secondary reservoir role of domestic animals other ...

Published by A.P.E.

Research and Reviews in Parasitology, 57 (I): 39-46 (1997)

© 1997 Asociaci6n de Parasitologos Espaiioles (A.P.E.)

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SECONDARY RESERVOIR ROLE OF DOMESTIC ANIMALS OTHER THAN SHEEP AND CATTLE IN FASCIOLIASIS TRANSMISSION IN THE NORTHERN BOLIVIAN ALTIPLANO S. MAS-COMA', A. RODRIGUEZ1, M.D. BARGUES1, M.A. VALEROI, J.R. COELLd & R. A GLES3 I

Deportamento de Parasitologia, Facultad de Farmacia, Universidad de Yatencia, AI'. Yicente Andres Estelles

Sill,

46100 Burjassot

- Yalencia, Spain

2LaboralOrio de Biologia, Colegio "Sail Calixto», Casilla 283, La Pa; Bolivia 3Ullidad de Parasitologia, lnstltuto Nacional de l.aboratorios de Salud «Nestor Morales Yillaton» (INLASA). Pasaje Rafae/ Subieta No. 1889, Miraj7ores, La Pat; Bolivia Received 29 March 1997; accepted 25 May 1997

REFERENCE:MAS-COMA(S.), RODRIGUEZ (A.), BARGUES (M.D.), VALERO(M.A.), COELLO(J.R.) & ANGLES(R.), 1997.- Secondary reservoir role of domestic animals other than sheep and cattle in fascioliasis transmission in the Northern Bolivian Altiplano. Research and Reviews in Parasitology, 57 (I): 39-46.

ABSTRACT:Research studies were undertaken to analyse the possible reservoir role played by pigs, donkeys, horses, llamas and alpacas present in the Fasciola hepatica human endemic region of the Northern Bolivian Altiplano. Coprological analyses showed liver fluke eggs in the stools of 27, I % of 59 pigs, and 15,4% of 65 donkeys. Egg output (eggs per g of faeces: epg) was 4-65 epg (mean: 21,6 epg) in pigs and 3-10 I epg (mean: 38,8 epg) in donkeys. No positive samples were found in horses, goats and llamas. A comparison with a coprological control study performed in sheep (61,5% of 26 sheep infected, with a range of 3-145 epg and a mean of 44,7 epg) and with literature data on alpacas and cattle from the same endemic region is carried out. According to the populations of pigs, donkeys. horses, goats, llamas and alpacas present in the endemic region, it may be concluded that only pigs and donkeys represent potential reservoir hosts of the parasite in the orthern Bolivian Altiplano. The need to take pigs and donkeys into account within preventive and control measures is pointed out. KEYWORDS:Fasciola Bolivian Altiplano.

hepatica.

transmission, pigs, donkeys. horses, goats. llamas, alpacas, prevalences, egg output. human fascioliasis, orthern

I TRODUCTION Besides its well-known veterinary importance (MALEK, 1980; BORAY, 1982), fascioliasis by Fasciola hepatica (Linnaeus, 1758) (Trematoda: Fasciolidae) has also proved to be a great worldwide health problem (CHE & MOTT, 1990; MAS-COMA et al., 1997; MAS-COMA, BARGUES & ESTEBAN, 1997). Recent papers estimate human infection up to 2,4 million (RIM et al., 1994) or even 17 million people (HOPKINS, 1992). Studies carried out in recent years have demonstrated that the most important human fascioliasis endemic regions are located in South America (MAS-COMA et al., 1995; WHO, 1995; HILLYER & APT, 1997; MAS-COMA, BARGUES & ESTEBAN, 1997). Among the human fascioliasis endemic regions, the Northern Bolivian Altiplano has shown to be the area in which the highest human prevalences and intensities are known. Different studies have reported human prevalences of up to 70% in coprological surveys (MAS-COMA et al.,1995;ESTEBA et al., 1997a,b;A GLESetal., 1997) and even higher in immunological surveys (HILL YER et al., 1992; MAS-COMA et al., 1995; BJORLAND et al., 1995; STRAUSS et al., 1997). lntensities in Bolivian children, measured as egg output in stools, ranged from 24 to 5064 eggs per gram (epg), with arithmetic and geometric means of 474-100 I and 201-309 epg, respectively (ESTEBAN et al., 1997b). Moreover, the human fascioliasis problem in the Altiplano is increased by the presence of many other pathogenic protozoan and helminth spe-

