Parasitol Res (2007) 101:16571661 DOI 10.

1007/s00436-007-0709-2

ORIGINAL PAPER

Hematological and biochemical changes in organically farmed sheep naturally infected with Fasciola hepatica
K. Matanovi & K. Severin & F. Martinkovi & M. impraga & Z. Janicki & J. Barii

Received: 10 July 2007 / Accepted: 30 July 2007 / Published online: 13 August 2007 # Springer-Verlag 2007

Abstract A naturally occurring outbreak of fasciolosis in a group of 20 Merinolandschaf (German Merino) sheep was studied. Hematological and blood biochemical values in sheep spontaneously infected with liver fluke Fasciola hepatica were compared with equivalent values in 20 parasite-free sheep from organically farmed flock. Investigated animals were kept in outdoor system, on pastures covered with swamps, which remain flooded after rainy season. Significantly lower red blood cell (RBC) count, lymphocytes, hemoglobin (Hb), packed cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine, and albumin were recorded in sheep from the infected herd, whereas white blood cell (WBC) count, eosinophil, segmented and band neutrophil count, mean corpuscular volume (MCV), gamma-glutamyl transferase (GGT), concentrations of glucose, and globulins were significantly higher than in the parasite free herd. No significant correlation between the investigated blood parameters and the number of F. hepatica eggs in the feces was
K. Matanovi (*) : M. impraga Department of Physiology and Radiobiology, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia e-mail: kmatanov@vef.hr K. Severin : Z. Janicki : J. Barii Department of Game Biology, Pathology and Breeding, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia F. Martinkovi Department of Parasitology and Parasitic Diseases with Clinic, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia

detected. This study shows that hematological and biochemical values can be useful in early diagnosis and prognosis of sheep fasciolosis.

Introduction Fasciolosis is a common disease of domestic ruminants with important implications for animal health and welfare and farming economics. In sheep production, the disease causes severe losses because of reduced growth and productivity, immune suppression, and death of heavily infected animals (Boray 1985, Mulcahy et al. 1999). In temperate climates, it is related to common liver fluke (Fasciola hepatica) and is most prevalent in swamp areas and pastures that remain flooded during the rainy season (Haroun et al. 1986). Control strategies based on a routine anthelmintic treatments and soil drainage are routinely used in conventional farming systems, but are contrary to the basic guidelines of rapidly growing organic farming: maintaining soil fertility, environmental protection, animal welfare, and the production of good quality animal products based on nonchemical prevention of diseases (Cabaret et al. 2002). Parasite control procedures for gastrointestinal nematodes based on grazing management can be only partially used to control fasciolosis in organic sheep flocks so the best way is to evade infested pastures. Chronic fasciolosis is commonly detected by clinical diagnosis and confirmed by positive fecal egg counts along with elevated level of gamma-glutamyl transferase. During the acute fasciolosis, diagnosis is made by measurement of serum activities of hepatic enzymes that escape from the damaged hepatic cells. Detection of antibodies to the excretory-secretory antigen by ELISA is widely used for the diagnosis and is positive 24 weeks after infection (Price et al. 1993), however says nothing about

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the severity of infection. The shortage of indirect ELISA is that it demonstrates only the previous exposure to antigen so newer methods for detection of serum circulating antigens have been recently proposed (Paz-Silva et al. 2003). Previous studies explored hematological and biochemical changes during single experimental infections which eliminate all factors except the number of metacercariae. However, natural infections are much more complicated with several dependant factors such as feed deficiencies or concurrent mixed infection with gastrointestinal nematodes. The aim of this study was to compare hematological and biochemical findings between F. hepatica naturally infected and noninfected sheep flocks raised on organic farms in Croatia. We discuss about the usefulness of these findings as diagnostic tools and for the estimation of severity of fasciolosis in affected animals.

concentration (MCHC), and number of platelets (PLT) were calculated using an automated blood cell counter Coulter Act-Diff (Beckman-Coulter, USA) with veterinary software. Differential blood counts were performed on Pappenheim-stained blood smears. Biochemical assays Blood was drawn from jugular vein into serum separating tubes. Sera were frozen in plastic tubes at 20C. Serum activities of aspartate aminotransferase (AST) and gammaglutamyl transferase (GGT) and concentrations of glucose, blood urea nitrogen (BUN), creatinine, total protein (TP), and albumin were measured according to standard procedures using automatic analyzer Tehnicon RA-1000 (Tehnicon Instruments, Tarrytown, NY, USA) with commercial kits Randox (Randox Laboratories LTD, Crumlin, Antrim, UK). Serum enzyme activities were determined at 37C. The concentration of globulins was calculated. Fecal egg counts Fecal samples were collected from the rectum. The number of F. hepatica eggs was determined by flotationcentrifugation method according to modified McMaster technique using ZnSO4 solution (1.18 specific gravity). Necropsy Despite the started treatment, two ewes from the infected herd have died and were submitted to the Department of General Pathology and Pathological Morphology for autopsy. Liver samples were taken from both carcasses, fixed in 10% formalin and routinely processed for histological examination. Statistical analysis Data were analyzed using Statistica 7.1 for Windows (StatSoft, Tulsa, USA). Results are expressed as means SD (standard deviation). Significance of difference between herds was determined by Students t test. Correlation between fecal egg counts (FEC) and hematological and biochemical values was tested using regression analysis Pearsons correlation coefficient. Values of P <0.05 were considered significant.

