The effect of disc thickness and trauma on disc surface friction in the porcine temporomandibular joint.

The pathomechanics of osteoarthritis in the human temporomandibular joint (TMJ) are unknown. Compromised lubrication is a potential factor, but, lubrication within even the normal TMJ is not understood completely. Weeping lubrication is a concept that may be applicable to the TMJ. A characteristic of weeping lubrication is a slow increase in friction during static loading. The rate of increase in friction is related to the rate of lateral movement of synovial fluid away from the loading area. The TMJ disc is expected to be the main source of TMJ lubrication. This study tested two variables, disc thickness and magnitude of trauma to the disc, as factors that can affect the rate of flow of synovial fluid and thus alter lubrication of the disc surfaces. To test these variables, TMJ disc surface friction was measured before and after an impulse load. Before the impulse load, all discs demonstrated a gradual increase in friction during light static loading. The rate of increase in friction was inversely related to the disc thickness (R(2)=0.75). After an impulse load of known magnitude and peak force, disc surface friction was higher. The magnitude of this surface friction was correlated with the magnitude of the impulsive blow (R(2)=0.89) and the area of surface damage (R(2)=0.85). Disc thickness was a significant factor in determining the minimal impulse needed to produce higher surface friction (R(2)=0.99). These results confirm that disc thickness and trauma to the disc affect surface friction in the TMJ, and therefore may be important factors in compromised lubrication and the development of osteoarthritis.

The sequence of Giardia small subunit rRNA shows that voles and muskrats are parasitized by a unique species Giardia microti.

The small subunit ribosomal RNA (eukaryotic 16S rRNA) gene from Giardia trophozoites, isolated from 8 different prairie voles and 8 different muskrats, was amplified by the polymerase chain reaction. The 16S rDNA was sequenced in its entirety for 2 prairie vole and 2 muskrat Giardia. In addition, the 5' 500 nucleotides of the 16S rDNA from Giardia isolates from each of 6 voles and 6 muskrats were amplified and sequenced. The results show that Giardia from voles and muskrats are very similar to each other but differ substantially from Giardia isolated from humans. We believe that the Giardia isolate from voles and muskrats constitutes a distinct species, which will be referred to as Giardia microti. These results suggest that both voles and muskrats are parasitized by the same species of Giardia, that this species is different from the Giardia that parasitizes humans, and that voles and muskrats do not contribute to the zoonotic character of human giardiasis.

Scanning electron microscopy of target cells and molecular weight determination of a bacteriocin produced by Lactococcus lactis D53.

A bacteriocin, lactococcin D53, from Lactococcus lactis strain D53 was partially purified by precipitation with ammonium sulfate and dialysis against deionized water, at which time it precipitated from solution. A native molecular weight was determined by gel filtration, where bacteriocin was detected in two fractions which were measured at 104 and 6.7 kDa. A molecular weight of 7.0 kDa under denaturing conditions was determined by Tricine-SDS-polyacrylamide gel electrophoresis. The molecular weight determinations implied that lactococcin D53 complexed with other macromolecules in its native state in solution. Scanning electron micrographs of Lactobacillus D17 target cells treated with lactococcin D53 showed considerable differences from untreated control cells. The bacteriocin-treated cells had rougher, more granular-looking outer surfaces than untreated cells, which appeared smooth. Counts of viable cells in buffer solution rapidly declined by about one log in target cells treated with bacteriocin.

Suppression of resistance to Giardia muris and cytokine production in a murine model of acquired immune deficiency syndrome.

Peyer's patch (PP) T cells through the production of appropriate cytokines foster the development of immunity to the intestinal protozoan parasites such as Giardia. T cell destruction by the human immunodeficiency virus precedes the development of acquired immune deficiency syndrome. Thus, HIV may increase susceptibility to intestinal parasite infections. Therefore, we measured the resistance and T cell cytokine responses to Giardia in C57B1/6 mice infected with the retrovirus LP-BM5 which produces a murine AIDS (MAIDS). Mice with MAIDS and controls were intragastrically challenged with 1 x 10(5) G. muris cysts. Fecal counts were measured weekly following challenge. Also, PP T cell production of interleukin (IL)2, IL3, IL4, and Interferon-gamma in response to G. muris trophozoite antigens displayed on antigen presenting cells were measured at these times. Prior to day 14 of the infection, the number of Giardia cysts in the retrovirus group paralleled that in controls. However, by day 21 after Giardia infection, mice with MAIDS failed to clear the Giardia cysts from the intestine while the control mice were completely free of cysts. IL2 and IL4 production in response to Giardia trophozoites by unfractionated PP lymphocytes were severely depressed in the retrovirus infected group, while IFN-gamma production was increased. Depressed cytokine production was most likely due to depressed PP T cell numbers. When fractionated enriched T cells were adjusted to a uniform concentration in in vitro immunization cultures, the production of IL2 and IL4/IL5 were similar between retrovirus infected compared with control mice. Recoverable PP T cells were lower in mice with MAIDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Infectivity of cryopreserved Giardia cysts for Mongolian gerbils (Meriones unguiculatus).