cies concomitantly parasitizing F. hepatica-infected human subjects (ESTEBAN et al., I 997a, c, d). Multidisciplinary studies were undertaken to understand how this digenean parasite is able to reach such high transmission rates in the orthern Bolivian Altipiano, a region of a very high altitude of 3800-4100 m. Up to the present, the Altiplanic liver fluke has shown no significant difference when compared to European F. hepatica, both at genotypic (MARQUEZ & MAS-COMA, 1993) and phenotypic levels (VALERO et al., 1997). Concerning the intermediate snail host, despite initial reports of two different Iymnaeid transmitting species (Lymnaea viatrix d'Orbigny, 1835 and Lymnaea cubensis Pfeiffer, 1839 variety) in the Bolivian Altiplano (UENO et al., 1975), it has been recently demonstrated that only one transmitting species is there. Shell morphology (OVIEDO, BARGUES & MAS-COMA, 1995a; ROUMEGOUX et al., 1997), visceral mass anatomy (OVIEDO, BARGUES & MAS-COMA, 1995a; ROUMEGOUX et al., 1997), molecular (BARGUES & MAS-COMA, 1997; BARGUES et al., 1997) and isoenzyme (JABBOUR-ZAHAB et al., 1997) studies have proved that this species is the European Lymnaea truncatula (MUlier, 1774), most probably introduced by Spanish colonizers long ago. Veterinary surveys performed by both coprological and serological tests have shown that sheep and cattle present high F. hepatica infection prevalences and intensities in the Altiplanic endemic zone (UENO & MORALES, 1973; UE 0 et al., 1975; MAS-COMA et al., 1995; BUCHON & MAS-COMA, 1995; HILLYER et al., 1996; BUCHON et al.,

40 1997). At any rate, these prevalences and intensities found in Altiplanic sheep and cattle are similar to those known in other non-human endemic regions, that is, not as high as to constitute a very large environmental contamination with liver fluke eggs. It must be taken into account that Iymnaeid snail populations in the Altiplano do not appear to be numerous and dense (OVIEDO, BARGUES & MAS-COMA, I995b) enough to explain by themselves the very high human infection rates. With the aim to investigate other factors that can improve liver fluke transmission in the orthern Bolivian Altiplano, research studies were undertaken to analyse the possible reservoir role which could be played by other domestic animal species present in the endemic zone. In the Altiplano, Aymara inhabitants markedly depend upon livestock, owing to the agricultural difficulties proper to the high altitude. That is why numerous other domestic mammal species are present in the region (MAS-COMA et al., 1995): large mammals such as pigs, donkeys, horses, goats, llamas and alpacas; small mammals such as lagomorphs (rabbits, hares) and even rodents (guinea pigs). All these species have already demonstrated their capacity to be successfully infected by F. hepatica (BORAY, 1982; MAS-COMA et al., 1987, 1988).

S. MAS-COMA et al. To consider a mammal species able to play a significant role in the transmission of the liver fluke in a given zone, different aspects must converge: A) the parasite must be able to develop, mature and produce eggs that are expelled with faeces; B) prevalences must be sufficient and not sporadic in time; C) egg output must be sufficiently high; D) populations of the mammal species in question must be sufficiently numerous and dense; E) parasite eggs shed with stools must prove to be viable, that is, able to embryonate, hatch, the miracidium capable to infect a susceptible Iymnaeid snail, intramolluscan larval stages be able to develop, cercariae to be produced, shed in sufficient number and metamorphose into encysted metacercariae, and the latter be able to successfully infect a susceptible definitive host species. The present paper deals with aspects A, B, C and 0 concerning large mammal species. Studies being presently carried out on aspect E in large mammals, as well as on all aspects concerning small mammals, will be the objectives of further papers.

MATERIAL AND METHODS Animal samples: Animals surveyed were in localities where the presence of Iymnaeid snails assuring F. hepatica transmission was

Fig. 1.- Map of the fascioliasis human endemic region in the Northern Bolivian Altiplano showing localities in which domestic animal stool samples were obtained. I = Queroni; 2 = Huacullani; 3 = Chojasihui; 4 = Lacaya Baja; 5 = Chiripujo; 6 = Korila; 7 = Caleria; 8 = Chijipata Alto; 9 = Batallas; 10= Chirapaca; II = Palcoco; 12 = Ancocagua: 13 = Tambillo.

