Susceptibility of spiny rats (Proechimys semispinosus) to Leishmania (Viannia) panamensis and Leishmania (Leishmania) chagasi

The role of Proechimys semispinosus as reservoir of Leishmania (Viannia) panamensis on the Colombian Pacific coast was experimentally evaluated. The susceptibility to L. chagasi also was assessed to determine the utility of this rodent as a model for studying reservoir characteristics in the laboratory. Wild-caught animals were screened for natural trypanosomatid infections, and negative individuals were inoculated intradermally (ID) in the snout or feet with 10(7) promastigotes of L. panamensis. L. chagasi was inoculated intracardially (10(7) promastigotes) or ID in the ear (10(8) promastigotes). PCR-hybridization showed that 15% of 33 spiny rats were naturally infected with L. Viannia sp. Animals experimentally infected with L. panamensis developed non-ulcerated lesions that disappeared by the 7th week post-infection (p.i.) and became more resistant upon reinfection. Infectivity to sand flies was low ((1/2)0-(1/4)8 infected/fed flies) and transient, and both culture and PCR-hybridization showed that L. panamensis was cleared by the 13th week p.i. Animals inoculated with L. chagasi became subclinically infected and were non-infective to sand flies. Transient infectivity to vectors of spiny rats infected with L. panamensis, combined with population characteristics, e.g., abundance, exploitation of degraded habitats and high reproductive rates, could make them epidemiologically suitable reservoirs.     

Metastatic capability of Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis in golden hamsters

The pattern and kinetics of internal dissemination and frequency of cutaneous metastatic lesions resulting from experimental infection of golden hamsters with Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis were examined. Nineteen strains were evaluated: 16 L. (V.) panamensis isolated from patients and 3 L. (V.) guyanensis, 2 isolated from human cases and 1 WHO reference strain originating from a sandfly vector. Lymphatic dissemination occurred within 3 mo and was observed for 16 of 16 (100%) of L. (V.) panamensis and 3 of 3 (100%) of L. (V.) guyanensis. Parasites were cultured infrequently from liver and spleen: 3 of 125 (2%) L. (V.) panamensis and 1 of 22 (5%) L. (V.) guyanensis. Decreased frequency of isolation from the inoculation site and draining lymph nodes over time was accompanied by increased frequency of isolation from distant lymph nodes. Dilution of triturated tissue samples resulted in an increased efficiency of parasite culture. Both primary lesions and secondary cutaneous metastatic lesions were more severe in hamsters infected with L. (V.) guyanensis than with L. (V.) panamensis. Cutaneous metastatic lesions were produced more frequently by L. (V.) guyanensis, 24 of 46 hamsters (52%), than by L. (V.) panamensis, 28 of 252 hamsters (11%). Individual Leishmania strains displayed distinctive propensities to produce cutaneous metastases, manifested as a reproducible phenotype. Metastatic pathogenicity was independent of the inoculum dose, supporting the dissociation of infectivity and pathogenicity.     

Leishmania braziliensis: protein, carbohydrate, and antigen differences between log phase and stationary phase promastigotes in vitro

When Leishmania species are grown in vitro, parasites from the stationary phase differ from those in log phase growth in being more infective and more resistant to complement and macrophage mediated killing. In the present study, log phase and stationary phase promastigotes of Leishmania braziliensis panamensis were compared at the molecular level. Differences in polypeptide and glycoprotein composition and antigenicity between log and stationary phase promastigotes of L. b. panamensis were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the former showed that two polypeptides were unique to log phase promastigotes and one was unique to stationary phase promastigotes. There were also differences in surface lectin binding characteristics of log and stationary phase promastigotes. Live stationary phase promastigotes bound more concanavalin and lentil lectin than log phase promastigotes, indicating a greater number of mannose residues on their surfaces.     

