Interaction of entomopathogenic nematodes (Steinernematidae) with selected species of ixodid ticks (Acari: Ixodidae).

Entomopathogenic nematodes, currently used for biological control of various insect pests, were tested for their ability to penetrate and kill replete females of several species of ticks including Dermacentor variabilis (Say), Rhipicephalus sanguineus (Latreille), Amblyomma maculatum Koch, and A. cajennense (F.). These species were found to be susceptible to the entomopathogenic nematodes, Steinernema feltiae (Filipjev) or S. riobravus (Cabanillas & Poinar), shown in previous studies in our laboratory to be attracted to and kill replete A. americanum. S. riobravus killed D. variabilis (96%), R. sanguineus (89%), A. maculatum (24%), and A. cajennense (88%), and S. feltiae killed D. variabilis (91%) and R. sanguineus (71%). Of the ticks that survived mean egg mass weights were significantly lower than those of the unexposed controls. When nematode-exposed ticks were examined with light microscopy, nematodes were found to have entered ticks but did not multiply or produce subsequent generations of infective juveniles. The nematodes were separated from surrounding tissues by a clear space, suggesting that they produced protective compounds. Bacteria, thought to be symbiotes released from the nematodes, multiplied initially in the hemocoel of the tick and subsequently were found throughout the degenerating tick tissues. These bacteria eventually filled the tick and appeared to be the cause of tick death. Nematode guts were filled with the bacteria, suggesting that the bacteria were a food source. When ticks were exposed to nematodes while feeding on cattle, partially engorged females were most susceptible to the nematodes. Tick mortality and reduced egg production resulted when the ticks had fed 6 and 9 d before nematode exposure but not when ticks were exposed after 3 d of feeding. Exposure of feeding female ticks demonstrated that the nematodes were able to penetrate tick orifices other than via the hypostome, which was embedded in the bovine epidermis for the duration of the feeding process.

Entomopathogenic nematodes as a potential biological control method for ticks.

Entomopathogenic nematodes have been used for biological control of certain insect pests. In these studies the nematodes were tested as a possible biological control agent for engorged female ticks. Five species of infective juveniles (IJs) were tested initially for their ability to penetrate and kill ticks, including Steinernema glaseri (SG), S. riobravus (SR), S. carpocapsae (DT), S. feltiae (SF) and Heterorhabiditis bacteriophora (HP88). Infective juveniles (IJs) of SRs and SFs appeared to be the most effective in killing ticks and invaded and killed 30 to 100% of replete females. These two nematode species were tested on several tick species including Amblyomma americanum, A. cajennense, A. maculatum, Dermacentor variabilis and Rhipicephalus sanguineus. Although the killing rate of each tick species varied, the nematodes did not appear to be host specific and were able to kill ticks of all species tested. Egg mass weights of exposed ticks of each species were significantly lower than those of the controls. Ticks were examined with microscopy to determine whether nematodes entered and multiplied inside ticks. Partially fed female Amblyomma americanum and Dermacentor variabilis exposed to 5000 IJs in petri dishes were collected at 8, 24, 48 and 96 hrs (Trial 1) and 1, 2, 3, 4, 7 and 9 days (Trial 2) post-exposure, and fixed, processed and embedded in resin for microscopy studies. Only a few nematodes were seen in the hemocoel and tissues and they were surrounded by a clear space. Bacteria, released from the nematodes, were present in the exposed ticks and appeared to increase daily causing a generalized infection. Degeneration of tick tissues and death of the ticks appeared to result from bacterial proliferation. Nematodes did not multiply within ticks as they do in insect larvae. In these controlled laboratory studies, exposure of ticks to nematodes resulted in tick mortality and reduced egg production. Entomopathogenic nematodes appear to have potential as a biological control agent of ticks, but future studies will be required to determine whether nematode/tick interactions will occur in the field.

In vitro characterization of Acanthamoeba castellanii cytopathic effect.

This study examined the mechanism of the cytopathic effect (CPE) of Acanthamoeba castellanii on human target cells. Pathogenic Acanthamoeba trophozoites were incubated with human ocular melanoma (OCM1) cells for 30 min, 1 hr, and 3 hr. The amoebae were treated with a calcium ionophore (A23187), phorbol myristate ester (PMA), calcium channel blocker (Bepridil), cytochalasin D, and L-leucyl-L-leucine methyl ester (leu-leu-OMe). Cytolysis was quantified using a spectrophotometric assay. Cocultures of amoeba and cells were also observed by transmission electron microscopy at 1, 2, and 3 hr. Results show that trophozoites formed pseudopodia that made intimate contact with the target cell membrane. Neither amebostomes nor phagocytosis was seen. The calcium ionophore A23187 increased the cytopathic effect of the trophozoites on the cultured OCM1. In contrast, cytochalasin D, Bepridil, and PMA reduced the cytopathic effect. Leu-leu-OMe did not result in killing of Acanthamoeba trophozoites. The results suggest that the cytopathic effect of Acanthamoeba trophozoites involves calcium channels and cytoskeletal elements. There was no evidence of trogocytosis or phagocytosis as sometimes occurs in cytolysis by other free-living amoeba. Although Acanthamoeba-mediated CPE in some ways resembles CPE produced by cytotoxic lymphocytes, the mechanisms are not identical.

