Giardia duodenalis associated with other gastrointestinal parasites in sheep in the North of the Brazilian state of Parana

The sheep industry, an important economic activity in the Brazilian State of Parana, can be affected by gastrointestinal parasites. This study aimed to evaluate the occurrence of gastrointestinal parasites in sheep raised in the North of said state. Fecal samples were collected from 69 animals and processed through flotation and sedimentation. Parasites were found in 63 (91.3%) animals. The most frequent parasites were strongylid eggs (81.2%), followed by Eimeria sp (42%) and Giardia duodenalis (10.1%). To the best of our knowledge, this is the first report of Giardia duodenalis in sheep raised in the State of Parana. These findings indicate that sanitary management must be improved in order to control parasite infections and increase productivity.


Introduction
Sheep farming is an activity widely exploited in tropical countries, where sheep are raised for their meat, milk and wool (Vieira, 2003). In developing countries, gastrointestinal parasitic diseases are one of the main factors in herd productivity reduction (Amarante et al., 2004;Sweeny et al., 2012).
Sheep are usually raised in small overcrowded areas, which causes intense pasture contamination. Even in extensive breeding, gastrointestinal parasites can become a major problem when they are associated with malnutrition, management errors, and anthelmintic therapy inefficiency (Amarante et al., 2004;Sweeny et al., 2012).
Reduced productivity and mortality are directly related to helminthiasis in sheep (Amarante et al., 2004). The protozoan Giardia duodenalis is relevant in this context. This flagellate has been reported as the cause of diarrhea and decreased weight gain in lambs (Aloisio et al., 2006). In addition, the fact that domestic ruminants and other animals are sources of infection for humans has garnered the scientific community interest in animal giardiasis (Olson, et al., 1995;Feng;Chiao, 2011;Coelho et al., 2017).
To propose adequate control, knowledge of the main gastrointestinal parasites is essential, since the indiscriminate and repetitive use of different chemical treatments results in the selection of resistant parasite populations (Cunha Filho et al. 1998;Melo et al., 2003;Almeida et al., 2010). The objective of this study is twofold: 1) to provide the first report of Giardia duodenalis, and 2) investigate gastrointestinal parasitism by both helminths and protozoa in sheep raised in the North of the Brazilian State of Parana.

Material and methods
The study was conducted in the metropolitan area of Londrina, in the municipalities of Cambé (23º16'33"S, 51º16'42"W) and Ibiporã (23º16'09"S, 51º02'53"W), both located in the north of Paraná State. The animals were Vet The fecal samples were chemically conserved in acetic formaldehyde solution and sent to the laboratory for further processing and analysis. They were examined for cestode, nematoid and intestinal protozoa through centrifugal flotation in zinc sulfate solution with density of 1.180 g/mL (Faust et al, 1938) and spontaneous sedimentation (Hoffmann et al, 1934). Samples were examined in an optical microscope, and the parasites were morphometrically identified. It was not possible to perform fecal culture and oocyst sporulation due to the use of chemical conservation.

Results and discussion
A total of 69 sheep were studied (25 from Farm A, 44 from Farm B): 69.6% (48) Santa Ines, 19.4 % (12) Texel, 13.0% (9) cross-breed (SI and ½ Texel). Ten animals (14.5%) were younger than 6 months old while 59 (85.5%) were adults, i.e., over 6 months old. All animals had good corporal score and normal feces. The sheep industry in Paraná State is concentrated in small areas, with high levels of pasture contamination (Nieto et al., 2003). Grasses of stoloniferous habit are traditionally used in Brazilian pastures for feeding sheep, which form a dense plant mass, preventing the direct incidence of sunlight and forming a microclimate that is favorable for the survival of helminth larvae and the viability of eggs and oocysts. This leads to a high infection rate, as observed in both properties in this study.
Polyparasitism was found in 30 (47.6%) of the positive samples, and the most frequent association involved strongylid eggs and Eimeria sp. oocysts ( Figure 1).   This is the first report to date of natural infection by Giardia duodenalis in sheep in the State of Parana, with a rate of 10.1% positive animals (7/69). This parasite was only found in young animals, which is probably related to their developing immune system and elimination of large numbers of cysts in the feces Herd, 1994;Koudela;Vitovec, 1998).  The presence of domestic fowl and dogs in the farm B may also have contributed to the transmission of parasites, since these animals may be mechanical transmitters of parasitic structures (Traub et al., 2003;Majewska et al., 2009). Besides, dogs are hosts of zoonotic Giardia duodenalis genotypes, acting as a vessel of this protozoan. This fact contributes to environmental contamination, being a risk factor for parasite transmission (Bomfim et al., 2005;Sudré;Couto;Bonfim, 2012;Coelho et al., 2017).
The actual frequency of protozoa such as Giardia and Cryptosporidium is probably under diagnosed in the databases due to the parasitological techniques used in the routine control of small ruminants. Co-diagnostic methods allow greater sensitivity to research these agents in these animals.
In addition, all animals in this study were asymptomatic, which may further contribute to the zoonotic risk of transmission.