Age determination of Phlebotomus papatasi by detection of cuticular growthlines

166

TRANSACTIONSOF THE ROYAL.SOCIETYOF TROPICALMELX~NE AND HYGIENE(1987) 81, CORRESPONDENCE

This work was supported by grants from the Minis&e de la Coop&ation (France), the Secretariat

d’Etat g la Recherche Scientifique et Technique (Senegal), INSERM and the FondaGon de France. We thank the SenegaleseGovernment, without whose support this work-would

have been impossible.

We

also thank the Medical Officers working in the Fatick region, the Chiefs and Elders, as weir as the entire local population, for their help and cooperation. B. YVONNET P. F. P. M. C. J. I.

COURSAGET DECIRON VINCELOT SARR DIOUF P. CHIRON DIOP-MAR

Institut de Virologie de Tours and Laboratoire de Microbiologic, Fact& de Pkarmacie, 2 his Boulevard Tonnell& 37032 Tours, France; and Fact& de Mbdecine et de Pkarmacie de Dakar, Senegal References Cabau. N.. Lew. F. M.. Relweld. E. H.. Labusauiere. R., @o&r, A:; Rake, 6. & khambbn, L. (1970): Diptheria-tetanus vaccination by calcium phosphateadsorbed toxoids administered by hvo one-year-interval injections. Annales de l’lnszirut Pasteur, 119,663-670. Coursaget, P., Chiron, J. P., Barin, F., Goudeau, A., Yvonnet, B., Denis, F., Correa, P., N’Doye, R. & Diop-Mar, I. (1983). Hepatitis B vaccine: immunization of children and newborns in an endemic area (Senegal). Developmentsin Biological Standardization, 54, 245-257. Coursaget, P., Yvonnet, B., Relyveld, E. H., Barres, J. L., Diop-Mar, I. & Chiron, J. P. (1986). Simultaneous administration of diphtheria-tetanus-pertussis-polio and hepatitis B vaccines in a simplified immunization program: immune response to diphteria toxoid, tetanus toxoid, pertussis and hepatitis B surface antigen. In&tion and Immunity, 51, 784-787. Maupas, P., Chiron, J. P., Barin, F., Coursaget, P., Goudeau, A., Perrin, J., Denis, F. & Diop-Mar, I. (1981). Efficacy of hepatitis B vaccine in prevention of early HBsAg carrier state in children. Controlled trial in an endemic area (Senegal). Lancet, i, 289-292. Sureau, P., Fabre, J., Bedaya N’Garo, S., Come Butor, S., Poulougou, M. M. & Relyveld, E. H. (1977). Simultaneous tetanus and poliomyelitis vaccination of infants in the tropics. Bulletin of the World Health Organization, 55, 739-746. Yvonnet, B., Coursaget, P., Petat, E., Tortey, E., Diouf, C., Barin, F., Denis, F., Diop-Mar, I. & Chiron, J. P. (1984). Immunogemc effect of hepatitis B vaccine in children: comparison of two and three dose protocols. Journal of Medical Virology, 14, 137-139.

Accepted

for publication 2nd August, 1986.

Age determination of Phlebotomus DaDatasi by detection of cuticular growth&es The demands of eoidemioloaical and ecological

studies on phlebotomihe sandfX&, vectors of L&hmanif, have created a need for an accurateagegrading

techmque. Of the existing techniques for age grading insects (see review by TYNDALE-BISCOE, 1984), only

Polovodova’s method of counting follicular dilatations has been successfully applied to several phlebotomine species, with the limitation of discriminating solely between parous and nulliparous females (WILKES & RIOUX,

