Comparison of in vitro-cultured and wild-type Perkinsus marinus. I. Pathogen virulence

Perkinsus marinus is a highly contagious pathogen of the eastern oyster Crassostrea virginica. Until recently, transmission studies have employed wild-type parasites isolated directly from infected oysters. Newly developed methods to propagate P. marinus in vitro have led to using cultured parasites for infection studies, but results suggest that cultured parasites are less virulent than wild-type parasites In this paper, we report results of experiments designed to quantify differences between wild-type and cultured P. marinus virulence and to test the following hypotheses: (1) in vitro-cultured parasites are less virulent than wild-type parasites; (2) virulence decreases gradually during in vitro culture; (3) virulence of in vitro cultures can be restored by in vivo passage; (4) virulence changes with culture phase. Our results demonstrate that parasites freshly isolated from infected hosts are much more virulent than those propagated in culture, indicating a potential deficiency in the culture medium used. Virulence was lost immediately in culture and, for that reason, the practice of repassing cultured cells through the host to restore virulence does not work for P. marinus. Virulence was also associated with culture phase: log-phase parasites were significantly more virulent than those obtained from lag- or stationary-phase cultures.     

Characterization of human cytotoxic lymphocytes directed against cells infected with typhus group rickettsiae: evidence for lymphokine activation of effectors

An in vitro culture and assay system was used to determine whether cytotoxic lymphocytes are generated in humans after rickettsial infection. Peripheral blood mononuclear cells (PBMC) were obtained from six individuals with serologic evidence of prior infection with typhus group rickettsiae and from six nonimmune individuals. After PBMC from immune individuals were stimulated in vitro for 7 days with rickettsial antigen, they were capable of lysing typhus group rickettsia-infected, autologous phytohemagglutinin (PHA)-induced blasts, but not uninfected PHA-blasts. No cytotoxic effector cells were generated when either PBMC from immune individuals were placed in culture for 7 days without antigenic stimulation, or when PBMC from nonimmune individuals were stimulated in vitro with antigen for 7 days. Freshly isolated PBMC from immune donors were also unable to lyse typhus group rickettsia-infected autologous PHA-blasts or an autologous rickettsia-infected lymphoblastoid cell line (LCL). Neither supernatants from antigen-stimulated cultures of PBMC from immune donors nor recombinant human interferon-gamma were capable of significantly lysing typhus group rickettsia-infected PHA blasts by this assay. Populations of cytotoxic effector cells depleted of OKT3, OKT4, or OKT8-positive cells by treatment with the respective monoclonal antibodies and complement were assayed for their cytotoxic capacity. The results suggest that the cytotoxic effector cell population is predominantly OKT3 and OKT8-positive, but OKT4-negative. Positive selection with the use of a fluorescence-activated cell sorter also suggested that most of the cytotoxic effector cells are OKT8-positive. PBMC from immune donors after in vitro stimulation with rickettsial antigen were capable of significantly lysing infected autologous LCL or infected HLA-mismatched LCL as compared with the respective uninfected controls. In addition, PBMC from either immune donors or nonimmune donors after stimulation in vitro for 7 days with media containing purified lymphokines were capable of significantly lysing autologous infected LCL as compared with the uninfected autologous control. We conclude that lysis of cells infected with typhus group rickettsiae is mediated by a lymphokine-activated killer.     

An in vitro assay system for the identification of potential antimalarial drugs

Current models for antimalarial drug screening generally measure the survival of drug-treated rodents infected with Plasmodium berghei. Modifications of existing continuous culture methods for P. falciparum allow the rapid, accurate and economical determination of drug effects directly against the human pathogen. Parasite cultures can be maintained in RPMI 1640 medium supplemented with human or rabbit serum or with hypoxanthine-supplemented bovine serum. The antiparasite effects of four drugs, chloroquine, chloramphenicol, clindamycin, and halofuginone, are identical in these sera; drugs can be screened routinely against P. falciparum grown in bovine serum supplemented with hypoxanthine. Drug effects may be rapidly and accurately determined by monitoring the incorporation of 3H-hypoxanthine into parasite nucleic acids. Results obtained with this technique are highly correlated with those derived from visual counting of parasites in thin blood films. Compounds with antimalarial activity in culture may be further screened by measuring the effects of serum obtained from drug-treated rabbits on parasites in culture. The advantages of this system over models currently used for antimalarial screening are discussed.     

