Giardiasis in dairy calves: effects of fenbendazole treatment on intestinal structure and function

International Journal for Parasitology 31 (2001) 73±79

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Giardiasis in dairy calves: effects of fenbendazole treatment on intestinal structure and function R.M. O'Handley a, A.G. Buret b, T.A. McAllister c, M. Jelinski d, M.E. Olson a,* a

Department of Gastrointestinal Sciences, University of Calgary, Calgary, Alberta T2N 4N1, Canada b Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada c Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge Alberta T1J 4B1, Canada d Hoechst Roussel Vet, Regina, Saskatchewan S7N 5B4, Canada Received 15 September 2000; received in revised form 9 November 2000; accepted 9 November 2000

Abstract Twelve Giardia duodenalis-infected Holstein dairy calves were allocated into a treatment (n ˆ 6) and placebo group (n ˆ 6) according to pre-study faecal cyst counts. Calves in the treatment group received an oral dose of 5 mg/kg fenbendazole once daily for 3 days, while placebo calves received a sterile saline solution. Calves were euthanised 7 days following the initiation of treatment and intestinal were collected and prepared for trophozoite quantitation, histology, electron microscopy, and disaccharidase assays. In all calves treated with fenbendazole, intestinal trophozoites were below detection limits, while in saline-treated calves, trophozoites were observed in all intestinal segments. Histologically, no signi®cant difference was observed between treatment groups with respect to intestinal villus height or crypt depth. However, a signi®cant decline in the number of intraepithelial lymphocytes (IEL) was observed in fenbendazole-treated calves when compared with placebo-treated calves in the duodenum (13.9 ^ 1.2 vs. 17.0 ^ 1.1 IEL/100 enterocytes) and jejunum (21.6 ^ 0.8 vs. 30.7 ^ 1.0 IEL/100 enterocytes). In addition, measurements from TEM micrographs demonstrated a signi®cant increase in microvillus surface area in the jejunum of fenbendazole-treated calves compared with saline-treated calves (31.2 ^ 10.2 vs. 22.8 ^ 7.6 mm 2). This increase in microvillus surface area was also associated with an increase in jejunal maltase activity in fenbendazole-treated calves compared with calves treated with saline. These results demonstrate that fenbendazole is an effective treatment for giardiasis in calves. fenbendazole treatment eliminated Giardia trophozoites from the small intestine of calves resulting in increased microvillus surface area and greater intestinal enzyme activity. This study also demonstrates that the pathogenesis of giardiasis in calves is similar to that observed in humans and laboratory animals, and provides further evidence that Giardia is a pathogen of cattle with potential economic importance. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Giardia; Giardiasis; Trophozoite; Calves; Fenbendazole; Pathogenesis; Intestine; Histology; Microvilli; Disaccharidase

1. Introduction The protozoan parasite Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) has recently emerged as an important parasite of domestic livestock. Many reports indicate Giardia infections occur in lambs and calves with a prevalence of up to 100% (Buret et al., 1990; Taylor et al., 1993; Xiao and Herd, 1994; Olson et al., 1997a,b; O'Handley et al., 1999). Calves as young as 4 days of age can become infected with Giardia, and infections can persist for over 4 months (Xiao and Herd, 1994; O'Handley et al., 1999). Infected calves and lambs can develop diar* Corresponding author. Animal Resources Centre, University of Calgary Faculty of Medicine, 3330 Hospital Drive, N.W., Calgary, Alberta T2N 4N1, Canada. Tel.: 11-403-220-6836; fax: 11-403-270-0954. E-mail address: [email protected] (M.E. Olson).

