Lipid and carbohydrate metabolism of Giardia lamblia.

The lipid and carbohydrate metabolism of Giardia lamblia was studied using trophozoites isolated from a human and axenically grown in vitro in medium containing fetal bovine serum. The phospholipid, fatty acid and neutral lipid composition of the G. lamblia trophozoites was similar to that of the medium. Phosphatidylethanolamine, phosphatidylcholine and sphingomyelin were the major phospholipids detected; monoacyl-, diacyl-, triacylglycerides, sterols, and sterol esters were the major neutral lipids found. Several unidentified glycolipids were also detected. Glucose and threonine were readily incorporated by the trophozoites, but not into cellular phospholipids or sterols. However, approximately 86% of the glucose incorporated into the trophozoites was found in the nucleic acids, and 38% of the threonine incorporated was detected in the cellular proteins. Small amounts of the glucose and threonine were incorporated into glycolipid-containing fractions. Glycerol and acetate were not appreciably incorporated into trophozoites while glycerol 3-phosphate incorporation was not detected. Cholesterol was readily assimilated by the trophozoites; 98% of the incorporated was found in the sterol fraction. Radiorespirometric data suggest that the major routes of glucose metabolism in G. lamblia are via Embden- Meyerhof-Parnas and pentose phosphate pathways. However, endogenous acetate (as acetyl-CoA) formed during the metabolism of glucose is not used for lipid biosynthesis. These findings suggest that G. lamblia trophozoites are incapable of synthesizing cellular phospholipids or sterols de novo, but rather, utilize lipids already present in the medium.

Giardia cyst destruction: effectiveness of six small-quantity water disinfection methods.

None of the available chemical methods for disinfecting drinking water has ever been tested for its ability to destory Giardia cysts. We tested the ability of six such methods to act against Giardia, using excystation as the criterion of viability. Two water qualities (cloudy and clear) and two temperatures (3 and 20 degrees C) were tested. At 20 degrees C, using cloudy and clear water, all of the method proved completely effective. However, at 3 degrees C, in cloudy water one method ("saturated" iodine) was less than completely effective, and in clear water four methods (bleach, Globaline, tincture of iodine and "saturated" iodine) failed to destory all of the cysts. The failure of these methods appears to be related to either an insufficient halogen residual or contact time. This study underlines the importance of considering water temperature when employing halogen disinfection methods.

Dipetalonema from the eye of a man in Oregon, U.S.A. A case report.

A 21-mm filarial worm appeared suddenly in the anterior chamber of the right eye of a 32-year-old man in western Oregon. By a simultaneous irrigation-aspiration procedure, it was removed alive and only slightly damaged and was identified as a female Dipetalonema in the fourth stage of development. It was the third such case to be reported from western Oregon. In this and one other case the worms were morphologically similar to adult worms identified as Dipetalonema arbuta Highby 1943 from the body cavity of the porcupine (Erethizon dorsatum) and a similar species, Dipetalonema sprenti Anderson 1953, from the body cavity of the beaver (Castor canadensis).

A comparison of isozymes of five axenic Giardia isolates.

The relative mobilities of six enzymes from the trophozoites of five axenically-cultured isolates of Giardia from human, cat, and guinea pig hosts were compared by starch and polyacrylamide gel electrophoresis. The six enzymes compared were malate dehydrogenase (NAD+) (MDH) (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (ME) (EC 1.1.1.40), hexokinase (EC 2.7.1.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6P) (EC 1.1.1.49), and alpha-glycerophosphate dehydrogenase (EC 1.1.1.8). The latter three enzymes have not been previously reported in Giardia. On the basis of zymogram patterns, the five Giardia isolates were divided into three zymodemes. Zymodeme I comprised human-1/England, human-1/Bethesda, and cat-1/Portland, Zymodeme II the guinea pig-1/Portland isolate, and Zymodeme III the human-1/Portland isolate. These zymodemes were further substantiated when several physical and kinetic properties of three of the enzymes, MDH, ME, and G6P, were examined. Our results, in which Giardia isolated from different mammalian hosts share multiple isoenzymes, question the validity of the practice of assigning Giardia species names on the basis of the animal host from which the protozoan was obtained.

A morphometric comparison of five axenic Giardia isolates.

The length and width of trophozoites from axenic cultures of 5 Giardia isolates were measured both live and after fixation and Giemsa staining. These isolates, as named on the basis of host source, are classified as G. lamblia (3 isolates), G. felis (1 isolate), and G. caviae (1 isolate). The size of live, unstained trophozoites from the 5 isolates, measured without regard to the presence or absence of median bodies, showed only occasional significant differences in length. Statistically significant differences in length and/or width were observed for all comparisons when stained preparations of the isolates were compared. These size differences occurred between isolates assigned to different species as well as among the 3 G. lamblia isolates. These data and previously reported isozyme studies of these isolates most appropriately led to a re-examination of the presently utilized criteria for Giardia speciation.

