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Little information is currently available on the occurrence and genetic diversity of pathogenic and commensal protist species in captive and semi-captive non-human primates (NHP) resident in zoological gardens or sanctuaries in low- and medium-income countries. In this molecular-based study, we prospectively collected individual faecal samples from apparently healthy NHP at the Abidjan Zoological Garden (AZG) in Côte d’Ivoire, the Tacugama Sanctuary (TS) in Sierra Leone, and the Quistococha Zoological Garden (QZG) in Peru between November 2018 and February 2020. We evaluated for the presence of pathogenic (Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi, Balantioides coli) and commensal (Entamoeba dispar, Troglodytella abrassarti) protist species using PCR methods and Sanger sequencing. Giardia duodenalis was the most prevalent species found (25.9%, 30/116), followed by Blastocystis sp. (22.4%, 26/116), and E. dispar (18.1%, 21/116). We detected E. bieneusi (4.2%, 1/24) and T. abrassarti (12.5%, 3/24) only on NHP from AZG. Cryptosporidium spp., E. histolytica, and B. coli were undetected at the three sampling sites investigated here. Sequence analyses revealed the presence of zoonotic sub-assemblages BIII (n = 1) in AZG and BIV (n = 1) in TS within G. duodenalis. We identified Blastocystis subtype ST3 (100%, 6/6) in AZG, ST1 (80.0%, 12/15), ST2 (6.7%, 1/15), and ST3 (13.3%, 2/15) in TS, and ST2 (80.0%, 4/5) and ST3 (20.0%, 1/5) in QZG. The only E. bieneusi isolate detected here was identified as zoonotic genotype CAF4. Our PCR-based data indicate that potentially pathogenic protist species including G. duodenalis, Blastocystis sp., E. bieneusi, and B. coli are present at variable rates in the three NHP populations investigated here. The identification of zoonotic genotypes within these species indicates that human-NHP transmission is possible, although the extent and directionality of these events need to be elucidated in future molecular surveys., Graphical abstract Image 1, Highlights • Giardia and Blastocystis are highly prevalent in confined non-human primates. • Diarrhoea-causing Cryptosporidium and Entamoeba histolytica were undetected. • First description of Enterocytozoon bieneusi genotype CAF4 in non-human primates. • Confined non-human primates harbour protist species with zoonotic potential. • Cross-species (including human) transmission is possible in zoos and sanctuaries.

Acanthamoeba spp. is a widespread protozoan that has been isolated from air, dust, soil, water and biological samples. An opportunistic pathogen of humans and animals, it may cause ocular keratitis, encephalitis, and even multisystem disease. The frequency of Acanthamoeba in animals is unknown. The aim of present study was determine the presence of Acanthamoeba spp. in immunocompromised stray cats - animals possibly more likely to harbour the infection given their immunocompromised status and frequenting of contaminated environments. Of 307 cats examined, 55 were positive for feline immunodeficiency virus and/or feline leukaemia virus and therefore included in the study. Corneal scrapings were obtained to isolate Acanthamoeba spp. by culture and molecular detection by conventional and real time PCR. None of the samples examined directly by molecular methods were positive for Acanthamoeba spp. However, two (3.6%) cases of the cultured samples provided positive results, which were confirmed by subsequent molecular analysis. Sequencing assigned one isolate to genotype T4 and the other to T2. Since Acanthamoeba spp. may also infect animals and humans, the present findings may raise some public health and veterinary concerns.

Domestic dogs and cats may act as natural reservoirs of a large number of zoonotic pathogens, including the enteric parasites Giardia duodenalis and Cryptosporidium spp., the most relevant protozoan species causing gastrointestinal disease worldwide. A cross-sectional epidemiological study aiming to assess the prevalence and molecular diversity of G. duodenalis and Cryptosporidium spp. was conducted in an animal rescue centre in the province of Álava (Northern Spain). A total of 194 and 65 faecal dropping samples from individual dogs and cats, respectively, were collected between November 2013 and June 2016. G. duodenalis cysts and Cryptosporidium spp. oocysts were detected by direct fluorescence microscopy and PCR-based methods targeting the small subunit ribosomal RNA gene of these parasites. Overall, G. duodenalis and Cryptosporidium spp. were detected in 33% (63/194) and 4.1% (8/194) of dogs, and 9.2% (6/65) and 4.6% (3/65) of cats, respectively. G. duodenalis and Cryptosporidium co-infections were observed in 1.5% (3/194) of dogs, but not in cats. No significant differences in infection rates could be demonstrated among dogs or cats according to their sex, age group, status, or geographical origin. Multi-locus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of G. duodenalis allowed the characterization of 19 canine isolates that were unambiguously assigned to sub-assemblages AII (n=7), BIII (n=1), and BIV (n=7), and assemblages C (n=3) and D (n=1). Two feline isolates were genotyped as assemblages A and F, respectively. No mixed assemblage or sub-assemblage infections were identified. C. canis (n=5) and C. hominis (n=1) were the Cryptosporidium species found in dogs, whereas C. felis (n=1) was identified in cats. The finding of G. duodenalis sub-assemblages AII, BIII, and BIV circulating in dogs (but not cats) may have zoonotic potential, although most of the AII and BIV isolates sub-genotyped corresponded to genetic variants not previously found in Spanish human populations. Dogs may also act as novel suitable hosts for C. hominis. We recommend to considerer companion animals as sentinel surveillance system for zoonotic giardiasis and cryptosporidiosis in order to minimize the risk of spreading of these parasitic diseases among the human population.

