Experimental reproduction of the Jorge Lobo's disease in BALB/c mice inoculated with Lacazia loboi obtained from a previously infected mouse

Long-term maintenance of Lacazia loboi in the laboratory has not been reported. We report here the use BALB/c mice to maintain the Lacazia loboi for extended period of time. Eight to ten week-old mice were inoculated intradermally in both hind footpads with a fungal suspension from a macerated footpad obtained from an original mouse previously infected with the fungi and sacrificed 8 months after inoculation. The inoculated animals were sacrificed at different time intervals, footpads were excised, the right one was submitted to histopathological examination and the left one was macerated in sterile saline for fungal count and viability index determination. The inoculated animals presented the histopathological picture identical to the mice previously inoculated with material from human lesion. Granulomatous infiltrates with predominance of macrophages and giant cells were observed. The granulomas evolved progressively as observed in the different times of sacrifice. After 7 months of inoculation, macroscopic lesions were observed, and the number of fungi obtained from macerated footpads was higher than the number of inoculated fungi. The pattern of lesion development was similar to what was observed in animals infected with a fungal suspension obtained from a human lesion. Considering the histopathological findings, the clinical manifestations, and the finding of a higher number of fungi obtained than the inoculated into footpads of each mice, we believe the BALB/c mice strain is as an excellent way to maintain L. loboi in laboratory. Moreover, even after serial passages of the fungi, the granulomatous lesions are reproduced consistently in laboratory conditions.This revised version was published online in October 2005 with corrections to the Cover Date.     

Comparative experimental infection of Lacazia loboi in BALB/c and B10.A mice

Both hind foot pads of BALB/c and B10.A mice strains, were inoculated with a fungal suspension of Lacazia loboi obtained from a Jorge Lobo's disease patient. The suspension had 9 x 105 cells/ml and its viability index was 45%. The animals were sacrificed at different time periods varying from 24 h to 18 months after inoculation. The BALB/c mice developed an extensive granulomatous infiltrate, similar to the disease in humans, that progressively evolved. The number of fungal elements also increased as the disease progressed, and after the seventh month of inoculation, macroscopic changes of the foot pads were evident. Although the B10.A mice developed an exuberant granulomatous infiltrate, macroscopic changes were not detected. The number of fungal cells in the infected tissues increased in number, but they were lower then the numbers found in the BALB/c strain. The viability indexes were also lower for the B10.A strain. Considering the histopathological findings, the presence of macroscopic changes and the great amount of fungal cells in the infected tissues, the authors concluded that the BALB/c mice strain was more susceptible to L. loboi infection than the B10.A strain.     

Tissue responses against Cladosporium trichoides and its parasitic forms in congenitally athymic nude mice and their heterozygous littermates

The tissue responses against Cladosporium trichoides and its parasitic forms were studied using nude (nu/nu) mice and their heterozygous (nu/+) littermates of BALB/c background.1.0,0.1 and 0.01% cell suspensions were prepared from a culture broth which had been inoculated with the C. trichoides and cultured with reciprocal shaking at 27 ° C for 7 days. Sixty nu/nu or 60 nu/+ mice were divided into three groups consisting of 20 each which was allotted to one of the three cell suspensions. Each mouse was inoculated intravenously with 0.1 ml of either the cell suspensions. Two mice from each of the six groups were sacrificed at adequate intervals until 30 days after inoculation and histopathologic sections stained with H & E or by PAS were prepared from their visceral organs.There were no characteristic findings in the nu/nu and nu/+ mice inoculated with the 0.01% cell suspension. When inoculated with the 1.0% cell suspension, the brain was the favorite target organ in both groups of mice and the kidney was the second. When inoculated with the 0.1% cell suspension, brain lesions were observed only in the nu/nu mice. The susceptibility of the nu/nu mice was higher than that of the nu/+ mice.The parasitic forms in the brain of the nu/nu and nu/+ mice were slender septate true hyphae with or without polymorphonuclear leucocyte infiltrate, while in the liver, spleen and lung of both groups of mice the parasitic forms were short thick hyphae, moniliform hyphae, chlamydospores or round cells (sclerotic cells). Many giant cells containing fungal elements appeared in the liver of the nu/nu mice.     

