Immunological characteristics of experimental murine infection with Leishmania (Leishmania) amazonensis

The murine models of Leishmania infection are well-studied and suitable models for studying this disease, which, despite its incidence of nearly 2 million new cases worldwide per year and its prevalence of 12 million cases, has been a somewhat neglected disease. Data obtained using such models are important for a better understanding of the disease in humans due to similarities in physiology and the advantage provided by the uniform infection profile within each mouse strain. In this review, we focus on studies of experimental murine infection with Leishmania (Leishmania) amazonensis, a species that has been associated with infections exhibiting various clinical features in humans. Mainly, we point out and discuss reports on: the effects of variations of the inoculum (such as strain, site, and size) in the establishment and development of the infection; characteristics of the infection in distinct mouse strains; and, the effects and subversions of the infection on components of the host innate and adaptive immune responses. The results obtained in these studies show that L. (L.) amazonensis infection in mice presents some unique features and immunoregulatory mechanisms, making it an interesting model for obtaining further knowledge of potential drugs targets and immunotherapy in Leishmania infection.     

Extracellular matrix alterations in experimental Leishmania amazonensis infection in susceptible and resistant mice

ABSTRACT: Leishmania is inoculated, by the bite of an infected sandfly, into the skin of the host, where the promastigotes are phagocyted by dermal macrophages. The dermal region comprises cells and abundant extracellular matrix. Studies show that matrix metalloproteinases play an important role in host defense responses against pathogens in mammals and that their activities lead to the production of antimicrobial peptides. The aim of this study is to evaluate the changes in the distribution of fibronectin and laminin as well as in the elastic system fibres during the course of infection caused by Leishmania amazonensis in mice with distinct genetic backgrounds of susceptibility to this parasite. The results showed that BALB/c presented an enhancement of fibronectin during the course of infection when compared to their control group while the infected or non-infected C3H.He showed a decrease of this protein at end of the experiment. Laminin, on the other hand, remained unaltered in both strains. Also in both BALB/c and C3H.He mice the elastic and elaunin fibres remained unchanged while the oxytalan fibres decreased along the experiment. Ninety days after the infection C3H.He mice had recovered their capacity to produce oxytalan fibres.     

Histopathological studies of visceralized Leishmania (Leishmania) amazonensis in mice experimentally infected

BALB/c, C57BL/6, and DBA/2 mice were subcutaneously infected in the left footpad by injecting 10(4) Leishmania (Leishmania) amazonensis amastigotes. Mice were sacrificed 20, 30, 40, 60 and 90 days post-infection. Fragments of liver, kidney, spleen, skin, and draining lymph node were collected for histological examination. Light microscopy showed that at 20 days after infection BALB/c mice presented discrete inflammatory infiltrates in the skin made up of eosinophils, lymphocytes, and rare parasitized macrophages. Ninety days post-infection, the dermis showed necrotic tissue, large numbers of mononuclear cells and vacuolated macrophages filled with amastigotes. Forty days post-infection, the draining lymph nodes showed hyperplastic germinal centers, increase of high endothelial venules and apoptosis in germinal center cells. After the first 3 months post-infection, the involvement of spleen, kidney and liver was discreet, being characterized by multifocal inflammatory infiltrates. Eight months after infection, the animals presented metastatic lesions in the contralateral footpad and nose. In deep dermis, there was remarkable proliferation of fibroblasts associated with collagen fibers. The liver showed multifocal granulomas and mononuclear infiltrate around the blood vessels, but no parasites were observed, except in one animal. In some mice there were immature cells of the hematopoietic lineage. Both BALB/c and C57BL/6 mice presented osteonecrosis, which is characterized by pycnotic osteocytes and empty lacunae at the point of inoculation and subsequently, replacement of this tissue by fibrous connective tissue and colonization of the bone marrow. A diffuse inflammatory process composed of mononuclear cells and rare parasites were seen in the kidneys. In one mouse, bone marrow cells were observed in the renal medulla along with where free amastigotes. DBA/2 mice developed a mild infection and they did not visceralize. In conclusion, our data demonstrates that in susceptible mice L. (L.) amazonensis, a causative agent of tegumentary leishmaniasis, causes pathological changes similar to those produced by Leishmania (L.) infantum in both humans and canids.     

