Transactions of the Royal Society of Tropical Medicine and Hygiene (2008) 102, 374—379


Parasite contamination of soil in households of a Mexican rural community endemic for neurocysticercosis
M. Huerta a,c, R. Avila b, H.I. JimŽnez b, R. DŽ a, J. DŽ c, M.E. DŽ Huerta a, e A±az A±az A±az d e f a,c o M. HernŽndez , J.J. Martinez , T. Garate , E. GŽmez , T. Abad a, a d g d, G. Fragoso , A. Fleury , E. Sciutto
a

Facultad de Medicina, BenemŽrita Universidad AutŽnoma de Puebla, 13 Sur No. 2702, e o Col. Volcanes, Puebla, Pue. Mexico b Escuela de Medicina Veterinaria y Zootecnia, BenemŽrita Universidad AutŽnoma de Puebla, 13 Sur No. 2702, e o Col. Volcanes, Puebla, Pue. Mexico c Unidad Medico Familiar 57 IMSS, Puebla, Mexico d Instituto de Investigaciones BiomŽdicas, Universidad Nacional AutŽnoma de MŽxico, Apartado 70228, e o e 04510 MŽxico D.F., Mexico e e Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional AutŽnoma de MŽxico, o e Circuito Escolar Exterior s/n, 04510 MŽxico D.F., Mexico e f Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Crta. Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain g Instituto Nacional de NeurologŽ y NeurocirugŽ Insurgentes Sur 3877, Col. A Fama, DelegaciŽn Tlalpan, A±a A±a, o 14269 MŽxico D.F., Mexico e Received 2 July 2006; received in revised form 21 December 2007;accepted 21 December 2007

KEYWORDS
Neurocysticercosis; Taenia; Parasitic diseases; Soil microbiology; Rural communities; Mexico

Summary High neurocysticercosis (NC) prevalence was recently determined by a computed tomography (CT) scan study in the community of Tepetzitzintla, State of Puebla, Mexico. The aim of the present work was to evaluate the magnitude of fecal and parasite contamination by Taenia spp. in the soil of households of this community during the four seasons of the year. The toilet, backyard, kitchen, washboard, water containers and corrals of 14 to 26 households were sampled during each season. High Taenia spp. egg intensity was found in 24.2% of the sampled areas. The highest percentage was detected in Spring and the lowest in Summer. Significantly higher levels of Taenia spp. eggs were present in kitchen soil samples. A significant correlation was found between the presence of Taenia spp. eggs in household soil during the Summer, and NC diagnoses of the inhabitants by CT scan. Coproparasitological examinations and anticysticercal antibodies were determined in a cohort of inhabitants of the sampled households. Antibody levels and coproparasitological results were not associated with NC. Overall, these

∗

Corresponding author. Tel.: +52 55 5622 3153; fax: +52 55 5622 3369. E-mail address: edda@servidor.unam.mx (E. Sciutto

0035-9203/$ — see front matter © 2008 Royal Society of Tropical Medicine andHygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2007.12.009


Parasite contamination of soil in Mexican households

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results illustrate the high degree of fecal contamination of potential risk to human health in rural communities and could be of use for control programmes. © 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

1. Introduction
In Mexico, as in other endemic countries, soil contamination with Taenia solium eggs is of special interest considering the high prevalence of pig and human cysticercosis reported in rural communities, and the great impact of this disease on human health. Taenia solium eggs are deposited in the environment through defecation by humans, the only known definitive hosts. Human infection with T. solium occurs by accidental ingestion of eggs in soil, contaminated vegetables, water, etc. In two independent computed tomography (CT) scan epidemiological studies performed in two endemic rural communities, 9.1% of asymptomatic neurocysticercosis (NC) cases were detected (Fleury et al., 2003, 2006). No exposure or socio-economic factors were found to be associated with the infection. Considering this, the present study evaluated the relationship between parasite contamination of the soil and the presence of NC and included identification of Taenia spp. eggs in the soil samples to determine the usefulness of thistechnique for epidemiological purposes.