Defense mechanisms of mice against Fonsecaea pedrosoi infection

Defense mechanisms of a host against Fonsecaea pedrosoi infection were studied histopathologically using athymic nude (nu/nu) mice of BALB/c background and their heterozygous (nu/+) littermates. Thirty male nu/nu and 30 nu/+ mice, weighing 16–19 g, were employed in this experiment. The nu/nu or nu/+ mice were divided into 3 groups consisting of 10 each. Furthermore, 4 nu/nu mice were supplemented to investigate effects of lymph node cell transfer. Subglobose cells of F. pedrosoi Tsuchiya strain were obtained from a culture in brain heart infusion glucose (1%) broth with reciprocal shaking at 37 °C for 17 days, and then 0.02, 0.1 and 0.5% cells suspensions were prepared. Each cell suspension was allotted to one group of the nu/nu or nu/+ mice. 0.1 ml of the cell suspension was inoculated into a tail vein, then one mouse from each group was sacrificed 1, 2, 4, 6, 8, 10, 14, 18, 21 and 25 days after inoculation. In both the nu/nu and nu/+ mice, the brain, kidneys and heart were affected severely with the strain in that order. Histopathologically, the defense mechanisms of the nu/+ mice against the fungus infection consisted chiefly of 2 steps: first, of non-immune phagocytosis by polymorphonuclear leucocytes (PMNs), and second, of granuloma formation induced by cell-mediated immunity. Those of the nu/nu mice consisted only of one step: phagocytosis by PMNs. A difference in susceptibility to the strain between the nu/nu and nu/+ mice changed according to the amount of the fungal cells inoculated. When inoculated with the 0.02% cell suspension, the resistance of the nu/nu mice was stronger than that of the nu/+ mice. In contrast, when inoculated with the 0.5% cell suspension, the former was affected more severely than the latter. There were little differences in the susceptibility to the strain between the nu/nu and nu/+ mice inoculated with the 0.1% cell suspension. These data seem to indicate that the phagocytic function of PMNs of the nu/nu mice was more active than that of the nu/+ mice, and the nu/nu mice inoculated with the 0.5% cells suspension (beyond the phagocytic capacity) lost resistance against the fungus infection. When the nu/nu mice were transferred with lymph node cells before inoculation of the strain, granulomata were formed to prevent hyphae from growing freely in the tissue.     

Lacazia loboi and Rhinosporidium seeberi: a genomic perspective

In the past five years, with the use of molecular strategies the phylogenetic affinities of the two more resilient pathogens studied in medical mycology, Lacazia loboi and Rhinosporidium seeberi were finally deciphered. These studies found that L. loboi was the sister taxon to Paraccidioides brasiliensis, and R. seeberi was closely related to protistan spherical aquatic fish pathogens, located at the point were animals diverged from the fungi, in the class Mesomycetozoea. These initial studies indicated that a molecular strategy was the ideal approach to further understand these anomalous pathogens. However, the limited amount of information gathered so far from few DNA sequences, although crucial to place these organisms in the tree of life and to take a glance to their ecological preferences, did not provide answers to other important traits. In the following pages we discuss a genomic perspective for both pathogens and the benefit that such information could generate to understand more about these two uncultivated pathogens.     

Granuloma formation and killing functions of granuloma in congenitally athymic nude mice infected with Blastomyces dermatitidis and Paracoccidioides brasiliensis