Although Giardia species trophozoites have been cryopreserved successfully, no report on the successful cryopreservation of cysts could be found. Using infectivity to Mongolian gerbils (Meriones unguiculatus) as a measure of cyst viability, we tested the viability of 4 strains of Giardia cysts that had been cryopreserved for 1-67 wk. Cysts were frozen in either Keister's medium or physiological saline, both containing 5% or 7.5% dimethylsulfoxide as the cryoprotectant. Viability of cryopreserved cysts was dependent upon the number of cysts inoculated, the length of time cysts were held at 4 C before cryopreservation, and the cryopreserving medium. Infection was established in gerbils by inoculating them with cysts that had been cryopreserved for up to 67 wk, cysts that had been held for less than 30 days before cryopreservation, and cysts frozen in Keister's medium. Saline appears to be unsuitable as a freezing medium for the cryopreservation of Giardia cysts.

Excystation of Giardia muris induced by a phosphate-bicarbonate medium: localization of acid phosphatase.

Giardia muris cysts were incubated briefly in an aqueous induction medium of 0.1 M potassium phosphate with 0.1, 0.2, or 0.3 M sodium bicarbonate. High rates of excystation (91.1-96.7%) were recorded within 5 min after the cysts were placed in trypticase-yeast extract-iron-serum (TYI-S) medium. Substitution of phosphate-buffered saline for TYI-S as the excystation medium resulted in high rates (95.9%) of excystation but required an incubation of 15 min. Excystation was inhibited by the presence of 4-4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS), a specific inhibitor of vacuolar and lysosomal acidification. Microscopic observation showed the loss of the peritrophic space and a change in the refractile nature of the cyst wall prior to excystation. Histochemical studies demonstrated a reaction product of acid phosphatase activity in the lysosomelike peripheral vacuoles in induced cysts and in the peritrophic space of cysts placed in excystation medium. Staining with acridine orange suggested that the peripheral vacuoles become acidified during induction. This staining was inhibited also by DIDS. These studies show that in vitro excystation can be produced at high rates by easily prepared media without exogenous enzymes, low pH, reducing agents, or complex components. The data also suggest that excystation may be stimulated by the bicarbonate-phosphate medium accompanied by acidification of the peripheral vacuoles and the release of their contents into the peritrophic space.

Axenic culture and characterization of Giardia ardeae from the great blue heron (Ardea herodias).

Trophozoites of Giardia ardeae were obtained from the great blue heron (Ardea herodias) and established in axenic culture using the TYI-S-33 medium. The generation time in culture for G. ardeae was 22-25 hr, which was 3-fold longer than for Giardia duodenalis (WB strain). A morphological comparison of trophozoites in the original intestinal isolate to those grown in culture revealed that they were identical for the following characteristics: a pyriform-shaped body, a ventral adhesive disc with a deep notch in the posterior border, teardrop-shaped nuclei, pleomorphism in median body structure ranging from a round-oval appearance (Giardia muris type) to that of a clawhammer (G. duodenalis type), and a single caudal flagellum on the right side (as viewed dorsally) with the left one being rudimentary. Analysis of the chromosomal migration patterns was performed by orthogonal-field-alternation gel electrophoresis and demonstrated that the pattern for G. ardeae was distinctly different from that for G. duodenalis (Portland 1-CCW strain). Bacterial symbionts were seen attached to trophozoites in the original isolate but could not be detected in cultured trophozoites using scanning electron microscopy, fluorescence light microscopy using the Hoechst 33258 dye for DNA localization, or by standard microbiological techniques using nonselective media for growing aerobic or anaerobic bacteria. This study demonstrated that avian-derived Giardia could be grown in axenic culture; based on morphological criteria and chromosomal migration patterns, that G. ardeae should be considered a distinct species; and that rationale for determining Giardia spp., based on median body structure alone, should no longer be considered adequate for classification at the species level.

A comparison of Giardia microti and Spironucleus muris cysts in the vole: an immunocytochemical, light, and electron microscopic study.

We have shown that cysts of the genus Spironucleus share many common morphological features with Giardia cysts including: 2-4 nuclei, flagellar axonemes, a distinct cyst wall, and they even display the same immunostaining as Giardia cysts when labeled with antibodies specific for Giardia cyst wall. A direct comparison of Spironucleus muris and Giardia microti cysts have revealed that cysts of S. muris are significantly smaller than cysts of G. miroti. At the ultrastructural level, the cyst walls are similar in fibrillar appearance, but the width of the S. muris cyst wall is significantly less than that of G. microti. The cysts of S. muris also differ from G. microti in that they contain a striated rootlet fiber, flagellar sheath, and numerous glycogen rosettes. Characteristic features of Giardia include the adhesive disc and median body. Although the cysts of Spironucleus and Giardia are similar in appearance, these unique morphological features can be used to distinguish between the 2 protozoa and should be employed in the detection of Giardia cysts in water samples.

Ultrastructural evidence for the presence of bacteria, viral-like particles, and mycoplasma-like organisms associated with Giardia spp.