2. Materials and methods
2.1. Study area
This study was performed in the rural community of Tepetzitzintla, Puebla, Mexico, with 1782 inhabitants. The community, located in a region called mixteca poblana, was selected because of the poor prevailing sanitary and socioeconomic conditions that promote the life cycle of T. solium (open-air defecation, absence of toilets and drainage, poor hygienic and dietary habits, rustically bred pigs, consumption of meat without inspection). Most of the houses have soil floors, and water is provided only twice a week. In each of the 26 houses tested, soil samples were taken from the outhouse (a place on the patio used for defecation over the floor), the backyard (open patio between the areas most commonly used for specific purposes, in which the children and animals dwell), the kitchen (a barely roofed shack in the backyard mainly used to cook with wood close to the floor, in which domestic animals roam freely), around the washboard (a place on the patio in which women manually wash clothes), the water containers (open containers in which water is collected when available) and the corral (a place on the patio in which the pigs and cows spend the night). Animals walk around freely inside the houses. A total of 123 dogs, 46 cats, 92 pigs, 32 cows and 15 rabbits were registered in the 26 tested houses, all with free access into the premises.

2.2. Studydesign
The study was performed in two stages. During the first stage, from August 2000 to July 2001, a head CT scan without contrast was performed on 115 volunteers living in the

26 houses at the Puebla General Hospital to diagnose NC. Informed consent was obtained from all participants or their guardians. In addition, three fecal samples were collected from 75 inhabitants and one blood sample from 96 inhabitants. A coproparasitological study was done using direct microscopical examination and the Faust and Graham method (Faust et al., 1938; Graham, 1941). IgG antibody levels against T. solium cysticerci were determined in the collected plasma samples by ELISA using T. solium cyst fluid following the procedure reported by Chavarria et al. (2003). A sample was considered positive if the optical density (OD) values were higher than 0.4 (mean of the values obtained with Not-NC samples from the inhabitants of the same endemic community plus 2 SD). During the second stage, soil samples were obtained in the Autumn, Winter, Spring and Summer of 2001 to 2003 (Table 1). For additional analysis, a house was considered positive if Taenia spp. eggs were found in at least one sample. The surface soil of each house was swept, and samples of approximately 200 g soil were collected in plastic bags at 2—10 cm depth from different parts of the house. Twenty gram soil samples were kept under refrigeration and processed within a maximum period of72 h after collection. Each sample was dried and passed through a 4 mm2 mesh to remove debris. The remaining soil was cleaned by suspending it in purified water and then centrifuging it at 300 g for 3 min. The sediment was washed with 500 ml 1% Tween 80 solution in PBS and then shaken for 2 h and left to rest for 24 h until the supernatant was clear. Subsequently, twothirds of the supernatant was removed. The sediments were suspended with the remaining supernatant; samples were divided into two aliquots, placed into 15 ml tubes, and centrifuged at 838 g for 10 min. Helminth eggs from soil samples were recovered by a flotation method (Matsuo and Kamiya, 2005). Briefly, one tube of the sediment was suspended in 1.18-specific gravity zinc sulfate, stirred during 2 h in a high-speed vortex and sieved through a 1 mm mesh. The suspension was centrifuged at 1000 g for 5 min. The process was repeated twice. After this, samples were transferred to 15 ml tubes and centrifuged again at 1000 g for 15 min. Tubes were filled to the top, and a coverslip (24 × 24 mm) was placed on top of the tube. Coverslips were examined under a light microscope 2 h later. The tube was covered with a new coverslip and left overnight to collect the remaining eggs. For routine egg examination, the ether sedimentation technique (Ritchie et al., 1960) was applied to the remaining sediment in the aliquot tube. Briefly, the sediment was suspended in 10 ml 10% formalin,emulsified and allowed to fix for at least 10 min. After that, 5 ml ether was added; tubes were then stopped, inverted, vigorously shaken for 30 s and centrifuged at 425 g for 2 min. Four layers were formed: solvent at the top, a debris plug, formalin, and sediment at the bottom. The debris plug was loosened by ringing


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Table 1 Frequency of parasites and parasite intensity found in the soil of sampled households in the community of Tepetzitzintla, State of Puebla, Mexico Spring (n = 109) Summer (n = 116) Autumn (n = 113) Winter (n = 53)

Frequency of Parasite Frequency of Parasite Frequency of Parasite Frequency of Parasite parasitesa intensityb parasitesa intensityb parasitesa intensityb parasitesa intensityb Hymenolepis nana Hymenolepis diminuta Giardia spp. Ascaris spp. Taenia spp. Entamoeba spp. Enterobious vermicularis Sarcocystis spp. Trichuris trichiura Entamoeba coli Balantinium coli
a b