We did this experiment to clarify the mechanism of granuloma formation and the killing functions of granuloma in nude mice against Blastomyces dermatitidis and Paracoccidioides brasiliensis infections. B. dermatitidis A-295 and P. brasiliensis B-1183 were the cultures used. Congenitally athymic nude (nu/ nu) mice and their heterozygous (nu/ +) littermates of BALB/ c background were the test animals. From culture A-295, 0.1% and 1% cell suspensions (wet weight) were prepared and from culture B-1183 0.2% and 2% cells suspensions were prepared. Ten nu/ + and 10 nu/ nu mice were allotted to each of four cell suspensions. For experimental blastomycosis each mouse was inoculated intravenously with 0.2 ml of the cell suspension of A-295 and for experimental paracoccidioidomycosis, with 0.15 ml of the cell suspension of B-1183. Two mice from each of the four groups were killed at 5, 8, 12, 18 and 25 days after inoculation, and histopathologic sections, stained with H&E or by PAS, were prepared from various internal organs.In the nu/ nu mice inoculated with B. dermatitidis A-295 granuloma was formed in the brain tissue after the 12th day. However, mononuclear cells, which formed the granuloma, did not kill the fungal cells, and the fungal cells continued to multiply in the granuloma. On the other hand, in the heart, kidney and fat tissue, their histopathological findings after the 18th day were clumps of fungal cells with slight cell reactions. In these organs the exertion of cell-mediated immunity was necessary for granuloma formation against the fungal infection.In the nu/ nu mice infected with P. brasiliensis B-1183, granuloma appeared in the brain and kidney after the 18th day and fungal cells continued to multiply within the granuloma as well as in those inoculated with culture A-295.These results show that the exertion of cell-mediated immunity plays an important role as the defense mechanisms of hosts against these fungal infections. However, PMNs also play an important role in the mouse's defense mechanisms against these fungal infections.We assume that the defense mechanisms of immunocompetent mice against B. dermatitidis or P. brasiliensis infection consist chiefly of two steps: in the first step phagocytosis by PMNs occurs and in the second step cell-mediated immunity enters into play.     

Experimental pulmonary candidiasis in modified rabbits

The present experiment was performed in order to analyze and compare the histopathological features of Candida infection in various states of host defense capacity. The pulmonary lesions were induced by an intratracheal inoculation of 10⁸ live cells of Candida albicans into each of the rabbits in the following 4 groups: 1) untreated controls, 2) animals sensitized non-specifically to bovine serum albumin (BSA), 3) those sensitized specifically to formalin-killed C. albicans cells, and 4) those treated with cyclophosphamide and mitomycin C. The animals were sacrificed at appropriate intervals up to 16 days after inoculation. At autopsy, the lungs were cultured and then subjected to histopathological, electron microscopic and enzyme cytochemical examinations.In the healthy control animals, the fungal lesions consisted of polymorphonuclear leukocytes (PMN) at the initial stage, gradually changing to granulomatous inflammation, which contained no peroxidase-positive macrophages. In the animals sensitized non-specifically to BS A exudative macrophages appeared in the lesions, the nature of which did not differ from that of the control animals. In the animals sensitized specifically to Candida cells, more extensive infiltration of PMN was observed at the initial stage, a fact which may suggest the participation of the Arthus phenomenon in the development of the lesions. Furthermore, an epithelioid cell transformation of the macrophages in the granulomatous lesions may also suggest that immune complexes contributed to their formation. In the drug-treated animals, the lesions consisted of necrotic or less prominent cellular foci which correspond to the features of human candidiasis in debilitated states, and the inoculated fungi grew progressively to form pseudohyphae. An asteroid structure protruding radially from the surface of the fungal cells and attaching to primary lysosomes in the phagocytes was observed occasionally. This structure seems to be formed when the function of the phagocytes in the defense mechanisms against the fungi is suppressed to some extent.From the results of the present experiment, we would emphasize that PMN play an initial role in the elimination of C. albicans cells in the lung, and that macrophages then contribute to the formation of the lesions immunologically or non-immunologically.     

