in hamster skin and/or blood-agar culture medium, was in each case identified by both biological characteristics and a monoclonal antibody specific for promastigotes of L. (L.) amazonensis. Of the 62 patients, 46 (74.2%) presented with a single cutaneous lesion, and on no occasion was evidence found indicating metastatic spread to either the naso-pharyngeal mucosae or the viscera. Recent claims that this parasite may be responsible for both mucocutaneous leishmaniasis and typical visceral leishmaniasis are discussed. Meglumine antimoniate (Glucantime) proved highly efficient in the treatment of all patients. Of the 62 patients examined by the Montenegro skin test, only 32 (51.6%) gave a positive reaction. The significance of this finding is considered and the hypothesis made that the parasite itself may induce an immunoinhibition. Field studies amply confirmed the role of Lutzomyia flaviscutellata as the major sandfly vector of L. (L.) amazonensis in Amazonia.     

An epitope-specific PCR test for diagnosis of Leishmania donovani infections

The carboxy-terminal region of recombinant heat shock protein 70 of Leishmania donovani has been shown to have an immunodominant epitope. We designed a PCR assay using a new set of primers encompassing the gene sequence from 457bp to 927bp, which amplified a segment of the L. donovani genome in a species-specific manner. The assay was sensitive enough to detect 0.5pg of parasite DNA, which increased 10-fold (0.05pg) when an internal probe (583-609bp) was used in the Southern blot. The assay was able to detect parasite DNA from the liver and spleen of L. donovani-infected hamsters as well as lesion aspirates from parasitologically confirmed post-kala-azar dermal leishmaniasis (PKDL) and bone marrow aspirates from visceral leishmaniasis (VL) patients. No amplification was seen with axenically cultured promastigotes of L. infantum, L. tropica, L. major, L. mexicana, L. aethiopica or other intracellular organisms such as Entamoeba histolytica, Mycobacterium tuberculosis, Plasmodium vivax or human peripheral blood mononuclear cells. This PCR provides a specific tool for the diagnosis of VL and PKDL with simultaneous species identification.     

Delayed culture of Leishmania in skin biopsies

Between January 1997 and October 1998, 16 skin biopsies collected from 13 patients with cutaneous leishmaniasis in French Guiana were inoculated in culture medium after travel for 3-17 days from the place of biopsy to the culture laboratory in France. Each biopsy fragment was introduced near the flame of a Bunsen burner into the transport medium (RPMI medium supplemented with 10% fetal calf serum) which was maintained at ambient temperature during postal delivery to France. In France the biopsies were ground in sterile saline before being inoculated into NNN culture tubes. The cultures were incubated at 25 degrees C and subcultured every week until the 5th week. The cultures were positive in 9 cases, remained negative in 4, and were contaminated in 3 cases. Positive results were obtained at all seasons and for 3 different Leishmania species. The study indicates that delayed culture can yield useful results from biopsies taken in field conditions.     

Human urine stimulates growth of Leishmania in vitro.

Enhancement of trypanosomatid metacyclogenesis by insect urine components led us to test the effect of human urine as a culture additive. The addition of 1-5% urine to Schneider's Drosophila medium containing 10% foetal calf serum enhanced the growth of 11 Leishmania strains representing 8 different taxonomic groups. Cell division was stimulated in cultures with non-dividing organisms. Peak cell density was increased, as was the efficiency with which L. donovani could be isolated from infected hamsters. Preliminary work suggested that the modified medium would be useful for field isolation of L. donovani and L. braziliensis. The active nutrients or growth factors are not known.     