rhoea (O'Handley et al., 1999; Xiao et al., 1993; Olson et al., 1995). Therefore, Giardia infections in livestock may be important from an animal health perspective, as well as an economic perspective as infections may result in production losses. In fact, a previous study demonstrated that speci®c pathogen free lambs experimentally infected with Giardia experience signi®cantly decreased weight gain, impaired feed ef®ciency, and reduced carcass weight compared with non-infected lambs (Olson et al., 1995). Even though domestic livestock have emerged as important hosts for Giardia, few studies have examined the pathogenesis of the disease in food-producing animals. In humans and laboratory animals, villus atrophy, reduced brush border surface area, decreased disaccharidase activity, impaired nutrition and water absorption, increased intestinal transit, and an in®ltration of intraepithelial lymphocytes have been observed during Giardia infections (Buret et al., 1990b,

0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(00)00148-X

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1991, 1992; Farthing, 1993; Deselliers et al., 1997; Scott et al., 2000). These pathophysiological changes culminate in the malabsorptive diarrhoea associated with the disease. In calves, Giardia infections may be associated with similar pathophysiological changes, including villus atrophy and intraepithelial lymphocyte in®ltration (Ruest et al., 1997). However, giardiasis in calves has yet to be examined beyond the level of histology. A number of studies have shown that the anthelmintic benzimidazoles may be effective anti-Giardia agents. Benzimidazoles are effective against Giardia trophozoites in vitro (Chavez et al., 1992; Morgan et al., 1993), and treating infected animals, including calves, with albendazole and fenbendazole can eliminate the production of faecal Giardia cysts (Barr et al., 1994; Xiao et al., 1996; O'Handley et al., 1997; Zajac et al., 1998). We recently demonstrated that treating calves for giardiasis with fenbendazole can signi®cantly reduce the incidence of diarrhoea (O'Handley et al., 2000). Studies have yet to examine the effects of treating calves with benzimidazoles on intestinal colonisation by trophozoites in vivo. By eliminating trophozoites from the intestines of calves through treatment, insight could be gained into the pathogenic mechanisms of giardiasis in calves, as well as the mechanisms by which treatment results in clinical improvement. In this study, the effectiveness of fenbendazole at eliminating intestinal Giardia trophozoites from infected calves housed within a commercial dairy setting was evaluated. In addition, the mechanisms by which the parasite may cause intestinal malfunction in calves, as well as the mechanisms by which treatment may result in clinical improvement were examined.

2. Material and methods 2.1. Animals and housing Twelve, 2-week-old dairy calves were purchased from various commercial dairies for this study. Upon arrival at Agriculture and Agri-food Canada's dairy facility in Lethbridge, Alberta, calves were placed in individual pens consisting of concrete ¯oors and solid wooden walls, which prevented contact between animals. The pens were cleaned and fresh bedding, consisting of wood shavings, was provided daily. Calves were fed milk from the dairy (10% body weight/day), were provided ad libitum access to 0.5 kg of feed concentrate per day as well as loose alfalfa hay. Calves were given trace minerals and had ad libitum access to water (¯occulated, ®ltered and chlorinated municipal water, Lethbridge, Alberta). All workers followed Agriculture and Agri-food Canada's recommended code of practice for the care and handling of dairy cattle. All animal handling procedures were in accordance with recommendations of the Canadian Council on Animal Care.