Materials and methods Animals Two groups of 20 ewes each, of German Merino breed and from 2 to 4 years of age, were included in the study. The first group came from the infected flock which numbered totally to 100 ewes. They were kept on lowland pasture, surrounded with oak trees, which was flooded several times during the late summer/early autumn season, particularly because of poorly maintained water-drainage canals. Pasture had no fencing so wild animals could easily enter and graze. Because of the above facts and extremely rainy conditions, pastures were heavily contaminated with metacercariae which infected ewes. The second group was selected from the noninfected (control) herd of the approximately same size, kept under similar meteorological conditions. Sheep were kept on pasture which was surrounded by swamps but had significant slope which prevented water retention. As a result of continuous grazing, grass was low, allowing easy drying of soil surface and direct sunlight. Both herds were kept according to organic farming principles and without any preventive anthelmintic treatment; however, only the noninfected farm was registered in organic producers register and was under regular control by veterinarians. In the infected herd, neither the health control program nor the preventive parasite control measures were maintained. Hematology Blood samples were taken from the jugular vein into evacuated EDTA tubes and stored at +4C. Samples were analyzed within 12 h. Total white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin

Results Hematology Hematological parameters of F. hepatica infected and control sheep are summarized in Table 1. Significantly

Parasitol Res (2007) 101:16571661 Table 1 Hematological parameters of F. hepatica infected and control sheep (mean SDa) Parameter WBC (109/l) RBC (1012/l) Hb (g/l) PCV (l/l) MCV (fl) MCH (pg) MCHC (g/l) Lymphocytes (109/l) Seg. Neu. (109/l) Band Neu. (109/l) Eosinophils (109/l) Monocytes (109/l) Basophils (109/l) Infected (n =20)b 13.763.85a 3.901.54a 40.3516.63a 0.130.05a 33.943.87a 10.361.09a 305.0014.32a 4.761.37a 7.033.65a 0.200.42a 1.621.37a 0.150.16a 0.000.00a Control (n =20) 10.202.04b 10.830.91b 111.358.22b 0.330.02b 30.751.49b 10.300.39a 334.9515.57b 7.601.87b 2.170.82b 0.000.00b 0.280.25b 0.150.15a 0.000.00a

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Discussion To our knowledge, this is the first time that fasciolosis was compared between organically farmed ewes. Organically farmed sheep are much more sensitive to parasite infections while being kept on pasture without any supplemental feed whereas sheep in conventional systems, fed on diets higher in protein, can boost their immunity to raise the parasite threshold. Consequently, data obtained from experimental infections in conventionally farmed sheep are hardly clinically applicable in those organically farmed. In this study, ewes from both herds were infected with common gastrointestinal nematodes but coprological tests confirmed that FEC was below the economic threshold. In this study, hematological and biochemical parameters in the noninfected control sheep were within the reference ranges (Kramer 2000; Kaneko et al. 1997), except for the concentration of albumin and BUN which were slightly elevated probably as a result of grazing on forage legumes. On the other hand, sheep from the F. hepatica-infected herd showed severe normocytic hypochromic anemia, leukocytosis with neutrophilia, and eosinophilia, elevated serum GGT, hypoalbuminemia and hyperglobulinemia and a lower concentration of creatinine. Our results are in agreement with previous studies about effect of F. hepatica on hematological and biochemical parameters. Sykes et al. (1980) also found significantly lower RBC and hemoglobin together with higher MCV during chronic subclinical fasciolosis in sheep, compared to noninfected sheep. Hawkins (1984) suggested hemoglobin and PCV to be useful in predicting the size of a fluke burden and to indicate the likelihood of death or survival of infected sheep. He has found that the death of infected sheep occurred when their PCV fell to 15%; however, in our work, mean PCV for the infected herd was 13%, while nine ewes had PCV below 10%, yet none of them had died. In spontaneous infections, sheep ingest metacercariae during several days or
Table 2 Biochemical parameters of F. hepatica infected and control sheep (mean SDa) Parameter AST (U/l) GGT (U/l) Glucose (mmol/l) BUN (mmol/l) Creatinine (mol/l) Total protein (g/l) Albumin (g/l) Globulins (g/l) Infected (n =17)b 84.3523.55a 93.2424.81a 4.071.17a 5.181.64a 71.7614.51a 72.0618.24a 17.544.99a 54.5215.40a Control (n =20) 113.0016.67b 53.709.00b 2.860.27b 10.521.03b 134.3511.98b 68.904.62a 38.852.76b 30.055.45b

