Slov Vet Res 2011; 48 (2): 51-56 UDC 636.4.09:616.993.1:615.283
Case Report
EFFECTIVE TREATMENT OF GIARDIOSIS IN PIGS BY ALBENDAZOLE
Marina Stukelj1*, Zdravko Valencak1, Aleksandra Vergles Rataj2, Janez Posedi2
institute for Health Care of Pigs, Veterinary Faculty, University of Ljubljana, Cesta v Mestni log 47, institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbiceva 60, 1000 Ljubljana, Slovenia
'Corresponding author, E-mail: marina.stukelj@vf.uni-lj.si
Summary: Giardiosis is a parasitic disease prevalent worldwide that affects humans, domestic and wild animals and can be responsible for chronic diarrhoea. Diarrhoea started in the time of wintering at the stables of the Institute for the Health Care of Pigs. Two sows (Krsko polje breed), and four piglets aged two months (the Krsko polje breed) were affected. Rectal smears were sent for bacteriological examination. Faecal samples were examined by microscopy using the SAF method for concentrating protozoa and by direct immunofluorescence (DIF).
No pathogenic bacteria were isolated at the first bacteriological examination but the second examination confirmed the presence of Campylobacter coli. Giardia sp. was detected by SAF and DIF at the first test. The pigs were treated with Monil®5% (Pliva d.d., Croatia) with 20 mg/kg albendazole, peros, once daily, for 3 days.
After 3 days therapy the diarrhoea stopped and the pigs were clinically healthy and remained without diarrhoea for the next four months. Seven days, ten days and eleven days after the treatment all tested samples were negative by SAF and DIF.
Key words: pig; Giardia; treatment; albendazole
Introduction
Giardiosis is a worldwide parasitic disease that affects humans, domestic and wild animals (1) and can be responsible for chronic diarrhoea (2, 3). Giardia is a protozoan, which is bilaterally symmetrical, with eight flagella, and has a large adhesive disk on the body's ventral surface (trophozoit). The disk enables attachment to the epithelial cells of the intestinal mucosa. The simple life cycle of Gia-rdia, involving an environmentally resistant cyst, provides many opportunities for the parasite to be transmitted directly from one infected individual to another, or indirectly through contamination of the environment or water and food (4). The cysts are more common in pens with solid floors, and piglets and weaners appear to be infected by the sows (5). The parasite colonizes the small intestine of animals and may lead to moderate to severe diar-
Received: 25 October 2010 Accepted for publication: 13 May 2011
rhoea (6) or clinical signs may be absent (5). Giardia infections have been reported in pigs in all groups from nursing piglets to boars and sows from Europe, Australia, Asia and North America with prevalence ranging between 0,1% and 20%, but usually not associated with clinical illness (7, 8, 9, 10). Giardia cysts were identified in 3.8% of piglets, 9.8% of weaners, 10.8% of growers, 15% of finishers, 5.7% of boars and 4.1% of sows (6). Diagnosis of Giardia by traditional microscopic methods following the application of faecal concentration techniques, especially zinc sulphate flotation and centrifugation (11) which remains a reliable indicator of infection. However, the detection of Giardia by microscopy or faecal ELISA is of limited epidemiological value, especially in terms of the source of infection. The development of direct immunofluorescence microscopy has generally improved the sensitivity of detecting and quantitating faecal Giardia cysts and may allow for more accurate determination of prevalence rates and cyst excretion intensities than conventional microscopy (12). With Giardia,
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molecular techniques, particularly PCR-based procedures are more sensitive and specific than 'conventional' diagnostics that rely on microscopy and/ or immunodiagnostics (13). Infections with Giardia stimulate humoral immunity that results in self-limiting infection in many animal species (6). Unfortunately, it may take several months for the host to produce protective antibodies that can eliminate the parasite. Chemotherapy may be highly effective in eliminating infection (4). TWo benzimidazole drugs, albendazole and mebendazole, have been suggested to exhibit clinical efficacy against human giardiosis (14, 15, 16, 17). Although the claims for mebendazole were disputed (18,19, 20), in vitro studies have shown that albendazole, mebenda-zole and fenbendazole are much more effective against Giardia trophozoites than metronidazole, tinidazole, or quinacrin (21, 22, 23, 24, 25). Benz-imidazoles are well known as inhibitors of the polymerization of tubulin to microtubules. Because microtubules are major components of the four pairs of flagella, the median body, and the ventral disk of Giardia trophozoites, it is likely that these drugs exert their activities against Giardia through the inhibition of their attachment to the intestinal mucosa (24, 26, 25).
In this report, the efficacy of albendazole against Giardia infection of pigs is presented.
Materials and methods
Pigs
TWo sows of the Krsko polje breed, two years old, one pregnant, were taken to the stables of the Institute for health care of pigs for wintering from the Centre for sustainable recultivation, Vremscica where pigs are kept outdoors.