牛miR-29b影响牛病毒性腹泻病毒感染BALB/c小鼠的作用

前期研究发现高表达miR-29b能显著抑制牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)在体外的复制,而miR-29b过表达是否影响BVDV在体内的复制尚未见有报道。研究设计扩增牛pre-miR-29b基因片段的引物,以MDBK基因组为模板,PCR扩增pre-miR-29b并克隆至慢病毒载体pLL3.7。将阳性质粒pLL3.7-pre-miR-29b与包装质粒共转染HEK-293T细胞,包装慢病毒并测定慢病毒滴度,同时包装pLL3.7空质粒的慢病毒作为阴性对照。将4～5周龄BALB/c小鼠随机分成5组,每组6只,连续2次尾静脉注射2.5×10~7 IU慢病毒悬液pLL3.7-pre-miR-29b或pLL3.7,并于慢病毒注射后96 h通过滴鼻途径攻毒BVDV毒株NADL(1.68×10~5 TICD_(50)/只),于攻毒后不同时间(0,2,4,10,15 d)处死BALB/c小鼠,采集心脏、肝脏、脾脏、肺脏、小肠和血液,提取总RNA,使用荧光定量RT-PCR检测不同组织中BVDV拷贝数;同时制备病理组织切片观察各组织病变情况。结果显示,成功构建pLL3.7-pre-miR-29b质粒;成功包装pLL3.7-pre-miR-29b和pLL3.7慢病毒;使用荧光定量RT-PCR检测发现,pLL3.7-pre-miR-29b感染能显著性降低BVDV拷贝数;与pLL3.7-pre-miR-29b感染的处理组相比较,pLL3.7感染的对照组中各组织病变情况较为严重。结果表明,BALB/c小鼠体内过表达miR-29b能明显抑制BVDV的复制,减轻BVDV感染造成的病变,为以后研发抗BVDV的有效策略和方法提供了理论依据。     

猪圆环病毒2型感染裸鼠及BALB/c小鼠的研究

用猪圆环病毒2型（PCV2）经腹腔注射BALB/c小鼠及裸鼠各15只,每隔2周1次,共感染3次.BALB/c小鼠初次、再次感染后均未出现明显的临床症状,仅有2只小鼠出现病理变化;从血清中可检测到PCV2核酸及其ORF2抗体;同时淋巴细胞增殖反应显著增强,NK和CTL细胞杀伤率显著升高;CD4^＋、CD8^＋、CD3^＋T淋巴细胞及CD19^＋B淋巴细胞数量显著减少.裸鼠也未出现明显的临床症状,从血清中可检测到PCV2核酸,但未检测到PCV2 ORF2抗体;除NK细胞杀伤率显著升高外,其余免疫学指标无显著改变.结果表明,BALB/c小鼠比裸鼠对PCV2易感,各项免疫学指标变化与PCV2感染猪一致,但不表现临床症状,仅个别BALB/c小鼠出现病理变化,说明BALB/c小鼠对PCV2不如猪易感.     

蜥蜴利什曼原虫在小鼠体内的分布及增殖研究

通过腹腔途径用体内含有荧光蛋白的蜥蜴利什曼原虫感染BALB/c小鼠,感染后采其脏器做冰冻切片,荧光染料染色后用荧光显微镜观察,并经PCR鉴定,结果显示蜥蜴利什曼原虫感染小鼠后,主要分布于心脏、肝脏、脾脏、肺脏、肾脏。另外,成功建立了利什曼原虫荧光定量PCR检测方法,并采用该方法检测感染蜥蜴利什曼原虫后BALB/c小鼠体内利什曼原虫的增殖情况,结果显示,感染13 d内,BALB/c小鼠体内蜥蜴利什曼原虫呈波浪状增殖。这一结果为研究蜥蜴利什曼原虫感染人和动物的致病机理和免疫方法及疫苗研制等方面提供了基础理论依据。     

Identification of a virulence factor for Brucella abortus infection in BALB/c mice

Immunogenic or pathogenic factors of recombinant proteins (rBCSP20, rBCSP-31, and rBCSP45 of Brucella abortus strain 19) for mice were compared with factors of a proteinase K-treated lipopolysaccharide extracted from B abortus strain 2308. Mice were vaccinated with 4 products, using different inoculation schedules and were challenge exposed with a virulent culture of B abortus strain 2308. Blood samples were collected 2 weeks after vaccination and at necropsy and sera were obtained. Spleens were cultured for B abortus at necropsy (3 to 4 weeks after challenge exposure). Mice given proteinase K-treated lipopolysaccharide alone or in conjunction with rBCSP20 or rBCSP45 proteins were protected, but mice given rBCSP31 on the same day as challenge exposure were not. Vaccination with recombinant proteins alone neither provide protection nor significantly (P greater than 0.05) increase the pathogenic effect of the challenge-exposure culture. Seemingly, rBCSP31 might be a virulence factor of B abortus.     

