A cross-sectional study combining different serological and molecular techniques for the detection of Ehrlichia species in dogs and their ticks was carried out with data from all regions of Costa Rica. A seroprevalence of 32.1% (131/408), and infection with E. canis of 3.2% (13/407) was found, whereas 6.9% (9/130) of ticks attached to the dogs were PCR positive to E. canis. Higher prevalences were found outside the Greater Metropolitan Area (GMA). Risk factors associated with E. canis seropositivity were age, between 2 and 7 years (RR: 1.6, 95% CI: 1.2-2.2) and 8-15 years (RR: 1.8, 95% CI: 1.2-3.0), number of dogs/total of households [Dogs per Household Ratio (DHR) ≥3.1 (RR: 2.0; 95% CI: 1.4-3.0)], number of dogs infested with at least one tick/total of dogs sampled [Tick Infestation Prevalence (TIP)≥31% (RR: 2.1; 95% CI:1.3-3.3)] and living outside the GMA (RR: 1.7; 95% CI: 1.2-2.4) and being a mixed-breed dog (RR: 1.5; 95% CI: 1.1-2.1). Risk factors for E. canis PCR positive dogs were a depressive attitude (OR: 11.2; 95% CI: 1.1-115.9), fever (OR:4.8; 95% CI:1.2-19.3), DHR≥3.1 (OR: 5.7; 95% CI:1.7-19.2)], number of ticks/total of dogs sampled [Tick Distribution Ratio (TDR) ≥2.1 (OR: 6.5; 95% CI: 1.3-31.8)], and TIP≥40% (OR: 5.7; 95% CI: 1.7-19.2). This paper describes E. canis seroprevalence, PCR prevalence and tick analysis in dogs from Costa Rica, with associated clinical signs and owner perceptions. In summary, most of the E. canis infections in dogs in our country seemed to pass unnoticed by owners. Since most of the seropositive dogs (97.7%, 131/134) were negative for E. canis DNA in their blood, it is important to determine in future studies if these dogs recovered from the E. canis infection without any medication, or are persistently infected, and will develop chronic disease.

Although vector-borne diseases are globally widespread with considerable impact on animal production and on public health, few reports document their presence in Central America. This study focuses on the detection and molecular identification of species belonging to selected bacterial genera (Ehrlichia, Anaplasma and Rickettsia) in ticks sampled from dogs in Costa Rica by targeting several genes: 16S rRNA/dsb genes for Ehrlichia; 16S rRNA/groEL genes for Anaplasma, and ompA/gltA/groEL genes for Rickettsia. PCR and sequence analyses provides evidences of Ehrlichia canis, Anaplasma platys, and Anaplasma phagocytophilum infection in Rhipicephalus sanguineus s.l ticks, and allow establishing the presence of Rickettsia monacensis in Ixodes boliviensis. Furthermore, the presence of recently discovered Mediterranean A. platys-like strains is reported for the first time in Central America. Results provide new background on geographical distribution of selected tick-transmitted bacterial pathogens in Costa Rica and on their molecular epidemiology, and are pivotal to the development of effective and reliable diagnostic tools in Central America.

A cross-sectional study was carried out in four indigenous communities of Costa Rica to detect presence and prevalence of Babesia caballi and Theileria equi and to investigate factors associated with presence of these hemoparasites. General condition of horses (n = 285) was evaluated, and hematocrits and hemoglobin were determined from blood samples of 130 horses, which were also analyzed using blood smears, nested polymerase chain reaction (PCR), and immunosorbent assay (c-ELISA). The general condition of the horses (n = 285) in terms of their body and coat was between regular and poor, and hematocrit and hemoglobin average values were low (19% and 10.65 g/dL, resp.). Erythrocyte inclusions were observed in 32 (24.6%) of the samples. Twenty-six samples (20.0%) gave positive results for B. caballi and 60 (46.2%) for T. equi; 10 horses (7.7%) showed mixed infection, when analyzed by PCR. Using c-ELISA, it was found that 90 (69.2%) horses had antibodies against B. caballi and 115 (88.5%) against T. equi, while 81 (62.3%) showed mixed reactions. There were no factors associated with the presence of B. caballi and T. equi. These results contrast with results previously obtained in equines in the Central Valley of Costa Rica.

The trematode Paragonimus mexicanus is the etiological agent of paragonimiasis, a food-borne zoonotic disease in Latin America. This species, as well as Paragonimus caliensis, have been reported from Costa Rica, but it is not known if the two are synonymous. Two types of Paragonimus metacercariae from freshwater pseudothelphusid crabs from several localities in Costa Rica were recognized by light microscopy. Morphologically, these corresponded to descriptions of P. mexicanus and P. caliensis. Metacercariae of the former species lacked a membrane or cyst and their bodies were yellow in color. Those of P. caliensis were contained in a transparent thin cyst and were pink in color. Morphotypes of metacercariae were determined using scanning electron microscopy (SEM). Based on the number and distribution of papillae in the ventral sucker, three morphotypes were found for P. mexicanus and two for P. caliensis. Analysis of DNA sequences (nuclear ribosomal 28S and ITS2 genes, and partial mitochondrial cox1 gene) confirmed the presence of P. mexicanus and provided the first molecular data for P. caliensis. The two species are phylogenetically distinct from each other and distant from the Asian species. The confirmation of P. caliensis as a separate species from P. mexicanus raises several questions about the ecology, biological diversity, and epidemiology of the genus Paragonimus in Costa Rica.