Apoptosis as a mechanism of cytolysis of tumor cells by a pathogenic free-living amoeba.

Previous studies have shown that trophozoites of the pathogenic free-living amoeba Acanthamoeba castellanii rapidly lysed a variety of tumor cells in vitro. Tumor cells undergoing parasite-mediated lysis displayed characteristic cell membrane blebbing reminiscent of apoptosis. The present investigation examined the role of apoptosis (programmed cell death) in Acanthamoeba-mediated tumor cell lysis. The results showed that more than 70% of tumor cell DNA was fragmented following exposure to Acanthamoeba cell extracts. By contrast, only 7% of untreated control cells underwent DNA fragmentation. DNA fragmentation increased significantly in a dose-dependent fashion following concentration of the parasite extract. Apoptosis was also confirmed by DNA ladder formation. Characteristic DNA ladders, consisting of multimers of approximately 180 to 200 bp, were produced by tumor cells exposed to Acanthamoeba cell extracts. The morphology of tumor cell lysis was examined by light and scanning electron microscopy. Tumor cells exposed to parasite extract displayed morphological features characteristic of apoptosis including cell shrinkage, cell membrane blebbing, formation of apoptotic bodies, and nuclear condensation. By contrast, similar effects were not found in tumor cells exposed to extract similarly prepared from normal mammalian cells (i.e., human keratocytes). The results suggest that at least one species of pathogenic free-living amoeba is able to lyse tumor cells by a process that culminates in apoptosis.

In vitro and in vivo tumoricidal properties of a pathogenic/free-living amoeba.

The chemotactic and tumoricidal properties of the pathogenic/free-living amoeba Acanthamoeba castellanii were examined in vivo and in vitro. A. castellanii trophozoites displayed strong positive chemotactic responses to human melanoma (OCM-1) and murine melanoma (D5.1G4) cells. Although the parasites typically invade and destroy the corneal epithelium and stroma, positive chemotactic responses were not detected against extracts of either of these corneal elements. In vitro studies revealed that viable parasites, as well as cell-free parasite lysates, produced swift and extensive cytolysis of a wide variety of tumor cells. The tumoricidal properties of A. castellanii were examined in vivo and revealed that injection of viable parasites or cell-free parasite lysates into progressively growing subcutaneous melanomas resulted in 83% and 53% reductions in the tumor masses compared with untreated controls. The feasibility of utilizing the tumoricidal properties of pathogenic/free-living amoebae and their cell-free products in the treatment of drug-resistant or radioresistant tumors warrants further investigation.

Characterization and pathogenic potential of a soil isolate and an ocular isolate of Acanthamoeba castellanii in relation to Acanthamoeba keratitis.

Acanthamoeba castellanii, one isolate from the eye and one from the soil, were compared on the basis of: (a) pathogenic potential; (b) plasminogen activator activity; (c) chemotactic activity; (d) cytopathic effects; (e) collagenolytic activity; (f) binding ability to contact lenses; and (g) and binding ability to corneal buttons. The ocular isolate of A. castellanii was found to be pathogenic based on its ability to produce corneal infections in Chinese hamsters. By contrast, the soil isolate produced only mild lesions in a single Chinese hamster. Amoebae from the ocular isolate bound to corneal epithelium in greater numbers than the soil isolate counterparts. Moreover, ocular isolate organisms displayed plasminogen activator activity that was not detected in cultures from soil isolates of A. castellanii. Although neither the soil isolate nor the ocular isolate amoebae responded chemotactically to epithelial or stromal components, the ocular isolate displayed a curious and reproducible positive chemotactic response to endothelial extracts. Both A. castellanii isolates produced cytopathic effects on pig corneal epithelium, however the cytotoxicity from the ocular isolate was significantly greater than that of the soil isolate. The results indicate that the pathogenic potential of A. castellanii is correlated with the parasite's capacity to bind to corneal epithelium, respond chemotactically to corneal endothelial extracts, elaborate plasminogen activators, and produce cytopathic effects on corneal epithelium.