Exploring the life cycle of Besnoitia besnoiti—Experimental infection of putative definitive and intermediate host species

Abstract

The biology of Besnoitia besnoiti, the cause of bovine besnoitiosis, is poorly understood. Its definitive host is unknown, and information on potential intermediate hosts is scarce. In order to investigate potential definitive and intermediate hosts for European isolates of B. besnoiti, domestic dogs, cats, rabbits, guinea pigs (Cavia porcellus), gerbils (Meriones unguiculatus), common voles (Microtus arvalis) and NMRI-mice were inoculated with B. besnoiti isolated from naturally infected German cattle. Dogs and cats were fed 5 × 10⁶ B. besnoiti tachyzoites (isolate Bb-GER1), or tissue cysts containing at least 2 × 10⁷ B. besnoiti bradyzoites obtained from the skin of a naturally infected Limousin cow from the same herd where strain Bb-GER1 was isolated. Rodents and rabbits were subcutaneously inoculated with either 5 × 10⁵ Bb-GER1 tachyzoites or 5 × 10⁵ bradyzoites. Groups of 2–4 non-inoculated animals of each species were monitored as negative controls. Feces from all dogs and cats were daily examined by a sedimentation–flotation technique for at least 11 weeks after inoculation but no B. besnoiti oocysts were identified. Cats fed tachyzoites and dogs did not seroconvert, but specific antibodies to B. besnoiti tachyzoites were detected by IFAT (titer ≥ 100) in 2 out of 3 cats fed tissue cysts since 5–7 weeks post infection. By immunoblot, these two cats exhibited a reaction pattern against tachyzoite antigens similar to that observed in naturally infected cattle. Antibodies against B. besnoiti tachyzoites were detected in all inoculated rodent species and rabbits by both, IFAT and immunoblot since 3 weeks post-inoculation. Rabbits and rodents, subcutaneously inoculated with same doses of inactivated bradyzoites remained serologically negative (IFAT titer < 50). Clinical signs observed in the inoculated rabbits included fever, serous conjunctivitis and transient swelling of the testes. No clinical abnormalities were noticed in the other tested animal species. Voles developed pneumonia as observed by histological examination. B. besnoiti-DNA was detected by PCR in blood from rabbits, gerbils and voles at 9 days post-infection, and in skin, heart, lung, striated muscle and kidney tissues from voles at 19–21 weeks post-infection.

Domestic dogs and cats could not be shown to be definitive hosts of B. besnoiti, but cats seroconverted after feeding on B. besnoiti tissue cysts indicating that B. besnoiti stages had invaded the cats’ tissues. The molecular and serological results from this study indicate that European B. besnoiti isolates may infect cats, rabbits, guinea pigs, gerbils, mice and voles; however a persistence of the parasite could be demonstrated only in voles.

Introduction

Besnoitia besnoiti (Protozoa, Apicomplexa) is a cyst-forming coccidian parasite, closely related to Toxoplasma gondii and Neospora caninum. It is the cause of bovine besnoitiosis, an economically important disease in some countries of Africa, Asia and Europe, which may severely affect body condition in cattle and lead to temporary or irreversible infertility in bulls. The disease is characterized by fever, nasal and ocular discharge, salivation, stiff gait, orchitis and subcutaneous edema during the acute stage of the infection. In the chronic stage, affected animals may show thickening, hardening and folding of the skin, hyperkeratosis and alopecia as well as atrophy of testes. Nevertheless, the infection usually remains subclinical. Characteristic thick-walled tissue cysts containing many thousands of bradyzoites are mainly found in cutis, subcutis, conjunctiva, genital and respiratory mucosa, fasciae and blood vessels, and may be macroscopically evident due to their size of up to 600 μm in diameter (Bigalke, 1968, Eckert et al., 2008, Jacquiet et al., 2010, Majzoub et al., 2010, Stöber, 2006).

The European Food Safety Authority (EFSA) reported recently that bovine besnoitiosis is re-emerging in Europe (http://www.efsa.europa.eu/en/scdocs/scdoc/1499.htm). In the last years spread of the disease was observed, accounting for outbreaks also in countries traditionally free of bovine besnoitiosis, like Germany and Italy (Gollnick et al., 2010, Schares et al., 2009). Although the trade of animals from endemic European regions was most probably involved in the spread of the disease, there are still many aspects of the epidemiology of bovine besnoitiosis that are poorly understood. It has been suspected that B. besnoiti, like other cyst-forming coccidia, has an indirect life cycle, with a carnivore as definitive host and cattle and wild bovids (i.e. antelopes) representing intermediate hosts. However, the complete life cycle of B. besnoiti is still unknown and a definitive host, which sheds oocysts after ingestion of infected tissues, has not been identified yet (Diesing et al., 1988, Rommel, 1975). Of the named Besnoitia species, life cycles of only B. darlingi, B. wallacei, B. oryctofelisi and B. neotomofelis are known, and domestic cats are their definitive hosts (Dubey et al., 2003a, Dubey and Yabsley, 2010, Frenkel, 1977, Smith and Frenkel, 1977). For the remaining Besnoitia species (B. bennetti, B. jellisoni, B. caprae, B. tarandi and B. akodoni) including B. besnoiti, the definitive host is still unknown (Dubey et al., 2003a, Dubey et al., 2003b). The only experimentally confirmed modes of transmission among cattle so far are mechanically through blood-sucking insects and iatrogenically through hypodermic needles (Bigalke, 1968); also the transmission through direct contact seems to be possible. It is not known if there are differences in the host specificity among B. besnoiti isolates from different parts of the world, or if other intermediate hosts than bovids, are involved in the epidemiology of bovine besnoitiosis. In Europe, thus far B. besnoiti infections were only confirmed in cattle; however, the EFSA recently stated that wild ruminants and rodents should not be disregarded as reservoirs of the parasite (http://www.efsa.europa.eu/en/scdocs/scdoc/1499.htm).

The aims of the present study were to determine if domestic dogs and cats may act as definitive hosts of B. besnoiti, and to evaluate the susceptibility of rabbits, gerbils (Meriones unguiculatus), common voles (Microtus arvalis), NMRI-mice, and guinea pigs (Cavia porcellus) to European B. besnoiti isolates obtained from natural cases of bovine besnoitiosis in Germany, using in vitro-cultured B. besnoiti tachyzoites and tissue cysts or bradyzoites isolated from cattle by parasitological, serological, histopathological and molecular methods.