Giardia trophozoites and cysts, isolated from mammalian and avian hosts, were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and by fluorescent light microscopy for the presence of microbial symbionts. Mycoplasma-like organisms were observed on the surfaces of trophozoites isolated from the prairie vole, laboratory rat, and beaver. Intracellular bacteria were observed by TEM in the trophozoites and cysts of G. microti and by fluorescence microscopy in trophozoites and cysts of Giardia spp. isolated from beaver, muskrat, great-blue heron, and the green heron. Trophozoites of G. muris from rat small intestine contained viral-like particles measuring 60 nm in diameter. These observations suggest that biological associations between Giardia spp. and diverse microbes may be more common than formerly appreciated. It also raises the possibility of transmission of these apparent symbionts, via the Giardia cyst, to other mammalian hosts including man.

Morphology of the cyst of Giardia microti by light and electron microscopy.

Cysts of Giardia microti, isolated from feces and intestinal contents of Microtus ochrogaster, were examined by light and electron microscopy. These cysts differed morphologically from cysts of other G. duodenalis morphological types in that these cysts often contained two apparently differentiated trophozoites with mature ventral discs. Cysts more closely resembling those reported for G. lamblia and G. muris were in greater abundance in preparations made from intestinal contents and were interpreted as immature cysts. "Multiple fission" cysts, reported in G. muris and G. microti by earlier workers, were not observed; however, endosymbiotic bacteria were found in the cysts of G. microti and may have been responsible for reports of multiple fission in the cysts of Giardia.

Localization of acid phosphatase activity in Giardia lamblia and Giardia muris trophozoites.

Numerous membrane-bounded vacuoles are found adjacent to the plasma membrane of the pathogenic protozoan Giardia lamblia. The function of these vacuoles has been discussed by several authors. Approximately 100-400 nm in diameter with a core of low electron density, they have been suggested to be mitochondria, mucocysts, lysosomes, and endocytotic vacuoles. Enzyme cytochemical localization for acid phosphatase activity using cerium as a capturing agent demonstrates reaction product in these vacuoles as well as in the endoplasmic reticulum and nuclear envelope cisternae. The distribution of reaction product suggests the vacuoles are lysosome-like; however, their function and development remain in question.

Morphology of Giardia agilis: observation by scanning electron microscopy and interference reflexion microscopy.

The flagellated protozoan, Giardia agilis, was isolated from tadpole small intestine and examined by scanning electron microscopy and interference reflexion microscopy. The general morphology of the G. agilis trophozoite is similar to G. muris and G. duodenalis, but with modifications that reflect its elongated form. Interference reflexion microscopic analysis of attachment of G. agilis reveals a pattern of focal contacts by the lateral crest of the ventral disc, the ventrolateral flange, the lateral shield, and by numerous microvillus-like appendages found along the lateral border of the trophozoite. The pattern of focal contacts was observed to be dynamic; trophozoites were observed to make and break the focal contacts in a relatively short time and to glide along the surface of the substratum without breaking focal contacts.

Effect of cytochalasin-B, low Ca++ concentration, iodoacetic acid, and quinacrine-HCl on the attachment of Giardia trophozoites in vitro.

Giardia sp. trophozoites were isolated directly from the small intestines of rats and permitted to attach to polystyrene Petri dishes incubated at 37 C. Attached trophozoites were treated in vitro with various agents which inhibit cell motility (cytochalasin-B, low Ca++, colchicine) and metabolism (iodoacetic acid, 2,4-dinitrophenol) and chemotherapeutic agents (quinacrine-HCl, metronidazole). Trophozoite attachment was sensitive to cytochalasin-B, low Ca++, iodoacetate, and quinacrine-HCl. Contractile proteins (actin, myosin) have been demonstrated previously in the periphery of the ventral disc. The effect of cytochalasin-B and low Ca++ concentrations on trophozoite attachment is consistent with the suggested role of contractile proteins in the mechanism of attachment. The effect of iodoacetate suggests that energy for attachment is derived from glycolysis. The effect of quinacrine-HCl on attachment was rapid (less than 10 min with 10.0 micrograms/ml). Its mode of action on attachment is not understood.

Isolation and purification of Giardia trophozoites from rat intestine.

A method for the isolation of Giardia trophozoites based on their ability to attach to warm surfaces has been developed. Mucosal scrapings were obtained from the small intestine of rats infected with Giardia and suspended in Hanks' balanced salt solution (HBSS). Trophozoites were concentrated by centrifugation and allowed to attach to the surfaces of polystyrene petri dishes incubated at 37 C. Incubation temperature significantly affected the recovery of trophozoites. After attachment at 37 C, trophozoites were separated from contaminating intestinal debris by incubation at cold temperature. The trophozoites detach at 4 C, whereas the intestinal debris remain adherent. Then the detached trophozoites were isolated by reattachment at 37 C. Examination by scanning electron microscopy revealed a marked reduction in contamination of attached trophozoites and dish surfaces after the use of cold temperature detachment and reattachment at 37 C. Viability of trophozoites as measured by erythrosin-B dye exclusion, remained above 90% up to 120 min after isolation. This method of isolation facilitates the recovery of this protozoan directly from small intestine for morphological and experimental study.