Low-pathogenicity Mycoplasma spp. alter human monocyte and macrophage function and are highly prevalent among patients with ventilator-acquired pneumonia.

BACKGROUND: Ventilator-acquired pneumonia (VAP) remains a significant problem within intensive care units (ICUs). There is a growing recognition of the impact of critical-illness-induced immunoparesis on the pathogenesis of VAP, but the mechanisms remain incompletely understood. We hypothesised that, because of limitations in their routine detection, Mycoplasmataceae are more prevalent among patients with VAP than previously recognised, and that these organisms potentially impair immune cell function. METHODS AND SETTING: 159 patients were recruited from 12 UK ICUs. All patients had suspected VAP and underwent bronchoscopy and bronchoalveolar lavage (BAL). VAP was defined as growth of organisms at >10(4) colony forming units per ml of BAL fluid on conventional culture. Samples were tested for Mycoplasmataceae (Mycoplasma and Ureaplasma spp.) by PCR, and positive samples underwent sequencing for speciation. 36 healthy donors underwent BAL for comparison. Additionally, healthy donor monocytes and macrophages were exposed to Mycoplasma salivarium and their ability to respond to lipopolysaccharide and undertake phagocytosis was assessed. RESULTS: Mycoplasmataceae were found in 49% (95% CI 33% to 65%) of patients with VAP, compared with 14% (95% CI 9% to 25%) of patients without VAP. Patients with sterile BAL fluid had a similar prevalence to healthy donor BAL fluid (10% (95% CI 4% to 20%) vs 8% (95% CI 2% to 22%)). The most common organism identified was M. salivarium. Blood monocytes from healthy volunteers incubated with M. salivarium displayed an impaired TNF-α response to lipopolysaccharide (p=0.0003), as did monocyte-derived macrophages (MDMs) (p=0.024). MDM exposed to M. salivarium demonstrated impaired phagocytosis (p=0.005). DISCUSSION AND CONCLUSIONS: This study demonstrates a high prevalence of Mycoplasmataceae among patients with VAP, with a markedly lower prevalence among patients with suspected VAP in whom subsequent cultures refuted the diagnosis. The most common organism found, M. salivarium, is able to alter the functions of key immune cells. Mycoplasmataceae may contribute to VAP pathogenesis.

Efficacy of heartworm preventatives against ascarids and hookworms in client-owned dogs: a retrospective case control study.

The efficacy of heartworm preventatives against ascarids and hookworms was assessed in a retrospective analysis of 1329 dogs that received a fecal examination and were surveyed about heartworm preventative use upon presentation to the veterinary teaching hospital at the University of Pennsylvania. To remove confounding due to age, patients under 6 months old were analyzed separately from the remaining population. Although there were no reported cases of ascarids or hookworms in patients under 6 months old receiving monthly heartworm prevention, the prevalence reached 5.2% and 11.7%, respectively, in patients that were not using any products. For patients over 6 months old, there were no apparent associations between parasites and heartworm preventative use. Of the 75.5% of dogs that were administered heartworm preventatives, 16.1% reported seasonal use and 83.9% reported using the products year round. Patients using heartworm preventatives seasonally were no more likely to be harboring nematode parasites than patients using preventatives year round (OR = 0.70, 95% CI: 0.19-2.55). Overall efficacy rates were consistent with prior studies on the active ingredients. Heartworm preventative have the greatest value for controlling nematode endoparasites in patients under 6 months old.

Efficacy of major anthelmintics for reduction of fecal shedding of strongyle-type eggs in horses in the Mid-Atlantic region of the United States.

In the last decade there have been numerous reports of anthelmintic resistant cyathostomins in many parts of the world. The objective of the present study was to evaluate the efficacy of the commercially available anthelmintics against cyathostomin egg shedding in the Mid-Atlantic region of the United States. A total of 989 horses from 67 different farms located in southeastern Pennsylvania, northern Delaware, and northeastern Maryland were treated with fenbendazole, oxibendazole, pyrantel pamoate, ivermectin, or moxidectin at their recommended dosages. Fecal egg count reduction testing was used to determine the efficacy of each anthelmintic on those horses with fecal egg counts of ≥ 200 eggs per gram on the day of treatment (272 horses). Decreased efficacy (reduction of strongyle-type fecal egg counts by less than 90%) was found for fenbendazole, oxibendazole, and pyrantel pamoate, with only 6%, 21% and 43% of horses showing reductions of greater than 90%, respectively. The macrocyclic lactones showed high efficacy in all horses sampled in this study. The decreased anthelmintic efficacy detected in this study adds further evidence for the existence of resistant cyathostomins throughout much of the eastern United States. Findings from this study can be used to create a more sustainable approach for parasite control programs.