H.pylori抗体鸡蛋制品对H.pylori感染BALB/c小鼠的实验研究

目的通过幽门螺杆菌(Helicobacter pylori,H.pylori)标准株的接种,建立BALB/c小鼠感染H.pylori胃炎动物模型,评价H.pylori抗体鸡蛋制品对小鼠感染性胃炎的预防效果。方法将灭活的H.pylori国际标准菌株(NCTC 11637)作为抗原,对产蛋鸡进行免疫。免疫后收集鸡蛋,对达到效价的鸡蛋,无菌采集卵黄。BALB/c小鼠60只,适应性喂养1周后,随机分为5组,Ⅰ组为胃炎模型组,Ⅱ组为生理盐水组,Ⅲ、IV、V组分别为低剂量、中剂量和高剂量卵黄抗体组,每组12只。Ⅰ组予H.pylori菌液灌胃造模,Ⅱ组先予生理盐水灌胃后再予H.pylori菌液灌胃对照,Ⅲ、IV、V组分别用不同剂量抗H.pylori卵黄抗体灌胃后再予H.pylori菌液灌胃造模。小鼠均于距最后一次灌胃后8周全部处死,用微需氧细菌培养检测H.pylori定植;HE染色观察小鼠胃黏膜病理组织学改变。结果在接种H.pylori后第8周,Ⅰ组和Ⅱ组小鼠胃内均有大量H.pylori定植,感染率为91.7%,Ⅲ组的感染率是58.3%,IV和V组的感染率均小于30%。结论 H.pylori抗体鸡蛋制品可以抑制BALB/c小鼠感染H.pylori,抗体的保护作用与抗体的剂量呈正相关。     

Relationship between proteolytic activity of Aspergillus fumigatus and the fungus' invasiveness of mouse brain

This experiment was carried out to determine whether proteolytic activity of Aspergillus fumigatus was enhanced in the mouse brain during advance in growth of hypae, and if any relationship might be demonstrated between the proteolytic activity of the fungus and its invasive ability for the mouse brain.The K-2 strain of A. fumigatus and male mice of ddy line weighing 22±1 g were used in this experiment. Each mouse was inoculated intravenously with 5×10⁶ conidia of the strain suspended in 0.2 ml. phosphate buffer solution supplemented with 0.01 per cent Tween 80. Mice were sacrified at 4 hours' intervals up to 40 hours after inoculation and at an hour's intervals later. Two mice were assigned for each time. Each brain obtained was fixed in 10 per cent formalin and was cut into three sections from which histopathological specimens were prepared.The brains over 40 hours after inoculation were markedly swollen and abundant hypae were observed in the specimens. The extent of the fungal growth in the specimens from the mouse brain was 1.60, 7.71 and 10.84 per cent at 32,45 and 49 hours after inoculation, respectively.For assay of the proteolytic activity in the mouse brain, three groups of fifteen mice each were tested with the K-2 strain. These groups of mice were inoculated with 5×10⁶conidia of the strain and sacrificed 0,32 and 45 hours after inoculation, respectively. Then the brains pooled in each group and four volumes of phosphate buffer solution was added. The material was homogenized and used as test samples (enzyme solution). The proteolytic activity was measured quantitatively by a modification of Anson's method.The proteolytic activity in the mouse brain sacrificed 0,32 and 45 hours after inoculation was 0.00, 0.08 and 0.13, respectively, as expressed by the optical density.In conclusion, it was possible to confirm that proteolytic activity increased in the mouse brain in proportion to the growth of hyphae in it.     

Diffuse Alveolar Lesion in BALB/c Mice Induced with Human Reovirus BYD1 Strain and its Potential Relation with SARS.

The objective of this study was to investigate the pathogenicity and associated lesions of a new reovirus (ReoV) isolated from patients with Severe Acute Respiratory Syndrome (SARS) in China. Twenty-five four-week-old BALB/c female mice inoculated intranasally with either ReoV (strain BYD1) alone, or ReoV combined with SARS-CoV (strain BJF) displayed ejecting fur and loss of body weight compared with control animals. ReoV and SARS-CoV were isolated from most postmortem tissues. The histopathological features of ReoV infected animals consisted of diffuse alveolar damage, with scattered hemorrhage, hyaline membrane formation and interstitial pneumonia. A typical type II pneumocyte hyperplasia and fibrogranulomatous tissue formation in the alveolar septae were observed both in the animals inoculated simultaneously with these two viruses and in the animals inoculated firstly with SARS-CoV, followed by ReoV. The animals inoculated firstly with ReoV, followed with SARS-CoV displayed scattered hemorrhage in the alveolar septa. Furthermore, other lesions in above two combination groups included depletion of lymphocytes in the germinal center of lymph nodes in the lung hilus and the spleen, hemorrhagic necrosis in white pulp of spleen, hydroid degeneration, and fatty degeneration in the liver and kidney. Mice induced with SARS-CoV alone did not display clinical signs, characteristically hyaline membrane formation, hemorrhage and early pulmonary fibrosis in lung tissue. This study demonstrated that the newly isolated ReoV might be a virulent pathogen for BALB/c mice. Mice infected firstly with SARS-CoV, followed with ReoV developed a typical diffuse alveolar lesion.     

