PhD Thesis defense by Mª Victoria Ortega García at the VISAVET Centre of the Complutense University of Madrid

July 27^th, 2021

Leishmaniasis is a group of parasitic diseases of zoonotic and anthroponotic origin caused by protozoa of the genus Leishmania, within the Trypanosomatidae family. Transmission to the vertebrate host occurs through the bite of female sandfly, blood-sucking insects belonging to the genera Phlebotomus (Old World) or Lutzomyia (New World), within the Psychodidae family. Among the parasitic diseases, leishmaniasis ranks second as the most frequent cause of death in the world behind malaria. In addition, this disease is included in the group of neglected tropical diseases (NTD), and is present in more than 98 countries, with about 350 million people at risk and about 12 million cases.

The main reservoir of leishmaniasis in the Mediterranean area is the dog and the etiologic agent is Leishmania infantum. In the last decade, it has been demonstrated that wild rabbits and hares can act as hosts, evidencing a change in the epidemiology of this disease. The largest outbreak of human leishmaniasis reported in Europe began in 2009 in the Community of Madrid (Spain). Since then, more than 700 cases have been reported, and for the first time, wild Leporidae have been proved to be competent reservoirs of the parasite. This outbreak had a multifactorial aetiology attributable, in part, to the urban modifications developed in the affected areas in recent years. There was a greater density of vectors in the area and an increase in leporid populations, probably due to the lack of predators, among other causes. Taking into account these antecedents and the relevance that is being given to the epidemiological control in animals that are not currently considered as important, the present study has tried to contribute to a model that allows better surveillance to prevent new outbreaks. To achieve this goal, the detection of Leishmania spp. in uncommon reservoirs and the molecular characterization of L. infantum isolates from these reservoirs have been performed. The work carried out is described in this doctoral thesis, presented in four experimental studies, grouped into three chapters.

In the first experimental study, carried out on hair, skin and spleen samples from European rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis), the presence of the parasite was detected by a quantitative real-time PCR (qPCR), previously validated for the detection of L. infantum in dogs, and by a nested PCR. The obtained results by both methods of aetiological diagnosis were compared with those obtained by indirect immunofluorescence (IFAT) and isolation of the parasite by in vitro culture. The qPCR was found to be more sensitive than the nested PCR in all the samples analysed, with a limit of detection for L. infantum genomic DNA of 143 fg/reaction (3.9 parasites). Furthermore, the percentage of positive animals detected by qPCR was at least 1.4 times higher than those detected by IFAT and by this technique 6.7 times higher than by isolation. If isolation and in vitro culture is considered as the gold stardard for our first study, the best agreement with it was obtained by nested PCR in spleen/skin samples and with qPCR in spleen samples. Thus, nested PCR is less sensitive and therefore closer to that obtained by isolation and culture where infection cases are clearly underestimated, and on the other hand, only qPCR-positive animals in spleen samples would be considered truly infected. These results demonstrate that qPCR is a suitable method to detect Leishmania genomic material in samples of different nature, in addition to confirm hair as an optimal matrix for the direct, reliable and non-invasive diagnosis of leishmaniasis in wild animals.

In the second experimental study, the pathology of natural infection by L. infantum in lagomorphs was described. The obtained results in the pathological study by direct immunofluorescence antibody (DFA) test were compared with those of the serological diagnosis (IFAT) and the aetiological diagnosis by qPCR. Histological analysis revealed only small lesions compatible with subclinical leishmaniasis, without other inflammatory signs. The presence of amastigotes was confirmed by DFA in all types of tissues analysed: spleen, lymph node, bone marrow, gastrointestinal tract, liver, pancreas, cardiac striated muscle, skeletal striated muscle, lung, kidney, meninges and in the skin, which gave an idea of the degree of spread of the parasite during infection. In 21% of the rabbits and 100% of the hares previously diagnosed as positive by DFA, the presence of L. infantum was confirmed by qPCR on spleen and/or skin samples. Thus, in this work DFA is confirmed as a useful diagnostic method for leishmaniasis, also helping to precisely localise the parasite in different tissues, serving as a guide to molecular detection tests such as qPCR.

In the third experimental study, four cases of equine cutaneous leishmaniasis were described in Costa Rica, a condition little described at the time we carried out the study. The pathological study included the identification of the parasite by immunohistochemistry (IHC), using a rabbit polyclonal antibody produced against Leishmania spp. In the lesions, together with the amastigotes, a granulomatous inflammatory reaction was observed in the dermis with multinucleated macrophages, giant cells, lymphocytes, and few neutrophils and eosinophils. Our results emphasize the importance of Leishmania spp., not only as a causal agent of equine cutaneous leishmaniasis, but also, the need to include these species in the epidemiological control of the disease. Leishmaniasis is one of the prevalent public health parasitic problems throughout the world and Equidae, being domestic animals, can play a very important role in the epidemiology of the disease.

Finally, in the fourth experimental study, the characterisation of the restriction fragments was carried out by combining the polymerase chain reaction and polymorphisms in their length (PCR-RFLP), in parasites from wild hares and rabbits both inside and outside the outbreak area, analysed in parallel by polyacrylamide gel electrophoresis and capillary electrophoresis. The obtained results showed that there are no differences between the parasites from different origins, not even with the promastigotes from the axenic culture of the referemce L. infantum isolate (MCAN/ES/97/10 445, zymodeme ZM/MON-1). Therefore, it was demonstrated that, independently of the technique applied, there is a single strain of L. infantum responsible for the infection in wild lagomorphs and that they sustain the sylvatic cycle of the disease, both in and outside the outbreak area.

As a general conclusion, it can be said that for the epidemiological surveillance of leishmaniasis in atypical reservoirs (domestic and wild), in order to prevent the appearance of future outbreaks, qPCR in hair samples is a reliable, rapid, sensitive, non-invasive, and specific technique, that can be complemented with other methods such as DFA or IHC when pathological studies are carried out.

- Estudian reservorios atípicos de Leishmania en España para prevenir brotes - animalshealth.es