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Ejection of malaria sporozoites by feeding mosquitoes
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE
AKD HYGIENE (1992) 86, CORRESPO~.DENCE
1Correspondence Ejection quitoes
of malaria
spohoites
by feeding mos-
In discussing the results of their ingenious and revealing experiments on the fate of malaria sporozoites, Ponnudurai and colleagues (1991: Transactions, 85, 175-180) speculated that the reason they did not see a positive correlation between magnitude of salivary gland infection and number of sporozoites ejected, as had been reported earlier (Rosenberg et al., 1990: Transactions, 84, 209-212), was because the earlier work used an enzymelinked immunosorbent assay (ELISA) to measure gland infection. The ELBA, they suggested, may give a false positive correlation because a ‘variable’ amount of circumsporozoite protein is shed in and around the glands. I doubt this as an explanation; it seems improbable that protein shedding will produce a strong positive correlation in a sample of 46 if such a correlation does not already exist. A simpler explanation is that, in fact, no discrepancy exists between their observations and ours; each measures a different aspect of the same phenomenon. In our experiment the correlation referred to by Ponnudurai et al. was found among the 50% highest salivating mosquitoes; those most likely to have cleared all the sporozoites lying in the common ducts. There was little correlation in the 50% lowest salivating, presumably because salivation in those mosquitoes was incomplete. The Nijmegen mosquitoes, which stopped salivating when they encountered blood, are unlikely to have ejected all available sporozoites; they therefore were comparable to the lower 50% in the Walter Reed study. One might summarize by saying that Rosenberg et al. found a strong correlation between the size of gland infection and the number of sporozoites available in the ducts, while Ponnudurai et al. found no correlation between gland infection and number of sporozoites ejected at any one initial feed. These are not contradictory findings. Theoretically, this could be tested by forcing individual mosquitoes to make a number of separate, closely sequential probes. I suspect that a positive correlation would then emerge and the median number of ejected parasites would rise slightly. One additional, minor point. The Nijmegen group credited us with stating that sugar feeding ‘might influence entry of sporozoites into the duct system’. Apparently this was a misreading of our caveat that loss of sporozoites during sugar feeding or probing immediately before an experiment might exaggerate the variability of ejected sporozoite counts. We did not speculate on how sporozoite ejection might influence invasion of the ducts by sporozoites from the acinar cells. Ronald Rosenberg Entomology Department US Army Medical Component 31516 Rajvithi Road Bangkok 10400 SJuly 1991 Thailand
Ejection of malaria sporozoites by feeding mosquitoes: a reply One of the central questions we wished to answer regarding feeding infected mosquitoes was: is the sporozoite inoculum related to the sporozoite gland load? This is an important epidemiological question. Using an experimental design which approached as closely as possible normal feeding behaviour of a mosquito, the data gave an unequivocal, clearly negative answer (see statistical analyses on page 177 and Fig. 3, page 178 of the Transactions, 85). There was no relationship between the gland sporozoite load and the sporozoite inoculum. The conclusion we came to was also based on other very sound experimental data. We demonstrated in our
109
paper that the explanation for our results lay in the architecture of the salivary glands and the distribution of the sporozoites relative to the distal opening of the salivary duct (see Fig. la and b of our paper). Our conclusion was not based on the fact that the earlier work of Rosenberg et al. (1990: Transactions, 84, 209-212) used an ELISA to assess the number of gland sporozoites. We still believe the widely held view that ELBA k a poor dternatiVe t0 VkUdy COUIIting sporozoites, although this may not explain the discrepancy in full. The fact that another experimental design, not related to normal feeding, indicated that the sporozoite inoculum was correlated with gland load perhaps merely reflects the constraints of that design. Clearly, Rosenberg et al. (lot. cit.) and our paper cannot both be right at the same time. As to the minor point that was raised, our misinterpretation in part of the sentence (p. 179) ‘Rosenberg et al. (1990) stated that individual behaviour, such as feeding on sugar or probing, might influence the enty of sporozoites into the duct system [italics added] and their subsequent loss’, we simply made the assumption that sporozoites with the saliva would have to enter the duct system to be subsequently lost during probing. It was an innocent error and is regretted. Thivi Ponnudurai Institute of Medical Parasitology University of Nijmegen Geert Grooteplein zuid 24 Postbus 9101 6500 HB Nijmegen The Netherlands 223~ly1991