Inhibition of in vivo and in vitro infectivity of Leishmania donovani by tunicamycin.

Leishmania donovani 2S strain promastigotes were rendered non-infectious to mice and mouse peritoneal macrophages by treatment with tunicamycin, an inhibitor of N-linked protein glycosylation. Concentrations of tunicamycin (1-10 micrograms ml-1) that reduced promastigote infectivity to 2% or less of control levels had little or no measurable effect on the in vitro growth of the promastigotes. Tunicamycin has no apparent effect on the entry of promastigotes into macrophages. These results indicate that the sugar residues of glycoproteins are important to the promastigote during the early stages of macrophage infection.

Strongyloides stercoralis: histopathology of uncomplicated and hyperinfective strongyloidiasis in the Mongolian gerbil, a rodent model for human strongyloidiasis [corrected].

Tissues from corticosteroid-treated gerbils hyperinfected with Strongyloides stercoralis were compared grossly and microscopically to similar tissues from animals with uncomplicated strongyloidiasis. Gerbils with hyperinfection developed severe pulmonary alveolar haemorrhage with a variable degree of subacute eosinophilic interstitial pneumonia associated with numerous alveolar, vascular and interstitial larvae. Hyperinfection induced by corticosteroids, given either before inoculation of S. stercoralis larvae or after a chronic Strongyloides infection was established, produced similar lesions. In contrast, lungs from gerbils with uncomplicated Strongyloides infection had severe eosinophilic perivasculitis and vasculitis with very little haemorrhage, no pneumonia and no larvae. Sections of adult worms were present in the proximal part of the intestinal tract, lodged in spaces between mucosal epithelial cells. Adult worms were not associated with inflammation and were more common in the corticosteroid-treated gerbils. In corticosteroid-treated gerbils only, there were numerous larvae in the distal intestinal tract, throughout the intestinal wall and adjacent mesentery, within interstitial tissues and in lymphatic vessels. Significant inflammation with associated larvae was only present in the caecum and mesenteric lymph nodes, suggesting that the caecum was the main site for initiation of parenteral migration with subsequent invasion of the lymphatic system and lungs. The lesions in these gerbils were similar to those found in humans. Infection of gerbils with S. stercoralis is the best rodent model of human strongyloidiasis.

Strongyloides stercoralis: oral transfer of parasitic adult worms produces infection in mice and infection with subsequent autoinfection in gerbils.

Oral transfer of parasitic adult Strongyloides stercoralis produced patent infections in gerbils, C57BL/6J and SCID mice. In gerbils receiving adult worms, 7.3% of the transferred worms established and autoinfective L3 were found beginning on day 5 post-transfer, with peak numbers seen on days 6 and 7 post-transfer and few seen by 9 days post-transfer. These results suggest that development of autoinfective L3 in the gerbil is limited by the immune response of the host. When given orally to mice, between 7.2% (C57BL/6J) and 19.5% (SCID) of the adult worms established. These levels are higher than those previously obtained by the subcutaneous infection of SCID mice with infective larvae. No autoinfective larvae were found in infected mice and the ratio of L1/adult worms was small compared with that seen in gerbils. Thus, mice infected orally can be used as a model to study the interaction between the adult worm and the host, and since autoinfection has not been seen in the murine model, as developed to date, orally infected mice may be useful as a model to study mechanisms preventing autoinfection.

Large-cell "mixed-phenotype" lymphoma in AIDS. Identification of a CD5-expressing subset of B-cell non-Hodgkin's lymphoma.

We have characterized cells from an AIDS-associated non-Hodgkin's lymphoma (NHL). We found a malignant CD5+ B cell (approximately three-fourths of cells), with the balance of cells comprised of T cells having an activated phenotype. The lymphoma was unusual, in that all cells bear "T-lineage" markers, CD3 and CD5, and "B-lineage" markers, CD19 and CD20. Thus, conventional immunohistologic techniques were insufficient to type this mixture of cells; single-cell analysis was required. These "mixed-phenotype" lymphomas may occur frequently in AIDS-associated NHL. Our results show striking similarity to those obtained in the analysis of certain murine CD5+ B-cell lymphomas, suggesting that the study of lymphomagenesis in the mouse may aid in the understanding of AIDS-associated NHL.

Experimental infections of the multimammate rat (Mastomys natalensis) with Leishmania donovani and Leishmania major.

The susceptibility of the multimammate rat, Mastomys natalensis, to experimental infections with Leishmania donovani and L. major was examined. Inoculations of L. major promastigotes into the skin resulted in nonulcerating lesions in which parasites could be detected for more than 30 weeks later. Intravenous inoculations of L. donovani promastigotes produced visceral infections characterized by a continuing increase in splenic parasite burdens and liver parasite burdens which peaked during the first few weeks of infection and gradually decreased as the disease became chronic. L. donovani could be isolated from the blood throughout the infection, and promastigotes were cultured from the spleens of rats inoculated intradermally. Thus, the multimammate rat appears to be a good reservoir host for these parasites.