Expressed resistance to black shank among tobacco callus cultures

Summary Quantitatively inherited resistance to the black shank pathogen (Phytophthora parasitica var. nicotianae) was expressed among callus tissue cultures of tobacco (Nicotiana). Tissue cultures of genotypes known to posses polygenic mechanisms for black shank resistance expressed that resistance in vitro when challenged by the viable pathogen. Callus of a susceptible cultivar was readily parasitized in culture. Furthermore, single gene resistance to the common pathogen race was also shown to operate in vitro. Nongenetic factors examined did not contribute significantly to the observed differences. Disease expression in vitro appeared to be highly correlated with its expression at the whole plant level.Screening for quantitative disease resistance can be complicated at the whole plant level by variable hostpathogen reactions and by significant genotype × environment interactions. Since quantitatively inherited mechanisms of black shank resistance are expressed in tobacco callus cultures, an in vitro host-pathogen system may be useful in screening tobacco lines for black shank resistance.The research reported in this paper (No. 82-3-6) is in connection with a project of the Kentucky Agr. Exp. Stn., and the paper is published with the approval of the director     

Immunofluorescence in the Study of Marek''s Disease: I. Detection of Antigen in Cell Culture and an Antigenic Comparison of Eight Isolates 1

The indirect fluorescent-antibody (FA) test was applied to the detection of Marek's disease (MD) antigen in cell culture and antibody in the serum of birds. For the detection of antigen, sera were obtained from birds hyperimmunized with the JM strain of MD. MD antigen could be detected in the nucleus and in the cytoplasm of duck and chick embryo fibroblasts and in those of chick kidney cells infected with material known to contain the MD virus. Uninoculated cultures of chicken cells were always free of MD antigen. When chick kidney cells were infected with a stock cellular preparation of MD virus, infected cells could be detected after 24 hr with the FA test. At this time no cytopathological areas were seen by conventional light microscopy. By 7 days after infection, the same number of infected areas were detected by both methods, and the fluorescent areas coincided with the cytopathological areas. This indicates that the fluorescent areas and the areas with cytopathology are caused by the same agent. A straight-line relationship between the dilution of inoculum and the number of fluorescent or morphological foci obtained indicates that one infectious unit produced one fluorescent or morphological focus. In addition, this time sequence study confirmed the cell association of the virus and demonstrated the cell-to-cell spread of infection. Cell cultures inoculated with eight different isolates of MD were tested in all combinations with sera prepared against the same isolates. The antigens were indistinguishable from one another, indicating that either the strains are antigenically identical or there is a common antigen or contaminant in all of them so that they stained equally well. The FA test can detect MD antigen before cytopathological areas develop in cell culture; however, the small size of the area usually examined precludes its use in initial isolations in which only a small number of infectious units are present in the inoculum. MD-infected cells contain a heat-stable antigen similar to that found in herpes simplex-infected cells.     

Cannibalism and the stage-dependent transmission of a viral pathogen of the Indian meal moth, Plodia interpunctella

1. The transmission of an insect pathogen by cannibalism was studied by a series of choice and no-choice experiments.
2. Infection of Plodia interpunctella larvae with a granulosis virus occurred through cannibalism of infected larvae.
3. Depending on the larval instars of the cannibal and the victim relative to each other, preferential cannibalism of both infected and healthy larvae was observed.
4. Larvae cannibalise healthy, less vulnerable larvae preferentially, but it is argued that there is no evidence that they are avoiding infection.
5. The victim cannibalised can be explained as a balance between the reduced risk of injury and the ease of cannibalism of moribund infected individuals on the one hand and the greater food resource value of healthy individuals on the other.
6. The implications for the insect population dynamics of the transmission of the pathogen via cannibalism and the effective removal of infective particles through cannibalism by immune stages are discussed.     

