On the efficacy of preventive toltrazuril treatments and the diagnosis of Cystoisospora suis infections in intensively raised piglets in farms from southeast Spain.

This study investigated the presence and abundance of Cystoisospora suis oocysts in faecal samples from 131 one- to three-week-old pig litters belonging to eight intensively raised, indoor herds in Spain. Seven herds used preventive anticoccidial toltrazuril treatments administered orally or by intramuscular injection, and one did not use preventive anticoccidial treatments. The diagnosis was performed using two oocyst flotation-concentration methods, Bailenger's method in every herd and the more recent Joachim's method in four herds. Oocysts were detected in every farm, the proportion of oocyst-positive samples was higher with Bailenger's technique, and the estimated overall prevalence (95% confidence interval) was 40 (32-49)%, including 47 (29-65)% in non-medicated litters, 52 (38-67)% in orally medicated litters and 28 (16-40)% in intramuscularly medicated litters (p < 0.05). However, mixed logistic regression models indicated that the risk of infection was not significantly associated with preventive anticoccidial treatments (p > 0.05), and increased with age, was higher in herds with partially compared to fully slatted dung floors in farrowing pens and in litters with pigs with diarrhoea (p < 0.05). The median (range) oocysts per gram of faeces (OpG) in infected litters by Bailenger's method was 623 (35-49048) and mixed negative binomial models revealed no significant association between infection intensity in positive litters and pen's floor type and piglets age, faecal consistency and treatment status (p > 0.05). The apparent low efficacy of Toltrazuril suggests treatment administration failures, reduced residual efficacy or low susceptibility of C. suis strains in study farms and needs further investigation.

Effect of high-volume insecticide spraying on sand fly vectors in household gardens in Spain.

This study investigated the efficacy of high-volume spraying with the adulticide α-cypermethrin alone and in combination with the larvicide diflubenzuron on the density of sand flies in gardens of three detached households in periurban areas in southeast Spain. Treatments were applied four times between June and August 2016, and four nearby sites, two households and two non-urbanized sites, were untreated controls. The number of sand flies collected between May and October 2016 using sticky interception and light attraction traps, was 4446 specimens. Species identified morphologically included Sergentomyia minuta (n = 2101; 48%), Phlebotomus perniciosus (n = 1922; 44%), Phlebotomus papatasi (n = 173; 4%), Phlebotomus sergenti (n = 161; 4%) and Phlebotomus ariasi (n = 36; 1%). Sand flies were detected in both treated and untreated sites. The proportion of positive sticky traps and the median (range) density of sand flies in positive traps were 61% traps and 7 (2-172) sand flies/m2 /day in untreated sites, and 43% traps and 4 (1-56) sand flies/m2 /day in treated sites (p < 0.05). Similarly, for light traps, it was 96% traps and 30 (3-168) flies/trap/day, and 83% traps and 3 (1-12) sand flies/trap/day, respectively (p < 0.05). However, sand fly density followed a comparable seasonal pattern in untreated and treated sites and did not consistently decrease following insecticide applications. These results were confirmed with mixed negative binomial modelling of sand fly density adjusted for time since application, month, environmental setting and site. The limited efficacy of the treatments, added to their cost, the impact of insecticides on non-target organisms and human health, and the risk of development of insecticide resistance, should dissuade similar outdoor applications to control sand fly vector populations in residential areas.

Clinical, diagnostic and epidemiological implications of Hepatozoon spp., Babesia spp. and Leishmania infantum infection in cats and dogs in a Mediterranean periurban setting.

Hepatozoon spp., Babesia spp. and Leishmania infantum are common parasites of dogs in Mediterranean countries and are less frequent in cats, particularly Babesia spp. and L. infantum. Moreover, there is limited information on coinfections between these parasites and on L. infantum's distribution in blood, skin and lymphoid tissue in cats. We used PCR and DNA sequencing to investigate the prevalence of these parasites and the aetiology of Hepatozoon spp. and Babesia spp., in blood, skin, spleen and lymph node samples from up to 212 stray cats and 82 abandoned dogs in southeast Spain. All except 2 dogs were healthy; instead, 112 cats had clinical signs. The estimated PCR prevalences (95% confidence interval) were 25% (19-31%) Hepatozoon felis in cats, 13% (6-21%) Hepatozoon canis in dogs, 1% (0-4%) Babesia vogeli in dogs, 0% Babesia spp. in cats and 21% (15-26%) and 44% (33-55%) L. infantum in cats and dogs, respectively, and infections were not associated with each other. Leishmania infantum prevalence in lymphoid tissue was significantly higher in dogs than in cats (p < 0.001), and dogs had higher parasite loads than cats (p = 0.012). Moreover, L. infantum prevalence was significantly higher in the skin and lymphoid tissue compared to blood in infected, asymptomatic animals but it was similar in cats with clinical signs, which also had higher parasite loads compared to infected, asymptomatic cats (p < 0.05). The study highlights significant differences between sympatric dogs and cats with respect to the parasite infections investigated, as well as the need to examine both lymphoid tissue and skin samples to maximise the sensitivity of L. infantum infection diagnosis.

