Discovery of a granulovirus of Pandemis pyrusana (Lepidoptera: Tortricidae), a leafroller pest of apples in Washington

Pandemis pyrusana (Kearfott) is an important leafroller pest of apples in Washington. Surveys for natural enemies discovered a pathogen infecting Pandemis leafrollers in an apple orchard in central Washington. The pathogen was propagated in the laboratory and light microscopy using an azan stain demonstrated that it infected fat body, epidermis, and tracheal matrix cells. The virus was identified morphologically as a granulovirus using electron microscopy and designated PpGV. Rates of infection were determined for each generation in an apple orchard for three years. Infection rates were variable and ranged from 2.6 to 67% of individuals collected from each generation.     

Transovarial and transphasic transmissions of Borrelia by the taiga tick Ixodes persulcatus (Ixodidae)

Three generations of the taiga tick Ixodes persulcatus, the descendants from naturally infected females, have been examined by means of dark field and phase contrast microscopies and indirect immunofluorescent reactions with monoclonal antibodies. Location of borreliae in oocytes was examined by means of electron microscopy. The examined ticks derived from 9 females collected in the Novgorod Province, from 6 females of 1st laboratory generation and 5 females of the 2nd generation. In total, 250 larvae, 178 nymphs, 59 females and 70 males of three consequent generation have been examined. Almost 100% of descendants of naturally infected females were infected with Borrelia burgdorferi s. l. and similar infection rate was observed in unfed tick larvae collected in field conditions. The borreliae received transovarially to larvae of the 1st generation then were transmitted to 100% nymphs and imago of this generation and two next generations.     

Transmission Ecology of Sin Nombre Hantavirus in Naturally Infected North American Deermouse Populations in Outdoor Enclosures

Sin Nombre hantavirus (SNV), hosted by the North American deermouse (Peromyscus maniculatus), causes hantavirus pulmonary syndrome (HPS) in North America. Most transmission studies in the host were conducted under artificial conditions, or extrapolated information from mark-recapture data. Previous studies using experimentally infected deermice were unable to demonstrate SNV transmission. We explored SNV transmission in outdoor enclosures using naturally infected deermice. Deermice acquiring SNV in enclosures had detectable viral RNA in blood throughout the acute phase of infection and acquired significantly more new wounds (indicating aggressive encounters) than uninfected deermice. Naturally-infected wild deermice had a highly variable antibody response to infection, and levels of viral RNA sustained in blood varied as much as 100-fold, even in individuals infected with identical strains of virus. Deermice that infected other susceptible individuals tended to have a higher viral RNA load than those that did not infect other deermice. Our study is a first step in exploring the transmission ecology of SNV infection in deermice and provides new knowledge about the factors contributing to the increase of the prevalence of a zoonotic pathogen in its reservoir host and to changes in the risk of HPS to human populations. The techniques pioneered in this study have implications for a wide range of zoonotic disease studies.     

嗜肺巴氏杆菌外膜蛋白和脂多糖抗原在血清学诊断中的意义

目的评价嗜肺巴氏杆菌外膜蛋白(OMP)和脂多糖(LPs)作为血清学诊断抗原的敏感性和特异性.方法用OMP、LPS和全菌(WC)作为Western blot和ELISA的诊断抗原检测自然感染和实验感染嗜肺巴氏杆菌小鼠相应的IgG抗体滴度,同时测定3种抗原与实验动物常见致病菌的交叉反应.结果与嗜肺巴氏杆菌自然感染和实验感染小鼠血清的ELISA反应中,不同时期,LPS作为诊断抗原时血清抗体阳性率最高,WC次之,OMP最低.自然感染小鼠群中,出生4周LPS抗体阳性率即可达80%,而同期的WC和OMP仅为25%和20%,故LPS敏感性最高.与实验动物常见致病菌免疫血清和阴性种鼠血清的ELISA反应中,WC抗原表现出较高的吸光度(A)值,经Western blot证实,其反应为非特异性反应,LPS抗原特异性最强,OMP抗原次之.结论混合多株具有型或种特异性的OMP或LPS作为ELISA的诊断抗原,无论从特异性和敏感性上均高于全菌抗原.     