Bacteriological
studies on snakes
I read with interest the paper by Theakston et al. (1990: Transactions, 84,875-879) on the bacterial flora of wild Malayan pit vipers (Calloselasma rhodostoma). They stressed that secondary bacterial infection can be a complication of snake-bite and drew attention to the need for clinicians to be familiar with the bacterial flora of venomous snakes. Over the past 10 years there has also been interest in the bacterial flora of snakes amongst veterinary clinicians and comparative pathologists, in this case primarily because captive reptiles are prone to stomatitis (‘mouth rot’), an inflammatory disease in which bacteria usually play a significant role (Cooper, 1981: Diseases of the Reptilia. London: Academic Press). Theakston et al. referred to several published papers on the oral bacteria of snakes, including that by Draper et al. (1981: Journal of the American Veterinary Medical Association, 179, 1223-1226), but there have been a number of other especially in the veterinary and zoological ~~~~;f~t?e: for example, Cooper (1973: British Journal of Herpetology, 5, 368-374), Cooper et al. (1985: Journal of Zoology, 207, 521-526), Kennedy (1973: Canadian Journal of Comparative Medicine, 37, 325-326), Mayer & Frank (1974: Zentralblatt fiir Bakteriologie und Parasitenkunde (1 Abteilung, originale, A), 229, 470-481)., Roggendorf & Miiller (1976: Zentralblatt fiir Baktenologie und Parasitenkunde (Z Abteilung, originale, A), 236, 22-35) and Sheridan et al. (1989: Journal of Herpetology, 23, 202-205). In some of these studies the emphasis was on gut, cloaca1 or skin isolates rather than oral bacteria but the findings are important because little is known of the ‘normal’ flora of snakes and organisms found elsewhere on, or in, the body which may contribute to contamination of saliva and thus indirectly to bite wounds. An even larger volume of literature exists on the bacterial isolates associated with stomatitis in snakes. This again is relevant to human snake-bite because most of the organisms involved are believed to be opportunists, bacteria that are normally present in the buccal cavity or the prey of the snake, and as such may be considered permanent or transient members of its oral flora. In addition, however, a proportion of ‘wild’
AKD HYGIENE (1992) 86, CORRESPO~.DENCE
1Correspondence Ejection quitoes
of malaria
spohoites
by feeding mos-
In discussing the results of their ingenious and revealing experiments on the fate of malaria sporozoites, Ponnudurai and colleagues (1991: Transactions, 85, 175-180) speculated that the reason they did not see a positive correlation between magnitude of salivary gland infection and number of sporozoites ejected, as had been reported earlier (Rosenberg et al., 1990: Transactions, 84, 209-212), was because the earlier work used an enzymelinked immunosorbent assay (ELISA) to measure gland infection. The ELBA, they suggested, may give a false positive correlation because a ‘variable’ amount of circumsporozoite protein is shed in and around the glands. I doubt this as an explanation; it seems improbable that protein shedding will produce a strong positive correlation in a sample of 46 if such a correlation does not already exist. A simpler explanation is that, in fact, no discrepancy exists between their observations and ours; each measures a different aspect of the same phenomenon. In our experiment the correlation referred to by Ponnudurai et al. was found among the 50% highest salivating mosquitoes; those most likely to have cleared all the sporozoites lying in the common ducts. There was little correlation in the 50% lowest salivating, presumably because salivation in those mosquitoes was incomplete. The Nijmegen mosquitoes, which stopped salivating when they encountered blood, are unlikely to have ejected all available sporozoites; they therefore were comparable to the lower 50% in the Walter Reed study. One might summarize by saying that Rosenberg et al. found a strong correlation between the size of gland infection and the number of sporozoites available in the ducts, while Ponnudurai et al. found no correlation between gland infection and number of sporozoites ejected at any one initial feed. These are not contradictory findings. Theoretically, this could be tested by forcing individual mosquitoes to make a number of separate, closely sequential probes. I suspect that a positive correlation would then emerge and the median number of ejected parasites would rise slightly. One additional, minor point. The Nijmegen group credited us with stating that sugar feeding ‘might influence entry of sporozoites into the duct system’. Apparently this was a misreading of our caveat that loss of sporozoites during sugar feeding or probing immediately before an experiment might exaggerate the variability of ejected sporozoite counts. We did not speculate on how sporozoite ejection might influence invasion of the ducts by sporozoites from the acinar cells. Ronald Rosenberg Entomology Department US Army Medical Component 31516 Rajvithi Road Bangkok 10400 SJuly 1991 Thailand