A function-based treatment for school refusal behavior using shaping and fading.

A function-based behavioral treatment package was developed for an 18-year-old male with mild mental retardation admitted to a specialized inpatient unit for treatment of severe destructive behaviors associated with school refusal behavior. Teacher and school attendance reports, parent interview, and unstructured observations led to the hypothesis that the subject's school refusal behavior was maintained by positive reinforcement. The treatment goals included increasing the subject's compliance with a morning hygiene routine and attending school (shaping), increasing the involvement of the subject's parents in managing the subject's problem behaviors (fading), and generalizing treatment from the hospital to the home. All three treatment goals were achieved in a relatively brief period of time and follow-up data indicated that the treatment effects were durable. The authors concluded that function-based assessment and treatment can be successfully utilized to treat school refusal behavior in children with developmental disabilities.

Domestic violence--battered women syndrome.

Domestic violence is extremely widespread and often goes unrecognized by physicians. The problem is discussed and ideas for dealing with it are outlined.

Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water.

AIM: To determine whether batch solar disinfection (SODIS) can be used to inactivate oocysts of Cryptosporidium parvum and cysts of Giardia muris in experimentally contaminated water. METHODS AND RESULTS: Suspensions of oocysts and cysts were exposed to simulated global solar irradiation of 830 W m(-2) for different exposure times at a constant temperature of 40 degrees C. Infectivity tests were carried out using CD-1 suckling mice in the Cryptosporidium experiments and newly weaned CD-1 mice in the Giardia experiments. Exposure times of > or =10 h (total optical dose c. 30 kJ) rendered C. parvum oocysts noninfective. Giardia muris cysts were rendered completely noninfective within 4 h (total optical dose >12 kJ). Scanning electron microscopy and viability (4',6-diamidino-2-phenylindole/propidium iodide fluorogenic dyes and excystation) studies on oocysts of C. parvum suggest that inactivation is caused by damage to the oocyst wall. CONCLUSIONS: Results show that cysts of G. muris and oocysts of C. parvum are rendered completely noninfective after batch SODIS exposures of 4 and 10 h (respectively) and is also likely to be effective against waterborne cysts of Giardia lamblia. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that SODIS is an appropriate household water treatment technology for use as an emergency intervention in aftermath of natural or man-made disasters against not only bacterial but also protozoan pathogens.

The epidemiology of Giardiasis.

Giardiasis is now known to occur not only endemically, but also as an epidemic disease spread primarily via drinking water. Giardia from different animals, although morphologically indistinguishable, vary in host specificity. This raises the possibility that lower animals may harbour representatives of this genus which are transmissible to humans. In this article, Ernest Meyer discusses our present understanding of the epidemiology of Giardia, related to problems of speciation in this genus.

Citrate cycle and related metabolism of Listeria monocytogenes.

The growth response of Listeria monocytogenes strains A4413 and 9037-7 to carbohydrates was determined in a defined medium. Neither pyruvate, acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, nor malate supported growth. Furthermore, inclusion of any of these carbohydrates in the growth medium with glucose did not increase the growth of Listeria over that observed on glucose alone. Resting cell suspensions of strain A4413 oxidized pyruvate but not acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, or malate. Cell-free extracts of strain A4413 contained active citrate synthase, aconitate hydratase, isocitrate dehydrogenase, malate dehydrogenase, fumarate hydratase, fumarate reductase, pyruvate dehydrogenase system, and oxidases for reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. The alpha-ketoglutarate oxidation system, succinate dehydrogenase, isocitrate lyase, and malate synthase were not detected. Cytochromes were not detected. The data suggest that strain A4413, under these conditions, utilizes a split noncyclic citrate pathway which has an oxidative portion (citrate synthase, aconitate hydratase, and isocitrate dehydrogenase) and a reductive portion (malate dehydrogenase, fumarate hydratase, and fumarate reductase). This pathway is probably important in biosynthesis but not for a net gain in energy.

Opsonization in vitro of Giardia lamblia trophozoites.

The ability of peritoneal rabbit macrophages from immunized and nonimmunized animals to phagocytose Giardia lamblia trophozoites in the presence of serum was studied and compared in an in vitro system. The rabbits which served as the source of immune serum and macrophages were injected repeatedly at multiple sites (intramuscularly, subcutaneously, and intradermally) with a mixture of G. lamblia trophozoites and Freund complete adjuvant. In the presence of normal rabbit serum, a low level of phagocytosis of Giardia trophozoites by normal and immune macrophages was observed. In the presence of hyperimmune rabbit serum, an increased phagocytic activity of both types of macrophages occurred. The opsonic activity was similar whether whole serum or purified immunoglobulin G was used and whether or not these were heat inactivated. G. lamblia trophozoites in suspension were shown to be agglutinated in the presence of hyperimmune serum. Tests employing serial dilutions of hyperimmune serum resulted in a parallel loss of opsonifying and agglutinating activities. It is suggested that opsonization in vivo may play a role in the ability of the host to limit infection by these organisms.