茄病镰刀菌致ICR小鼠局部皮肤感染的实验研究

目的建立茄病镰刀菌感染皮肤的ICR小鼠模型。方法将茄病镰刀菌三个不同浓度的菌悬液分别接种于正常和免疫抑制ICR小鼠受损及正常未受损的皮肤,于接种后第7、14、21、28天处死动物,取皮疹和各脏器进行真菌镜检、培养和组织病理学检查。结果正常和免疫抑制组皮肤受损的小鼠均出现皮疹,而免疫抑制组皮疹程度重,持续时间长,且真菌镜检、培养及组织病理学大都可见菌丝。皮肤未受损的小鼠不发生皮疹。实验小鼠均未出现系统播散。结论接种109CFU/mL或1010CFU/mL茄病镰刀菌的孢子悬液于免疫抑制ICR小鼠擦伤的皮肤,可有效造成ICR小鼠的茄病镰刀菌皮肤感染。     

Acute oral intragastric pathogenicity and toxicity in mice of <Emphasis Type="Italic">Paecilomyces fumosoroseus</Emphasis> isolated from whiteflies

Paecilomyces fumosoroseus, monospore culture EH-506/3, isolated in Mexico from Bemisia tabaci whitefly was tested for acute oral intragastric pathogenicity and toxicity in CD-1 mice. Animals were inoculated by gavage with only one dose (10⁸ conidia/animal) of viable (72 mice), heat-killed (24 mice) fungus and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies at days 3, 10, 17, and 21 after the inoculation. No mice were clinically ill or died. At the end of the study, their mean weight corresponded to healthy adults. Positive fungal cultures of feces were obtained only 24 h after inoculation. Positive cultures were found in 15 out of 360 organs (liver, spleen, kidney, brain, lung) in 12 of 72 mice inoculated with viable conidia. Gross pathology exhibited splenomegaly and liver paleness in mice inoculated with viable and heat-killed fungus. Non-germinated conidia were observed in studied organs, without any pathological tissue reaction, suggesting no mycological or histopathological evidence of fungal multiplication. The fungus was able to persist, but did not cause permanent damage to the host. This study supports the non-pathogenic/toxic status of P. fumosoroseus EH-506/3 when administered intragastrically in mice.     

Experimental infection studies of Pasteurella pneumotropica and V-factor dependent Pasteurellaceae for F344-rnu rats.

To investigate the pathogenicities of P. pneumotropica (Pp) and V-factor dependent Pasteurellaceae (VFDP) in immunodeficient rats, experimental infections of F344-rnu rats were performed using 3 strains (ATCC 35149, CNP 160 and RPZ) of Pp and 4 strains (V6, V7, V8 and V9) of VFDP. Four animals per experimental group were inoculated twice on day 0 and post-inoculation day (PID) 14 with bacterial suspension intranasally. Two animals from each group were sacrificed on PID 60 and 120, and examined. In the animals inoculated with strains of Pp, sneezing was observed in some animals inoculated with strains ATCC 35149 and CNP 160 until PID 31. No clinical signs were observed in other animals. The strains were mainly isolated from the nasal cavity and trachea on PID 60, and the nasal cavity, trachea and lung on PID 120. Inflammation and necrosis of nasal cavity mucosa were observed in all animals inoculated with strains ATCC 35149 and CNP 160 in a histopathologic examination. No histopathological changes were observed in any other animal. In the animals inoculated with strains of VFDP, neither clinical disorder nor histopathological change was observed. The strains were mainly isolated from the trachea on PID 60, and from the trachea and lungs on PID 120. From these results, the pathogenicity of Pp in immunodeficient rats appears to differ by strain, and VFDP appears to be non-pathogenic in immunodeficient rats.     

