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.

Stray cats live in high-density colonies in urban areas and pose a health hazard to household cats and humans. In Portugal, information on the parasitic fauna of stray cats is limited and relies mostly on results from faecal analysis. The present survey aimed to determine the prevalence, diversity and intensity of parasites in stray cats from the urban area of Lisbon by means of parasitological necropsy. Internal organs were collected from 162 cats captured in different areas of the city and systematically subjected to parasitological dissection. Helminths were identified by macro- and microscopic examination and protozoa by faecal floatation and sedimentation techniques. The overall prevalence of parasites was 90.7% (95% confidence interval (CI): 85.3-94.6%). A total of 12 parasite species was recorded: Cystoisospora felis (14.2%), Cystoisospora rivolta (46.3%), Sarcocystis sp. (1.2%), Ancylostoma tubaeforme (19.1%), Toxocara cati (38.3%), Ollulanus tricuspis (30.9%), Aelurostrongylus abstrusus (12.4%), Eucoleus aerophilus (0.6%), Taenia taeniaeformis (3.1%), Dipylidium caninum (53.1%), Joyeuxiella pasqualei (15.4%) and Diplopylidium nölleri (3.7%). Overall mean species richness was 2.36 ±  1.52. Helminth mean intensity was highest for O. tricuspis (285.8), followed by D. caninum (42.4), J. pasqualei (14.4), A. tubaeforme (8.1) and T. cati (5.9). The prevalence and variety of parasites found in our sampling are substantially higher than the numbers previously reported in Portugal. Some of the parasites, including T. cati and A. tubaeforme, are zoonotic, which emphasizes the need for parasite control strategies based on demographic containment of stray cat populations in urban areas to promote public health protection.

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Bovine besnoitiosis, caused by the apicomplexan parasite Besnoitia besnoiti is considered an emergent disease in Europe. This study aimed to determine the prevalence and geographic distribution of B. besnoiti in cattle herds in continental Portugal and to identify potential spatial clustering of infection. A stratified two-stage cross-sectional serological survey was carried out between March 2012 and May 2013 with the five administrative NUTS II regions, Norte, Centro, Lisboa, Alentejo, and Algarve, as the stratification level. Sera from 391 herds in 220 parishes and 83 municipalities were analyzed by a serial testing strategy, with the modified agglutination test (B-MAT) as the first screening assay and the immunofluorescent antibody test (IFAT) as the confirmatory test. Within-herd prevalence of positive herds varied between 0.7 and 72.4% and was ≥10.3% in half of the infected herds. Using a Bayesian approach, the true prevalence of B. besnoiti in cattle herds was determined to be 5.1% (confidence interval (CI), 3.1-7.8%) and the mean within-herd prevalence of positive herds was 33.0% (CI, 20.3-46.0%). The sensitivity and specificity of the B-MAT were estimated to be 96.9% (CI, 93.7-98.8 %) and 99.7% (CI, 99.6-99.8%), whereas those of the IFAT were 89.6% (CI, 86.0-92.5%) and 99.7% (CI, 98.5-99.9%), respectively. Spatial scan statistics analysis identified one spatial cluster covering the majority of the Alentejo region. Seropositive herds were detected for the first time outside Alentejo, in the region Centro and in the northeast of Portugal. Further epidemiological research is needed to identify eco-biological factors, which could explain the geographic clustering of B. besnoiti in Portugal.

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