3.7 0.9 2.7 52.2 43 34.8 8.2 1.8 10.1 7.3 31.1

0.04 0.009 0.05 1.28 0.43 0.92 0.13 0.10 0.12 0.23 0.53

0 0 0 19 7.8 0 0 0 10.3 0.9 1.7

0 0 0 0.31 0.08 0 0 0 0.13 0.01 0.03

21.2 3.5 14.1 41.5 29.2 31.8 19.5 10.6 19.5 37.2 17.6

0.36 0.07 0.32 0.88 0.30 0.49 0.42 0.20 0.31 0.88 0.37

9.4 7.5 11.3 24.5 17 20.7 15 9.4 26.4 28.3 26.4

0.11 0.09 0.23 0.41 0.17 0.30 0.32 0.15 0.45 0.60 0.74

Percentage of positive sampled areas = (number of positive areas/total number of sampled areas) × 100.Parasite intensity = number of parasite eggs/number of sampled areas.

with an applicator stick, and then the top three layers were decanted. The sediment was mixed and removed with a disposable pipette and one drop of sediment was covered with a 22 mm2 cover slip. A single unstained wet mount was prepared from each concentrate. The entire coverslip area was examined with an optical microscope at low power (×10); high power (×40) was used in a similar fashion to identify and count eggs.

which includes the rainy months in Mexico. Ascaris spp. and Taenia spp. were the main genera among the parasites detected.

3.2. Soil contamination with Taenia spp.
As Table 2 shows, the kitchen was the most infected place in terms of parasite intensity and percentage of contaminated samples.

2.3. Statistical analysis
Data were processed in Excel 7.0 (Microsoft Corp., Redmond, WA, USA) and SPSS 10 (SPSS Inc., Chicago, IL, USA). Distribution of independent variables was compared by 2 tests with Yates’ correction, two-tailed Fisher’s exact tests or Student’s t test, using 95% CI.

3.3. Relation between NC and Taenia spp. soil contamination
CT scans were performed on 69 women and 46 men aged 5—79 years. Individuals were classified as silent-NC or nonNC according to whether their CT scan images showed lesions compatible with NC, as previously described (Fleury et al., 2003). In 14 individuals (five men and nine women), CT scans showedcalcified granuloma. Twelve individuals had a single parasite, whereas two had multiple parasites. The 14 positive individuals lived in 12 households (in one house three NC cases were diagnosed), while the 101 NC-negative subjects lived in the remaining 14 houses. Table 3 shows that only in the Summer was a significant correlation between the presence of Taenia spp. eggs in soil and NC found (P = 0.05). Interestingly, as shown in Table 2, it was during the Summer when the lowest percentage of soil samples with Taenia spp. eggs was detected (nine eggs in the 116 samples tested, 7.8%).

3. Results
3.1. Parasite contamination of soil
Two different parameters were defined to measure parasite contamination: the percentage of positive sampled areas = (number of positive areas/total number of sampled areas) × 100; and the parasite intensity = number of parasite eggs/number of sampled areas. A total of 391 soil samples was collected from the soil of different areas of the sampled houses (Table 1). The total number of households sampled was 26 in Autumn 2001, 14 in Winter 2001, 24 in Spring 2002 and 23 in Summer 2003. Households in which eggs were detected were randomly distributed in the community with no significant geographical aggregation (data not shown). Table 1 shows the different parasites found in the soil of the sampled households throughout the four seasons, and their frequency. The lowest percentage of positive sampled areasand parasite intensity was found in the Summer,

3.4. Relation between intestinal parasites and soil contamination
Coproparasitological studies were performed on the inhabitants of the 26 houses included in this study. The following parasites were found: Ascaris lumbricoides (9.3%), Entero-


Parasite contamination of soil in Mexican households

377

Table 2 Frequency of Taenia spp. eggs found in the soil of sampled households in the community of Tepetzitzintla, State of Puebla, Mexico Outhouse Spring Frequency of parasitesa Parasite intensityb Summer Frequency of parasitesa Parasite intensityb Autumn Frequency of parasitesa Parasite intensityb Winter Frequency of parasitesa Parasite intensityb Total frequency of parasitesa
a b