Characterization of pathology and parasite load in outbred and inbred mouse models of chronic Neospora caninum infection

Here, we analyzed histological findings and parasite burden in chronic Neospora caninum infection in BALB/c and ICR mice and studied the correlation between lesion severity and parasite load in brain. To obtain a better understanding of the infection, we examined the influence of various host pathogen factors. Groups of outbred (ICR) and inbred (BALB/c) mice were inoculated using several NC-1 parasite doses (4 x 10(5), 10(6), and 5 x 10(6) tachyzoites), inoculation routes (intraperitoneal and subcutaneous), and 3 immunosuppressive treatments (methylprednisolone, cyclophosphamide, and vinblastine). Lesion severity was analyzed in the liver, lung, heart, and brain tissues, and parasite load was measured by real-time polymerase chain reaction in brain tissue. The results indicated more severe cerebral lesions and higher brain parasite burdens in inbred than in outbred mice. Hepatic tissue was the primary lesion site in immunosuppressed ICR mice. We also observed that increased inoculum size was reflected in greater lesion severity and a higher cerebral parasite load. No difference was observed with respect to inoculation route. The study also showed an association between brain parasite burden and severity of cerebral lesions in BALB/c mice.     

Facilitated expansion of pneumococcal colonization from the nose to the lower respiratory tract in mice preinfected with influenza virus.

A strain of Streptococcus pneumoniae, when inoculated intranasally in 2 microl of suspension into BALB/c mice preinfected with influenza virus, colonized first in the nose, and several days thereafter also colonized significantly in the trachea and lungs with purulent inflammation. Pneumoccocal colonization was also observed in the noses of normal mice after the same bacterial inoculation, but not apparently in the lower respiratory tract. These results suggest that pneumococcal infection may develop from the upper to the lower respiratory tract as a possible sequence preferentially in influenza virus-infected subjects.     

Effects of tacrolimus (FK506) on encephalomyocarditic virus-induced diabetes in mice

The effects of tacrolimus on insulin-dependent diabetes mellitus (IDDM) induced by the D-variant of encephalomyocarditis virus (D-EMCV) have been investigated. Male BALB/c mice were treated with tacrolimus before viral inoculation, and then were inoculated with 10 plaque forming units (PFU) of DEMCV. The mice continued to be treated with tacrolimus until the animals were sacrificed. D-EMCV-infected mice, which were treated with saline as controls, showed abnormal glucose tolerance test (GTT) values, whereas all infected mice with tacrolimus pretreatment were normal on 7 days-post inoculation (DPI). Histological observations revealed that non-treated tacrolimus D-EMCV-infected mice and which developed diabetes showed severe insulitis in their islets of Langerhans. On the other hand, D-EMCV-infected mice treated with tacrolimus were normal. In D-EMCV-infected mice, viruses in the pancreata were detected at the same level regardless of treatment with tacrolimus or saline. Expressions of TNF-alpha and IFN-gamma mRNA in spleens of tacrolimus-treated D-EMCV-infected mice were lower than that of non-treated tacrolimus DEMCV-infected mice on 7 DPI. The results suggest that tacrolimus suppresses expressions of TNF-alpha and IFN-gamma mRNAs to prevent the onset of D-EMCV-induced IDDM.     