Development of Leishmania (Viannia) panamensis lesions and relationship of numbers of amastigotes to lesion area on antimony-treated and untreated hamsters

Young adult (60-70-g) male golden hamsters (Mesocricetus auratus) each were injected intradermally at the dorsal base of the tail with 15 x 10(6) promastigotes of Leishmania (Viannia) panamensis (MHOM/PA/83/WR539), and progression and regression of subsequent lesions were evaluated for up to 17 wk postinfection (PI) as to area, weight, and number of amastigotes within lesions in untreated hamsters and in hamsters treated with meglumine antimoniate (Glucantime). In untreated hamsters total area of lesion, weight, and numbers of amastigotes generally increased rapidly and concomitantly up to 3-4 wk PI. Amastigote numbers tended to decrease from 4 to 11 wk PI and subsequently the numbers of amastigotes within the lesions decreased rapidly, whereas relatively little change occurred in the area and weight of the lesions. Meglumine antimoniate treatment of cutaneous hamster lesions resulted in marked concomitant decrease in size of the lesions and numbers of amastigotes within the lesions examined 1 wk after treatment. Measurement of the area of cutaneous leishmanial lesions thus would appear to be a valid method of evaluating the efficacy of promising compounds against L. panamensis in hamsters when measurements are taken 3-5 wk after experimental infection and reflects the number of amastigotes present in the lesion.     

Leishmania braziliensis: development of primary and satellite lesions in the experimentally infected owl monkey, Aotus trivirgatus

Twelve male and 8 female feral owl monkeys, Aotus trivirgatus, were inoculated intradermally at the dorsal base of the tail with 2 X 10(7) promastigotes (strains WR 128 or WR 539) or 5 X 10(5) amastigotes (strain WR 128) of Leishmania braziliensis panamensis, and the progression and regression of subsequent lesions were examined for up to 13 or 54 weeks after inoculation. Three of these monkeys had been infected previously with L. donovani, had been treated with meglumine antimoniate, and had recovered clinically from visceral leishmaniasis. All monkeys developed a cutaneous nodule at the inoculation site, but the size of the nodule varied (maximum 78 to 326 mm2 between 4 and 16 weeks after inoculation.) The initial nodule became ulcerated after 4 to 8 weeks in 17 of the 20 monkeys, and the ulcers persisted for 4 to 16 weeks until covered by a crust. Primary lesions disappeared by 17 to 52 weeks after inoculation, but satellite lesions, of similar morphology to the primary lesions but smaller, developed after 4 to 21 weeks in 14 of the monkeys. The primary nodule was excised in 4 monkeys at 6 weeks and did not recur nor did satellite lesions subsequently develop. The satellite lesions (median total number of 4, range 1 to 25) were adjacent to or at a maximum distance of 6 cm from the primary lesion, varied in size from 3 to 117 mm2, and persisted for 10 to 37 weeks. At 6 and 8 weeks after inoculation, tissue from the cutaneous leishmanial lesions from five monkeys was excised and examined. The granulomatous leishmanial lesions, located primarily in the dermis and subcutis, consisted of macrophages containing parasites, lymphocytes, plasma cells, and occasionally eosinophils. Satellite lesions at 14 weeks after inoculation were similar grossly and microscopically to the initial nodule. No significant differences were observed between promastigote or amastigote derived infections, between the two strains of L. b. panamensis, or between the course of infection based on the sex, age, karyotype, or country of origin of the owl monkeys. Cutaneous lesions developed when 5 X 10(5) amastigotes of L. b. panamensis (strain WR 128) were inoculated intradermally into the dorsal base of the tail, the upper eyelid, and the thorax of three monkeys. Leishmanial nodules which developed on the thorax regressed rapidly (after 2 to 5 weeks) whereas those on the upper eyelid and at the dorsal base of the tail persisted for 5 to 45 weeks after inoculation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental respiratory infection with Pasteurella multocida and Bordetella bronchiseptica in rabbits.

Eight-to-10-wk-old offspring of a colony of specific pathogen free [Eda:(NZW x FG)F1BR] rabbits were exposed to cultures of Pasteurella multocida and Bordetella bronchiseptica. Two groups of 9 animals each were exposed to cultures of either species of bacteria intranasally and killed 2, 7, 14, and 21 da postinoculation. Five of 9 rabbits in each group developed a mucopurulent nasal discharge 4-7 da postinoculation. The remaining 4 rabbits in each group failed to develop clinical signs. The gross and microscopic lesions did not differ in character or distribution among the inoculated rabbits. The infection was characterized by an acute upper respiratory syndrome accompanied by a mild bronchopneumonia.     