Backyard 48 (11/23) 1.74

Kitchen 55 (11/20) 2.05

Washboard 26.7(4/15) 0.47

Bucket 30.8 (4/13) 0.46

Corral 43.7 (7/16) 1.06

Total 43 (47/109)

45.4 (10/22) 1.64

10.5 (2/19) 0.16

10 (2/20) 0.10

5.3 (1/19) 0.16

5.3 (1/19) 0.053

10 (2/20) 0.15

5.3 (1/19) 0.053

7.8 (9/116)

28.6 (6/21) 0.81

14.3 (3/21) 0.29

41.2 (7/17) 0.94

47 (8/17) 1.35

31.6 (6/19) 0.47

22.2 (4/18) 0.33

30.1 (34/113)

12.5 (1/8) 0.375 27.1 (19/70)

7.7 (1/13) 0.154 22.1 (17/77)

25 (2/8) 0.75 32.8 (21/64)

18.2 (2/11) 0.182 24.2 (15/62)

14.3 (1/7) 0.29 22 (13/59)

33.3 (2/6) 0.33 23.7 (14/59)

16.9 (9/53)

Percentage of positive sampled areas = (number ofpositive areas/total number of sampled areas) × 100. Parasite intensity = number of parasite eggs/number of sampled areas.

bius vermicularis (7.3%), Hymenolepis nana (4.6%), Trichuris trichiura (1.3%), Sarcocystis hominis (1.3%), Entamoeba coli (21.2%), Entamoeba histolytica (11.9%), Giardia lamblia (6.6%), Lodamoeba buetschlii (1.3%). No intestinal parasites were found in the inhabitants of only three of the houses. In these three houses a smaller number of eggs was found in the soil with respect to the number of parasites in the respective season (i.e. in one house negative for coproparasites, only four parasites were detected in the Spring, while 11 parasites were found in the overall soil inspection).

two different houses: one lived alone and revealed a positive CT scan, while in the other house, all inhabitants showed negative CT scans. Thus, coproparasitological studies did not correlate with NC diagnosis (P = 0.70).

3.6. Anti-cysticercal antibodies do not correlate with CT scan diagnosis
Serum ELISA was performed on the plasma of 96 inhabitants of the community. As previously reported (Fleury et al., 2003), no association was found between CT scan and seropositivity (P = 0.18), although a significant difference in optical densities (P = 0.008) was determined between inhabitants with calcified NC (0.32 ± 0.24) and without NC (0.19 ± 0.11).

3.5. Taenia spp. eggs in coproparasitological studies do not correlatewith CT scan diagnosis
Coproparasitological studies found Taenia spp. eggs in only two of the 75 inhabitants tested. These two subjects lived in

Table 3 Relation between computed tomography (CT) scan results and presence of Taenia spp. eggs in the different seasons of the year in the community of Tepetzitzintla, State of Puebla, Mexico Taenia spp. eggs in soil Spring + CT scan households Positive Negative 11 9 − 1 3 P 0.59 Summer + 5 1 − 5 12 P 0.05 Autumn + 11 10 − 1 4 P 0.33 Winter + 3 3 − 4 4 P 1


