Este é um Blog educacional, dedicado a discussões acadêmicas sobre a Ecologia Evolutiva. Contém chamadas específicas relacionadas às disciplinas de Ecologia da Universidade Federal de Ouro Preto, e textos didáticos gerais.
V. cholerae 01 in Bangladesh, 1987 to 1990
In order to determine more definitely the source and host of V. cholerae in the environment, an extensive environmental studywas conducted in Bangladesh during 1987 to 1990 (71). Sampleswere collected from 10 fixed stations comprising two river sitesand eight ponds in villages surrounding the Matlab area, located46 km southeast of the capital city of Dhaka, Bangladesh, in thedelta formed by the Meghna and Ganges rivers. One of the ponds,a protected pond that was relatively free of human use, was includedin the study as a control. Water and plankton samples were collectedat the 10 stations every 2 weeks, from February 1987 through January1990.
Water samples were collected in pre-sterilized glass bottles. Plankton samples were collected by filtering 50 liters of waterthrough a plastic sampler fitted with a 0.77 mesh net, achievinga 1000-fold final concentration. From the concentrated planktonsamples, which were 50 ml in final volume, 10 ml were transferredinto each of three different vials. Directly after sampling andwhile in the field, the samples were preserved in formaldehyde,to a final concentration of 4%. From the remaining 20 ml of eachsample, 10 ml were homogenized, using a teflon-tipped, tissuegrinder (StedFast Stirrer, Model 300, Fisher Scientific) and enrichedby addition of alkaline peptone broth for isolation of V. choleraeby conventional culture methods (51).
The fluorescent antibody (FA) technique was used to screen formaldehyde-preserved plankton samples for V. cholerae 01, asdescribed by Brayton et al. (54). Temperature, dissolved oxygen(DO), pH, and a variety of chemical parameters were measured atthe time of collection, using field instruments (Yellow Springs,Ohio, Model YSI 58 and HACH Chemical Co., Ames, Iowa, Model HachOne). Organisms were identified and grouped as adult copepods,juvenile copepods, nauplii copepods, cladocerans, and "other,"in the case of zooplankton. For phytoplankton, the groups included:green algae, diatoms, dinoflagellates, volvox, "other colonialalgae," and cyanobacteria. Where possible, each of the above wereanalyzed to species level.
Monthly means for each station for pH, temperature, iron, salinity, and geometric means of counts of copepods for nauplii,juvenile and adult stages, diatoms, dinoflagellates, as well aspercent of samples positive by FA were computed. It was hypothesizedthat copepods provide a suitable host environment for V. cholerae.Therefore, on the basis of earlier data, an association of copepodnumbers with presence of V. cholerae could be predicted and detectableby fluorescent antibody (FA). For the statistical analysis, theconditional logistic regression model (72) was used in which
log pt1 − pt = &bgr;0 + &bgr;1 (zt−1)" type="#_x0000_t75">
&ggr;iwi + &bgr;2 log(x) + &bgr;3log(x)2" type="#_x0000_t75">
where pt is the probability of observing a positive FA reading at time t and zt is an indicator or lag variable for whetherFA was positive for the previous reading at the same site. Theterms i are (dichotomous) variables, defined to be 1 if the observationis from the ith pond and 0 otherwise. The control site was arbitrarilydefined as pond 0. We allowed x to stand for numbers of adultcopepods in some analyses and for nauplii or juveniles in otheranalyses. We used the logarithm of the untransformed copepod numbersbecause the distributions were highly skewed. The indicator variableZt was included to account for the degree of correlation thatmay be observed in successive observations at the same location.A quadratic term [that is, log(x)2] along with a linear term [that is, log(x)] was used to testwhether above some concentration of copepods, the probabilityof a positive FA reading declined. All the models were fit byway of maximum likelihood, using the SAS procedure (PROC Logistic,SAS Institute, Cary, North Carolina).
When the quadratic term was insignificant, we interpreted a positive coefficient (that is, 2 > 0) to imply that as x increasedthe risk of positive FA increased. One overall model was fit tothe eight ponds and a separate model was fit to each river site,in part because descriptive statistics indicated that the riversites were distinct from each other and the ponds, but the pondsappeared to be similar. For the river sites, i was omitted fromthe model. We assessed lack of fit subjectively by fitting thesame model to each study site, and also by comparing observedand expected proportions of FA positive examples for differentlevels of copepods.