2.2. Experimental protocol When calves ®rst arrived at the dairy facility, a faecal sample (1±5 g) was collected directly from the rectum of each calf using a disposable latex glove. Faecal samples were examined for the presence of Giardia cysts and Cryptosporidium oocysts using a sucrose gradient centrifugation procedure, followed by immuno¯uorescence microscopy as previously described (O'Handley et al., 1999). All calves not shedding faecal Giardia cysts at this time were orally inoculated with approximately 10 5 G. duodenalis cysts obtained from a naturally infected calf. Seven days following inoculation, a second set of faecal examinations con®rmed successful establishment of Giardia infection in all 12 calves. Calves were then ranked according to their cyst counts, blocked, and randomly assigned to a treatment (n ˆ 6) and a placebo (n ˆ 6) group. After allowing calves to adapt to their diets (day 10 after arrival), the following treatments were initiated. Calves in the treatment group were administered an oral dose of fenbendazole at 5 mg/kg once daily for 3 consecutive days. This treatment regimen was previously shown to eliminate Giardia cysts from the faeces of calves (O'Handley et al., 1997). Calves in the placebo group received a 5-ml oral dose of sterile saline solution, once daily for 3 days. Seven days after the initiation of treatment, faecal samples were once again collected from each calf for Giardia cyst enumeration and examination for the presence of Cryptosporidium oocysts. Calves were then euthanised by an i.v. injection of sodium pentobarbital. Immediately following euthanasia, the entire small intestine was removed and divided into four sections: duodenum, de®ned as the segment between the pylorus and ligament of Treitz; proximal jejunum, the proximal one-third of the remaining intestine; distal jejunum, the mid one-third of the remaining small intestine, and ileum, distal one-third of the remaining intestine. Through careful measurement, samples 10 cm in length were removed from the mid-point of each intestinal segment and prepared for the following procedures. 2.3. Trophozoite extraction and enumeration Segments measuring 1 cm in length were removed from duodenum, proximal jejunum, distal jejunum, and ileum. Each segment was split longitudinally and placed in a 10ml centrifuge tube containing 5 ml of sterile PBS. Segments were incubated with shaking for 30 min at 378C. After incubation, a drop of the suspension was removed from the centrifuge tube, placed on a haemocytometer, and Giardia trophozoites were counted at 400£ magni®cation on a light microscope. 2.4. Histology Additional 1-cm segments, taken from the duodenum, proximal jejunum, and ileum, were cut along the mesentery then ®xed for 24 h in a scintillation vial containing Carnoy's

R.M. O'Handley et al. / International Journal for Parasitology 31 (2001) 73±79

®xative. Following dehydration in ethanol, the tissue was in®ltrated and embedded in JB-4 plastic medium according to the manufacturer's instructions (Polysciences Inc.). Sections of approximately 1.5 mm were cut, mounted on glass slides, and stained with Lee's methylene blue and basic fuchsin. Villus and crypt measurements were made from 10 villus-crypt units per slide under a light microscope using a calibrated micrometer at 100£ magni®cation. In addition to villus-crypt measurements, the number of enterocytes and intraepithelial lymphocytes (IELs) were counted along the villus from sections of the duodenum and jejunum at 400£ magni®cation. At least 500 enterocytes were counted on each section, and the number of IELs counted per 100 enterocytes was calculated. 2.5. Disaccharidase assay In order to assess intestinal disaccharidase activities, which are often impaired during giardiasis (Buret et al., 1990b, 1991; Scott et al., 2000; Belosevic et al., 1989), segments from the proximal jejunum were removed and placed on glass plates. The segments were opened along the mesentery and the mucosa scraped using a pre-weighed microscope slide. The scraping was then weighed and placed in a scintillation vial containing 10% w/v of 2.5 mM EDTA. The sample and solution were then homogenised and 1-ml aliquots were transferred to microcentrifuge tubes. The samples were ¯ash frozen in liquid nitrogen and stored at 2708C until they could be assayed. After thawing, samples were assayed for maltase and lactase activity (Dahlqvist, 1964), and protein was measured according to the method of Lowry. Disaccharidase activity was then calculated in units per gram of protein. 2.6. Transmission electron microscopy Epithelial ultrastructure of the jejunum was examined from three calves in the fenbendazole treatment group and three calves in the placebo group. Segments from the proximal jejunum measuring approximately 10 mm 2 were removed and immediately placed in scintillation vials containing 5% glutaraldehyde in 0.1 M cacodylate buffer. The specimens were cut into 1-mm 2 cubes while immersed in the ®xative, then stored overnight at 48C. After washing the specimens in cacodylate buffer, they were post®xed in 1% OsO4 for 2 h and dehydrated in ethanol. Following dehydration, the specimens were cleared with propylene oxide, in®ltrated, and ®ve specimens per animal were embedded in Spurr's low viscosity medium (J.B. EM Services Inc., Dorval, Quebec). Approximately 20 thin sections were obtained from three embedded specimens per calf. The thin sections were stained with uranyl acetate in 50% ethanol, followed by 0.4% lead citrate (Venable and Coggeshall, 1965). Sections were examined in a Hitachi H7000 TEM at 75 kV, and mid-villus regions were identi®ed under low magni®cation. After mid-villus regions were located, micrographs of the microvillus border were taken at