Infections by members of the protozoan genus Cryptosporidium are among the most common causes of human gastrointestinal illness worldwide. In Spain cryptosporidiosis is not a compulsory notifiable disease, so the actual burden of the infection in both clinical and general populations remains largely unknown. We present here data on the diversity and frequency of the Cryptosporidium species and sub-genotypes identified in symptomatic individuals seeking medical care in two major hospitals in Madrid, Spain, between December 2013 and January 2015. Initial detection of the parasite was conducted on a total of 122 stool samples collected from 120 patients by microscopy with modified Ziehl-Neelsen and/or immunochromatographic tests. We used immunofluorescence, PCR-based methods and sequence analyses of the 60-kDa (GP60) glycoprotein and the small subunit ribosomal RNA (SSU rRNA) genes for confirmatory purposes and to characterize Cryptosporidium isolates. A total of 110 patients were confirmed with cryptosporidiosis. Overall, 101 isolates were successfully sub-genotyped at the GP60 locus, and an additional seven at the SSU rRNA locus. The analyses of all amplicons defined 10 distinct sequence types representing the GP60 family sub-genotypes IbA10G2 (78.7%), IeA11G3T3 (3.7%) of C. hominis, and the GP60 family sub-types IIaA15G2R1 (5.6%), IIaA18G6R1 (0.9%), IIcA5G3a (0.9%), IIdA18G1 (0.9%), IIdA19G1 (0.9%), IIdA21G1 (0.9%), and IIdA22G1 (0.9%) of C. parvum. A single isolate was assigned to C. felis (0.9%), two C. parvum isolates (1.9%) could not be characterized at the sub-genotype level and an additional four isolates (3.7%) were not typable. These results strongly suggest that transmission of cryptosporidiosis is mostly anthroponotic in origin in the clinical sample under study. We expect that our molecular epidemiological data will make a significant contribution to unravel the actual epidemiological situation of cryptosporidiosis in Spain, providing health care and policy makers with solid baseline information to unavoidably improve the national surveillance system and allocate additional resources to research, diagnosis, and treatment of cryptosporidiosis.

Resumen Introduccion Durante los ultimos anos se ha registrado un aumento creciente de la poblacion inmigrante y de viajeros procedentes de areas endemicas de filariasis producidas por Loa loa (L. loa) , Mansonella perstans (M. perstans) y Wuchereria bancrofti (W. bancrofti) . Esta situacion epidemiologica ha hecho necesario el desarrollo de tecnicas especificas de diagnostico molecular, alternativas al metodo parasitologico clasico. Por lo tanto, el objetivo planteado en este trabajo ha sido la utilizacion de las tecnicas moleculares optimizadas en nuestro laboratorio, nested-PCR e ITS1-RFLP, en el diagnostico de filariasis importadas, y la comparacion de los resultados obtenidos con los derivados del diagnostico parasitologico. Material y metodos Se ha estudiado mediante la tecnica de concentracion de Knott, nested-PCR e ITS1-RFLP un total de 523 muestras (517 sangres, 5 helmintos adultos y un humor vitreo) que fueron remitidas al Servicio de Parasitologia del Centro Nacional de Microbiologia-Instituto de Salud Carlos III, entre los anos 2006 y 2009, por 47 centros sanitarios de las comunidades autonomas. Resultados Las tecnicas moleculares utilizadas demostraron ser mas sensibles que el metodo de concentracion de Knott, tanto en el diagnostico de L. loa (n = 12 frente a n = 4) como en el de M. perstans (n = 57 frente a n = 25) en el total de muestras de sangre estudiadas. Conclusion Los metodos de PCR utilizados permiten un diagnostico especifico y mas sensible de L. loa y M. perstans en muestras clinicas de poblacion inmigrante y viajeros procedentes de areas endemicas, donde estas especies de filarias son coendemicas. El metodo de concentracion de Knott debe emplearse como tecnica complementaria siempre que sea posible.