Leishmaniosis in Rodents Caused by Leishmania infantum: A Review of Studies in the Mediterranean Area

Leishmaniosis infection begins when a phlebotomine sand fly vector inoculates pathogenic protozoan parasites of the genus Leishmania into a mammalian host. In the case of Leishmania infantum, the domestic dog is considered to be the main parasite reservoir, and canine leishmaniosis (CanL) has a high mortality rate in untreated dogs. Hundreds of cases of human leishmaniosis (HL) are reported in the world each year, the incidence in Europe being relatively low. Leishmaniosis control is primarily focused on the dog, combining methods that prevent sand fly bites and boost host resistance to infection. However, these measures are only partially effective and new solutions need to be found. One of the main factors limiting CanL and HL control is the existence of a sylvatic Leishmania transmission cycle that interacts with the domestic cycle maintained by dogs. It is suspected that the main reservoir of infection in wildlife are rodents, whose expansion and rapid population growth worldwide is increasing the risk of human and zoonotic pathogen transfer. The aim of this review is therefore to analyze reports in the literature that may shed light on the potential role of rodents in the leishmaniosis transmission cycle in the Mediterranean area. Following the general methodology recommended for reviews, six databases (Google Scholar, Ovid, PubMed, Science Direct, Scopus and Web of Science) were explored for the period January 1995 to December 2020. The results extracted from 39 publications that met the established inclusion criteria were analyzed. It was found that 23 species of rodents have been studied in nine countries of the Mediterranean basin. Of the 3,643 specimens studied, 302 tested positive for L. infantum infection by serology, microscopy and/or molecular techniques

Detection of specific antibodies against Leishmania infantum in canine serum and oral transudate using an in-house ELISA

Canine leishmaniosis caused by the protozoan Leishmania infantum is a complex infection due to its variable clinical signs and laboratory findings. Therefore, a broad range of techniques is available for diagnosis. Testing for specific antibodies in serum is the most commonly used technique, although the testing of other body fluids, such as oral transudate (OT), can be an alternative as its collection is non-invasive and testing can be performed by untrained personnel. The aim of this study was to assess and compare the detection of L. infantum -specific antibodies in paired samples of serum and OT collected from apparently healthy dogs and dogs with clinical leishmaniosis using an in-house enyzme-linked immunosorbent assay (ELISA). Serum and OT were collected from 407 dogs, which varied in breed, sex, age, lifestyle and clinical status, by many practicing veterinarians in Spain. The main geographical areas of sampling included Barcelona (n = 110), Mallorca (n = 94), Cadiz (n = 54) and Asturias (n = 47). The majority of infected dogs were apparently healthy (89.9%) while 41 presented clinical signs and/or clinicopathological abnormalities compatible with L. infantum infection and subsequently diagnosed with leishmaniosis (10.1%). An in-house ELISA was performed to quantify the anti- Leishmania antibodies in serum and OT. The L. infantum infection rate determined by the in-house ELISA was 37.1% in serum samples and 32.7% in OT samples. Serum and OT ELISA results showed a positive correlation (Spearman's correlation coefficient r = 0.6687, P < 0.0001). The percent agreement between the serum and OT ELISA results was 84%, while agreement according to Cohen's kappa statistic (κ) was substantial (0.66) when all samples were analyzed. The highest percent agreement (92.1%) between both tests was found in dogs from low endemicity regions and from sick dogs, with both groups presenting almost perfect agreement according to Cohen's κ agreement test (0.84). Few seronegative dogs (n = 23) tested positive by the OT ELISA. The agreement between serum and OT went from almost perfect to moderate when the geographical distribution and clinical status were analyzed. The results of this study demonstrated an almost perfect to moderate agreement between OT and serum samples tested using the in-house ELISA. These results are particularly promising in sick dogs with high antibody levels while the results seem less optimal in apparently healthy dogs with low antibody levels

Underestimation of Human Cutaneous Leishmaniasis Caused by Leishmania infantum in an Endemic Area of the Mediterranean Basin (Balearic Islands)

Leishmaniasis is an infectious zoonotic disease caused by protozoan parasites of the genus Leishmania. In the Mediterranean basin, leishmaniasis is caused by Leishmania infantum and transmitted by bites of sandflies of the genus Phlebotomus, with the dog as the main reservoir host. The most common form is cutaneous leishmaniasis (CL), although visceral cases also occur. The aim of this study was to assess the underestimation of CL in an endemic Mediterranean region. Thus, a retrospective study was performed on all CL cases diagnosed and treated in the Dermatology Service of Manacor Hospital (Majorca, Balearic Islands), and the data obtained were compared with those of local government epidemiological bulletins for the same period. The different clinical presentations were compiled, and data related to sex, age, and lesion type and number were analyzed. The results reveal a clear sub-notification, which indicates that the real incidence of human CL in this area is unknown. Keywords: Leishmania; cutaneous leishmaniasis; Majorca; subnotification of case