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12 000£ magni®cation. Microvillus border surface area was then calculated (Phillips et al., 1979). Measurements were made in blind fashion from nine micrographs per animal (27 per group) which were coded to eliminate observer bias. 2.7. Statistical analysis Giardia cyst and trophozoite counts were natural log transformed and are expressed as geometric means. Counts were evaluated by analysis of variance (ANOVA) and compared using Newman±Keul's multiple comparison of means. All other results are expressed as mean ^ SEM and were compared by Student's t-test. All analyses were performed using a statistical software package (Instat, Graphpad Inc.). Values of P , 0:05 were considered significant. 3. Results Three days prior to the initiation of treatment, Giardia infections were con®rmed in all 12 calves by the presence of cysts in the faeces. Cryptosporidium oocysts were not observed in post-treatment faecal samples collected from any of the calves. The geometric mean numbers of cysts per gram of faeces were similar prior to treatment, values being 1820 and 1826 cysts per gram of faeces for the fenbendazole and placebo groups, respectively. However, 7 days after the initiation of treatment, the geometric mean number of cysts per gram of faeces was 2.15 for calves treated with fenbendazole, signi®cantly lower (P , 0:01) than calves in the placebo group which had a geometric mean of 13 401 cysts per gram of faeces. Cryptosporidum oocysts were not observed in faecal samples collected from calves in either treatment group. Coinciding with the decline in faecal cyst excretion, the number of Giardia trophozoites observed in the small intestine of calves was signi®cantly reduced (P , 0:01) following fenbendazole treatment (Table 1). Trophozoites were not observed in any intestinal segments from calves treated with fenbendazole (detection limit 5 £ 103 trophozoites per cm 2), while in the placebo group, trophozoites were observed in all sections of the intestine. During histological examination, trophozoites were observed attached to the epithelium and within the lumen of intestinal segments taken from calves in the saline-treated group. No trophozoites were observed during histological examination of intestinal segments from any calves 7 days following treatment with fenbendazole. Based on morphometric measurements made from histologic sections, there was no difference (P . 0:05) between the two treatment groups with respect to villus height or crypt depth within the duodenum, jejunum, or ileum (Fig. 1). However, the number of intraepithelial lymphocytes observed per 100 enterocytes was signi®cantly reduced in the duodenum (P , 0:05) and jejunum (P , 0:01) of calves

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Table 1 Giardia duodenalis trophozoites per cm 2 of small intestine from infected calves treated with fenbendazole and saline a Treatment

Duodenum

Proximal jejunum

Distal jejunum

Ileum

Saline Fenbendazole

11 411 (9.34 ^ 0.78) BD*

18 711 (9.84 ^ 2.04) BD*

17 381 (9.75 ^ 2.06) BD*

3873 b (8.26 ^ 1.79) BD*

Data are presented as geometric mean and (mean natural log ^ SEM) trophozoites per cm 2 of intestinal segment. BD, below detection limits …5 £ 103 †. *Signi®cantly different from saline group (P , 0:01). b Number of trophozoites were BD in one calf and considered as 0 for calculations. a