海南普通野生稻稻瘟病的抗性鉴定与评价

在苗期应用自然诱发鉴定法对海南普通野生稻(Oryza rufipogonGriff.)41个居群的410份材料进行了2年的稻瘟病(rice blast)抗性鉴定,结果表明:经过初鉴和复鉴,410份海南普通野生稻中有21份表现高抗,占5.1%,117份表现抗,占28.5%,说明海南普通野生稻具有较好的稻瘟病抗性。     

The hantaviral load in tissues of naturally infected rodents

Hantaviruses cause a lifelong and asymptomatic infection in naturally infected hosts as well as in experimentally infected rodents. Understanding the ecology and pathogenesis of hantaviruses requires an interdisciplinary research approach, which links laboratory experiments with results gained from field studies. Although several studies report hantavirus persistence and tissue infection patterns for experimentally infected rodents, field data is very limited. For this reason, the aim of our study was to investigate Puumala, Dobrava and Saaremaa virus RNA loads and tissue infection patterns in their natural reservoirs. Hantavirus RNA was demonstrated in all tested internal organs and blood samples of 14 naturally infected rodent hosts. However, the concentration of a specific virus differs depending on the virus, the host and the organ tested. Above all, the Dobrava virus showed a considerably higher viral load in all internal organs and blood samples of infected Apodemus flavicollis hosts. Results obtained in the study support the thesis that virus RNA load reaches its peak in the first month after infection, presumably after the virus has spread throughout all internal organs. This also implies that recently infected rodents are more important for transmission of the virus in the community.     

Human cytomegalovirus infection elicits a glycoprotein M (gM)/gN-specific virus-neutralizing antibody response

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen that infects 40 to 90% of adult human populations. HCMV infections are often asymptomatic in healthy individuals but can cause severe organ and life-threatening disease in immunocompromised patients. The antiviral antibody response to HCMV infection is complex and is known to include virus-neutralizing antibody production against surface glycoproteins encoded by HCMV. We have investigated the human antibody response to a complex of HCMV surface glycoproteins composed of glycoprotein M (gM)/gN, the gene products of the UL100 and UL73 open reading frames. Mouse monoclonal antibodies generated against gM/gN have previously been shown to neutralize HCMV infection of human fibroblasts in vitro. To determine whether human antibodies reactive with the gM/gN complex possess virus-neutralizing properties, we isolated human antibodies reactive with gM/gN from pooled human HCMV hyperimmune globulin by affinity purification using recombinant gM/gN. The affinity-purified human anti-gM/gN antibodies reacted specifically by immunofluorescence with HCMV-infected human fibroblasts and with cells transiently expressing gM/gN, but not with cells transfected with plasmids encoding other immunogenic HCMV proteins. The anti-gM/gN antibodies also reacted specifically only with gM/gN in immunoblot assays using lysates of transfected cells expressing specific HCMV proteins. Last, human anti-gM/gN antibodies efficiently neutralized infectious HCMV in vitro with a capacity comparable to that of human anti-gB antibodies. These data indicated that gM/gN can elicit a virus-neutralizing antibody response in humans infected with HCMV and therefore should be considered a potential candidate for inclusion in prophylactic CMV vaccines.     

Shedding and intracage transmission of Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus) model

The mechanism(s) by which Sin Nombre (SN) hantavirus is maintained in deer mouse populations is unclear. Field studies indicate that transmission occurs primarily if not exclusively via a horizontal mechanism. Using an experimental deer mouse infection model in an outdoor laboratory, we tested whether infected rodents shed SN virus in urine, feces, and saliva, whether infected mice transmit infection to na?ve cage mates, and whether infected dams are able to vertically transmit virus or antibody to offspring. Using pooled samples of urine, feces, and saliva collected from mice infected 8 to 120 days postinoculation (p.i.), we found that a subset of saliva samples, collected between 15 and 90 days p.i., contained viral RNA. Parallel studies conducted on wild-caught, naturally infected deer mice showed a similar pattern of intermittent positivity, also only in saliva samples. Attempts to isolate virus through inoculation of cells or na?ve deer mice with the secreta or excreta of infected mice were uniformly negative. Of 54 attempts to transmit infection by cohousing infected deer mice with seronegative cage mates, we observed only a single case of transmission, which occurred between 29 and 42 days p.i. Dams passively transferred antibodies to neonatal pups via milk, and those antibodies persisted for at least 2 months after weaning, but none transmitted infection to their pups. Compared to other hantavirus models, SN virus is shed less efficiently and transmits inefficiently among cage mates. Transmission of SN virus among reservoir rodents may require factors that are not required for other hantaviruses.     