378

M. Huerta et al. Moreover, it is also possible that Taenia spp. eggs can survive in the environment for weeks, as has been reported for other taeniids (Gemmell and Johnstone, 1976), spreading the disease by overdispersion. Many epidemiological studies performed in rural areas of Mexico (Diaz Camacho et al., 1991; Sarti-GutiŽrrez et al., 1988) and other endemic e countries (Ferrer et al., 2003; GarcŽ et al., 2003) have A±a determined risk factors related to human cysticercosis seroprevalence, i.e. history of seizures, human behavior (particularly poor hygiene), pig-rearing practices, household use of toilets and the presence of a tapeworm carrier in the house (Sarti et al., 1992). However, the identification of risk factors related to human NC infection has remained elusive. In two epidemiological studies based on CT scans performed in Mexican rural communities, none of the above risk factors was found to be associated withNC (Fleury et al., 2003, 2006). In the present study, we found, for the first time, a high level of Taenia spp. egg contamination in the different household areas tested, pointing to the relevance of appropriate standardized techniques such as PCR to specifically diagnose the source of the detected Taenia spp. eggs. Such techniques will allow the detection of endemic areas with high transmission of the respective cestodiasis. Nevertheless, the finding of such a high level of contamination with Taenia spp. eggs is a clear indicator of the need to improve hygienic conditions in rural areas. The detection of Taenia spp. eggs in soil and water has been reported previously only by Diaz Camacho et al. (1991). However, these authors found only one soil sample with Taenia spp. eggs. The procedure used for egg detection in soil, as well as the different living standards and the lower T. solium prevalence between Northern and Central Mexico, could underlie the differences between that study and the present study. Herein, a significant correlation between CT scan results and detection of eggs in soil was found in the Summer season. The absence of correlation in the other seasons, in this part of Mexico, could be the consequence of the larger number of eggs and their more effective dispersion during the drier seasons. Interestingly, detection of Taenia spp. eggs in the households was found to be a more predictive parameter to identify NCinhabitants than the detection of Taenia spp. eggs in feces, as coproparasitological studies have not proved to be a predictive parameter of NC cases. This could be due to the short period that tapeworm infections last, as was reported by GarcŽ et al. (2003). In addition, the levA±a els of anti-cysticercal antibodies were also found to be a predictive parameter to identify NC cases. The present results may indicate a high risk of infection with Taenia spp. within the household, with important potential impact on human health, which should be further analyzed. The high level of parasite contamination of the soil may also serve to alert authorities and the population of the need to control the environment to prevent human infections.

3.7. Taenia spp. eggs in coproparasitological studies and plasma Ab levels do not correlate with Taenia spp. eggs in soil
A house was considered serologically positive when it was occupied by at least one inhabitant with positive plasma Ab levels (OD > 0.4). No significant relation was found between Ab levels and the presence of Taenia spp. eggs in soil (P = 0.91 in Spring, P = 0.95 in Summer, P = 0.25 in Autumn and P = 0.21 in Winter). An additional analysis was performed considering a house as positive according to the coproparasitological examination if Taenia spp. eggs were detected in at least one of its inhabitants. No significant relation was found between coproparasitological results and thepresence of Taenia spp. eggs in soil.

4. Discussion
This study clearly documents the significant soil contamination in rural areas by parasites that can potentially affect human health. Results are in accordance with the high frequency of intestinal parasites found in the inhabitants of this community. Indeed, in only three out of the 26 houses, coproparasitological studies detected no intestinal parasites. Several factors can contribute to the high level of parasite contamination in the soil: many domestic animals walking freely in the house, in particular dogs, cats and pigs; the lack of compartmentalization of the houses and the presence of soil floors; and the fact that most of the inhabitants have intestinal parasites. Considering the hyperendemicity of T. solium in the rural community in which this study was performed, the finding of Taenia spp. eggs in the soil was emphasized. There are higher proportions of Taenia-positive soil samples and parasite intensity in Spring, which could indicate that the environmental conditions are favorable for egg development during this season. Several factors, such as climatic and environmental conditions, the texture of the analyzed soil and the presence of domestic animals, may have contributed to this finding. The seasonal dynamics of parasitic infections has been reported previously for different parasites (Theodoropoulos et al., 2000; Vlassoff et al., 2001). Although the most positivesamples were found in the kitchen, it is worth noting the high Taenia spp. egg prevalence in all the tested household areas. The high presence of Taenia spp. eggs in the superficial layer of soil supports the notion that this contamination focus is a crucial instrument in its dissemination. Of particular interest is the high frequency of households with Taenia spp. eggs in the soil (99 of the 391 soil samples, 25.3%) compared with the apparently rare tapeworm carriers (two of the 75 fecal samples, 2.7%) according to coproparasitological studies. It is possible that the detected Taenia spp. eggs were from other cestodes that can infect domestic animals. The appearance of eggs in the soil does not allow distinction between taeniids, in particular between T. solium, T. saginata, T. pisiformis and Echinococcus spp. It is highly possible that most of the eggs found in soil were from the many dogs and cats that have free access to all areas of the houses.