Sensitization of offspring of Leishmania donovani-infected hamsters to immunization and of offspring of immunized hamsters to challenge.

Female hamsters, infected intracardially (i.c.) with 1.0--2.0 x 10(5) amastigotes of Leishmania donovani produced offspring, following mating, which, when immunized subcutaneously with 1.0 x 10(7) amastigotes at 8 weeks of age, were more resistant to i.c. challenge 6 weeks later than were hamsters born to non-infected mothers. Offspring of mothers infected with as many as 6.0 x 10(6) amastigotes demonstrated no greater capacity for immunization than did those of mothers infected with 1.0 x 10(5) amastigotes. Sensitization of offspring of infected mothers apparently is transplacental since the effect could only be seen in offspring of infected mothers and not in those of normal mothers weaned by infected dams. Offspring of female hamsters immunized by footpad inoculation of 1.0 x 10(7) amastigotes exhibited reduced spleen parasite burdens when challenged at 8.5 weeks of age and reduced spleen and liver parasite burdens when challenged at 16 weeks of age, compared to offspring of nonimmunized hamsters; this effect was not noted following challenge of offspring of the two groups within 1 day of weaning. The passage of parasites from mother to young during gestation and/or nursing apparently does not occur since, at 125 days of age, no parasites were observed in spleen or liver impression smears of offspring of hamsters infected i.c. with 6.0 x 10(6) amastigotes while parasites were seen in such smears of hamsters infected, 120 days previously, with 10 amastigotes i.c. Thus, sensitization to immunization in offspring of infected hamsters and to challenge of offspring of immunized hamsters is apparently transplacental, effected either by soluble leishmanial antigen, soluble lymphocyte (or transfer) factor, or by cells.

Chronicity in Strongyloides stercoralis infections: dichotomy of the protective immune response to infective and autoinfective larvae in a mouse model.

Strongyloidiasis is an intestinal disease that can last for decades due to the occurrence of autoinfective larvae (L3a) in an infected person, which contribute to the maintenance of the population of adult worms in the intestine. The goal of the present study was to determine if L3a are susceptible to the protective immunity that targets the infective stage of the worm, the third-stage larvae (L3). Mice immunized and challenged with Strongyloides stercoralis L3 kill more than 90% of challenge larvae contained within diffusion chambers. The L3 do not remain antigenically static in mice, however, but undergo some degree of antigenic change before they are killed, becoming host-activated larvae (L3+). The L3/L3+ are killed in this model system by the combined effects of both parasite-specific IgM and eosinophils. Mice immunized with L3 were able to kill L3/L3+, but did not kill L3a, in challenge infections. Eosinophils were, however, present in diffusion chambers containing L3a, and IgM bound to the surface of L3a. We hypothesized that differential IgM recognition of soluble L3a, L3, and L3+ antigens is the reason why the immune response generated against L3 could not kill L3a. Many common antigens on L3, L3+, and L3a were recognized by serum from mice immunized with L3, as determined by immunoblotting. However, several unique L3, L3+, and L3a antigens were also recognized by immune serum, thus indicating that antigen recognition with IgM antibodies is different between the L3, L3+, and L3a stages. This difference in antigen recognition could explain why L3a are able to evade the immune response that targets L3/L3+ in chronically infected hosts.

Time series analysis of the prevalence of endoparasitic infections in cats and dogs presented to a veterinary teaching hospital.

The apparent prevalence of endoparasitic infections of cats and dogs presented to the small animal Veterinary Hospital of the University of Pennsylvania was measured between 1984 and 1991. Two thousand feline and 8077 canine fecal samples were examined along with 6830 canine blood samples. The overall mean monthly prevalence of feline infections was 16% for ascarids, 0.9% for hookworms, 4.0% for tapeworms, 2.4% for Giardia spp. and 4.2% for coccidia. The overall mean monthly prevalence of canine infections was 5.7% for ascarids, 9.7% for hookworms, 9.7% for whipworms. 1.8% for tapeworms, 4.7% for Giardia spp. and 3.1% for coccidia. There was a significant downward trend in the prevalence of hookworms and heartworms in dogs (P < 0.001 in both cases). There was a significant upward trend in the prevalence of tapeworms in cats (P < 0.05). There were no significant long-term trends in any of the other time series. The smoothed data were analyzed for seasonal trends. None of the autocorrelation analyses gave incontrovertible evidence of seasonality. The repeated peaks at the 6, 12 and 24 month lags in the case of ascarid infections were suggestive of a 12 month seasonality with a peak prevalence in December, but the results were not statistically significant at the 5% level. Hookworms and whipworms in dogs occurred together more than would be expected by chance in 4 out of the 6 years for which data were available.