Secondary

reservoirs

of fascioliasis

in the Bolivian

Altiplano

previously verified (Fig. I). Localities in areas where human prevalences and intensities have proved to be high were preferentially surveyed, such as the Chirapaca-Batallas-Chijipata Alto-Cutusuma area in the so-called «corridor of Batallas» (MAs-CoMA et al .. 1995; ESTEBAN et al., 1997b; ANGLES et al., 1997; STRAUSS et al., 1997), and the Chiripujo-Chojasihui-Lacaya Baja-HuacullaniQueroni area in the so-called «corridor of Huacullani» (ESTEBAN et al., 1997a). Stool samples from llamas could only be obtained at a weekly trade fair of llamas in the locality of Paicoco. Estimates of the populations of all domestic animal species were made during numerous visits to all zones of the Northern Bolivian Altiplano endemic region carried out in the 1992-1997 period. Stool collection and preparation: Stool samples were obtained from pigs, donkeys. horses, goats and llamas in the month of March 1997 (March is usually the last month of the rainy season). For comparison purposes, several sheep faeces samples from the same localities were obtained from different months of the year. No stool samples could be obtained from alpacas; hence, prevalence and egg output data from alpacas are taken from previous work carried out by other authors in the same endemic region. Fresh faeces were collected off the ground as animals were observed defecating. Stool samples were put in a plastic bag and transported to the I LASA Institute of La Paz within the following 5 hours. At the INLASA Institute, a qualitative technique was first applied to all stool samples. Those which showed to be positive (detection of F. hepatica eggs) were appropriately fixed and transported to the Parasitology Department of the University of Valencia, Spain. for a quantitative analysis. Coprological techniques: For a first qualitative analysis, a small but sufficient amount of faeces was homogenized with 10% formaline. The mixture was then strained through a funnel with a sterile gauze into an assay tube. The assay tube was afterwards allowed to stand for 2 h for sedimentation. Finally, several drops of the sediment were taken with a Pasteur pipette and deposited on 2 microscope slides. After covering each sample with a cover slide. both preparations were examined under 10x magnification with a microscope. In cases in which F. hepatica eggs were found. 3 g from the remainder of the original stool sample were fixed with 50 ml of 10% formaline for a later quantitative study. For the quantitative analysis, the sedimentation test of DENNls, STONE & SWANSON (1954) was performed with the modification of using water without detergent as described by CHERIUYOT & JORDAN (1990). The final sediment was placed in a 7 cm-diameter Petri dish and was examined under 30x magnification with a stereoscopic microscope.

RESULTS Among the Aymara Indians there is the strong tradition that each family sustains its own livestock, including large mammals such as sheep, cattle, pigs, donkeys, horses, llamas or alpacas. Official data of the Ministry (MACA, 1988) refer to estimations of about 5350000 sheep, 500000 cattle, 350000 pigs, 1400000 llamas, and 150000 alpacas (no data on donkeys and horses could be found among official census reports) living in the whole Bolivian Altiplano. In the endemic area, observations in the field concerning domestic animal populations yielded data varying according to Aymara families and sometimes also according to the different zones within the endemic region.

41

Although familial herds comprising only one animal can be observed (most usually sheep or cattle, sometimes pigs, rarely donkeys), they almost always include more than one species (two, sometimes three, rarely four different species together), among sheep, cattle, pigs and donkeys (Fig. 2 B, D, H). In the endemic zone a family has approximately 1-2 cattle, sheep, pigs, and 4-5 birds. After official data, the estimated animal population includes 6 sheep and 3,4 cattle per family. In the locality of Ancocagua, for instance, familial herds include 0-50 sheep (mean 14,6), 0-17 cattle (mean 8, I), and 0-4 donkeys (mean 0,9). In Chijipata Alto, herds sometimes comprise up to 4 sheep, I I cattle, 9 pigs and 3 donkeys. In Cullucachi, there is an average of 0,08 sheep, 1,28 cattle, 1,4 pigs and 0,12 donkeys per family. In given places of the Northern Altiplano endemic region the number of animals owned by an Aymara family was pronouncedly higher 10 years ago, sometimes even surpassing 200-250 sheep; whereas today it does not reach 15-20 animals (see review of MAS-COMA et al., 1995). These data do not significantly differ from similar data from the Central Bolivian Altiplano except in the absence of llamas. In the locality of Huaraco, 130 km south of La Paz on the route to Oruro, each family owns a mean of 210 cattle, about 20-50 sheep, as well as several donkeys; but certain families also have 5-15 llamas (FISEL, 1989).