Evaluation of immune responses and analysis of the effect of vaccination of the Leishmania major recombinant ribosomal proteins L3 or L5 in two different murine models of cutaneous leishmaniasis

Four new antigenic proteins located in Leishmania ribosomes have been characterized: S4, S6, L3 and L5. Recombinant versions of the four ribosomal proteins from Leishmania major were recognized by sera from human and canine patients suffering different clinical forms of leishmaniasis. The prophylactic properties of these proteins were first studied in the experimental model of cutaneous leishmaniasis caused by L. major inoculation into BALB/c mice. The administration of two of them, LmL3 or LmL5 combined with CpG-oligodeoxynucleotides (CpG-ODN) was able to protect BALB/c mice against L. major infection. Vaccinated mice showed smaller lesions and parasite burden compared to mice inoculated with vaccine diluent or vaccine adjuvant. Protection was correlated with an antigen-specific increased production of IFN-γ paralleled by a decrease of the antigen-specific IL-10 mediated response in protected mice relative to non-protected controls. Further, it was demonstrated that BALB/c mice vaccinated with recombinant LmL3 or LmL5 plus CpG-ODN were also protected against the development of cutaneous lesions following inoculation of L. braziliensis. Together, data presented here indicate that LmL3 or LmL5 ribosomal proteins combined with Th1 inducing adjuvants, may be relevant components of a vaccine against cutaneous leishmaniasis caused by distinct species.     

Treatment of visceral leishmaniasis in Kenya by aminosidine alone or combined with sodium stibogluconate

The treatment of leishmaniasis, as currently conducted in Kenya with sodium stibogluconate, is unsatisfactory as it is expensive, resistance and relapses may occur, and major adverse effects have been reported. Recently, aminosidine (paromomycin) sulphate has shown good antileishmanial activity on its own as well as synergism with pentavalent antimony, administered concurrently. The present study was designed to assess the effectiveness of parenteral aminosidine, alone or combined with sodium stibogluconate, in visceral leishmaniasis, compared to treatment by stibogluconate alone. 53 patients were allocated to the 3 therapeutic regimes. The presenting signs and symptoms of leishmaniasis were those commonly seen in the visceral form of the disease, particularly in Kenya. At termination, clinical cures were achieved in all 53 patients with no difference between treatment groups. Spleen aspirates revealed the best parasitological results in patients receiving the combined treatment, with only 13% failures (partial cures + relapses), as opposed to 21% failures with aminosidine alone and 45% with stibogluconate alone. Treatment with aminosidine alone was the cheapest and safest regime.     

Experimental cutaneous leishmaniasis with Leishmania tropica in albino hairless mice, Mus musculus.

Cutaneous leishmaniasis was produced in 90 hairless mice, Mus musculus, with cultures of two strains of Leishmania tropica (one isolated from an exogenous human case in Texas, and the other a gerbil strain from the USSR). The mice were inoculated intradermally in five areas of the dorsal side of the body. Most of the inoculated mice developed visible cutaneous leishmaniasis. The early lesions at the sites of inoculation were small red papules appearing within one to two months following inoculation; some of these papules disappeared after a few weeks, others became nodular or pustular. These lesions gradually enlarged and became ulcerated, measuring about 5 to 10 mm in diameter, and some persisted as long as 386 days. The edges of the lesions were usually raised, indurated, and pink; the adjacent skin showed no visible reaction. The centre of the ulcer was either moist or covered with a dry brownish-grey crust. In a few cases the lesions were very extensive and merged with each other, covering nearly two thirds of the dorsal body surface. Smears from the edges of the lesions were positive for the amastigote (aflagellar) form of L. tropica, which produced infections when inoculated into other hairless mice and grew readily in promastigote (flagellar) form on NNP-2 medium (Packchanian, 1959) with suitable antibiotics (Packchanian, 1957). Although mice were inoculated intradermally with L. tropica, several animals with extensive skin lesions of cutaneous leishmaniasis also developed generalized leishmaniasis. This was proved by positive blood culture tests for promastigotes (from 15 mice) and by demonstration of the amastigote form of L. tropica in various organs such as the spleen, liver, kidney, heart and lymph nodes. On the other hand, heart blood from 18 mice with small skin lesions when tested during 62 to 269 days following infection, yielded negative results for promastigotes. The mice were kept under observation (60 to 478 days) before they were killed or died. The results of this study have demonstrated that the hairless mouse, Mus musculus, is a suitable laboratory animal for studying experimental cutaneous leishmaniasis.     