1980; READY et al., 1984). SCHLEIN & GRATZ

(1972) first determined exact age in several dipterans by counting daily growth layers in inner muscle attachments (apodemes),and this method has recently proved useful for exact age determination of Drosophila (JOHNSTON & ELLISON, 1982). We report here that these layers can be detected in P. papatasi, the main vector of cutaneous leishmaniasis in the Old World, thus providing a quite accurate age grading technique for this insect. The-procedure used for determining the growth lavers (adanted from SCHLEIN & GRATZ. 1972: SCHLEIN, i979) was as follows. The heads and abdomens of laboratory-bred

fries of both sexes were

removed under a dissecting microscope. The thoraces were then olaced for 12 h in 4% sodium hvdroxide solution in order to remove muscular tissue. When this process was completed the thoraces were thoroughly washed in distilled water and the third thoracic segment dissected out under a dissecting microscope, and split at the ventral side, between the coxae of the hind legs (pair 3). This segment was then stained by immersion for 5 minutes in a lactophenolcotton blue dye containing 0.2% aniline blue. The phragma was then flattened by distending the coxaein distilled water and the preparation was further flattened and then dehydrated in 70% and 100% ethanol, passed to xylol and mounted in Canada balsam. Preparations were then examined under a phase-contrast microscope. Preparations of male and female flies aged 1,2,4,6 and 10 days (+ 12 h) were mounted on individual slides which were coded so that when examined there was no knowledge of their actual age. Each author then examined all the slides and determined their apparent age, which was then compared to the actual age. The averageproportion of correct determinations by the authors was 80%. Most of the mistakes were due to scoring 2-day-olds as 4-day-olds or vice-versa, and lo-day-olds as 6-dayolds. Each author made only one mistake of greater magnitude. A one way Anova showed no difference between the actual agesand those determined by the authors (F= 0.4, P not significant), and Pearson’stest showed a highly significant correlation between these values (R= 0.83, 0.77; P
d

than that of laboratory-grown

flies as the line of

eclosion was much clearer in the field-captured Ilies, as were the successivegrowth lines. This may be due to the fact that the laboratory-grown flies grew at a constant temperature, which has been shown to affect adversely the appearanceof the daily growth layers in other insects (SCHLEIN & GRATZ 1972; SCHLEIN, 1975; JOHNSTON

& ELLISON,

1982).

It must be noted that this-technique has several drawbacks, the first being the difficultv involved in the &se&on due to the small size of pfilebotomines. Approximately 30% of the specimens were lost during

dissection, staining or mounting. Furthermore, the detection of the growth lines on a properly mounted

TRANSACTIONS OF THE

ROYALSOCIETYor TROPICAL MEDICINE AND HYGIENE

specimen requires some practice, as quite often they do not all appear at the same focal depth. Nevertheless, we now have a reliable technique for accurate age grading of P. papa&, and it would be interesting to see if the method is applicable to tropical phlebotomines, who are exposed to subtler circadian ambient variation. Acknowledgements. This study was supported by the project on the Epidemiology and Control of Vector-Borne Diseasesin Israel (REP-NIH-NIAIDAl-12669) and the UNDPWorld Bank/WHO Special Programme for Research and Training in Tropical Disease.. BOAZ YUVAL YOSEF SCHLEIN Dept. of Parasitology, Hebrew University-Hadassah Medical School, P-0. Box 1172, Jerusalem, 91010, Israel.

References

Johnston, J. S. & Ellison, J. R. (1982). Exactage deterinination in laboratory and field caught Drosophila.Journal of Insect Physiology~28, 773-779. Ready, P. D., Lamson, R., Wilkes, T. J. & KillickKendrick, R. (1984). On the accuracy of age-grading neotropical phlebotomines by counting follicular dilatations: hrst laboratory experiments using colonies of Lummyia jIaviscutellata (Mangaheira)and L. fwcuta (Mangabeira) (Diptera: Psychodidae). Bulletin of Entomologisul Research, 74, 641-646. Schlein, Y. (1975). Effects of U.V. light and temperature on the melanization and the formation of daily growth layers of Sarwpkugu fulculuta. 3oumal of Insect Physiology, 21, 1859-1863. Schlein, Y. (1979). Age grouping of anopheline malaria vectors (Diptera: Culicidae) by the cuticular growth lines. 3ownal of Medical Entomology, 16, 502-506. Schlein, Y. & Gratx, N. G. (1972). Age determination of some flies and mosquitoes by daily growth layers of skeletal apodemes.Bulletin of the World Health Orgunizution, 47, 71-75. Tyndale-Biscoe, M. (1984). Age grading methods in aduh insects: a review. Bulletin of EntomologicalResearch,74, 341-377. Wilkes, T. J. & Rioux, J. A. (1980). The application of Polovodova’s technique for the age determination of Phkbotomus (L4wou.hs) ariasi. Transactionsof the Roy41 Society of Tropical Medicine and Hygiene, 74, 119.