treated with fenbendazole compared with calves treated with saline (Table 2). In observations made from a number of TEM micrographs, the jejunal microvilli of fenbendazole-treated animals appeared to be longer and more numerous compared with microvilli from calves treated with saline (Fig. 2). Measurements con®rmed these observations, as the microvillus surface area per mm 2 of cell surface was signi®cantly increased (P , 0:01) in the jejunum of calves treated with fenbendazole compared with that of the placebo group (Table 2). Brush border maltase activity coincided with this increase in microvillus surface area in the jejunum (Table 2). Mean maltase activity in calves treated with fenbendazole was signi®cantly higher (P , 0:05) compared with calves treated with saline. While mean lactase activity was three times higher in calves treated with fenbendazole compared with the placebo group, this difference failed to reach signi®cance (P , 0:3). 4. Discussion Although benzimidazoles are effective anti-Giardia agents in vitro, and treating calves with fenbendazole eliminates faecal Giardia cysts, this is the ®rst study to examine the effects of fenbendazole on intestinal colonisation by Giardia trophozoites in vivo. In addition to eliminating Giardia cysts from the faeces of these calves, fenbendazole treatment brought small intestinal trophozoite numbers to undetectable levels via counts or microscopic assessment. The elimination of trophozoites associated with treatment resulted in physiological and morphological improvement within the small intestine of these calves. The increased epithelial brush border surface area and higher disaccharidase activities in treated animals may, in part, contribute to the clinical improvements observed in Giardia-infected calves treated with fenbendazole (O'Handley et al., 2000). These results also provide some insight into the pathogenesis of giardiasis in cattle. Previous studies demonstrate that Giardia infections in humans and laboratory animals are associated with a number of pathological characteristics. These include villus atrophy, decreased brush border surface area, reduced disaccharidase activity, impaired nutrient and water absorption, and increased numbers of IELs (Buret et al., 1990b, 1991, 1992; Farthing, 1993; Deselliers et al., 1997; Scott et al.,

2000; Koudela and Vitovec, 1998). Nevertheless, few studies have examined the pathophysiology of giardiasis in calves. The authors of one study observed villus atrophy and an increase in the number of IELs in calves infected with Giardia when compared with non-infected calves (Ruest et al., 1997). However, these authors did not examine intestinal enzyme activity or microvillus surface area following infection, both of which are important contributors to malabsorptive diarrhoea in a number of intestinal disorders, including giardiasis (Buret et al., 1990b, 1991, 1992). As up to 100% of conventionally raised dairy calves are naturally infected with Giardia (Xiao and Herd, 1994; O'Handley et al., 1999), the study presented here can not represent a true comparison between infected and noninfected calves. However, treating calves with fenbendazole was 100% effective at eliminating Giardia trophozoites from the intestinal tract. Whether treatment with fenbendazole corrected intestinal ultrastructure and function to levels one would observe in non-infected animals warrants further investigation. Although trophozoites were observed in all sections of the intestine in control animals, the jejunum was selected for comparison of intestinal disaccharidase activity and epithelial ultrastructure due to its importance in nutrient digestion and absorption. Compared with untreated infected

Fig. 1. Villus height and crypt depth in the duodenum, proximal jejunum, and ileum from Giardia duodenalis-infected calves treated with fenbendazole (A) or saline (B).

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Table 2 Mean number of intraepithelial lymphocytes, microvillous surface area, and intestinal disaccharidase activity in Giardia duodenalis-infected calves 7 days following treatment with fenbendazole or saline a Treatment

Saline Fenbendazole a b

Intraepithelial lymphocytes/100 enterocytes

Microvillus surface area (mm 2) b

Disaccharidase activity (U/g of protein)

Duodenum

Jejunum

Jejunum

Maltase

Lactase

17.0 ^ 1.1 13.9 ^ 1.2*

30.7 ^ 1.0 21.6 ^ 0.8**

22.8 ^ 1.5 32.1 ^ 2.1**

12.1 ^ 1.6 22.5 ^ 3.1*

10.4 ^ 3.3 31.1 ^ 12.8

Data are mean ^ SEM. *Signi®cantly different from saline group (P , 0:05); **signi®cantly different from saline group (P , 0:01). Surface area per mm 2 of cell surface in jejunum.

animals, brush border surface area increased in the jejunum of fenbendazole-treated calves. Microvilli, present on the luminal surface of intestinal enterocytes, are an adaptation

for increasing absorptive surface area. The reduction in brush border surface area during giardiasis is one of the major factors contributing to the malabsorptive diarrhoea

Fig. 2. Transmission electron micrographs of microvilli from proximal jejunum epithelia at the same magni®cation in Giardia duodenalis-infected calves 7 days following treatment with fenbendazole (A) or saline (B). Scale bar-1 mm.