The Krsko polje or "black belted" pig is the only Slovenian autochthonous breed of pig. It is an extensive breed, whose characteristics are resistance, good adaptability to poor rearing and feeding conditions, and excellent meat quality. These pigs have relatively large litters but too many stillborns and additional losses before the piglets are weaned (27).
Pigs, housing
The non-pregnant sow was housed in a box, size 1.2 x 4.2m with concrete floor. The pregnant sow was housed in the farrowing box size 1.3 x 3m,
with a concrete floor. The sow farrowed four piglets 14 days after transportation. The piglets were not weaned during the wintering. The temperature in the pen was between 16°C and 19°C, the farrowing box also had a heating lamp. Boxes were cleaned twice daily when pigs were fed. The piglets were fed with Pu-starter (Jata Emona, Ljubljana, Slovenia) ad libitum and they suckled. Sows were feed with S-doj (Jata Emona, Ljubljana, Slovenia) ad libitum. Water access from public water supply was ad libitum on water nipples in both boxes.
The pigs were reared according to the Council directive for minimum standards for the protection of pigs (2008/120/EC).
Clinical signs
While the sows were stabled they were examined clinically (rectal temperature, respiratory rate, faeces consistency). All tested parameters were in normal ranges. At the age of two months, diarrhoea was noted in all four piglets and also in the two sows. The diarrhoea was grey-green or grey-yellow in colour, and the backsides of pigs were smeared.
Collection of samples
Rectal smears and faecal samples were taken from both sows and piglets at the beginning of the diarrhoea, and 7, 10, and 11 days following the treatment. Rectal smears were sent to the laboratory for bacteriological examination. Faecal samples were sent for parasitological examination.
Bacteriological examination
Samples were inoculated on nutrient agar (Oxoid) supplemented with 5% of sheep blood and Drigalski agar (Oxoid) and incubated at 37°C for 24 hours.
For detection of Salmonella, samples were enriched in buffered peptone water (Biolife) at 37° C for 18 h and Rappaport Vassiliadis broth (Merck) at 41,5° C for 24 h, then subcultured onto solid selective media XLD agar (Biolife) and Rambach agar (Merck). Both were incubated at 37° C for 24 h.
Parasitological examination
SAF method for concentrating protozoa
2 to 5 g of faeces was diluted with 10 ml SAF (Sodium acetate 1.5 g, acetic acid, glacial 2.0 ml,
Effective treatment of giardiosis in pigs by albendazole
53
formaldehyde, 37 to 40% solution 4.0 ml, distilled water 92.0 ml). After homogenisation, the mixture was stood for 30 minutes, then passed through a filter (gauze) and centrifuged for 1 minute at 2000 rpm. The supernatant was removed, leaving 1 ml of sediment. 7 ml of physiological solution (9.0 g NaCl in 1000 ml solution) and 2 ml of ether were added to the sediment and centrifuged 3 minute at 2000 rpm. The supernatant was removed and a few drops from the 1 ml of sediment used for microscopic observation at 400-x magnification.
Direct immunofluorescence test (DIF)
The MerilFluor® Criptosporidium/Giardia, Direct Immunofluorescent Detection Procedure, Meridian, Bioscience, Inc. was used. Mixed concentrated sediment (10 |L) was smeared, with the transfer loop, in the well of the slide. 1 drop of detection reagent and 1 drop of counter stain were added, mixed gently, and incubated in a moist chamber. The slide well was rinsed with kit wash buffer. 1 drop of mounting medium was placed to close the cover slip. Wells were examined by fluorescence microscopy with FITC excitation/emission filters.
Therapy
Monil®5% (Pliva d.d., Croatia) containing 50 mg albendazole in 1 ml was used for treatment. The pigs were treated with 20 mg/kg albendazole, per os, once daily, for 3 days.
After the therapy faecal samples were sent for parasitological examination (SAF, DIF), to ascertain whether the therapy was effective.
Results
After 3 days of therapy the diarrhoea stopped and the pigs were clinically healthy. They remained without diarrhoea for the next four months till the end of wintering.
No pathogenic bacteria were isolated at the first bacteriological examination on nutrient agar supplemented with 5% of sheep blood and Drigalski agar, in enriched in buffered peptone water and Rappaport Vassiliadis broth, and then subcultured onto solid selective media XLD agar and Rambach agar.
The second examination confirmed the presence of Campylobacter coli.
Table 1: Results of the first SAF and DIF examination
	SAF	DIF	
	Giardia sp.	Cryptosporidium sp.	Giardia sp.
Sow 1	pos.	neg.	pos.
Piglet 1	neg.	neg.	neg.
Piglet 2	neg.	neg.	pos.
Piglet 3	pos.	neg.	pos.
Piglet 4	neg.	neg.	neg.
Sow 2	neg.	neg.	neg.