A subsequent exploratory analysis, using a stepwise logistic regression model, was used to examine the role of other environmental[air and water temperature, pH, and dissolved oxygen tension (DOT)],chemical (bromine, calcium, carbon dioxide, chloride, NaCl, color,conductivity, copper, fluoride, water hardness, iodine, iron,manganese, phosphorous, NO3, silicates, sulfates) and biologicalparameters (blue-greens, chladocerans, colonial algae, diatoms,dinoflagellates, green algae, volvox).
Because of the large number of variables, a preliminary analysis was done in which a Wilcoxon rank sum statistic was computedto compare the distribution of observations with a positive FAreading with those with a negative FA reading. If the variablewas significant for either the ponds or one of the two river sites,it was included in the stepwise part of the analysis. A significancelevel of .05 was used throughout.
The results show that the abundance of V. cholerae 01 increases with the abundance of copepods (71). This association appearsto be the basis of persistence of V. cholerae in the environment.Feeding action of many parasitic crustacea, such as copepods,effectively inoculate fish tissues with this pathogen (73).These findings, then, led us to examine seasonal distributionof copepods, ocean currents, and cholera epidemiology. The seasonalityof cholera epidemics in Bangladesh and of plankton showed interestingcorrelations. As noted above, results of studies of survival ofV. cholerae 01 in seawater microcosms revealed that it had thecapacity to remain in the culturable state in seawater for a relativelylong time, that is, sufficiently long to be carried by ocean currentsto widely distant geographical locations (74). Other studiesshowed that, when confronted with high concentrations of carbohydrate,but nitrogen and phosphorous limitation, V. cholerae enters theviable but nonculturable state (75). Thus, the viable but nonculturableV. cholerae could be transported in nutrient poor seawater and,in association with plankton, over several months and thousandsof kilometers, depending on currents and tides. Similarly, theorganism can persist within a given geographical location formany years, offering an explanation for reappearance of choleraafter years of quiescence or seeming absence.
Whether V. cholerae is a component of the commensal flora or a symbiont of a given plankton species remains to be determined.There are clues to potential roles of V. cholerae 01 in the environment.For example, V. cholerae produces chitinase and mucinase (76,77, 78) and most strains carry lux genes (79). Vibrio choleraestrains producing melanin have been isolated. Melanin and itsprecursors, including homogentistic acid have been implicatedin the induction of invertebrate larval settlement and development;for example, of barnacles, oysters and other invertebrates aswell as biofilm adhesiveness (80). Thus, the autochthonous natureof V. cholerae 01 in the aquatic environment takes on greatersignificance, with respect to function in the natural cycles ofaquatic ecosystems. Furthermore, it has been hypothesized thatcholera toxin may play a role in the osmoregulation of its environmentalhost (30).
The introduction of filtration sharply reduced the incidence of infectious disease in the United States. From 1900 to 1913,the population served with filtered water increased eightfold,and the typhoid death rate dropped by more than 55% (10, 81).In the early years of the 20th century, chlorine, with filtration,virtually eliminated waterborne infectious disease in the UnitedStates. The importance of filtration and disinfection in preventingthe spread of cholera cannot be overstated, considering the associationof V. cholerae with plankton in raw water supplies. Filteringwater at the time of collection and just before drinking is asuccessful means of removing cyclops, a planktonic crustaceancopepod and vector of the guinea worm, which causes dracunculiasis.The crustacean cyclops-associated worm is removed by filtrationwith polyester cloth and is now a recommended method of preventingdracunculiasis in Africa (82).
During severe flooding, which occurs every year in some areas of Bangladesh, living conditions deteriorate to those of meresurvival; building a fire to boil water is simply not possible.Using a filter constructed from either nylon net and one of severaldifferent types of sari material, the latter being very inexpensiveand readily available in villages in Bangladesh, V. cholerae attachedto plankton and comprising 99% of the V. cholerae, can be removedfrom water samples (83). From the results of extensive experimentsusing V. cholerae 01 and 0139 strains isolated from cholera victimsof epidemics in Bangladesh, Brazil, India, and Mexico, it wasfound that this simple filtration procedure, involving the useof domestic sari cloth, can reduce significantly the number ofcholera vibrios in raw water from ponds and rivers commonly usedfor drinking (83). Whether the number of cholera cases can bereduced by introducing this simple, low technology approach iscurrently under study.