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associated with this disease (Buret et al., 1990b, 1991, 1992; Scott et al., 2000; Koudela and Vitovec, 1998). Coinciding with the increase in brush border surface area in the present study, results demonstrated an increase in disaccharidase activity. Maltase activity was signi®cantly increased following fenbendazole treatment and mean lactase activity was almost threefold higher in fenbendazole-treated calves. The increase in lactase activity failed to reach signi®cance due to high standard errors, which were most likely the result of variability in calves of this age as they make a transition from pre-ruminant to ruminant (Le Huerou et al., 1992). Nevertheless, treatment appears to result in the recovery of the microvillus and brush border enzymes. This recovery would provide calves with a greater surface area and capacity for nutrient digestion. Previous research has shown that loss of brush border surface area correlates with reduced absorption of water and Na 21-coupled glucose (Buret et al., 1992). Therefore, intestinal recovery associated with the elimination of Giardia following treatment may result in clinical improvement and could have a potential impact on production. Treating calves for giardiasis also reduced the number of IELs present within the epithelium of the duodenum and jejunum. Increased numbers of IELs have been implicated in the pathophysiology of giardiasis (Farthing, 1993), but an increase in IELs is not always observed during giardiasis (Cevallos et al., 1995; Hardin et al., 1997). Intestinal lesions in calves infected with Cryptosporidium parvum are associated with a signi®cant increase in the number of IELs (Wyatt et al., 1997), and the pathology associated with intestinal disorders in humans, such as celiac disease, can also be associated with increased numbers of IELs (Oberhuber et al., 1996). T cells have recently been implicated in the pathogenesis of Giardia-induced loss of brush border surface area (Scott et al., 2000). However, whether or not IELs contribute to the pathology of disease remains unclear. Different subtypes of IELs may play different roles in contributing to pathology. For example, in coeliac disease, IELs expressing the gd T-cell receptor signi®cantly increase in number, but IELs expressing the ab receptor signi®cantly increase in number in calves infected with C. parvum (Wyatt et al., 1997; Oberhuber et al., 1996). Thus, further study is required in order to understand the role of IELs and speci®c IEL subtypes in the pathogenesis of giardiasis in calves. No changes were observed, with respect to villus height or crypt depth, between Giardia-infected calves treated with fenbendazole or saline. Although alterations in villus architecture have been previously observed in calves, experimentally infected goat kids, and laboratory animals with giardiasis (Buret et al., 1990b; Ruest et al., 1997; Koudela and Vitovec, 1998), changes in villus-crypt morphology do not always occur (Buret et al., 1992; Farthing, 1993; Hardin et al., 1997). It is possible that calves in this study were not given enough time between treatment and sacri®ce to allow for any changes in villus height and crypt depth to take

place. It is also possible that Giardia infection in these calves had no signi®cant effect on villus and crypt morphology. Due to the dif®culty in obtaining a non-infected control group for comparison in this study, the effects of giardiasis on villus architecture in these calves was not determined. The results of this study indicate that fenbendazole is an effective treatment for giardiasis in calves. Daily treatment for 3 consecutive days eliminates trophozoites from the entire small intestine, resulting in a signi®cant increase in brush border surface area and intestinal enzyme activity. These results also indicate that in cattle, the pathogenesis of giardiasis is similar to that observed in humans and laboratory animals. The study also provides further evidence that Giardia is an important pathogen of cattle with potential economic signi®cance.

Acknowledgements The authors wish to thank Liz Middlemiss, Nicole Guselle, Sandy Widder, and Carol Cockwill for technical assistance. This research was supported by the Natural Sciences and Engineering Research Council of Canada and Hoechst Roussel Vet.

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