Multiplex-PCR for detection of natural Leishmania infection in Lutzomyia spp. captured in an endemic region for cutaneous leishmaniasis in state of Sucre, Venezuela

We studied the natural infection of Lutzomyia (Lutzomyia) sp. with Leishmania in endemic foci of cutaneous leishmaniasis in the Paria peninsula, state of Sucre, Venezuela. Sand flies were collected between March 2001 and June 2003, using Shannon light-traps and human bait. Of the 1291 insects captured, only two species of phlebotomines were identified: L. ovallesi (82.75%) and L. gomezi (17.42%). A sample of the collected sand flies (51 pools of 2-12 individuals) were analyzed by using a multiplex-PCR assay for simultaneous detection of New Word Leishmaniaand Viannia subgenera. The results showed a total of 8 pools (15.68%) infected; of these, 7 were L. ovallesi naturally infected with L. braziliensis (2 pools) and L. mexicana (5 pools) and 1 pool of L. gomezi infected by L. braziliensis.     

Identification of immunoreactive extracellular proteins of Streptococcus agalactiae in bovine mastitis

Streptococcus agalactiae is a common pathogen that causes bovine mastitis. The aims of this study were to evaluate the antibody response against S. agalactiae extracellular proteins in the whey and serum of naturally infected bovines and to identify possible immunodominant extracellular antigens. IgG1 antibodies against S. agalactiae extracellular proteins were elevated in the whey and serum of naturally infected bovines. In the whey, the levels of IgG1 specific for S. agalactiae extracellular proteins were similar in infected and noninfected milk quarters from the same cow, and the production of antibodies specific for S. agalactiae extracellular proteins was induced only by infection with this bacterium. The immunoreactivity of extracellular proteins with bovine whey was clearly different in infected versus control animals. Group B protective surface protein and 5'-nucleotidase family protein were 2 major immunoreactive proteins that were detected only in the whey of infected cows, suggesting that these proteins may be important in the pathogenesis of S. agalactiae-induced mastitis. This information could be used to diagnose S. agalactiae infection. In addition, these antigens may be useful as carrier proteins for serotype-specific polysaccharides in conjugate vaccines.     

Serological evidence for naturally occurring transmission of Neospora caninum among foxes (Vulpes vulpes)

The study describes the time course of the Neospora caninum-specific antibody response in experimentally infected foxes, in naturally N. caninum-seropositive vixens and their litters. An immunofluorescence test, a tachyzoite surface antigen based ELISA and an immunoblot assay were established for this purpose. The immunoblot patterns of naturally seropositive and experimentally infected foxes revealed a high degree of similarity and resembled those reported for other intermediate host species. Reactions against immunodominant antigens of Mr 56, 68 and >94 kDa were observed which could be linked with a period of 14 days-2 months post experimental infection with tachyzoites. Cubs born by naturally seropositive vixens were found to be persistently or transiently seropositive, in the latter case, specific antibodies were detected only up to 44 days after birth. These antibodies may thus be of maternal origin. Differences between the immunoblot patterns of persistently positive cubs, those of their mothers and of transiently positive cubs, in particular the differential response to antigens of Mr 56 and 68 kDa, prove that cubs with persistent antibodies had actively mounted an antibody response. This result provides the first evidence for the postnatal or vertical transmission of N. caninum among naturally seropositive foxes.     

Natural breeding with bulls experimentally infected with Neospora caninum failed to induce seroconversion in dams

