Isolation of Besnoitia besnoiti from infected cattle in Portugal

Abstract

Besnoitia besnoiti, an obligate intracellular protozoan parasite belonging to the phylum apicomplexa, is the causative agent of bovine besnoitiosis. Besnoitiosis is responsible for significant losses in the cattle industry of Africa and Mediterranean countries due to the high morbidity rate, abortion and infertility in males. The acute stage of disease is associated with the proliferative forms (tachyzoites) and is characterized by fever, whimpery, general weakness and swelling of the superficial lymph nodes. During the following chronic stage, a huge number of cysts are formed mainly in the subcutaneous tissues. This process is non-reversible, and chronic besnoitiosis is characterized by hyper-sclerodermia, hyperkeratosis, alopecia and, in bulls, atrophy, sclerosis and focal necrosis that cause irreversible lesions in the testis. In this paper we report on the identification of large cysts in the skin of a cow and a bull in Portugal, which presented loss of hair and enlargement and pachydermis all over the body. The observation of a two-layered cyst wall within the host cell, the encapsulation of the host cell by a large outer cyst wall, and the subcutaneous localization of the cysts within the host, were characteristic for B. besnoiti. The parasites were isolated from the infected animals and successfully propagated in Vero cells without prior passages in laboratory animals. Morphological characterization of B. besnoiti tachyzoites and the amplification of the 149 bp segment from the internal transcribed spacer 1 (ITS1), aided with specific primers, confirmed the identification of B. besnoiti.

First in vitro isolation of Besnoitia besnoiti from chronically infected cattle in Germany

“…Effective parasite growth with an easily visible parasite-induced cytopathic effect in cell culture was only reached after 30 and 40 days p.i. for both the Portuguese isolates, Bb1Evora03 and Bb2Evora03, respectively (Cortes et al, 2006). Recently, it was shown that B. besnoiti growed rapidly in MARC-145 cells.…”

“…Such a characterization can only be achieved if many isolates from different countries become available for analysis. There are reports on the in vitro isolation of B. besnoiti in cell cultures, but only few permanently growing isolates Q2 are existing worldwide (Bigalke et al, 1967;Bigalke, 1968;Neuman, 1974;Gö bel et al, 1985;Shkap et al, 1987;Cortes et al, 2006;Ferná ndez-García et al, in press). For isolation purposes, a variety of cell lines have been tested.…”

“…For isolation purposes, a variety of cell lines have been tested. Vero cells are often used for the in vitro isolation and the long-term cultivation of B. besnoiti (Bigalke et al, 1974;Neuman, 1974;Gö bel et al, 1985;Shkap et al, 1987;Cortes et al, 2006). In the case reported here, however, the isolation and long-term cultivation of B. besnoiti from a total number of 13 tissue samples from eight cattle and from two of three peritoneal washings of infected GKO mice failed in Vero cells in all but one case, although numerous viable cystozoites (Table 2) or tachyzoites were visible in many inocula.…”

“…In the case reported here, however, the isolation and long-term cultivation of B. besnoiti from a total number of 13 tissue samples from eight cattle and from two of three peritoneal washings of infected GKO mice failed in Vero cells in all but one case, although numerous viable cystozoites (Table 2) or tachyzoites were visible in many inocula. Cortes et al (2006) also reported a delayed adaptation of their isolates to Vero cells when they used cystozoites to infect cell cultures. Effective parasite growth with an easily visible parasite-induced cytopathic effect in cell culture was only reached after 30 and 40 days p.i.…”

“…For IFAT, purified B. besnoiti Bb1Evora03 tachyzoites cultivated in Vero cells (Cortes et al, 2006) were used as antigen. The assay was performed essentially as described for N. caninum (Schares et al, 1998).…”