Efficacy of an ivermectin/pyrantel pamoate chewable formulation against the canine hookworms, Uncinaria stenocephala and Ancylostoma caninum.

The effectiveness of the combination of pyrantel pamoate (5 mg kg-1) and ivermectin (6 micrograms kg-1) against the canine hookworms Uncinaria stenocephala and Ancylostoma caninum was determined. This combination is intended for monthly use as a heartworm preventative and for treatment and control of canine hookworms. The formulation was found to be effective (99.6% reduction in worm burdens) against both species of hookworms in experimentally infected dogs. No adverse effects due to the drug combination were observed in any dog during the course of this study.

Eustrongylidiasis in eastern great blue herons (Ardea herodias).

The nematode Eustrongylides ignotus was found in peritoneal lesions of several great blue herons (Ardea herodias) submitted for necropsy from a wildlife rehabilitation center in northern Delaware. Prior to death, signs of disease included ataxia, emaciation, weakness, and anemia. Blood collection was not uniformly performed, but in cases where it was performed, affected birds demonstrated abnormal clinical hematology. Postmortem findings included numerous lesions associated with verminous peritonitis. Significant histologic granulomatous response to the presence of these organisms was noted, particularly in the proventricular specimens. Other organs involved included intestine, spleen, pancreas, and liver.

First report of the giant kidney worm (Dioctophyme renale) in a harbor seal (Phoca vitulina).

A male harbor seal (Phoca vitulina) was found moribund on the coast of New Jersey in January of 2003 and died a few hours later in the Marine Mammal Stranding Center. On necropsy, a single female Dioctophyme renale was recovered from the peritoneal cavity, and a tissue mass was found adjacent to the pelvic urethra and urinary bladder. Within this tissue mass were found D. renale ova. This is the first report of this nematode in the harbor seal and in a North American marine mammal.

Cryopreservation of human hookworms.

First-stage larvae (L1) of the human hookworms Ancylostoma duodenale and Necator americanus were frozen over liquid nitrogen after a 1-hr incubation in a cryoprotectant (10% DMSO and 10% dextran). Thawed larvae developed to the infective third stage (L3) on agar plates. In the case of A. duodenale, the larvae were infective to dogs. The infectivity of N. americanus L3 was not tested.

Cryopreservation of first-stage and infective third-stage larvae of Strongyloides stercoralis.

Infective third-stage larvae of Strongyloides stercoralis were frozen over liquid nitrogen and remained infective to dogs when thawed. Successful cryopreservation depended on a 30-60-min incubation in a cryoprotectant (10% DMSO and 10% dextran) before freezing and thawing the frozen larvae into RPMI. First-stage larvae could also be frozen by this method. Thawed first-stage larvae remained viable and continued their development to third-stage larvae, which were shown to be infective to dogs.

Hyperinfective strongyloidiasis: Strongyloides stercoralis undergoes an autoinfective burst in neonatal gerbils.

One of the unusual aspect of the life cycle of Strongyloides stercoralis is the occurrence of autoinfective third-stage larvae (L3a). These are the causative agents of severe hyperinfective strongyloidiasis. When 6-wk-old gerbils are infected with 1,000 infective third-stage larvae (L3i), no L3a are seen during the course of the infection. However, in neonatal gerbils (1-13 days of age) infected with 1,000 L3i, a burst of autoinfection takes place between 15 and 30 days postinfection (PI). Only occasional L3a can be found in neonatally infected gerbils after 4 wk PI. This autoinfective burst is not seen in neonatal gerbils infected with 200 L3i.

Strongyloides stercoralis host-adapted third-stage larvae are the target of eosinophil-associated immune-mediated killing in mice.

Host-adapted, transformed, Strongyloides stercoralis third-stage larvae (L3+) were previously found to be antigenically different from free-living, infective, third-stage larvae (L3). These antigenic differences were reproduced by transformation of free-living larvae in tissue culture medium at 37 C over 24 hr. Transformed L3 of both derivations were given as challenge infections in diffusion chambers to naive mice and mice immunized with S. stercoralis L3. Within 12 hr, the challenge infections were killed regardless of whether the L3+ were generated in vitro or in vivo. Eosinophils, previously found to be important in the immune response to S. stercoralis larvae, were recruited into the L3+ microenvironment within 12 hr of challenge infection in immune mice, which supports the previously proposed mechanisms of S. stercoralis larval killing. Thus, S. stercoralis L3+ appear to be targets of the immune response in mice instead of being involved in immune evasion.