Vertical transmission of Neospora caninum in BALB/c mice in both acute and chronic infection

To examine the frequency of congenital infection by Neospora caninum, BALB/c mice were inoculated intraperitoneally with tachyzoites of N. caninum either during pregnancy (Group 1) or 4 weeks or more before pregnancy (Group 2). Further, the mice inoculated during pregnancy were bred at 4 weeks or more after delivery to form Group 3. Congenital transmission was observed in 76% of the neonates of the mice in Group 1 and in 50% of the neonates of the mice in Group 2. Interestingly, congenital transmission was observed in 86% of the neonates from Group 3. These results suggest that chronically-infected BALB/c mice efficiently transmit N. caninum infection to their offspring.     

Influence of Neospora caninum infection in BALB/c mice during pregnancy in post-natal development

The influence of Neospora caninum infection during pregnancy on the post-natal period has been poorly investigated. In a previous study, we suggested that infection with N. caninum during pregnancy could affect the normal post-natal development of the offspring. For this reason, in the present work we evaluated the influence of N. caninum infection in pregnant BALB/c mice at days 0, 7 and 14 of gestation (groups A, B and C, respectively) on the post-natal development of the offspring from birth to day 60 post-partum (PP). Morbidity and mortality, vertical transmission, and histopathological lesions were investigated. The humoral immune response (IgG) of pups was also evaluated. Results showed that infection with N. caninum during pregnancy had fatal consequences for pups, especially during mid-gestation (day 7). Infection provoked a delay in the general development of neonates, clinical signs compatible with neosporosis and severe histopathological lesions. A high mortality rate was found in all infected groups. A 69% of mortality rate was found in group A, a 100% in group B and a 46% in group C. Necrotizing encephalitis and multifocal hepatocellular necrosis were the most severe lesions found. All neonates, except four animals from group C, had antibodies against N. caninum but the immune response was not sufficient to control parasite infection. We have demonstrated that extension of the observation period after N. caninum infection permits a more accurate study of vertical transmission, the major route of parasite transmission, and mortality rates. We propose that infection at mid-gestation (day 7) in BALB/c mice and its study during the post-natal period constitutes a valuable experimental model for testing new chemotherapeutic agents and vaccines designed to protect against congenital neosporosis, in order to select effective protocols before its use on bovine.     

Leishmania donovani-derived lipophosphoglycan plus BCG induces a Th1 type immune response but does not protect Syrian golden hamsters (Mesocricetus auratus) and BALB/c mice against Leishmania donovani

The efficacy of Leishmania donovani-derived lipophosphoglycan (LPG) plus Mycobacterium bovis Bacille Calmette-Guerin (BCG) as a vaccine candidate against visceral leishmaniosis in susceptible BALB/c mouse and Syrian golden hamster (Mesocricetus auratus) models was investigated. Following a triple vaccination with a total dose of 150 microl BCG plus 60 microg or 30 microg of LPG for hamsters and BALB/c mice respectively, there were no noticeable side effects both locally and systemically; implying that the molecule was safe at this dosage level. Vaccinated animals demonstrated an activation of both the humoral as well as cell-mediated responses to LPG, which correlated with resistance against the disease. Protection by LPG plus BCG, was however, poor as the remaining immunized animals showed disease progression leading to severity of the disease as illustrated by emaciation, mass loss and heavy splenic parasitaemia in hamsters. These data nevertheless suggest that it may be rewarding to further evaluate the potential of LPG as a vaccine candidate in leishmaniosis using other adjuvants, which may enhance its immunogenicity.     

Experimental infection with Toxocara cati in BALB/c mice, migratory behaviour and pathological changes

Experimental inoculations of 1000 Toxocara cati larval eggs were carried out in 18 BALB/c mice. The T. cati eggs used for inoculation were collected from the faeces of naturally infected cats. Euthanasia was performed on two mice on days 1, 2, 3, 4, 5, 6, 14, 21 and 28 post-inoculation (p.i.). Tissue samples were taken for digestion and histopathology. Larvae were recovered from all infected mice and the average of all larvae recovered was 28.3% (95%; CI: 14.1-42.4). Maximum number was obtained from liver on days 1 and 2 p.i.; from the lung on day 2 p.i. and from the brain on day 28 p.i. In muscle, the recovery was high as from day 3 p.i., with the maximum obtained on day 28 p.i. Superficial foci of congestion and haemorrhage were macroscopically observed in the lungs between days 2 and 5 p.i. and in the brain between days 3 and 6 p.i. Microscopic lesions were observed in the liver between days 2 and 14 p.i., with periportal and subcapsule inflammatory infiltrates. In the lungs, haemorrhages and inflammatory infiltrates can be observed in the alveolar parenchyma, close to bronchioles and large blood vessels. In the brain, congestive areas without inflammatory reactions were seen. In muscle, the presence of inflammatory infiltrates and degenerated muscle can be observed surrounding a parasite larva. These same lesions were observed in myocardium and pericardium. The kidneys were congested with inflammatory infiltrates. The inflammatory cells present in all the tissues studied were lymphocytes, neutrophils and a few eosinophils. Formation of granulomas or signs of larva encapsulation were not observed. The migratory pattern of T. cati larvae in BALB/c mice and its tendency to become concentrated in the muscle reinforce the importance of the mouse as a paratenic host for the parasite's cycle in the environment.     