Reduced Anthracnose Stalk Rot in Resistant Maize is Associated with Restricted Development of Colletotrichum graminicola in Pith Tissues

Resistance to anthracnose stalk rot (ASR) in maize was investigated for its effects on the development of Colletotrichum graminicola. ASR and fungal presence in pith tissues of resistant and susceptible genotypes, inoculated at time intervals after wounding in the first internodes, were assessed by rating tissue discoloration and by quantifying ergosterol production using high performance liquid chromatography (HPLC) and fungal recovery from tissues, respectively. Slices (30 μm thick) of pith cores (2 mm diam) of first internodes at late‐whorl and kernel blister stages were also inoculated with a suspension of fungal conidia immediately, 2 or 6 h after slicing. Fungal development was observed in tissues by light microscopy. ASR was markedly reduced in resistant genotypes when compared to susceptible genotypes and when inoculation was delayed after stalk wounding. Ergosterol content in tissues was associated with extent of discoloration due to ASR and fungal recovery. Conidial germination, germ tube elongation, appressorium formation and penetration of cortical cells were all markedly delayed in resistant genotypes, in both resistant and susceptible maize at vegetative stages, and by wound healing. C. graminicola macerated more rapidly and to a greater extent pith tissues of susceptible than resistant genotypes. Resistance mediated by maize genotype and ontogeny, and wound healing is expressed at early stages and subsequent events of host–pathogen interaction. Fungal structural development in detached pith tissues and the rapidity and extent of pith maceration in susceptible when compared to resistant genotypes was indicative of genotypic reaction to ASR in maize in the field. Laboratory inoculation and observation of detached pith tissues could be a useful and accurate tool for rapid screening of maize germplasm to identify ASR resistant genotypes that will function well in the field even where pathogen ingress occurs via wounds.     

H7N9禽流感病毒小鼠感染动物模型的建立

目的建立H7N9禽流感病毒小鼠感染模型。方法 1×108,1×107或1×106TCID50H7N9禽流感病毒原液(A/Anhui/1/2013)滴鼻感染BALB/c小鼠。主要观测指标:临床症状、死亡率、病理变化、病毒载量和血清抗体检测。结果被感染的小鼠表现为竖毛、弓背、体重下降;病理表现为间质性肺炎,感染后第2天开始在呼吸道脱落细胞中检测到病毒;免疫组化或病毒分离方法在肺、肾、脑、肠、脾等组织检测到病毒;感染后14 d在小鼠血清中血凝抑制试验特异性抗体效价达到160;淋巴细胞减少,中性粒细胞增多。结论 H7N9感染BALB/c小鼠模型与人类禽流感感染疾病的基本特征相似,为研究该病的发病机制及药物疫苗的研发提供了工作基础。     

Technical evaluation of nested PCR for the diagnosis of experimental sporotrichosis

BackgroundSporotrichosis caused by the dimorphic fungus Sporothrix schenckii can presents in a variety of clinical forms. Routine diagnosis is made by mycology and serology studies. Few investigations have been focused on the evaluation of the molecular diagnosis.AimTo determine the value of the nested PCR technique for the diagnosis of experimental sporotrichosis in organs of mice, and to compare the results with the established laboratory diagnostic procedures.MethodsBALB/c mice were inoculated with growing concentrations of the 2 morphological phases of the fungus. The infected animals were sacrificed one month later and specimens from liver, spleen, lung and testicle were obtained to perform wet mount, culture and molecular diagnosis by the nested PCR technique. Blood samples were obtained for determination of specific antibodies against S. schenckii by the double immunodiffusion procedure.ResultsThe pathogenicity observed with the different concentrations of the fungus inoculated and its isolation by culture, showed scarce differences in the study of specimens from organs infected with the 2 morphological phases of S. schenckii. Specimens from organs of mice inoculated with the mycelial phase when studied by wet mount and culture, showed a higher positivity (100 and 37.5%) than those from mice inoculated with the yeast phase (73 and 2%). However, diagnosis by the nested PCR molecular technique applied to the latter specimens showed a higher percentage of positivity (75%) and 43% of positive results coming from animals infected with the mycelial phase. Specific antibody detection was positive in 100% all groups of infected mice.ConclusionsIn the study of experimental sporotrichosis in mice, the culture, as well as the antibody detection, was an effective diagnostic procedure, while the nested PCR and microscopic studies had a lower diagnostic value.