Tumor necrosis factor induces resistance of macrophages to Legionella pneumophila infection

Legionella pneumophila is an ubiquitous opportunistic intracellular pathogen that replicates readily in thioglycollate-elicited peritoneal macrophages from genetically susceptible A/J mice. Treatment of macrophage cultures in vitro with tumor necrosis factor-alpha (TNF-alpha) induced resistance of the macrophages to infection by Legionella as compared with control macrophages treated with medium alone. Addition of small amounts of monoclonal antibody to TNF-alpha restored susceptibility of the macrophages. Furthermore, antibody to the proinflammatory cytokine interleukin-1 (IL-1) alpha/beta increased resistance, but recombinant IL-1 had little effect. Such decreased susceptibility to Legionella growth in anti-IL-1 antibody-treated cultures corresponded with enhanced levels of TNF-alpha in the supernatants of the treated cells. An antibody to another proinflammatory cytokine with known immunoregulatory properties (i.e., IL-6) had little or no effect on the ability of the macrophages to be infected by Legionella and, furthermore, treatment with recombinant IL-6, similar to recombinant IL-1, did not modify the ability of the cells to be infected in vitro. These results indicate that TNF-alpha is important in controlling L. pneumophila replication, and IL-1 can regulate TNF-alpha levels, affecting susceptibility of macrophages to infection with an intracellular opportunistic pathogen like Legionella.     

Immunohistochemistry based assay to determine the effects of treatments on Cryptosporidium parvum viability

Cell culture infectivity assays can provide an accurate means of detecting viable Cryptosporidium parvum oocysts from environmental samples or to test the effects of various treatments on oocyst infectivity. Cell culture assays can also be used to test candidate chemotherapeutic agents. The use of a human cell line provides a situation close to human infection. The present assay uses an anti-Cryptospordium primary antibody, combined with a biotinylated secondary antibody, and an immunoperoxidase detection system. Cryptosporidium parvum oocysts excysted in vitro when placed on monolayers of HCT-8 cells and developmental stages including schizonts and merozoites were visualized using light microscopy of the immunoperoxidase stained slides and by transmission electron microscopy of infected HCT-8 cell cultures. Because the immunoperoxidase system used gives a permanent preparation, the cell cultures can be retained and examined later. Dose titration of oocysts indicated that as few as 50 inoculated oocysts could be detected. The activity of paromomycin was evaluated in this system and 500 microg/ml produced a 97.8% reduction in infection.     

Experimental inoculation of meadow voles (Microtus pennsylvanicus), house mice (Mus musculus), and Norway rats (Rattus norvegicus) with Mycobacterium bovis

Mycobacterium bovis has a wide host range that includes several wildlife species, and this can hamper attempts to eradicate bovine tuberculosis from livestock. The purpose of this study was to determine if common rodent species, namely meadow voles (Microtus pennsylvanicus), house mice (Mus musculus), and Norway rats (Rattus norvegicus), that inhabit the bovine tuberculosis endemic area of Michigan, can be experimentally infected with M. bovis. The objectives of the study were: 1) to determine if these rodent species can be infected, and if so, to document attendant pathologic processes/pathogenesis; 2) to detect any fecal shedding of M. bovis; and 3) to evaluate the relative susceptibility of the three species to M. bovis infection. For each species (n=36) there were two treatment (n=12/group) and one or two control groups depending on species (n=6-12/group); the maximum study duration was 60 days. The meadow vole treatments consisted of high dose inocula that were given by oral or intranasal routes, whereas the house mice and Norway rats were given only oral inocula at either a high or low dose. Of the three species, meadow voles were most susceptible to M. bovis infection. Upon intranasal inoculation, all 12 voles were infected as determined by gross and microscopic lesions and culture of M. bovis from tissue and feces. Seven of the 12 meadow voles inoculated orally were infected. House mice also were susceptible; M. bovis was isolated from 14 of 24 animals. Only one Norway rat in the high dose treatment group was positive by culture and this was the only animal from which minimal attendant lesions were observed. Results of this study indicate that meadow voles and house mice can be infected with M. bovis and might serve as spillover hosts. Concerted efforts should, therefore, be made to reduce or eliminate these rodents on premises where M. bovis-infected livestock are present.     