Hybrid Genotype of Anisakis simplex (s.s.) and A. pegreffii Identified in Third- and Fourth-Stage Larvae from Sympatric and Allopatric Spanish Marine Waters

The sibling species Anisakis simplex (s.s.) and Anisakis pegreffii are parasites of marine mammals and fish worldwide and the main causative agents of human anisakiasis. In sympatric areas, a hybrid genotype between the two species has been identified, mainly in third-stage larvae, but rarely in fourth-stage and adult forms. The aim of this study was to confirm the presence of hybrid genotypes in larvae parasitizing fish caught in sympatric and allopatric Spanish marine waters, the North-East Atlantic and West Mediterranean, respectively, and to study possible differences in the growth behaviour between genotypes. Of the 254 molecularly analysed larvae, 18 were identified as hybrids by PCR-RFLP analysis of the rDNA ITS region, 11 of which were subsequently confirmed by EF1 α-1 nDNA gene sequencing. These results therefore indicate an overestimation of hybrid genotypes when identification is based only on the ITS region. We also report the detection of a hybrid specimen in a host from the West Mediterranean, considered an allopatric zone. Additionally, fourth-stage larvae with a hybrid genotype were obtained in vitro for the first time, and no differences were observed in their growth behaviour compared to larvae with A. simplex (s.s.) and A. pegreffii genotypes

Anisakid Presence in the European Conger, Conger conger, from Spanish Mediterranean Waters

The European conger, Conger conger, is a benthic marine fish species with a geographicaldistribution extending through the northeastern Atlantic and the Mediterranean. Despite beingextensively distributed and widely appreciated by Spanish consumers, studies regarding parasitepresence in this fish are scarce. In the present work, a hundred and eight specimens from theMediterranean coast of northeastern Spain (Catalan waters) were surveyed for the presence ofnematode parasites. Several species were morphologically identified: third-stage larvae of Anisakistype I (sensu Berland, 1961) (n = 131), third-stage larvae of Anisakis type II (sensu Berland, 1961)(n = 18), third- and fourth-stage larvae and adults of Hysterothylacium spp. (n = 48), adults ofCucullanus sp. (n = 391), and adults of Cristitectus congeri (n = 69). Moreover, some fish and decapodespecies were also observed as part of the host’s diet, with the most detected preys being Micromesistiuspoutassou, Sardina pilchardus, Macropipus sp., and Goneplax rhomboides. This represents the first surveyof nematode parasites infecting C. conger from the northeastern Spanish Mediterranean waters.Among the parasite species detected, the presence of Anisakis species should be highlighted as theingestion of C. conger parasitized with these larvae could potentially lead to anisakiasis in consumers</p

Leishmaniosis caused by Leishmania infantum in ferrets: Update review

Leishmaniosis in domestic ferrets (Mustela putorius furo) is a disease caused by Leishmania infantum, a parasite transmitted through the bite of an infected female phlebotomine sand fly. Among vertebrates, the dog is the primary domestic reservoir of the parasite; however, other domestic animals can be implicated such as cats. The first description of a clinical case of leishmaniosis in domestic ferrets was reported recently. As a result, new knowledge has been published including empirically based treatment protocols, confirmatory techniques to detect the presence of the parasite infection and seasonal variation in the antibodies against Leishmania in apparently healthy domestic ferrets. The most common clinical signs observed are enlargement of peripheral lymph nodes and skin lesions such as papular and/or ulcerative dermatitis. Additionally, the most frequent laboratory alterations seen are hyperproteinaemia with hyperglobulinaemia and biochemical analytes alterations depending on the affected tissue. Two different therapeutic protocols have been described to treat domestic ferrets with leishmaniosis: meglumine antimoniate plus allopurinol protocol or miltefosine plus allopurinol protocol. These treatment protocols seemed to be able to control the Leishmania infection, although the presence of xanthinuria could be detected. The susceptibility of domestic ferrets to Leishmania infantum, the clinical picture, treatment of infected animals and prevention are poorly understood, due to the scarcity of recent description in the literature. Different proposed diagnostic algorithms have been included for domestic ferrets with suspected leishmaniosis, clinically healthy domestic ferrets and animals as blood donors. In this sense, the present review provides updated data on scientific knowledge of leishmaniosis in ferrets

Signalment, Immunological and Parasitological Status and Clinicopathological Findings of Leishmania-Seropositive Apparently Healthy Dogs