Pigs Stool samples of 59 pigs were analysed: 6 pigs from the locality of Tambillo; 7 from Lacaya Baja; 18 from Chijipata Alto; I from Batallas; 14 from Queroni; 3 from Huacullani; 2 from Chiripujo; 5 from Chojasihui; 2 from Korila; I from Caleria. A total of 16 pigs (27, I %) presented F. hepatica eggs in their faeces: I from Tambillo; 12 from Chijipata Alto; 2 from Queroni; I from Chiripujo. The number of eggs per gram of faeces ranged between 4 and 65 epg, with a mean of 21,6 epg. According to general estimations of the amount of stools defecated by a pig per day (MORROS SARDA, 1967; GURTLER et al., 1979), the total number of F. hepatica eggs shed with faeces by a pig per day in the Northern Bolivian AItiplano may be between 2000 and 195000. Pigs are numerous in all zones of the endemic region, as they are usually part of the livestock of Aymara families. According to their number, it appears to be the third domestic animal species in importance in the Northern Bolivian Altiplano, after sheep and cattle. In most zones pigs run free and can usually be observed in or around fresh water bodies inhabited by Iymnaeid snails (Fig. 2 B).

Donkeys Stool samples of 65 donkeys were analysed: 35 donkeys from the locality of Ancocagua; 2 from Tambillo; IQ from Lacaya Baja; 7 from Queroni; 2 from Huacullani; 2 from Chiripujo; 6 from Chojasihui; I from Ko-

S. MAS-COMA et af.

42

Horses appear to be very scarce in the Northern Bolivian Altiplano. Most of the few living in the endemic region are found in the «corridor of Batallas», sometimes in pastures presenting water bodies inhabited by lymnaeids as in Chirapaca (Fig. 2 C). None could be seen in the «corridor of Huacullani» despite the numerous visits to this zone.

rila. A total of 10 donkeys (15,4%) presented F. hepatica eggs in their faeces: 3 from Ancocagua; 3 from Lacaya Baja; I from Huacullani; 3 from Chojasihui. The number of eggs per gram of faeces was from 3 to 101 epg, with a mean of 38,8 epg. According to general estimations of the amount of stools defecated by a donkey per day (deduced from animal's weight from data of MORROS SARDA, 1967 and GORTLERet al., 1979), the total number of liver fluke eggs expelled with faeces by a donkey per day in the Northern Bolivian Altiplano may be around 9000-808000. Donkeys are relatively numerous in all zones of the endemic region. A few donkeys are usually part of the livestock of Aymara families, although their numbers vary in the different zones. In Ancocagua, for instance, they are very numerous. It appears to be the fourth domestic animal species in number in the Northern Bolivian Altiplano, after sheep, cattle and pig. Donkeys are frequently found in pastures besides water collections presenting lyrnnaeid snails (Fig. 2 D).

Goats The goat may not be considered a domestic animal species present in the Northern Bolivian Altiplano. During the 6-year period of this study, only a very few goats were seen throughout the whole endemic region: 4 specimens in the area of Batallas (Fig. 2 A) and about 10 in the zone between Oquetiti and Iquiaca. A stool sample from I goat male from Batallas could be obtained and analysed. No F. hepatica eggs were found. Llamas Llamas can only very sporadically be observed in the orthern Bolivian Altiplano. Besides the few specimens present in the garden of the Titicaca Hotel and the Tiwanaku ruins, both for mere touristic purposes, only a few individuals can regularly be found in the «corridor of Pe-

Horses Faeces of only 4 horses could be analysed: I horse from the locality of Chijipata Alto and 3 from Chirapaca. All were negative in the coprological tests.

o. animals studied

Prevalence (%)

Pigs

59

27,1

4-65

21.6

Donkeys

65

15,4

3- [01

38,8

Horses

4

0

Goats

Intensity (epg) range mean

Stools/day' (kg) 0,5-3

15-23

0 30

0

Alpacas

22d 29'

59, Id 86,2'

2-75d < I0->30'

n.s." n.s."

26 1345'

61,5 0-100 (43,6)'·g

3-145 4- [203,·h

44,7 n.s. c

1-3 1-3

0-50 (34,Ol

1-88; 1-31 ; 1-20; [-30; 1-49; 1-96;

5,9; 6.9;

15-35 15-35 15-35 15-35 15-35 [5-35

Cattier Kallutaca zone Laja zone Tambillo zone Pucarani zone Huayrocondo zone Achacachi zone

2000-195000 9000-808000

Llamas

Sheep

o. liver fluke eggs/day"

569 80 351 646 303 1166

8,0-26,3 4,0-45,2 2.5-66,6 8,5-45,5 0-56,8

(17,W (31.9)g (7,8)g (14,8)g (23,8)g

5,2' 4S 5.6; 12,6;