A cross-sectional study combining serological and molecular techniques for detecting selected Anaplasma species was conducted between 2011 and 2012 in dogs and ticks sampled in all provinces of Costa Rica. Global Anaplasma spp. seroprevalence was 2.7% (11/408) by indirect immunofluorescence assay. The 16S rRNA PCR confirmed active A. phagocytophilum infection only in one dog (0.3%, 1/374); however, the same sample was negative to groEL PCR. Out of 122 Rhipicephalus sanguineus s.l. ticks analyzed, one (0.8%) was found positive to A. phagocytophilum 16S rRNA PCR but negative when tested by groEL PCR; this tick was collected from a seronegative and PCR negative dog. Both 16S rRNA sequences were 100% (510/510 bp) identical to A. phagocytophilum strains isolated in different countries from different hosts. The presence of A. platys was established in four dogs (1%, 4/374) by both 16SrRNA and groEL PCR. Ticks collected from the same dogs tested negative by PCR. The 16S rRNA sequences were 100% identical to the corresponding sequences of A. platys strains isolated from dogs in Croatia and Brazil, however groEL sequences showed variable similarity levels (99-100%) with different strains of A. platys isolated in Chile, Japan and Thailand, pointing out the possible presence of different variants in Central America. Collectively data indicate low prevalence of A. phagocytophilum and A. platys in dogs from Costa Rica. Furthermore, infections seem to occur without clinical signs but with some hematological changes, and seem to resolve without treatment.

Although the presence of rickettsial agents in ticks infesting wild birds in Costa Rica has been recently reported, information on strain diversity is limited to selected rickettsial species. In order to mine deeper into rickettsial agents of ticks infesting Costa Rica wild birds a total of 399 birds from the North Huetar Conservation Area of Costa Rica were captured, and 134 immature ticks (76 larvae and 58 nymphs) were recovered from 61 birds. Ticks were tested for the presence of Rickettsia spp. by conventional PCR and sequencing of the gltA, ompA, ompB, 17 kDa, and groEL genes. Six (11.3%) Amblyomma longirostre and Amblyomma geayi ticks collected from passeriform birds, yielded amplicons of the expected size. Amplicons were sequenced, and BLAST results collectively showed that all sequences had 99-100% nucleotide identity with Rickettsia amblyommatis (formerly, 'Candidatus Rickettsia amblyommii'). Three different R. amblyommatis strains were identified. Four new tick species-host associations and the first detection of R. amblyommatis in A. geayi in Costa Rica are also reported.

Dictyocaulosis and fasciolosis are parasitic diseases that cause considerable economic losses for owners of farm animals worldwide, with special relevance on fasciolosis because it is an emerging zoonosis. Indirect diagnosis of these diseases through analyses of bulk milk tank samples has allowed carrying out large-scale prevalence studies, while the use of geographical information systems has helped to visualize and determine those variables that affect distribution of these pathogens. This study is intended to describe the spatial distribution of Dictyocaulus viviparus and Fasciola hepatica in dairy herds from Costa Rica, as well as their associated environmental factors. Bulk milk tank samples from 526 dairy herds in the three most important dairy regions of Costa Rica were analyzed using enzyme immunoassays. Results from the farms were subjected to spatial analyses using Holdridge's life zones, relief and soil type environmental layers. Of the total bulk milk tank samples analyzed, 3.8% (n = 20) and 3.6% (n = 19) were positive for D. viviparus and F. hepatica, respectively. Moran's I analysis revealed the existence of potential cluster (Moran's I = 1.789, z = 12.726 p < 0.05) for D. viviparus. Consequently, Getis-Ord General G analysis showed that the spatial distribution of positive farms in the dataset was clustered (Observed General G = 0.015, variance = 0.000001, z = 12.823, p < 0.05). No significant positive spatial autocorrelation (Moran's I = 0.038, z = 0.286, p > 0.0.5) was observed for F. hepatica. Furthermore, a significant difference was detected in the spatial locations of both parasites (latitude p < 0.05, longitude p < 0.05), and about the spatial distribution of both D. viviparus negative and positive farms (latitude p < 0.05, longitude p < 0.05), as well as in F. hepatica negative and positive farms regarding on latitude (p < 0.05), but not on longitude (p > 0.05). In the case of environmental factors, significant differences were found for D. viviparus and F. hepatica with respect to types of soil, precipitation, altitudinal belts, life zones, biotemperature, and elevation.

Prosthenorchis elegans is a worm of the family Archiacanthocephala that infects non-human primates in the Americas, producing an intestinal pathology that may compromise the life of its hosts. Squirrel monkeys, Saimiri oerstedii citrinellus, were found with P. elegans in Costa Rica. Histopathological analysis revealed a severe pyogranulomatous response composed by macrophages, neutrophils, eosinophils, fibroblasts and lymphocytes. Morphological worm analyses revealed 36 hooks in the proboscis distributed in six rows; and total body, hook and lemnisci length were compatible to the original descriptions of P. elegans. In addition, phylogenetic, haplotype network and genetic distance analyses were done on cytochrome oxidase subunit 1, cox1, sequences obtained from the collected specimens. Sequences obtained herein clustered separately with high posterior probabilities in a Bayesian Inference tree and showed 8.12% nucleotide differences when compared to P. elegans from Colombia. This high divergence was confirmed in the TCS network that separated Colombian and Costa Rican sequences by 32 mutational steps, a genetic distance PCA which separated sequences from both geographical locations by 89.5% and an FST value of 0.655, indicating the presence of cryptic diversity in P. elegans. Additional studies from specimens collected from other definitive hosts and geographical locations are required to better understand the biodiversity of this species.