Visceral and cutaneous leishmaniasis (report of 2 cases)

Leishmaniasis is an endemic, sporadic infection in many parts of the world. Turkey is geographically unique in linking Asia and Europe. Of special interest is leishmaniasis, as various forms of this disease have long been reported in the surrounding regions. Visceral and cutaneous leishmaniasis are endemic in the western and southeastern parts of Turkey, respectively. Here, we report a cutaneous and a visceral leishmaniasis case, to draw attention to the increase in the incidence of leishmaniasis in Turkey. In the patient with cutaneous leishmaniasis, the ulcerative lesion on the cheek had persisted for two months before admittance to the hospital. Direct smears prepared from this lesion were negative for leishmania amastigotes whereas the promastigote forms were maintained in NNN (Novy-MacNeal-Nicolle) medium. The second patient was hospitalized with a prediagnosis of haematological malignancy, but the smears prepared from the bone marrow aspirates revealed leishmania amastigotes and promastigotes were seen on the smears from NNN cultures. These two reports mark the importance of inoculation of the specimens to NNN medium for the recovery of the promastigote forms. Cutaneous and visceral leishmaniasis have become endemic in considerable number of foci in Turkey, possibly due to the cessation of vector control programmes and increase in the agricultural and irrigation areas. These two reports also point out the increased prevalence of leishmaniasis in Turkey after 1980's.     

alpha-Difluoromethylornithine induces protective immunity in mice inoculated with Plasmodium berghei sporozoites

Mice inoculated weekly with Plasmodium berghei sporozoites while under treatment with alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, developed protective immunity against subsequent challenge with this parasite. The percentage of mice protected was similar whether DFMO alone (55%) or DFMO + chloroquine (65%) was used. With chloroquine alone, only 12% of mice were protected. This protection was long-lasting (at least six months). The immunity protected against sporozoites but not against erythrocytic form inoculation. It is suggested that this protection is induced by antigens released from exoerythrocytic schizonts whose further development is inhibited by DFMO.     

Comparison of parasitological and immunological methods in the diagnosis of leishmaniasis in Ethiopia.

The sensitivity and specificity of parasite demonstration methods (smear, culture and histology) and serological assays (enzyme-linked immunosorbent assay [ELISA], direct agglutination test and immunoblot) were compared in the diagnosis of leishmaniasis in Ethiopia. Culture was found to be the most sensitive diagnostic method, followed by ELISA, for the diagnosis of cutaneous leishmaniasis (CL). When the clinical type of CL was taken into consideration, serological and parasitological methods were equally good for the diagnosis of diffuse cutaneous leishmaniasis. Overall, the serological assays were not sensitive enough to diagnose all the parasitologically confirmed cases of localized cutaneous leishmaniasis. Both groups of diagnostic methods performed equally well in the diagnosis of visceral leishmaniasis patients. In cases of CL where clinical diagnosis was a problem and histology could not give a definitive diagnosis due to the absence of demonstrable parasites, one of the serological assays, preferably ELISA, was very useful in establishing the final diagnosis.     

Haemoculture of Leishmania (Viannia) braziliensis from two cases of mucosal leishmaniasis: re-examination of haematogenous dissemination.

Leishmania parasites were isolated from peripheral blood leucocytes of 2 patients with mucosal disease among a total of 23 parasitologically confirmed cases of leishmaniasis. One had had mucosal leishmaniasis for 4 years and active pulmonary tuberculosis was also diagnosed. The other patient presented a cutaneous lesion on his right leg of 3 months duration and asymptomatic mucosal involvement. He had received intravenous antimonials before isolation of parasites. Both patients had positive indirect fluorescent antibody and Montenegro skin tests. L. (Viannia) braziliensis was isolated from both patients. This culture of parasites from leucocytes provided direct evidence for metastatic spread of Leishmania via the blood.     