Four bulls and 56 heifers seronegative to Neospora caninum were used to determine the feasibility of venereal transmission in bovine neosporosis under natural conditions. Bulls were experimentally infected with 10⁸ live N. caninum tachyzoites. Two of them with the Nc-1 isolate and the other two with the Nc-Spain-7 isolate. After 13 months of initial infection, each bull was re-infected with the same isolate and dose. The experiments were carried out from March to September during 2006 and 2007 where groups of cyclic heifers were naturally mated by the experimentally infected bulls. In year 2006, two bulls infected with different N. caninum isolate serviced 12 heifers each. In year 2007, the same bulls serviced the same heifers a second time (now primiparous) and six new heifers were also added to each group. In addition, the other two bulls serviced 10 additional heifers each. Experimental animals were monitored for 30 weeks and serum samples were collected weekly and fortnightly in years 2006 and 2007, respectively to evaluate the presence of specific antibodies to N. caninum. Experimentally infected bulls showed a significant increase of specific IgG antibodies from 13 (Nc-SP-7) and 21 (Nc-1) days post-infection. Serum IgG antibody responses of individual animals were similar in kinetics but slightly different in magnitude. Serum samples from heifers were all negative. Pregnant rates were 100% in heifers and 91% in primiparous animals. Calves did not show precolostral specific antibodies to N. caninum. Venereal transmission of bovine neosporosis under natural grazing conditions is unlikely to occur.     

Environmental stress increases the prevalence and intensity of blood parasite infection in the common lizard Lacerta vivipara

Parasites affect the life-histories and fitness of their hosts. It has been demonstrated that the ability of the immune system to cope with parasites partly depends on environmental conditions. In particular, stressful conditions have an immunosuppressive effect and may affect disease resistance. The relationship between environmental stress and parasitism was investigated using a blood parasite of the common lizard Lacerta vivipara. In laboratory cages, density and additional stressors had a significant effect on the intensity of both natural parasitaemia and parasitaemia induced by experimental infection. Four weeks after infection, crowded lizards had three times more parasites than noncrowded lizards. After 1 month of stress treatment, naturally infected lizards had a significantly higher level of plasma corticosterone and a higher parasite load than nonstressed individuals. In seminatural enclosures, stress induced by the habitat quality affected both the natural blood parasite prevalence and the intensity of parasitaemia of the host.     

Virus load and sequence variation in simian retrovirus type 2 infection

The natural history of type D simian retrovirus (SRV) infection is poorly characterized in terms of viral load, antibody status, and sequence variation. To investigate this, blood samples were taken from a small cohort of mostly asymptomatic cynomolgus macaques (Macaca fascicularis), naturally infected with SRV type 2 (SRV-2), some of which were followed over an 8-month period with blood taken every 2 months. Provirus and RNA virus loads were obtained, the samples were screened for presence of antibodies to SRV-2 and neutralizing antibody titers to SRV-2 were assayed. env sequences were aligned to determine intra- and intermonkey variation over time. Virus loads varied greatly among cohort individuals but, conversely, remained steady for each macaque over the 8-month period, regardless of their initial levels. No significant sequence variation was found within an individual over time. No clear picture emerged from these results, which indicate that the variables of SRV-2 infection are complex, differ from those for lentivirus infection, and are not distinctly related to disease outcome.     

Candida albicans fibrinogen binding mannoprotein: expression in clinical strains and immunogenicity in patients with candidiasis.

A 58 kDa cell wall-associated fibrinogen binding mannoprotein (mp58), previously characterized by our group in a Candida albicans laboratory strain (ATCC 26555), was found to be also present in the cell wall of clinical isolates of this fungus. Most strains examined appear to have functional mp58 species, as detected by their ability to bind fibrinogen. Western immunoblot analysis, with a monovalent polyclonal antibody generated against the mp58 species from strain ATCC 26555, revealed differences in recognition patterns depending on the strain tested and the culture conditions used. Serum samples from normal and Candida infected individuals were examined for the presence of antibodies against mp58 by Western immunoblotting. None of the sera from control individuals and patients suffering from superficial candidiasis contained antibodies against mp58. However, positive reactivity with this antigen and other cell wall constituents was detected for all sera from patients with confirmed systemic candidiasis. Together, these results suggest that mp58 could play an active role during infection and may be useful as a specific antigenic marker for candidiasis.     