ESR和MDA两种方法检测BALB/c小鼠肺组织自由基的比较

自由基学说(free radical theory)是具有代表性的致衰学说之一.这一学说20世纪50年代中期由著名学者Harman首先提出,其后得到许多学者的共识.     

重组新城疫病毒HN基因乳酸菌抑制结肠癌作用评价

为评价重组新城疫病毒(NDV)血凝素-神经氨酸酶(HN)基因植物乳杆菌对鼠结肠癌(CRC)潜在的抑制作用,试验以前期构建的可表达NDV HN基因的植物乳杆菌(简称重组菌HN/Lab)为研究对象,通过肿瘤诱导建立小鼠CRC皮下移植瘤模型,通过腹部手术建立小鼠原位移植瘤模型,并在肿瘤诱导前后给荷瘤鼠灌胃重组菌和不表达HN基因的植物乳杆菌(L.p),通过监测肿瘤体积、荷瘤鼠的存活率以及肿瘤组织的病理学来评价重组菌对CRC的抑制作用。结果表明,试验分别成功建立了小鼠CRC皮下移植瘤和原位移植瘤模型;重组菌(CT26+HN/Lab组)能明显抑制荷瘤鼠皮下肿瘤的生长,分别在肿瘤诱导后25,30 d差异显著(P0.05),重组菌组小鼠的存活率和生存时间也显著高于CT26组(P0.05);重组菌对小鼠原位移植瘤模型抑制效果明显,在肿瘤诱导7 d后即能明显抑制肿瘤的生长(P0.05),抑瘤率为43.13%,与植物乳杆菌(CT26+L.p组)23.03%抑瘤率相比有了明显的提高;病理组织学观察结果显示,重组菌诱导肿瘤坏死明显,浸润的淋巴细胞数量增多,能够抑制肿瘤细胞向黏膜层转移。因此,NDV HN基因重组乳酸菌对鼠CRC模型具有较好的抑制作用。     

黄芩苷对BALB/c小鼠无乳链球菌性乳腺炎治疗作用的研究

在建立小鼠乳腺炎标准动物模型的基础上,通过细菌计数、HE染色、酶联免疫吸附试验等方法研究了乳腺炎模型中无乳链球菌感染诱发的乳腺免疫反应,并在此基础上研究黄芩苷对乳腺炎的治疗效果。结果表明,模型组母鼠乳腺内的无乳链球菌在攻菌后24h达到峰值,随后逐渐下降。模型组乳腺组织在攻菌后12h出现明显脂肪变性,并随感染时间延长有加重趋势。模型组IFN-γ水平在攻菌后72h达到峰值,和生理盐水组相比差异极显著（P〈0.01）;模型组IL-4水平在攻菌后48h达到峰值后逐渐下降。IL-4水平急剧升高可能是促使乳腺急性炎症的主要原因之一。治疗组乳腺内细菌数在攻菌后24h、48h极显著低于模型组（P〈0.01）,此后治疗组细菌数逐渐下降,乳腺炎症明显减轻,全身症状逐渐缓解;治疗组IFN-γ水平在感染后48h、72h极显著高于模型组（P〈0.01）;治疗组IL-4水平在感染后24h、48h极显著低于模型组（P〈0.01）;由此可推断,黄芩苷能够抑制无乳链球菌在体内增殖来降低乳腺感染程度,并通过调节Th1/Th2平衡来减轻乳腺局部的炎症表现,说明黄芩苷对试验性小鼠乳腺炎有良好的治疗作用。     

Therapeutic and prophylactic efficacy of imidocarb dipropionate on experimental Babesia ovis infection of lambs

