Changes in haematological parameters in wild ruminants experimentally infected with Haemonchus contortus

Parasitic infections in wild ruminants may cause high morbidity, and in the case of heavy infections, immunosuppression and poor body mass condition, they may even lead to death of the animal. The negative effect of parasitic infection depends on the host’s nutritional status, gender – males have a weaker immune system than females, age – young and elderly individuals are more susceptible to infection than adults (Jégo et al., 2014). Most studies have focused on the impact of parasites on the nutritional status of wild ruminants, but this has been confirmed as a nonspecific and slow indicator of parasitic infection (Irvine et al., 2006; Jégo et al., 2014). More specific and faster diagnostic indicator of the presence of parasitic infection may be host haematology parameters (Beldomenico & Begon, 2010). Changes in the health status of wild ruminants may also be reflected in haematological parameters (Ciberej et al., 2007; Mašek et al., 2009). Monitoring their dynamics contributes greatly to diagnosis, differential diagnosis, assessment of the effect of therapy as well as prediction of a course of diseases in animals. However, blood analysis may also indicate an accurate status of nutrition, trauma, and environmental stressors (Peréz et al., 2003). Haemonchus contortus is one of the hematophagous species that can cause haemorrhagic anaemia, especially in young and immunosuppressed animals. If the infection persists for a long time (chronic blood loss), it can lead to a rapid depletion of the erytrocyte stocks in the spleen and bone marrow and anaemia begins to develop, characterized by a decrease in all red blood component parameters (Doubek et al., 2003; Kolodziej-Sobocińska et al., 2016). Changes observed in the white blood cells in wild ruminants may result from an organism’s response to stress (stress leukocytosis) and inflammation as a result of parasitic and allergic reactions (Ciberej et al., 2007). The values of haematological parameters of both the red and white blood cells are dependent on many factors such as breed, age, sex, pregnancy and season (Küker et al., 2015).

Vengušt et al. (2006) and Casas-Diaz et al. (2008) also reported that the method of capture and fixation of wild ruminants can greatly affect the blood parameter values. Vengušt et al. (2006) in fallow deer recorded higher values ​in total erythrocyte count, haemoglobin and haematocrit in subjects immobilized by chemical methods compared to animals immobilized by physical methods. The aim of this study was to describe changes in total number of erythrocytes and leukocytes, haematocrit and haemoglobin and leukogram values ​(differential cell blood counts) in wild ruminants (mouflons, fallow deer, roe deer) experimentally infected with the model parasite H. contortus.

Three species of wild ruminants – six European mouflons (Ovis musimon), six fallow deer (Dama dama) and six roe deer (Capreolus capreolus) with an average age of 1.5 years were experimentally infected with the model parasite H. contortus. Each animal was housed individually, fed with meadow hay and a commercial concentrate with free access to water, mineral and salt licks.

All animals were treated per os with albendazole (10 mg / kg body weight, ALDIFAL 2.5 % susp. a.u.v., MEVAK, Slovakia) prior to the experiment for the purpose of utilising parasite free animals. Faecal samples from all experimental animals were examined and negative coprological findings were confirmed before the experiment. After a week, each animal was infected per os with 8000 L3 larvae of isolate of H. contortus (MHco4). MHco4 is a multi-resistant (ivermectin, BZ, rafoxanide, closantel) field isolate from South Africa (Van Wyk & Malan, 1988). The dynamics of haematological parameter values ​in all animals were monitored for 99 days. Blood samplings were performed on day (D) 0, day of infection, D16, D37, D58 and D77 days post infection (p.i.). On D77 p.i., levamizole was orally administered to all experimental animals with Ripercol Drench (Elanco, Germany) at a dose of 2 ml / 10 kg body weight. Blood was again collected on D99 p.i. of the experiment (D21 after treatment). Each animal was immobilized prior to the blood collection by applying Hellabrun mixture (125 mg xylazine hydrochloride 100 mg ketamine hydrochloride / 1 ml) at the following doses: mouflon 1.5 – 2.0 ml, fallow deer 2.0 – 2.5 ml and roe deer 0.2 – 0.3 ml. Blood was collected from the jugular vein into an ethylene-diamine-tetraacetic acid anticoagulant tube (EDTA) and processed on an IDEXX ProCyte DxTH (IDEXX Laboratory, Cymedica). The mean and ± statistical deviations were calculated using the Excel 2010 statistical program (Microsoft Int.).

Individual counts of eggs per gram (EPG) for each experimental animal were determined by a modified McMaster technique with a sensitivity of 50 EPG (Coles et al., 1992; 2004). Three grams of faeces from each animal were mixed with 42 ml of water and passed through a sieve. The filtrate was centrifuged at 605 g for 2 min. The sediment was mixed with a sugar solution with specific gravity 1.28 and centrifuged under the same conditions. One millilitre of the mixture was then transferred to McMaster chambers.

Tukey-Kramer test (one-way ANOVA) was used to evaluate the haematological parameters. A significance level of less than 5 % (P<0.05) was considered statistically significant.

Animal use and study design were approved by the Ethics Committee of the Institute of Parasitology of the Slovak Academy of Sciences in accordance with the national legislation in Slovakia − Animal Welfare Act No. 3/2009.