Giardia sp. was detected by microscopy examination (SAF, DIF) at first during diarrhoea.
Seven days, ten days and eleven days after the treatment all tested samples were negative by SAF and DIF examinations.
Discussion
Giardia spp. and Cryptosporidium spp. are commonly identified intestinal pathogens in humans and animals, causing asymptomatic to severe intestinal infections, depending on various factors (8).
Diagnosis of intestinal parasitic disease is confirmed by recovery and identification of protozoan cysts in the parasitological laboratory. The sodium-acetate acetic acid-formalin (SAF) fixative was used as a multipurpose fixative-preservative, permitting the recovery and identification of intestinal parasites for all diagnostic steps (28).
The most widely used assays for detecting Gia-rdia and Cryptosporidium are the direct immun-ofluorescence (DIF) assays (28). The sensitivity of the most commonly used commercial DIF test, the MerilFluor® Criptosporidium/Giardia, has been reported to be 95 to 100%, with a specificity of 99.8 to 100%, for both Giardia and Cryptosporidium (28, 29, 30, 31, 32). This test has a sensitivity greater than the traditional examination of permanent smears for Giardia (33) and equal to or greater than that of the traditional examination of permanent smears prepared from concentrated stool specimens for Cryptosporidium (30).
Giardiasis is currently treated with metronida-zole, tinidazole and quinacrine (34). The adverse effects and treatment failures of some of the currently recommended drugs (particularly 5-nitroim-idazoles) for giardia infection have raised the need
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for alternative anti-giardia agents. A recent study suggested that two benzimidazole drugs, alben-dazole and mebendazole, are clinically effective against human giardiosis (35). In vitro, albendazole inhibits the growth of trophozoites of Giardia and their adhesion to cultured intestinal epithelial cells and disturbs the activity of microtubules and mi-croribbons in the trophozoite's adhesive disk (36). Albendazole was successfully used for treating clinical giardiosis in dogs at a dosage of 25mg/kg per os, twice daily, for two days. It was found to be highly effective, 50x more so than metranidazole (37). In productive animal species, albendazol is highly effective for eliminating Giardia in house and range calves (35). In pigs, they treated giardiosis with 30 mg/kg of metronidazole, per os, once per day, for 3-5 days. All the contact piglets in the same pen received the same treatment. As a result, the clinical incidence decreased rapidly to 0, 8-1, 3% (38). Under the council Regulation (EEC) 2377/90 (Appendix IV) of 26 June 1990, the use of metronidazole as a veterinary medicine has been prohibited (39) and, for this reason, Albendazole was used for treating our pigs. Treatment with 20 mg/kg albendazole, per os, once daily, for 3 days stopped the diarrhoea in all treated pigs, which remained without diarrhoea for the next four months (all the period of wintering). Three weeks after the therapy, Giardia was absent from the faeces (negative SAF and DIF). This is the first use of albendazole to be reported for therapy of clinical giardiosis.
Acknowledgment
The authors thank Professor Roger Pain for review of English.
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UČINKOVITOST ALBENDAZOLA PRI ZDRAVLJENJU GIARDIOZE PRI PRAŠIČIH
M. Štukelj, Z. Valenčak, A. Vergles-Rataj, J. Posedi
Povzetek: Giardioza je zajedavska bolezen, ki se kaže v obliki kronične driske in je razširjena tako pri ljudeh kot pri domačih in divjih živalih po vsem svetu. V času prezimovanja prašičev krškopoljske pasme v hlevih Inštituta za zdravstveno varstvo prašičev smo pri dveh plemenskih svinjah in štirih pitancih opazili drisko. Za mikrobiološke preiskave smo uporabili rektalne brise. Vzorci blata so bili pregledana tudi na prisotnost zajedavcev z metodo koncentracije protozojev (SAF) in DIF (direct immuno fluorescent test). Rezultati prvih bakterioloških preiskav niso potrdili prisotnosti nobenih patogenih bakterij, ob ponovni bakteriološki preiskavi pa je bila ugotovljena prisotnost Campylobacter coli. Zajedavca Giardia sp. smo potrdili z metodo SAF in DIF ob prvi preiskavi, ko je bila prisotna tudi driska. Prašiče smo zdravili z zdravilom Monil®5% (Pliva d.d., Hrvaška) pri čemer je znašal odmerek albendazola 20 mg/kg. Zdravilo je bilo aplicirano per os, enkrat dnevno tri dni zapored. Po tridnevnem zdravljenju driska ni bila več klinično zaznavna in pri zdravljenih prašičih nismo zaznali nikakršnih kliničnih odstopanj. Pri zdrvaljenih prašičih nismo opazili driske še nadaljnje štiri mesece. Sedmi, deseti in enajsti dan po zdravljenju so bili vsi preiskani vzorci blata z metodo SAF in DIF negativni.
Ključne besede: prašič; Giardia; zdravljenje; albendazole