Fas2-ELISA in the detection of human infection by Fasciola hepatica

Fasciola hepatica has recently emerged as a major pathogen of humans from reports on areas of endemicity and hyper-endemicity for fascioliasis. This situation is aggravated by the lack of standard assays for the screen diagnosis of F. hepatica infection in humans living in endemic areas. Our laboratory has developed an enzyme-linked immunosorbent assay (Fas2-ELISA) based on the capture of IgG antibody by a purified protein Fas2, which is an adult fluke cysteine proteinase. Fas2-ELISA exhibited 95% sensitivity and 100% specificity in 38 individuals infected with F. hepatica diagnosed by finding eggs in stools and 46 serum samples from healthy volunteers. No cross-reaction was observed with 54 serum samples from patients with ten different parasitic infections including the trematodes Paragonimus westermani and Schistosoma mansoni. The high antigenicity of Fas2 is suggested by the fact that antibodies to Fas2 rise rapidly by 1-2 weeks of infection and rise until patency at 8 weeks of infection in experimentally infected alpacas. Field screening for human fascioliasis using Fas2-ELISA and coprology in three endemic locations of the Peruvian Andes resulted in 95.5% sensitivity, 86.6% specificity in a population of 664 children in an age range of 1 to 16 years old. These results provide evidence of the clinical potential of Fas2-ELISA to diagnose fascioliasis in humans exposed to liver fluke infection in endemic areas for this parasite. Fas2-ELISA is currently developed as a standard assay for both field screening for fascioliasis in people living in endemic areas and detecting occasionally F. hepatica infected patients in clinical laboratories.     

Studies on Tyzzer's disease: acquired immunity against infection and activation of infection by immunosuppressive treatment.

The correlation between serum antibody titre and resistance to challenge infection with Bacillus piliformis was studied in naturally infected mice, in experimentally infected but recovered mice, and in mice treated with antigen prepared from infected livers. Irrespective of the way in which the antibodies were acquired resistance to infection was found to be related to the immunofluorescence antibody titre found. Experimentally infected but recovered mice, as well as rats with persistent antibodies to Bacillus piliformis, were given prednisolone in order to activate a possible persistent infection. Bacillus piliformis was detected in the rats, but not in the mice.     

Serodiagnosis of cilia-associated respiratory bacillus infection by the indirect immunofluorescence assay technique

Antibody to cilia-associated respiratory (CAR) bacillus was detected by the indirect immunofluorescence assay (IFA) technique using tracheal sections of infected mice as antigen in serum samples collected from rats infected naturally and experimentally. Nine of 23 cases of natural infection were positive in IFA antibody, with titres ranging from 1:10 to 1:80, and all these antibody-positive cases were also histologically positive. The remaining 14 cases were negative in both IFA antibody and histological diagnosis, even though some of them were infected with Sendai virus and Mycoplasma pulmonis. In the experimental infection, serum samples collected from 18 rats on days 4, 7, 14, 21, 28 and 56 post-inoculation (PI) (three rats for each point) and examined for IFA antibody revealed that seroconversion occurred in one rat on day 14 PI and in three rats on day 21 PI. Antibody titres of 1:80 to 1:160 remained to the termination of the experiment. The IFA technique was useful for the diagnosis of CAR bacillus infection except in the early stage of the infection.     

显微注射青霉素G获得单感染Wolbachia和单感染Cardinium白背飞虱品系

Wolbachia和Cardinium都是广泛存在于节肢动物体内的一类母系遗传的共生细菌, 可以通过不同方式操纵寄主的生殖行为。Wolbachia和Cardinium感染同一寄主在自然界比较常见, 但是在某些可以同时感染Wolbachia和Cardinium的寄主中其单感染品系较难发现。本研究检测了云南文山(YN)、 海南三亚(HN)这2个不同地理种群中Wolbachia和Cardinium的感染情况; 以双感染Wolbachia和Cardinium的白背飞虱Sogatella furcifera海南种群为实验材料, 运用显微注射方法对双感染Wolbachia和Cardinium的白背飞虱若虫注射不同浓度青霉素G以获得单感染品系。结果表明： 白背飞虱自然种群中单感染Wolbachia比率极低, 本实验用到的海南种群未检测到单感染Wolbachia成虫; 通过显微注射青霉素G的方法可以从白背飞虱双感染品系中筛选获得单感染品系, 当青霉素G注射浓度为0.2%(w/v), 注射龄期为5龄时得到单感染品系效率最高; F5代的检测结果显示显微注射得到的单感染品系可以稳定遗传。本研究为获得单感染品系白背飞虱提供了快捷方法, 同时为其他双感染Wolbachia和Cardinium节肢动物不同感染品系的筛选提供参考。