Extracellular Amastigote-Like Forms of Leishmania panamensis and L. braziliensis. II. Stage- and Species-Specific Monoclonal Antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania . Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to >200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Leishmania braziliensis in the squirrel monkey: development of primary and satellite lesions and lack of cross-immunity with Leishmania donovani

Three female and 2 male adult laboratory-reared squirrel monkeys (Saimiri sciureus) that previously had been inoculated with Leishmania (Leishmania) donovani and had recovered from experimental visceral leishmaniasis were each inoculated intradermally at the dorsal base of the tail with 2.2 x 10(7) culture-derived promastigotes of Leishmania (Viannia) panamensis. The progression and regression of subsequent lesions were examined for 36 wk in all 5 monkeys after which 3 of the monkeys were killed (1 with a primary lesion and all with satellite lesions) and the 2 surviving monkeys (1 with primary lesion and both with satellite lesions) were treated with 104 mg/kg/day of meglumine antimoniate for 10 days. All of the monkeys developed a primary lesions at the site of injection of the parasite and later developed satellite lesions peripheral to the primary nodule. The primary lesions had disappeared from 3 of the 5 monkeys by 36 wk, whereas satellite lesions persisted on all at this time. Satellite lesions were present at 52 wk after treatment and persisted for 169 wk in the 2 surviving monkeys. The histopathologic appearance of the lesions was characterized as granulomatous inflammation. Our results indicated that squirrel monkeys that had recovered from visceral leishmaniasis remained susceptible to infection with L. (V). panamensis.     

Extracellular amastigote-like forms of Leishmania panamensis and L. braziliensis. II. Stage- and species-specific monoclonal antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania. Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to greater than 200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Lactobacillus reuteri protects epidermal keratinocytes from Staphylococcus aureus-induced cell death by competitive exclusion

Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P < 0.01) or simultaneously with (P < 0.01) infection with S. aureus but not when added after infection had commenced (P > 0.05). The protective effect of L. reuteri was not dependent on the elaboration of inhibitory substances such as lactic acid. L. reuteri inhibited adherence of S. aureus to keratinocytes by competitive exclusion (P = 0.026). L. salivarius UCC118, however, did not inhibit S. aureus from adhering to keratinocytes (P > 0.05) and did not protect keratinocyte viability. S. aureus utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P = 0.03). This suggests that the protective mechanism for L. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by S. aureus and thus aid in the prevention of infection.     

Vector competence of some Neotropical sandflies for the Leishmania (Viannia) braziliensis complex

Abstract. To evaluate the vector competence of some Lutzomyia spp. (Diptera: Psychodidae) for Leishmania (Viannia) spp. (Kinetoplastida: Trypanosoma-tidae), experimental infections of anthropophilic sandflies from the Colombian Pacific coast were performed, through membrane feeding and xenodiagnosis on hamsters infected with Le.(V.)braziliensis or Le.(V.)panamensis. Wild-caught or F, generation females of Lutzomyia gomezi, Lu.hartmanni, Lu.panamensis and Lu. trapidoi were allowed to feed on hamster lesions and then maintained at 26^oC and >80% r.h. on a sugar-water diet until dissection on the fifth day post-infection (p.i.).
Despite similar infection rates (range 37–44%) in both Lu.gomezi and Lu.trapidoi , infections were heavier (>100 parasites) in the latter species. Infections of Lu.trapidoi with Le.braziliensis ( n = 21) and Le.panamensis (n = 27) showed parasite migration toward the foregut, with promastigote colonization of the stomodeal valve and appearance of infective forms. In contrast, infections of Lu.gomezi with Le. braziliensis (n = 10) and Le.panamensis (n = 5) were light (<50 parasites) and usually restricted to the pylorus. In Lu.hartmanni , only a few promastigotes were found in the pylorus and midgut of 3/8 specimens infected with Le.braziliensis , and no Le.panamensis developed (n = 19). By day 5 p.i., promastigote colonization of the hind- and midgut by Le.panamensis was observed in 2/4 Lu.panamensis but not Le.braziliensis (n = 3).
It was concluded that Lu.trapidoi is a more efficient vector than Lu.gomezi for both Le.braziliensis and Le.panamensis , and that Lu.hartmanni and Lu.panamensis are of minor importance for Leishmania transmission in this endemic area.     