Ejection of malaria sporozoites by feeding mosquitoes: a reply One of the central questions we wished to answer regarding feeding infected mosquitoes was: is the sporozoite inoculum related to the sporozoite gland load? This is an important epidemiological question. Using an experimental design which approached as closely as possible normal feeding behaviour of a mosquito, the data gave an unequivocal, clearly negative answer (see statistical analyses on page 177 and Fig. 3, page 178 of the Transactions, 85). There was no relationship between the gland sporozoite load and the sporozoite inoculum. The conclusion we came to was also based on other very sound experimental data. We demonstrated in our
109
paper that the explanation for our results lay in the architecture of the salivary glands and the distribution of the sporozoites relative to the distal opening of the salivary duct (see Fig. la and b of our paper). Our conclusion was not based on the fact that the earlier work of Rosenberg et al. (1990: Transactions, 84, 209-212) used an ELISA to assess the number of gland sporozoites. We still believe the widely held view that ELBA k a poor dternatiVe t0 VkUdy COUIIting sporozoites, although this may not explain the discrepancy in full. The fact that another experimental design, not related to normal feeding, indicated that the sporozoite inoculum was correlated with gland load perhaps merely reflects the constraints of that design. Clearly, Rosenberg et al. (lot. cit.) and our paper cannot both be right at the same time. As to the minor point that was raised, our misinterpretation in part of the sentence (p. 179) ‘Rosenberg et al. (1990) stated that individual behaviour, such as feeding on sugar or probing, might influence the enty of sporozoites into the duct system [italics added] and their subsequent loss’, we simply made the assumption that sporozoites with the saliva would have to enter the duct system to be subsequently lost during probing. It was an innocent error and is regretted. Thivi Ponnudurai Institute of Medical Parasitology University of Nijmegen Geert Grooteplein zuid 24 Postbus 9101 6500 HB Nijmegen The Netherlands 223~ly1991
Bacteriological
studies on snakes
I read with interest the paper by Theakston et al. (1990: Transactions, 84,875-879) on the bacterial flora of wild Malayan pit vipers (Calloselasma rhodostoma). They stressed that secondary bacterial infection can be a complication of snake-bite and drew attention to the need for clinicians to be familiar with the bacterial flora of venomous snakes. Over the past 10 years there has also been interest in the bacterial flora of snakes amongst veterinary clinicians and comparative pathologists, in this case primarily because captive reptiles are prone to stomatitis (‘mouth rot’), an inflammatory disease in which bacteria usually play a significant role (Cooper, 1981: Diseases of the Reptilia. London: Academic Press). Theakston et al. referred to several published papers on the oral bacteria of snakes, including that by Draper et al. (1981: Journal of the American Veterinary Medical Association, 179, 1223-1226), but there have been a number of other especially in the veterinary and zoological ~~~~;f~t?e: for example, Cooper (1973: British Journal of Herpetology, 5, 368-374), Cooper et al. (1985: Journal of Zoology, 207, 521-526), Kennedy (1973: Canadian Journal of Comparative Medicine, 37, 325-326), Mayer & Frank (1974: Zentralblatt fiir Bakteriologie und Parasitenkunde (1 Abteilung, originale, A), 229, 470-481)., Roggendorf & Miiller (1976: Zentralblatt fiir Baktenologie und Parasitenkunde (Z Abteilung, originale, A), 236, 22-35) and Sheridan et al. (1989: Journal of Herpetology, 23, 202-205). In some of these studies the emphasis was on gut, cloaca1 or skin isolates rather than oral bacteria but the findings are important because little is known of the ‘normal’ flora of snakes and organisms found elsewhere on, or in, the body which may contribute to contamination of saliva and thus indirectly to bite wounds. An even larger volume of literature exists on the bacterial isolates associated with stomatitis in snakes. This again is relevant to human snake-bite because most of the organisms involved are believed to be opportunists, bacteria that are normally present in the buccal cavity or the prey of the snake, and as such may be considered permanent or transient members of its oral flora. In addition, however, a proportion of ‘wild’