Identification of Leishmania donovani as a cause of cutaneous leishmaniasis in Sudan

Eight patients with cutaneous ulcers were referred to the Institute of Endemic Diseases, Khartoum, Sudan, from June 2000 to March 2002 for the diagnosis of suspected cutaneous leishmaniasis (CL). Diagnosis was confirmed parasitologically by both positive Giemsa-stained smears and successful culture of Leishmania promastigotes in NNN medium. The eight parasite isolates were shown to belong to the Leishmania donovani complex by kDNA PCR. Isoenzyme typing of three isolates revealed that they were identical to the L. donovani MON-82 reference strain, and the gp63 PCR-RFLP profile showed similar patterns to a reference strain of MON-82. CL is endemic in most regions of Sudan and has been reported previously as being caused by L. major MON-74. The results of this study suggest that L. donovani is also a cause of CL in Sudan and that further study of isolates from Sudanese patients with cutaneous ulcers is warranted to ascertain whether L. donovani or L. major is the causative agent.     

Cutaneous leishmaniasis in Bolivia. A study of 185 human cases from Alto Beni (La Paz Department). Isolation and isoenzyme characterization of 26 strains of Leishmania braziliensis braziliensis [corrected

A clinical, serological, parasitological and therapeutic study of cutaneous leishmaniasis was carried out in a low sub-andean area (250-800 metres) of the La Paz Department, Bolivia. A team of seismic prospectors (350 workers) was surveyed for 12 months. Of 200 suspected cases of cutaneous leishmaniasis, 185 were serologically or parasitologically confirmed (incidence 52.8%). Those exposed to the greatest risk of infection were working in a virgin forest environment. Leishmanial organisms were isolated from 26 of the workers, either by in vitro cultivation or inoculation into hamsters. Isoenzyme characterization of the organisms by cellulose acetate electrophoresis showed them to be Leishmania braziliensis braziliensis [corrected]. The results of treatment of 168 patients with a pentavalent antimonial drug are also reported.     

Salmonella isolation with Rappaport's medium after pre-enrichment in buffered peptone water using a series of inoculum ratios.

The ability of malachite green/magnesium chloride broth (Rappaport''s medium) to isolate salmonellas from 25 ml quantities of sewage-polluted natural water was investigated. Samples were first pre-enriched in buffered peptone water and varying volumes of inoculum from the pre-enrichment culture were inoculated into Rappaport''s broth. Inoculum ratios in the range 1:2000 to 1:10 were examined. The inoculum ratio denotes the ratio of the volume of inoculum to the volume of fluid medium into which it is introduced. Optimum results were obtained with the 1:2000 ratio, although the salmonella isolation rate was only slightly less with the 1:500 and 1:100 ratios. The 1:2000 inoculum ratio was obtained with a graduated loop holding approximately 0.005 ml of fluid. Use of a loop for inoculation has advantages in speed of performance and safety of manipulation.     

Salmonella isolation with Rappaport's medium after pre-enrichment in buffered peptone water using a series of inoculum ratios

The ability of malachite green/magnesium chloride broth (Rappaport's medium) to isolate salmonellas from 25 ml quantities of sewage-polluted natural water was investigated. Samples were first pre-enriched in buffered peptone water and varying volumes of inoculum from the pre-enrichment culture were inoculated into Rappaport's broth. Inoculum ratios in the range 1:2000 to 1:10 were examined. The inoculum ratio denotes the ratio of the volume of inoculum to the volume of fluid medium into which it is introduced. Optimum results were obtained with the 1:2000 ratio, although the salmonella isolation rate was only slightly less with the 1:500 and 1:100 ratios. The 1:2000 inoculum ratio was obtained with a graduated loop holding approximately 0.005 ml of fluid. Use of a loop for inoculation has advantages in speed of performance and safety of manipulation.     