Genetic diversity and phylogenetic relationships between Leishmania infantum from dogs, humans and wildlife in south-east Spain.

Leishmania infantum causes human and canine leishmaniosis. The parasite, transmitted by phlebotomine sand flies, infects species other than dogs and people, including wildlife, although their role as reservoirs of infection remains unknown for most species. Molecular typing of parasites to investigate genetic variability and evolutionary proximity can help understand transmission cycles and designing control strategies. We investigated Leishmania DNA variability in kinetoplast (kDNA) and internal transcribed spacer 2 (ITS2) sequences in asymptomatically infected wildlife (n = 58) and symptomatically and asymptomatically infected humans (n = 38) and dogs (n = 15) from south-east Spain, using single nucleotide polymorphisms (SNPs) and in silico restriction fragment length polymorphism (RFLP) analyses. All ITS2 sequences (n = 76) displayed a 99%-100% nucleotide identity with a L. infantum reference sequence, except one with a 98% identity to a reference Leishmania panamensis sequence, from an Ecuadorian patient. No heterogeneity was recorded in the 73 L. infantum ITS2 sequences except for one SNP in a human parasite sequence. In contrast, kDNA analysis of 44 L. infantum sequences revealed 11 SNP genotypes (nucleotide variability up to 4.3%) and four RFLP genotypes including B, F and newly described S and T genotypes. Genotype frequency was significantly greater in symptomatic compared to asymptomatic individuals. Both methods similarly grouped parasites as predominantly or exclusively found in humans, in dogs, in wildlife or in all three of them. Accordingly, the phylogenetic analysis of kDNA sequences revealed three main clusters, two as a paraphyletic human parasites clade and a third including dogs, people and wildlife parasites. Results suggest that Leishmania infantum genetics is complex even in small geographical areas and that, probably, several independent transmission cycles take place simultaneously including some connecting animals and humans. Investigating these transmission networks may be useful in understanding the transmission dynamics, infection risk and therefore in planning L. infantum control strategies.

Understanding Phlebotomus perniciosus abundance in south-east Spain: assessing the role of environmental and anthropic factors.

BACKGROUND: Leishmaniosis is associated with Phlebotomus sand fly vector density, but our knowledge of the environmental framework that regulates highly overdispersed vector abundance distributions is limited. We used a standardized sampling procedure in the bioclimatically diverse Murcia Region in Spain and multilevel regression models for count data to estimate P. perniciosus abundance in relation to environmental and anthropic factors. METHODS: Twenty-five dog and sheep premises were sampled for sand flies using adhesive and light-attraction traps, from late May to early October 2015. Temperature, relative humidity and other animal- and premise-related data recorded on site and other environmental data were extracted from digital databases using a geographical information system. The relationship between sand fly abundance and explanatory variables was analysed using binomial regression models. RESULTS: The total number of sand flies captured, mostly with light-attraction traps, was 3,644 specimens, including 80% P. perniciosus, the main L. infantum vector in Spain. Abundance varied between and within zones and was positively associated with increasing altitude from 0 to 900 m above sea level, except from 500 to 700 m where it was low. Populations peaked in July and especially during a 3-day heat wave when relative humidity and wind speed plummeted. Regression models indicated that climate and not land use or soil characteristics have the greatest impact on this species density on a large geographical scale. In contrast, micro-environmental factors such as animal building characteristics and husbandry practices affect sand fly population size on a smaller scale. CONCLUSIONS: A standardised sampling procedure and statistical analysis for highly overdispersed distributions allow reliable estimation of P. perniciosus abundance and identification of environmental drivers. While climatic variables have the greatest impact at macro-environmental scale, anthropic factors may be determinant at a micro-geographical scale. These finding may be used to elaborate predictive distribution maps useful for vector and pathogen control programs.