It is well known that many species of gastrointestinal nematodes are common to domestic and wild ruminants due to their frequent common grazing on pastures. Little is known about the haematological profile of wild ruminants infected with H. contortus, one of the most pathogenic hematophagous gastrointestinal nematodes occurring in domestic and wild ruminants. Ferté et al. (2000) summarised and compared the prevalence of H. contortus in three species of wild ruminants (red, roe, and fallow deer) from surveys carried out across Europe. A total of 36 surveys in 17 European countries recorded the incidence of H. contortus in roe deer, with prevalence ranging from 0.3 to 85 %. H. contortus was found in red deer in nine countries, with prevalence between 5 and 25 %, and in fallow deer in four countries (4 – 7 %) (Ferté et al., 2000). Cerutti et al. (2010) confirmed the low host specificity of H. contortus by finding it in common populations of roe deer, chamois, alpine ibex, and domestic goats and sheep in various alpine areas. Mašek et al. (2009) noted the differences in blood analyses under physiological conditions between mouflon and sheep, Ciberej et al. (2007) described haematological profile of mouflons; however no data compares changes in blood parameters between three different species of wild ruminants during infection with hematophagous parasites.

The first sign of the H. contortus infection is a decrease in values in red blood cell count. The red and white blood count may interpret differences related to nutritional status, disease condition and environmental impact. The progression and prognosis of the disease depend on several factors: parasitic infection intensity, age, breeds and host health. The main pathogenic mechanism of H. contortus parasite lies in a direct damage to the gastric mucosa and subsequent blood sucking (Angulo-Cubillan et al., 2007). Haemorrhagic anaemia is a result of a strong infection characterized by pale gingiva and conjunctival sac. There is also a decrease in total erythrocyte count, hypoalbuminemia and total protein in haematological parameters (Alvarez et al., 2000). Lavin et al. (1997) described similar changes in red blood counts in wild Spanish goats with present H. contortus in the abomasum. Macrocytic hypochromic anaemia, anisocytosis, poikilocytosis and occasionally the Howell-Jolly body were diagnosed in the erythrocytes. The authors assumed that anaemia was caused by acute bleeding. Kolodziej-Sobocińska et al. (2016) reported similar changes in European bison infected with Ashworthius siedmi (Trichostrongylidae). Erythrocyte loss can be balanced by increased reticulocyte production, but if losses are too high anaemia occurs. Our results indicated that mild anaemia occurred only in mouflons and roe deer. The level of decline in red blood cell parameters correlated with increasing intensity of parasitic infection expressed in EPG values. After the deworming, the red blood cell values ​ were adjusted, confirming that irreversible changes did not occur in the early therapy of the disease in the blood and the organism was able to regenerate relatively quickly. Post-haemorrhagic anaemia in experimental wild ruminants was reversible as erythrocyte reserves in the bone marrow and spleen were sufficient to compensate for their loss in peripheral blood. Higher values of red blood cell parameters were noted in fallow deer, probably due to a lower sensitivity to infection with H. contortus. It can be assumed that the indicated parasitic infection rate in fallow deer was not high enough and long-term to cause a decrease in these values, respectively. Parasitic infection of animals in poor nutritional and condition status might also be of higher intensity because parasites would encounter less opposition to their survival. It is generally acknowledged that discrepancy in susceptibility is one reason why parasites tend to be concentrated in individual host (Beldomenico & Begon, 2009). Peinado et al. (1999) confirmed that the physiological values ​of total erythrocytes, haematocrit and haemoglobin are higher in young fallow deer. Similarly, Vengušt et al. (2006) reported that the total erythrocyte count, haemoglobin and haematocrit are also affected by the trapping method. Haematological values in fallow deer immobilized by chemical methods ​ were higher than in fallow deer immobilized by physical methods. The effect of catecholamine release is expected to cause spleen contraction and subsequent release of erythrocytes into peripheral blood. The results of haematological parameters may also be influenced by season, age, gender and pregnancy (Küker et al., 2015). In mouflons, leukopenia was recorded with neutrophilia and lymphopenia, eosinophilia and monocytosis, indicative of intense haemonchosis accompanied by post-haemorrhagic anaemia and suspected local acute mucosa inflammation in abomasum. Kraft & Dürr (2005) described pathological neutrophils with a regenerative left shift even during posthemorrhagic anaemia. In the fallow deer and roe deer, leukocytosis with neutropenia and lymphocytosis was recorded. Haematological parameters for roe deer and fallow deer did not indicate an acute inflammation associated with low parasitic infection intensity. Distinct eosinophilia accompanied by basophilia was recorded in the roe deer after D16 p.i. It can be assumed that an allergic reaction of the organism to the interaction of the parasitic infection and the immobilizing agent used has occurred. In comparison to the study of Peinado et al. (1999) our ​eosinophil granulocyte baseline values in roe deer group were increased. Eosinophilia can be caused by the chemicals used to immobilize experimentally infected wild ruminants. Similar changes in the white blood count in wild ruminants which can result from an organism’s response to local or total inflammation as a result of parasitic and allergic reactions were reported by Ciberej et al. (2007).

Our results indicated that changes in both red and white blood counts observed in infected wild ruminants were temporary. The degree of damage caused by H. contortus was dependent on the number of parasites present and wild ruminant’s species sensitivity. Changes in the red blood cell indicated a development of haemorrhagic anaemia. Changes in the white blood count could be attributed to the intensity of infection and the animal’s response to pathogenesis of H. contortus and its subsequent local inflammatory response with the presence of haemorrhages. These changes would be more pronounced in case of a severe parasitic infection, poor body condition or in immunosuppressed hosts.