Impact of myxomatosis in relation to local persistence in wild rabbit populations: the role of waning immunity and the reproductive period

Many diseases are less severe when they are contracted in early life. For highly lethal diseases, such as myxomatosis in rabbits, getting infected early in life can represent the best chance for an individual to survive the disease. For myxomatosis, early infections are attenuated by maternal antibodies. This may lead to the immunisation of the host, preventing the subsequent development of the lethal form of the disease. But early infection of young individuals requires specific demographic and epidemiological contexts, such as a high transmission rate of the pathogen agent. To investigate other factors involved in the impact of such diseases, we have built a stochastic model of a rabbit metapopulation infected by myxomatosis. We show that the impact of the pathogen agent can be reduced by early infections only when the agent has a long local persistence time and/or when the host subpopulations are highly connected. The length of the reproductive period and the duration of acquired immunity are also important factors influencing the persistence of the pathogen and thus, the impact of the disease. Besides confirming the role of classical factors in the persistence of a pathogen agent, such as the size of the subpopulation or the degree of connectivity, our results highlight novel factors that can modulate the impact of diseases whose severity increase with age.     

Evaluating bacterial pathogen DNA preservation in museum osteological collections

Reports of bacterial pathogen DNA sequences obtained from archaeological bone specimens raise the possibility of greatly improving our understanding of the history of infectious diseases. However, the survival of pathogen DNA over long time periods is poorly characterized, and scepticism remains about the reliability of these data. In order to explore the survival of bacterial pathogen DNA in bone specimens, we analysed samples from 59 eighteenth and twentieth century individuals known to have been infected with either Mycobacterium tuberculosis or Treponema pallidum. No reproducible evidence of surviving pathogen DNA was obtained, despite the use of extraction and PCR-amplification methods determined to be highly sensitive. These data suggest that previous studies need to be interpreted with caution, and we propose that a much greater emphasis is placed on understanding how pathogen DNA survives in archaeological material, and how its presence can be properly verified and used.     

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.     

Use of binomial group testing in tests of hypotheses for classification or quantitative covariables

In group testing, the test unit consists of a group of individuals. If the group test is positive, then one or more individuals in the group are assumed to be positive. A group observation in binomial group testing can be, say, the test result (positive or negative) for a pool of blood samples that come from several different individuals. It has been shown that, when the proportion (p) of infected individuals is low, group testing is often preferable to individual testing for identifying infected individuals and for estimating proportions of those infected. We extend the potential applications of group testing to hypothesis-testing problems wherein one wants to test for a relationship between p and a classification or quantitative covariable. Asymptotic relative efficiencies (AREs) of tests based on group testing versus the usual individual testing are obtained. The Pitman ARE strongly favors group testing in many cases. Small-sample results from simulation studies are given and are consistent with the large-sample (asymptotic) findings. We illustrate the potential advantages of group testing in hypothesis testing using HIV-1 seroprevalence data.     

Latex test used for diagnosis of dysentery caused by Shigella sonnei]

The aim of this study was to evaluate the usefulness of latex reagent coated with immunoglobulins specific for antigens of phase I and II of S. sonnei for detection of these antigens in primary, mixed bacterial cultures. The study was performed on 919 fecal samples from individuals with clinical symptoms of dysentery, convalescents and from contact individuals. Material used for the test was bacterial suspension collected from McConkey or SS agars and a culture from selenite F broth heated at 100C. The results of the latex test were compared with the results of isolation of S. sonnei from the same cultures. S. sonnei was isolated from feces of 140 individuals (15.2%), while the latex test was positive in 215 cases (23.4%). The highest testing effectiveness , significantly higher than when isolation of pathogen was performed, was obtained only when 18-20 hr culture on Selenite F medium was used for latex test. The correlation between efficacy of testing for S. sonnei and phosphate content of Selenite F and a mode of its preparation was found. The latex test allows to eliminate from further bacteriological studies cultures free of S. sonnei thus it gives measurable economical profits and it shortens significantly time period of bacteriological examination.     