Accepted

for publication

2nd August, 1986.

Apical ventricular aneurysm and myocarditis In tropical Africa left ventricular aneurysms of non-ischaemic origin are well described (LINTERMANS, 1977). Best known are the annular subvalvar aneurysms (POLTERA & JONES, 1973). We wish to

record here the case of a Zambian male tree-feller aged 44 in whom apparent myocarditis accompanieda massive apical aneurysm. Four months before admission to the Ndola Central Hospital in August 1972, substernal pain and fever commenced, later followed by palpitations, breathlessnessand cough with bloody sputum. On examination the patient was jaundiced, in biventricular failure with gallop and tricuspid regurgitation, BP 115/90. The chest X-ray showed an enormous heart shadow

(1987) 81, CORRESPONDENCE

167

due to enlargement of all chambers. The most prominent and, at the time, mystifying feature was a round calcified massthe size of a tennis ball in the left lower thorax or left upper abdomen which on fluoroscopy seemed &red. The ECG showed sinus rhythm at 80 per minute with multifocal ventricular extrasystoles, bi-atrial hypertrophy, Q waves in V2 and VS. ST sePmentselevated in I. II. aVF. VI-V< and T-waves inverted in I, aVL, ir,-‘V,. . - Haematological, serological and parasitological investigations were negative, but serum chemistry was abnormal: lactic dehydrogenasewas 383 Wacker units (normal less than 134), hydroxybutyric dehydrogenase was 148 HBD units (normal less than 74), creatine phosphokinase 144i.u. (normal less than 37), glutamic oxaloacetic transaminase66 i.u. (normal less than 25 units), glutamic pyruvic transaminase 70 i.u. (normal less than 30 units), alkaline phosphatase 10.4 King Armstrong units (normal range 3-13 units). Serum bilirubin was 102 umol/l (6.4 mg/dl, 1.8 mg direct). The serum electrolytes were Na 125 mmoY1, K 65 mmol/l, Cl 92 mmol/l. Urea was 816 mmoY1 (136 mg/dl). . At necropsy the heart weighed 1100 g. The main feature was a massive aneurysm containing laminated clot. The right atrium was aneurysmal and the atrio-ventricular valves were incompetent due to ring dilatation. The other valves were normal and the coronary arteries healthy. In the lungs the pleurae were adherent and the lower lobes consolidated. The abdominal viscera were congested. Histological examination was not performed, thus myocarditis cannot with certainty be diagnosed, but the serum enzyme levels strongly suggested inflammation or necrosis. In tropical practice the most frequent cause of apical cardiac aneurysm at post mortem is Chagasic myocarditis (OLIVEIRA et al., 1981), but clearly South American trypanosomiasis is not a consideration in this case. In the rare event of tuberculosis infecting the heart, an apical aneurysm has been found once in a series of 9 cases (KAPOOR et al., 1973). MARK N. LOWENTHAL Smoka Universirv Hosbital and Faculty of Healih Sci&ces, P.O. Box 151, Beer Sheba, 84101, Israel. IAN G. JONES F& tt;i Health Board, scotLld, U.K.

’

References

Kapoor, 0. P., Mascarenhas, E., Rananaware,M. M. & Cadgil, R. K. (1973). Tuberculoma of the heart: report of 9 cases. Am&curt Heurt Journal, 86, 334-340. Lintermans, J. P. (1977). L’aneurisme ventriculaire gauche chez le jeune africain. Arch&s M414dies du Coeur, 70, 129-134. Oliveira, J. S. M., De Oliveira, J. A. M., Frederigue, U., Filho, E. C. L. (1981). Apical aneurysm of Chagas’s heart disease. British Heart Journal, 44, 432-437. Poltera, A. A. & Jones, A. W. (1973). Subvalvar left

ventricular aneurysms.A report of 5 Ugaadancases.

British Heart 3ounta1, 35, 1085-1091. Accepted fm publication

I Ith August, 1986.