Leishmania panamensis: comparative inhibition of nuclear DNA topoisomerase II enzymes from promastigotes and human macrophages reveals anti-parasite selectivity of fluoroquinolones, flavonoids and pentamidine

Certain model inhibitors exerted selective action against the catalytic activity of nuclear DNA topoisomerase II (TOPII) of Leishmania panamensis promastigotes. The second-generation fluoroquinolones enoxacin and ciprofloxacin exhibited extraordinarily high anti-parasite selectivity displaying 582- and 40-fold greater potencies against L. panamensis TOPII as compared with the human macrophage enzyme. The flavonoids quercetin and ellagic acid showed inverse specificities, the former being 161-fold more potent against L. panamensis TOPII, and the latter 15.7-fold more active against macrophage TOPII. The protoberberine coralyne was a potent inhibitor of both Leishmania and macrophage TOPII. Bis-benzimidazoles and the diamidine diminazene aceturate exhibited uniformly high potencies against parasite and host TOPII, but a second diamidine pentamidine showed 17.6-fold greater specificity for Leishmania TOPII. The antimonial sodium stibogluconate was an ineffective inhibitor of parasite TOPII showing 4.3-fold greater potency against the macrophage enzyme. These findings suggest that the leishmanicidal activities of certain fluoroquinolones and pentamidine may be mediated partly through TOPII inhibition.     

Introductory Remarks: Symposium on Differentiation of Trypanosomatids

ABSTRACT. Leishmania differentiation in the gut of phlebotomine sand flies was evaluated based on five light and electron microscopic studies of natural (Leishmania panamensis/Lutzomyia gomezi, Leishmania chagasi/Lutzomyia longipalpis) and unnatural (Leishmania mexicana/Lutzomyia abonnenci, Leishmania panamensis/Phlebotomus papatasi, Leishmania major/Lutzomyia longipalpis) life cycles. In the bloodmeal, transformation of amastigotes into stumpy promastigotes occurred before or during division. Further division in pairs or rosettes resulted in the development of spatulate and/or elongate nectomonad (free-swimming) promastigotes. Elongate, short, and metacyclic nectomonad promastigotes, and nectomonad paramastigotes were present in the midgut lumen. Dividing short promastigotes predominated in the cardia, and appeared to generate metacyclic forms which were observed in three life cycles. Haptomonad (attached) forms of Leishmania panamensis in the hindgut were primarily spatulate promastigotes (natural host) or pear-shaped promastigotes (unnatural host); paramastigotes and dividing forms were rare. At the stomodeal valve, short haptomonad promastigotes predominated in unnatural hosts, while both short and pear-shaped haptomonads were abundant, along with paramastigotes in natural hosts. Haptomonad paramastigotes and pear-shaped promastigotes colonized the esophagus, while paramastigotes predominated in the pharynx. Metacyclics were free-swimming in the lumen of the foregut.     

I. Extracellular Cultivation and Morphological Characterization of Amastigote-Like Forms of Leishmania panamensis and L. braziliensis

. Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32°C for L. panamensis (WR442; L. braziliensis panamensis ) and at 28°C for L. braziliensis (M5052; L. braziliensis braziliensis ). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.     

Extracellular cultivation and morphological characterization of amastigote-like forms of Leishmania panamensis and L. braziliensis

Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32 degrees C for L. panamensis (WR442; L. braziliensis panamensis) and at 28 degrees C for L. braziliensis (M5052; L. braziliensis braziliensis). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.     

Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol

Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC₅₀ values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC₅₀ = 199 µM) compared to the parent strain (IC₅₀ = 45 µM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.     

Leishmania tropica infection in hamsters and a review of the animal pathogenicity of this species.

Four strains of Leishmania tropica s. st. from Afghanistan were found to be constantly infective to the golden hamster (Mesocricetus auratus), whether inoculated as culture promastigotes from early passages or as amastigotes from the cutaneous lesions. In total, 66 amastigote passages were carried out over 6 passage generations. The footpad lesions were chronic and nonulcerative infiltrates; their evolution is described up to 15 months. No self-healing was observed. Balb/c mice and gerbils were not susceptible. Earlier studies on L. tropica s. st. in laboratory animals, principally by Soviet and French workers, are critically reviewed. This new experimental model of chronic cutaneous leishmaniasis is of special interest for studies on the immunology and chemotherapy of the disease. Some advantages over the commonly used L. major model are discussed.     

