Dog are the main reservoir of Leishmania infantum, causing canine leishmaniasis, an incurable multisystemic disease that leads to death in symptomatic dogs, when not treated. This parasite causes visceral, cutaneous, and mucosal leishmaniasis in people in the Mediterranean Basin, North Africa, South America, and West Asia. This disease is mostly unknown by veterinarians outside the endemic areas, but the disease is expanding in the Northern Hemisphere due to travel and climate change. New methodologies to study the epidemiology of the disease have found new hosts of leishmaniasis and drawn a completely new picture of the parasite biological cycle. Canine leishmaniasis diagnosis has evolved over the years through the analysis of new samples using novel molecular techniques. Given the neglected nature of leishmaniasis, progress in drug discovery is slow, and the few drugs that reach clinical stages in humans are unlikely to be commercialised for dogs, but several approaches have been developed to support chemotherapy. New-generation vaccines developed during the last decade are now widely used, along with novel prevention strategies. The implications of the epidemiology, diagnosis, treatment, and prevention of canine leishmaniasis are fundamental to public health.

The diagnosis of cutaneous leishmaniasis (CL) might be difficult, in particular in endemic areas where different species of Leishmania can cause lesions of very similar appearance and where other skin diseases with similar clinical symptoms occur. Even today, the parasitological diagnosis of CL remains the gold standard and it is based on the direct identification of amastigotes in microscopy smears and/or culture of promastigotes from infected tissues. Although these techniques are highly specific, they are not sensitive enough. The objective of this study is to contribute to improving the diagnosis of CL and the identification of Leishmania species in Morocco by comparing three PCR-based assays applied directly on dermal samples. A total of 58 patients presenting with cutaneous lesions suggestive of CL were sampled for parasitological diagnosis by direct examination (DE), culture in NNN medium, two kinetoplast DNA (kDNA) PCRs (Lmj4/Uni21 and 13A/13B primers) and one rRNA gene internal transcribed spacer 1 (ITS1) PCR (LITSR/L5.8S primers). The techniques were statistically analyzed and compared. According to our consensus positive, 44 out of 58 samples were true positives. The 13A/13B-PCR and ITS1-PCR showed the highest sensitivities (100%). Parasite microscopy and culture detected 43% and 29% of the true positives, respectively, while culture and microscopy together improved sensitivity to 52%. PCRs 13A/13B and ITS1 were associated to four and one false positives, respectively, while the other assays were 100% specific. Furthermore, the ITS1-PCR-RFLP assay clearly identified the Leishmania species for all the true positives (44/44), whereas Lmj4/Uni21-PCR identified 35/44 samples. The comparison between the Leishmania molecular characterizations and the expected species according to the national data from the Ministry of Health indicate 7 discrepant results. The PCR-based assays tested on our samples increased the speed and sensitivity of the diagnosis of CL compared to the conventional techniques. Furthermore, we showed that we can not base the species identification on the national data from the Ministry of Health. Finally, we suggest the use of PCR-ITS1-RFLP for diagnosis and simultaneous identification of the species in the Moroccan epidemiological context, but also in similar areas of the Mediterranean Basin.

The Anisakis larvae presence in fish for human consumption is a health risk that needs to be monitored. The anchovy is a fish that is highly appreciated by consumers and that can harbour Anisakis. It is thus necessary to periodically evaluate the presence of anisakid larvae in them. So, anchovies from Iberian Peninsula coasts were analysed. Fish examination for macroscopic nematodes showed L3s of both Anisakis type I and Hysterothylacium aduncum. The Anisakis prevalence varies with the catching area and the fish size. The muscle prevalence was 7.45% (mean intensity 1.75; range 1-5). Molecular analysis showed 110 A. simplex s.s. (17 in muscle), 22 A. pegreffii (3) and 7 hybrid genotype individuals (1). Considering that most of the Iberian Peninsula coasts are a sympatry area between these two Anisakis species, it has been observed that A. simplex s.s./A. pegreffii ratio increases from south to north in a clockwise direction. Also, 19 larvae were detected on the fish surface from the Bay of Biscay, indicating the ability of these larvae to migrate after the fish death. The A. simplex s.s./A. pegreffii larvae proportion found on the anchovy surface is similar to the found in viscera and lower than in muscle, suggesting that most of the larvae migrating to the surface must have come from the visceral package. This confirms the importance of removing fish viscera immediately after capture, for those fish species where this is possible. As both species cause anisakiasis/anisakidosis, these data show a real risk to human health, especially in dishes highly prized in Mediterranean countries prepared with raw or semi-raw anchovies.

Leishmaniasis by Leishmania infantum has a zoonotic transmission cycle involving an increasing number of mammalian hosts, forming a cooperative network. The sand fly feeding on livestock is evidenced, but clinical confirmation regarding their infection is limited. We aimed to evaluate Leishmania seroprevalence in livestock to assess its impact on leishmaniasis epidemiology in an endemic area located in the Mediterranean region. A cross-sectional serological study screened livestock exposure to L. infantum and risk factors in Southern Spain. A total of 864 serum samples of clinically healthy sheep, goats, cattle, and pigs were examined by an indirect fluorescence antibody test, using a 1/80 cut-off titre to minimize cross-reactions. Global seroprevalence was 10.8%: 21.6% cattle, 15.4% sheep, 7.3% goats, and 0.6% pigs. Statistically significant differences in positive detection were observed among species (p < 0.001) and natural regions (p < 0.001). High positive reactions in cattle, goats, and sheep suggest livestock exposure to Leishmania spp. and potential asymptomatic infection. Livestock presence in biotopes could promote a dilution effect, reducing human leishmaniasis incidence. Further investigation is needed to confirm livestock roles in leishmaniasis maintenance and transmission.

Parasitism as a lifestyle is much more common in nature than it seems

The presence of third stage larvae (L3) of Anisakis spp. in wedge sole, Dicologlossa cuneata (Moreau, 1881), purchased in fishmarkets in the city of Granada (Andalusia, southern Spain) was assessed. The wedge sole were caught in two FAO zones: area 27.IXa NE Atlantic (SW Spain coast) and area 34.1.11 CE Atlantic (NW Morocco coast). Only Anisakis larvae, type I, were detected in the largest fish (>20 cm) from the CE Atlantic. These were molecularly identified as A. simplex s.s. The prevalence (P) of Anisakis in this area was 12.5% and the mean intensity (MI) was 1. The presence of Hysterothylacium spp. larvae was also detected in the fish from both areas, with the prevalence being approximately double in the CE Atlantic area (12.5 vs. 5.7). A comparison between the Anisakis-infected and non-infected fish from this area showed that the former were significantly longer than the latter (p < 0.01). These results show that Anisakis parasitization of wedge sole sold in the markets of the city of Granada is of low prevalence and intensity (P = 4.5, MI = 1), especially in those from area 27.IXa (P = 0), indicating that the risk of human infection is low, particularly as this fish is traditionally prepared by deep-frying in oil in Andalusia (southern Spain).