The sensitivity and specificity of the immediate-spin crossmatch.

The immediate-spin (IS) crossmatch is used to detect ABO incompatibility between donor red cells (RBCs) and the serum of the intended recipient. However, this test may be positive in the absence of ABO incompatibility (false positive) or it may be negative when ABO incompatibility exists (false negative). During a 25-month study, the rates of both false-positive and false-negative IS crossmatch results were evaluated, and the sensitivity and specificity of the IS crossmatch were determined. During the study period, 53,656 IS crossmatches were performed for patients without significant RBC antibodies. Fifty-five patients had positive IS crossmatches, and no false-negative reactions were found. In tests of 55 patients with positive IS crossmatches, 77 false-positive and 5 true-positive reactions were noted. The causes of the false-positive reactions were rouleaux (36 patients), cold-reactive antibodies (8 patients), a combination of rouleaux and cold-reactive antibodies (2 patients), fibrin clot (1 patient), and undetermined (3 patients). The sensitivity and specificity of the IS crossmatch were 100 and 99.86 percent, respectively. Laboratory personnel should be aware that the IS crossmatch may have false-positive or false-negative results, and they should develop written protocols to distinguish quickly between true-positive and false-positive reactions.

Effect of chlorine on Giardia lamblia cyst viability.

The effect of chlorine concentration on Giardia lamblia cyst viability was tested under a variety of conditions. The ability of Giardia cysts to undergo excystation was used as the criterion of viability. The experimental variables employed included temperature (25, 15, and 5 degrees C), pH (6, 7, and 8), chlorine-cyst contact time (10, 30, and 60 min), and chlorine concentration (1 to 8 mg/liter). In the pH range studied, cyst survival generally was observed to increase as buffer pH increased. Water temperature coupled with chlorination proved to be important in cyst survival. Results of these experiments at the three temperatures studied can be summarized as follows: at 25 degrees C, exposure to 1.5 mg/liter for 10 min killed all cysts at pH 6, 7, and 8. At 15 degrees C, 2.5 mg of chlorine per liter for 10 min killed all cysts at pH 6, but at pH 7 and 8 small numbers of cysts remained viable after 30 min but not after 60 min. At 5 degrees C, 1 mg of chlorine per liter for 60 min failed to kill all the cysts at any pH tested. At this temperature, 2 mg of chlorine per liter killed all cysts after 60 min at pH 6 and 7, but not at pH 8. A chlorine concentration of 4 mg/liter killed all the cysts at all three pH values after 60 min, but not after 30 min. A chlorine concentration of 8 mg/liter killed all Giardia cysts at pH 6 and 7 after contact for 10 min, and at pH 8 after 30 min. This study points up the role of temperature, pH, and chlorine demand in the halogen treatment of drinking water to destroy cysts. It also raises an epidemiological problem, namely: low water temperatures, where killing of Giardia requires relatively high chlorine concentrations and long contact times, are (i) to be expected in many areas where epidemic waterborne giardiasis has been reported and (ii) particularly conducive to the long-term survival of Giardia cysts.

In vitro excystation of Spironucleus muris.

In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia, were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.

Perception of odor mixtures by the spiny lobster Panulirus argus.

We used spiny lobsters (Panulirus argus) in a discrimination learning procedure with aversive conditioning to examine their behavioral discrimination of adenosine-5'-monophosphate (AMP), betaine, L-cysteine and their binary mixtures. Our results show that spiny lobsters can clearly discriminate among binary mixtures and their components. Lobsters aversively conditioned to avoid responding to a binary mixture continued to respond to that mixture's components, and lobsters that were aversively conditioned to avoid responding to a compound tended to continue to respond to binary mixtures containing that compound. Thus, responses of conditioned lobsters to binary mixtures were not usually intermediate between the responses to the mixtures' components, which might be expected for response-matched compounds. This result might arise from any of several factors. First, it might result from mixture interactions in the peripheral olfactory system, if the responses of olfactory receptor neurons to one component of a binary mixture were suppressed by the other component, making the response to the mixture more similar to the suppressing component. Electrophysiological data from a population of 50 singly-recorded olfactory receptor neurons (Daniel and Derby, 1994) do not consistently support this idea. A second possible reason for the behavioral response to a binary mixture not being intermediate between the responses to its components involves higher order processing, such as mixture interactions generated in olfactory interneurons in the CNS (which is known to occur: Derby et al., 1985; Ache, 1989), configural learning or associative processing.