Values in rows with different superscripts are significantly different (P <0.01). a SD Standard deviation b n Size of sample

lower number of RBC, lymphocytes, Hb, PCV, and MCHC were recorded in sheep from the infected herd whereas WBC, segmented (Seg. Neu.) and band neutrophils, eosinophils, and MCV were significantly higher than in the control herd. There were no significant changes in MCH, monocyte, and basophile count between the infected and noninfected herd.

Biochemical results Biochemical results are presented in Table 2. In sheep sera from the infected herd significantly higher activity of GGT and higher concentrations of glucose and globulins were detected, while serum AST activity, BUN, creatinine, and albumin values were significantly lower than in the noninfected sheep. No significant changes were detected for total protein concentration.

Pathological findings Both carcasses showed pronounced cachexia, marked icterus, and submandibular edema. Macroscopic findings include peritonitis and fibrosis of liver capsule with fibrinous exudate. Histology confirmed changes typical for acute fasciolosis. The bile ducts epithelium was hyperproliferative and immature flukes were found in the lumen. Hepatocytes were necrotic and degenerated, with mixed inflammatory component.

Values in rows with different superscripts are significantly different (P <0.01). a SD Standard deviation b n Size of sample

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weeks so immature flukes do not migrate through the liver at once, but according to the rate of uptake. Our data suggest that sheep can withstand infection with higher number of metacercariae if they were given during a prolonged period of time, probably as a result of induction of defense mechanisms and erythropoiesis. The Hawkinss method of prediction can only be used to evaluate fasciolosis induced by single experimental infections. In the present study, leukocytosis and eosinophilia were similar to those previously reported (Sykes et al. 1980; Sinclair 1967; Zhang et al. 2005). Zhang et al. (2005) observed no significant change in neutrophil counts in experimentally infected sheep whereas our results showed significantly higher neutrophil counts compared to noninfected sheep. Although several studies observed no effect of fasciolosis on feed intake (Akinbamijo et al. 1996) and BUN (Anderson et al. 1977; Sykes et al. 1980), our findings of significantly lower BUN compared with the noninfected sheep indicate a need for protein metabolism estimation during chronic fasciolosis. A significantly decreased creatinine concentration points to muscle loss caused by a deficiency of protein and is valuable in the prognosis of recovery from anemia. Hypoalbuminemia caused by a damage of liver parenchyma, hyperglobulinemia as a result of immune response, and decreased albumin/globulin ratio are commonly find during subacute (Scott et al. 2005) and chronic (Haroun et al. 1986; Sykes et al. 1980) fasciolosis in sheep, goats (Swarup et al. 1986), and cattle (Anderson et al. 1977). Results from our study support those observations and are consistent with pathological findings which may or may not be accompanied with clinical signs of fasciolosis. Significantly increased levels of serum GGT found in our study confirm the presence of flukes in the bile ducts. At the same time the activity of AST was because of a short half-life in the plasma, already within the reference limits for sheep, indicating young flukes have migrated through the liver parenchyma. Gonzalo-Orden et al. (2003) observed that AST activity returned to normal values 11 weeks postinfection while Ferre et al. (1995) reported AST to be significantly elevated even 14 weeks postinfection. On the other hand, serum activity of GGT still remained elevated at 18 weeks postinfection. No significant correlation between the investigated blood parameters and the number of F. hepatica eggs in the feces of infected ewes was detected (data not presented in this study). It can be concluded that serum enzyme activities together with BUN, albumin, and globulin concentration are reliable indicators of the stage and severity of fasciolosis in sheep. The increased serum GGT indicates the presence of flukes in the bile ducts; however, only the finding of eggs in the feces verifies the diagnosis of

fasciolosis. Serological methods based on the detection of antibodies to F. hepatica show only the previous exposure to the parasite so new methods that reveal the presence of serum circulating antigens (Paz-Silva et al. 2003) may be a better choice for demonstrating current fasciolosis, although the correlation between the concentration of antigen in serum and the number of adult flukes was not investigated. However, the estimation of the severity of disease and the prognosis of recovery of affected animals can only be made on the basis of clinical and laboratory data (hematological and biochemical blood parameters) as there is no significant correlation between the number of F. hepatica eggs and hematological and biochemical parameters of blood in naturally infected ewes.
Acknowledgements This work was supported by the projects TP3083 and 0053301 (Ministry of Science, Education and Sport, Croatia) and complies with the current laws for research on animals in Croatia.

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