The objective of this study was to evaluate the therapeutic and prophylactic efficacy of imidocarb dipropionate (IMDP) against babesiosis and to determine specific antibodies against Babesia ovis in experimentally infected lambs. Thirty-six 6-month-old splenectomized lambs were used. The lambs were randomly divided into six groups with six animals each, and were intravenously inoculated with 50 mL B. ovis-infected erythrocytes as follows: group I (therapy group) was treated with IMDP (1.2 mg/kg body weight) starting on the day of onset of clinical signs of babesiosis after the inoculation; group II (untreated control animals) was not treated with any therapeutic treatment after the inoculation; groups III, IV, V and VI (prophylaxis groups) were administered IMDP (2.4 mg/kg body weight) 1, 2, 3 and 4 weeks before the inoculation, respectively. The animals were housed in a tick-proof room with water and food ad libitum up to the 30th day post-inoculation (PI). The lambs were monitored from the first day PI by recording the manifestation of clinical disease, rectal temperature, and the degree of parasitaemia. All the lambs became infected with B. ovis, except five animals from group III, which were treated 1 week prior to experimental infection. Other animals showed signs of acute clinical babesiosis. The animals treated with IMDP (group I) were able to clear the parasite from the blood circulation after 48 h post-treatment. The recrudescence of B. ovis was observed in two lambs 7 days after treatment, and they were treated with the second similar dose of the drug. Six lambs (1, 1, 2 and 2 lambs in group III, IV, V and VI, respectively) from the prophylaxis groups died within 7-17 days after showing high parasitaemia and clinical symptoms of the disease. Regardless of the clinical symptoms, 83.30% and 66.66% of the lambs which were administered IMDP 1-2 and 3-4 weeks before, were determined to be protected against the virulent field strain of B. ovis.     

LPS对BALB/c小鼠血清及肝脏铁状态的影响

采用血清铁和组织铁测定及ELISA方法检测了脂多糖(LPS)作用不同时间点小鼠血常规、血清铁含量、组织铁含量及血清转铁蛋白(Tf)含量变化.结果显示,与对照组相比注射LPS组红细胞计数(RBC)和血红蛋白含量(HGB)均显著下降(P＜0.05),但平均红细胞体积(MCV)和HGB含量在注射后1.5h组与对照组相比均无显著差异.结果表明,在注射LPS后1.5h没有形成小细胞低色素性贫血,即缺铁性贫血(IDA),在此时间点适合进行铁代谢研究.而在其他作用时间点,可能是由于RBC和HGB显著下降,从而使MCV代偿性增加.另外,在注射LPS后1.5h组白细胞计数(WBC)与对照组相比显著下降,是临床革兰阴性细菌感染的特征,而LPS是革兰阴性细菌细胞壁内毒素的主要成分,可见其在对小鼠作用1.5h时起到了有效作用,而其他时间点WBC与对照组相比均没有出现显著差异.     

BALB/c小鼠结肠癌皮下转移模型的建立

体外培养人结肠癌细胞株HCT-8,将2×107个/mL人结肠癌细胞(HCT-8)悬液0.2 mL接种于BALB/c小鼠皮下,记录荷瘤生存时间,观察接种瘤生长及转移情况,死亡后解剖并做病理检查.结果显示,皮下转移发生率为94.00%(47/50),淋巴结转移率为96.00%(48/50).荷瘤鼠平均生存时间(60.20±7.60)d,病理结果提示,转移部位肿瘤细胞与接种部位肿瘤细胞结构相似,符合低分化腺癌的特征.本试验成功建立了小鼠结肠癌皮下转移动物模型.为结肠癌的相关研究提供参考.     

Histological and computed tomographic characteristics of the sinonasal structure of BALB/c mice

Mice models were used to study the pathogenesis of mice and human diseases. Although some mice models of allergic rhinitis and rhinosinusitis have been reported, no detailed anatomic, histological and computed tomographic comparative data of the normal murine sinus are available in the literature for new researchers to establish mice models. The purpose of this study was to clarify the histological and computed tomographic characteristics of the normal nasal sinus in BALB/c mice. Fifteen sinonasal specimens were collected. Five mice were subjected to micro-computed tomography imaging, and then dissected to observe its anatomic landmarks, and 10 mice were subjected to haematoxylin and eosin staining. Important anatomic landmarks were clearly demonstrated, including the ethmoturbinates, nasoturbinal, maxilloturbinate, ethmoid sinus, maxillary sinus, nasopharyngeal duct, nasolacrimal duct and vomeronasal organ. Full and typical sinonasal landmarks can be visualized by gross anatomy, micro-computed tomography imaging and haematoxylin and eosin staining, which will be useful for establishing the mouse models of nasal disease.