Elimination of cymbidium mosaic virus and odontoglossum ringspot virus from orchids by meristem culture and thin section culture with chemotherapy

Most commercially cultivated orchid plants are generally infected with cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV). Two methods were used in order to generate virus-free plants: meristem culture and thin section culture with chemotherapy. Meristems (0.10 mm to 1.00 mm) were excised from infected axillary shoots of an infected monopodial orchid hybrid (Mokara Char Kuan ‘Pink’) and cultured in modified Vacin and Went medium. Only larger meristem explants survived and the regenerated plantlets remained virus-infected. In contrast, high percentages of virus-free plantlets were obtained from thin section cultures of infected plantlets and protocorm-like bodies with ribavirin treatment. Interestingly, regenerants from thin section cultures without ribavirin treatment were also found to be free from CyMV and ORSV. All plantlets were tested by enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR).     

Transmission of Pepino mosaic virus by the Fungal Vector Olpidium virulentus

Transmission of Pepino mosaic virus (PepMV) by the fungal vector Olpidium virulentus was studied in two experiments. Two characterized cultures of the fungus were used as stock cultures for the assay: culture A was from lettuce roots collected in Castellón (Spain), and culture B was from tomato roots collected in Murcia (Spain). These fungal cultures were maintained in their original host and irrigated with sterile water. The drainage water collected from irrigating these stock cultures was used for watering PepMV‐infected and non‐infected tomato plants to constitute the acquisition–source plants of the assay, which were divided into six different plots: plants containing fungal culture A (non‐infected and PepMV‐infected); plants containing fungal culture B (non‐infected and PepMV‐infected); PepMV‐infected plants without the fungus; and plants non‐infected either with PepMV and the fungus. Thirty‐six healthy plants grouped into six plots, which constituted the virus acquisition–transmission plants of the assay, were irrigated with different drainage waters obtained by watering the different plots of the acquisition–source plants. PepMV was only transmitted to plants irrigated with the drainage water collected from PepMV‐infected plants whose roots contained the fungal culture B from tomato with a transmission rate of 8%. No infection was detected in plants irrigated with the drainage water collected from plots with only a fungus or virus infection. Both the virus and fungus were detected in water samples collected from the drainage water of the acquisition–source plants of the assay. These transmission assays demonstrated the possibility of PepMV transmission by O. virulentus collected from tomato crops.     

Differential ability of human immunodeficiency virus isolates to productively infect human cells

Isolates of HIV showed distinct differences in the ability to replicate in continuous human hematopoietic cell lines. Moreover, although all PMC cultures obtained from healthy individuals could be infected with HIV, considerable variation in the amount of virus released from different PMC cultures was observed. These biological properties of HIV could not be correlated with clinical state, binding properties of the virus isolates to target cells, or differences in target cell CD4 antigen expression. Some isolates of HIV that could not directly infect the HUT-78 cell line showed productive infection when PMC infected with these viruses were added to this human T cell line. These observations emphasize the importance of cell to cell contact in the spread of virus. The results demonstrate for the first time the differences in the host range specificity of HIV isolates in several individual PMC cultures, and indicate that the optimal isolation of HIV is achieved with normal human PMC rather than established human cell lines.     

Selective Isolation of Leptospiras from Contaminated Material by Incorporation of Neomycin to Culture Media

Incorporation of neomycin to the culture medium was found to be effective in inhibiting Escherichia coli contaminants without interfering with the growth of serotype L. autumnalis. The growth of 12 other Leptospira serotypes was unaffected by the addition of 300 mug of neomycin per ml to Ellinghausen medium or 5 mug/ml to Fletcher medium. Neomycin-containing medium was found to be of value in the isolation of leptospiras from cultures of blood from infected laboratory animals. A higher percentage of isolates was obtained in swine kidneys from an abattoir in medium containing neomycin than resulted from the same medium without antibiotic or with 5-fluorouracil. Contaminated leptospiral cultures growing in media with 5-fluorouracil were purified by subculturing into neomycin-containing media.