4000-300000 120000 3000-435000 800-72[800

3750-770000 3750-27 [250 3750-175000 3750-262500 3750-428750 3750-840000

Table 1.- Prevalences and intensities (egg output = eggs per gram = epg) of Fasciola hepatica infection in domestic animals in the orthern Bolivian Altiplano .• = general estimations of the amount of stools defecated by each domestic animal species per day, according to MORROS SARDA (1967) and GORTLER et al. (1979); b = only approximate, deduced from animal's weight; '= estimations of the total number of F. hepatica eggs shed with faeces by each domestic animal species per day in the Bolivian Altiplano; d = after UE 0 et al. (1975); e = see review by MAS-COMA et al. (1995); r = after studies carried out by BucHO et al. (1997); g = prevalence range according to different communities in the zone (with prevalence mean in parenthesis); h = number of eggs per 5 g of faeces; ; = number of eggs per 4 g of faeces; n.s. = not specified.

Secondary

reservoirs

of fascioliasis

in the Bolivian

Altiplano

iias» (Fig. 2 F) and the «corridor of Huancarani-Jesus de Machaca». However, the latter two corridors lie outside of the endemic region. At any rate, once a week llamas

43

concentrate in a trade fair specialized in this Andean eamelid in the locality of Palcoco (Fig. 2 E). They come from zones outside of the endemic region, such as from

Fig. 2.- Large domestic mammal species present in the fascioliasis human endemic region in the Northern Bolivian Altiplano: A) a very few goats in the zone between Batallas and Huarina, with the Lake Titicaca in the background; B) pigs in a water collection inhabited by Iymnaeid snails near the locality of Pucarani; C) a horse in the transmission focus of Chirapaca; D) donkeys in the endemic zone of Lacaya Baja; E) weekly trade fair of llamas in the locality of Palcoco; F) llamas in the «corridor of Pefias»; G) an alpaca in the «corridor of Pefias»; H) sheep and cattle in familial livestock.

S. MAs-CoMAet al.

44 the zone located north of the endemic region, at a higher altitude on the way to the Eastern Andean Chain, or from the Altiplanic zone located south of the endemic region, on the way to Oruro, where temperatures are colder owing to the absence of the climatic influence of Lake Titicaca (LORI 1& LIBERMA , 1983). A total of 30 llamas present in the Palcoco trade fair were surveyed. None showed liver fluke eggs in the stools. Sheep For comparison purposes, a total of 26 sheep were surveyed: 16 sheep from the locality of Batallas and 10 from Chijipata Alto. Stool samples from 16 sheep (6 I,5%) presented F. hepatica eggs: 13 from Batallas and 3 from Chijipata Alto. The number of eggs per gram of faeces ranged between 3 and 145 epg, with a mean of 44,7 epg. According to general estimations of the amount of stools defecated by a sheep per day (MORROS SARDA,1967; GURTLERet al., 1979), the total number of F. hepatica eggs shed with faeces by a sheep per day in the Northern Bolivian Altiplano may be between 3000 and 435000. Together with cattle, sheep are the most numerous domestic animals in all zones of the endemic region. Sheep usually constitute part of the livestock of Aymara families (Fig. 2 H).

DISCUSSIO In sheep (Table I), the prevalence found of 6 1,5% agrees with the results of previous coprological surveys carried out by different authors, which vary from 0 to 100% in different localities of the Northern Bolivian Altipiano, with a mean of 71,6% when including only localities presenting the parasite and a mean of 43,6% when also considering localities with prevalence 0% (see MASCOMAet al., 1995). Egg output data of a range of 3-145 epg and a mean of 44,7 epg found in our study also correspond with similar results of 4- I203 eggs/5 g previously reported by other authors in the same Altiplanic endemic region (see review in MAS-COMAet al., 1995). In the Bolivian Altiplano, cattle show F. hepatica infection rates somewhat under those reported in sheep (Table I). In the same zones analysed in the present paper (Tambillo, Pucarani, Huayrocondo and Achacachi zones) or in zones near to those here considered (Kallutaca and Laja zones), the prevalence of the different communities within the 6 zones taken into account ranges from 0 to 66,6% and the prevalence means of the same 6 zones range from 17,5 to 34,0% (data from BuCHONet al., 1997). Egg output data concerning epg, with individual ranges from I to 96 eggs/4 g and mean ranges between 4,5 and 12,6 eggs/4 g (data from BUCHONetal., 1997), are also less than in Altiplanic sheep, although they may be considered normal when compared to other world areas (see egg output after experimental infection