云南省普洱市思茅区大面积野鼠自毙疫情实验室检测结果与分析

目的 调查引起云南省普洱市思茅区大面积野鼠自毙疫情的病原体,以便为采取相应措施提供科学依据。方法 通过现场流行病学调查,采集119只自毙野鼠样本,解剖取脏器进行鼠疫耶尔森菌病原体分离、噬菌体裂解实验、F1抗原、caf1基因等病原学、免疫学及分子生物学检测,以排除鼠疫。同时,脏器样本接种绵羊血琼脂平板、巧克力平板、麦康凯平板、沙氏琼脂培养基等营养培养基和选择性培养基,分离培养细菌、真菌类病原体;选取10只组织腐败较轻的自毙鼠,按肺组织、脑组织、腹腔脏器组织分为3份样本后,研磨、离心,再以0.22μm滤膜过滤,收集离心上清液、滤膜滤出液和滤膜截留残渣共9份样本,腹腔接种雄性、体质量30~40g昆明小鼠,每组2只,每只0.5ml。另接种0.5ml Hank’s液2只鼠作对照。共接种昆明小鼠20只。死亡小鼠解剖观察病理变化,取脏器组织接种上述培养基进行细菌分离培养。结果 鼠疫耶尔森菌病原学、免疫学和分子生物学检测均为阴性;自毙野鼠脏器镜检、分离培养及接种实验动物均检出G- 短小杆菌,经梅里埃VITEK 2GN、GP卡、16S rRNA扩增鉴定为多杀巴斯德菌 (Pasteurella multocida,Pm) ,概率为99%;分离到的G+粗大杆菌为腊样芽孢杆菌,概率为99%。结论 多杀巴斯德菌可能是这次发生野鼠大面积死亡的病原菌。该病原体的生物学特性及危害值得进一步关注。     

Application of the indirect fluorescent antibody test in serodiagnosis of cutaneous leishmaniasis in experimentally infected mice and naturally infected Rhombomys opimus

The indirect fluorescent antibody test (IFAT) was used to detect leishmanial antibodies in experimentally inoculated mice with promastigote forms of Leishmania major, L. tropica and L. donovani infantum and in naturally infected Rhombomys opimus captured from the area endemic for cutaneous leishmaniasis in Esfahan, Iran. In the mice inoculated with L. major, the leishmanial lesion appeared at the site of inoculation and the leishmanial antibody level was much higher than in mice inoculated with other strains of Leishmania and in which no lesion was observed up to the 22nd week after inoculation. In R. opimus, the microscopical examination of the two smears prepared from the ears of each gerbil (one by the ordinary and the other by the sand-paper method) showed about 41% to be parasitologically positive. However, in IFAT about 84% were serologically positive. There was a good correlation between the percentage of thickened ears and leishmanial antibody titres in the gerbils.This investigation indicated that IFAT is a suitable serological technique for finding the reservoir hosts of leishmaniasis and determining leishmanial infection among rodents.     

妇科患者生殖道支原体属感染分布及药敏分析

目的 探究医院妇科门诊患者生殖道支原体属感染分布及耐药性,为临床诊治提供依据.方法 采集2011年1-10月医院就诊妇科感染性疾病患者宫颈管分泌物,接种到SP-4培养基作分离培养及药敏分析.结果 125例患者检出解脲脲支原体(Uu),检出率为15.4％,Uu对抗菌药物敏感率多西环素为74.4％、米诺环素为74.4％、交沙霉素为67.2％;对抗菌药物红霉素的耐药率为60.0％、氧氟沙星为47.2％、罗红霉素为31.2％.结论 妇科感染性疾病患者生殖道中广泛存在生殖支原体属感染,并且耐药性逐渐增强,临床诊治中应予关注,合理使用抗菌药物治疗.