Authors’ contributions: MHu, TG, GF, AF and ES designed the study protocol; AF carried out the clinical assessment; MHe carried out the immunoassay determination; JJM, GF, AF and ES carried out the analysis and interpretation of the data; RA and HHJ carried out the coproparasitoscopic studies; MHu, RD, JD and MEDH carried out the field work; EG


Parasite contamination of soil in Mexican households and TA provided laboratory support; GF, AF and ES drafted the manuscript. All authorsread and approved the final manuscript. AF and ES are guarantors of the manuscript. Acknowledgements: The authors are grateful to the authorities of the SecretarŽ de Salud del Estado de Puebla, A±a Puebla, Mexico, for the support to this project. We are also A±a grateful to Mercedes Baca for technical assistance and MarŽ Isabel PŽrez Montfort for the correction of the English vere sion of the manuscript. Funding: Consejo Nacional de Ciencia y Tecnologia (200401-040, 46953-M), Mexico, the Facultad de Medicina, Benemerita Universidad de Puebla, Puebla, Mexico, and the Howard Hughes Medical Institute, United States. Conflicts of interest: None declared. Ethical approval: The Ethical Committee of the National Institute for Neurology and Neurosurgery (Instituto Nacional de NeurologŽ y NeurocirugŽ Mexico City, Mexico A±a A±a),

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Fleury, A., Gomez, T., Alvarez, I., Meza, D., Huerta, M., Chavarria, A., Carrillo- Mezo, R.A., Lloyd, C., Dessein, A., Preux, P.M., Dumas, M., Larralde, C., Sciutto, E., Fragoso, G., 2003. High prevalence of calcified silent neurocysticercosis in a rural village of Mexico. Neuroepidemiology 22, 139—145. Fleury, A., Morales, J., Bobes, R.J., Dumas, M., YŽnez, O., Pi˜a, J., a n Carrillo-Mezo, R., MartŽ A±nez, J.J., Fragoso, G., Dessein, A., Larralde, C., Sciutto, E., 2006. An epidemiological study of familial neurocysticercosis in an endemic Mexican community. Trans. R. Soc. Trop. Med. Hyg. GarcŽ A±a,H.H., Gilman, R.H., GonzŽlez, A.E., VerŽstegui, M., a a Rodriguez, S., Gavidia, C., Tsang, V.C., Falcon, N., Lescano, A.G., Moulton, L.H., Bernal, T., Tovar, M., 2003. Cysticercosis Working Group in Peru, Hyperendemic human and porcine Taenia solium infection in Peru. Am. J. Trop. Med. Hyg. Gemmell, M.A., Johnstone, P.D., 1976. Factors regulating tapeworm populations: dispersion of eggs of Taenia hydatigena on pasture. Ann. Trop. Med. Parasitol. Graham, D.F., 1941. A device for the diagnostic on the Enterobius vermicularis infection. Am. J. Trop. Hyg. Matsuo, K., Kamiya, H., 2005. Modified sugar centrifugal flotation technique for recovering Echinococcus multilocularis eggs from soil. J. Parasitol. 91, 208—209. Ritchie, L.S., Lin, S., Moon, A.P., Frick, L.P., Williams, J.E., Asakura, S., Hishinuma, Y., 1960. The possible effects of pH and specific gravity on the ether-sedimentation procedure in concentrating eggs and cysts. Am. J. Trop. Med. Hyg. Sarti, E., Schantz, P.M., Plancarte, A., Wilson, M., Gutierrez, I.O., LŽpez, A.S., Roberts, J., Flisser, A., 1992. Prevalence and risk o factors for Taenia solium taeniasis and cysticercosis in humans and pigs in a village in Morelos, Mexico. Am. J. Trop. Med. Hyg. Sarti-GutiŽrrez, E.J., Schantz, P.M., Lara-Aguilera, R., GŽmez e o Dandoy, H., Flisser, A., 1988. Taenia solium taeniasis and cysticercosis in a Mexican village.Trop. Med. Parasitol. Theodoropoulos, G., Zervas, G., Kouneli, A., Martinez-GonzŽles, a B., Petrakos, G., Kostopoulos, J., 2000. Seasonal patterns of strongyle infections in grazing sheep under the traditional production system in the region of Trikala, Greece. Vet. Parasitol. Vlassoff, A., Leathwick, D.M., Heath, A.C., 2001. The epidemiology of nematode infections of sheep. NZ Vet. J. 49, 213—221.

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