in DE LEO & QUINONES,198 I). However, according to general estimations of the amount of stools defecated by a cattle per day (MORROSSARDA, 1967; GURTLERet al., 1979), it appears that their daily contribution to environmental contamination with fluke eggs is similar to that of sheep (Table I). According to the results of the present study (see Table I), it is evident that pigs and donkeys are able to develop the role of potential reservoirs of fascioliasis in the Northern Bolivian Altiplano endemic zone. Their individual egg output (epg) capacities do not greatly differ from those detected in Altiplanic sheep and cattle. Moreover, their daily egg output (eggs/day) capacities are sufficient to assure an important contamination of the environment. Owing to the smaller populations of pigs and donkeys compared to the more numerous and dense populations of sheep and cattle in the endemic region, pigs and donkeys may be considered as secondary reservoir hosts in fascioliasis transmission. This means that, if laboratory research work today under way demonstrates the viability of eggs expelled by Altiplanic pigs and donkeys, these two domestic animal species must be included in the control measures (infection and transmission prevention measures, treatment campaigns) to be applied in this endemic region. Horses, goats and llamas may be discarded as contributors to environmental contamination and fascioliasis transmission in the Northern Bolivian Altiplano, mainly because of the very scarce representatives of these three species in the endemic region. Concerning alpacas, besides a very few isolated individuals regularly found in the «corridor of Pefias» (Fig. 2 G), outside of the fascioliasis transmission area, in the Northern Bolivian Altiplano endemic region alpacas are only found on the farm of Belen, north of Achacachi. Previous coprological studies on alpacas from Belen farm (UENO et al., 1975; MAS-COMA et al., 1995) reported the following results: in one study 59,1 % of 22 alpacas were detected shedding F. hepatica eggs in stools, with averages of75, 16 and 2 eggs found in animals with high (n = 7), moderate (n = 2) and low (n = 2) infection, respectively; in another survey 25 alpacas among 29 studied showed eggs in their stools (86,2%), of which 22 alpacas presented fewer than 10 eggs, 2 alpacas showed between 10 and 30 eggs, and only I alpaca presented more than 30 eggs. According to these data, alpacas may be taken into account in the zone of the Belen farm, but can be discarded in the remainder of the endemic region.

ACKNOWLEDGEMENTS Study supported by a Project (Contract No. TS3-CT94-0294) of the STD Programme of the Commission of the European Communities (DG XII: Science, Research and Development), Brussels. EU. by the Programme of Scientific Cooperation with Latin America of the lnstituto de Cooperacion Iberoamericana of the Agencia Espafiola de Cooperacion Internacional (I.C.I.-A.E.C.I.), Madrid,

Secondary reservoirs

of fascioliasis

in the Bolivian

Altiplano

and by Project No. UE96-000 I of the Direcci6n General de Investigaci6n Cientffica y Tecnica (DGICYT), Spanish Ministry of Education and Science, Madrid. Or. J.G. Esteban and Or. M.V. Fuentes (Valencia. Spain) contributed in technical aspects. Or. R. Fons and M. J.P. Clara of the Laboratoire Arago. Universite P. et M. Curie de Paris VI (Banyulssur-Mer, France) participated in the collection of Faeces samples from the animals. Or. Maribel Acosta of the Facultad de Veterinaria. Universidad de C6rdoba (Cordoba, Spain) is acknowledged for information on aspects of domestic animal physiology. The authors want to acknowledge the facilities and collaboration received from the following institutions and centers. as well as their respective representatives or directors: Direcci6n Nacional de Relaciones Internacionales as well as the Direcci6n Nacional de Epidemiologfa of the Ministerio de Previsi6n Social y Salud PUblica (today become the Secretarfa Nacional de Salud of the new Ministerio de Desarrollo Humano) in La Paz; Cornite Regional de Zoonosis and Centro Piloto La Paz of the Unidad Sanitaria La Paz: Direcci6n Nacional de Producci6n Pecuaria and the Instituto Nacional de Biologfa Animal of Chasquiparnpa-Cota Cota both of the Ministerio de Asuntos Campesinos y Agropecuarios (M.A.C.A.) in La Paz; Granja de Mejoramiento Ganadero de Kallutaca related to the Programa de Fomento Lechero of the Corporaci6n Regional de Desarrollo de La Paz (CORDEPAZ); and Office of the Pan American Health Organization in La Paz.

REFERE

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