Canine leishmaniosis caused by Leishmania infantum is a disease with a wide range of clinical manifestations. Epidemiological serosurveys performed in Europe often lack a thorough assessment of clinical health status of studied dogs. The aim of this study was to evaluate signalment, immunological and parasitological status and clinicopathological findings of L. infantum-seropositive apparently healthy dogs (n = 212) living in endemic areas. Routine laboratory tests, endpoint inhouse ELISA to quantify the anti-Leishmania antibodies, blood Leishmania qPCR and IFN-ELISA were performed. All dogs enrolled were L. infantum-seropositive and were classified as healthy (n = 105) or sick (n = 107) according to LeishVet guidelines. The sick group presented a higher proportion of medium to high antibody levels and positive qPCR and lower IFN-concentration compared to the healthy group. Sick dogs were mostly classified in LeishVet stage IIa. Biochemical alterations (98%) were the most common clinicopathological findings, with fewer urinary tract (46%) and hematological (40%) alterations. Apparently healthy L. infantum-seropositive dogs can be classified between truly healthy dogs and sick dogs with clinicopathological findings. Sick dogs presented medium to high seropositivity and parasitemia and low IFN- concentrations, and their most common clinicopathological abnormalities were serum protein alterations followed by proteinuria and lymphopenia

First epidemiological survey of Leishmania infantum in the domestic ferret (Mustela putorius furo) in a canine leishmaniosis endemic area using serology and PCR

Leishmaniosis, a vector‑borne disease caused by Leishmania infantum, is one of the most important parasitic zoonoses in Europe. The transmission cycle of leishmaniosis is maintained by both domestic and wild animals. However, few data are available on the role of wild mammals in transmitting the parasite in the European Mediterranean basin. As feline leishmaniosis, diagnosis of the infection in ferrets can be a challenge, the use of different serological and molecular methods combined is a recommended approach. Our aim was to investigate the prevalence of infection of L. infantum in apparently healthy domestic ferrets (Mustela putorius furo) in an endemic region of Spain (Community of Valencia), using serological and molecular methods and to evaluate the results comparing the different techniques

Factors influencing the presence of sand flies in Majorca (Balearic Islands, Spain) with special reference to Phlebotomus pernicious, vector of Leishmania infantum

Background: Although the Mediterranean island of Majorca is an endemic area of leishmaniosis, there is a lack of up-to-date data on its sand fly fauna, the last report dating from 1989. The aim of the present study was to provide information on the current sand fly distribution, the potential environmental factors favoring the presence of Phlebotomus perniciosus and which areas are at risk of leishmaniosis. Methods: In July 2008 sand fly captures were carried out in Majorca with sticky castor oil interception traps. The capture stations were distributed in 77 grids (5x5 km2) covering the entire island. A total of 1,882 sticky traps were set among 111 stations. The characteristics of the stations were recorded and maps were designed using ArcGIS 9.2 software. The statistical analysis was carried out using a bivariate and multivariate logistic regression model. Results: The sand fly fauna of Majorca is composed of 4 species: Phlebotomus perniciosus, P sergenti, P. papatasi and Sergentomyia minuta. P. perniciosus, responsible for Leishmania infantum transmission, was captured throughout the island (frequency 69.4 %), from 6 to 772 m above sea level. Through logistic regression we estimated the probability of P. perniciosus presence at each sampling site as a function of environmental and meteorological factors. Although in the initial univariate analyses the probability of P. perniciosus presence appeared to be associated with a wide variety of factors, in the multivariate logistic regression model only altitude, settlement, aspect, drainage hole construction, adjacent flora and the proximity of a sheep farm were retained as positive predictors of the distribution of this species. Conclusions: P. perniciosus was present throughout the island, and thereby the risk of leishmaniosis transmission. The probability of finding P. perniciosus was higher at altitudes ranging from 51 to 150 m.a.s.l., with adjacent garrigue shrub vegetation, at the edge of or between settlements, and in proximity to a sheep farm

First Molecular Diagnosis of Clinical Cases of Gastric Anisakiosis in Spain

Anisakiosis is a fish-borne disease with gastrointestinal and/or allergic symptoms caused by the consumption of raw or undercooked fish parasitized with nematode larvae of the genus Anisakis. In Europe, Anisakis pegre i has been detected as the causative agent, although the sibling species Anisakis simplex sensu stricto (s.s.) is also known to cause the disease in other parts of the world, and discrepancies exist regarding their respective pathogenic potential. In Spain a high number of cases has been recorded, with marinated anchovies being the main source of infection, although no specific diagnosis has been documented in humans. In this study, we analyzed three cases of anisakiosis in patients from Barcelona (Spain) who had consumed undercooked hake. All patients described epigastric pain and several larval nematodes were removed endoscopically from their stomachs. Larvae were morphologically characterized as third-stage larvae of Anisakis simplex sensu lato (s.l.) and molecularly identified as A. simplex (s.s.) by means of PCR RFLP of the ITS region of the rDNA and sequencing of the elongation factor1 alpha1 (EF1 -1) nDNA gen. This study represents the first specific identification of Anisakis larvae in clinical cases of anisakiosis reported in Spain. Specific molecular diagnosis is of crucial importance for assessing the health risk of Anisakis sibling species. Hake consu