Increased susceptibility to Staphylococcus aureus colonization of the skin of the NOA mouse: a potentially useful animal model for evaluating antiseptic effects.

Isolation of bacteria from wet skin lesions was attempted using Naruto Research Institute Otsuka Atrichia (NOA) mice, which develop such lesions spontaneously at a high rate. As a result, Staphylococcus aureus was demonstrated to have colonized the wet skin lesions at high density. In addition, the isolated S. aureus was found to be similar to the strain of S. aureus thought to colonize the eczematous lesions seen in humans with atopic dermatitis. Furthermore, a survey of the S. aureus colonization status of NOA mice with no wet skin lesions confirmed colonization at higher density than in HR-1 mice as control, indicating that the skin of the NOA mouse has the novel characteristic of increased susceptibility to S. aureus colonization. Thus, by using changes in S. aureus counts as an index, the NOA mouse can be expected to serve as a useful animal model for evaluating the effects of topical antiseptics. The antiseptic effects of an ointment and a lotion containing chlorhexidine gluconate were confirmed using this animal model.     

Limitations in the use of Drosophila melanogaster as a model host for gram-positive bacterial infection

AIMS: To examine sensitivities of various Drosophila melanogaster strains towards human pathogenic and nonpathogenic gram-positive bacteria. METHODS AND RESULTS: The D. melanogaster Oregon R strain was infected by injecting the thorax with a needle containing Escherichia coli (negative control), Listeria monocytogenes, Staphylococcus aureus (both food-borne pathogens), Listeria innocua, Bacillus subtilis, Carnobacterium maltaromaticum, Lactobacillus plantarum or Pediococcus acidilactici (all nonpathogenic bacteria). Listeria monocytogenes and S. aureus killed the host rapidly compared with the negative control. Infection with L. innocua, B. subtilis or C. maltaromaticum also resulted in a high fly mortality, whereas Lact. plantarum and P. acidilactici resulted in a slightly increased mortality. Four additional D. melanogaster lines, three of which had been selected for heat, cold and desiccation resistance respectively, were subjected to infection by L. monocytogenes, S. aureus and E. coli. Mortality rates were comparable with that of the Oregon R strain. CONCLUSIONS: Use of the injection method shows the limitation of D. melanogaster as a model host for gram-positive bacteria as opportunistic infection by nonpathogenic gram-positive bacteria results in partial or high mortality. In addition, lines of fruit flies resistant to various stress exposures did not show an increased resistance to infection by gram-positive pathogens under the conditions tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the inadequacy of D. melanogaster infected by the injection method in order to distinguish between virulent and nonvirulent gram-positive bacteria.     

Immunoglobulin M synthesized by human lymphoblastoid cells: interaction with Staphylococcus aureus and protein A.

Immunoglobulin M synthesized by a human lymphoblastoid cell line, LA173, was found to bind specifically to the protein A-bearing Cowan I strain of Staphylococcus aureus. The (3H)-leucine-labeled, secreted IgM from these LA173 cells also formed precipitin complexes with purified protein A. Soluble complexes formed at high protein A/IgM ratios retained the ability to bind to the bacterial surface. Precipitin complexes also were observed in double diffusion Ouchterlony gels with a line of identity formed between the IgM, protein A, and anti-IgM in adjacent wells. Intracellular IgM species from detergent-lysed LA173 cells were bound to S. aureus. Labeled 19S pentamers, 8S monomers, and HL subunits were eluted from the bacteria and identified by velocity sedimentation and SDS agarose-acrylamide gel electrophoresis. In addition, several intermediates migrating between 8S and 19S were detected and shown to contain authentic H and L chains. Binding of the labeled IgM 19S pentamers to staphylococci was not inhibited by prior treatment of the bacteria with an excess of unlabeled human IgG. However, S. aureus saturated with unlabeled IgG did not bind either labeled IgM monomers or labeled IgG. The interaction of this human IgM with S. aureus exhibited a high degree of specificity with quantitative recovery of secreted 19S IgM. Intracellular IgM species were bound selectively by the bacteria with little if any contamination by other cytoplasmic proteins.