Ana Pérez-Creo

Prevalence and identity of Sarcocystis spp. in roe deer (Capreolus capreolus) in Spain: a morphological study.

Muscular samples from the oesophagus, diaphragm and heart of 101 roe deer (Capreolus capreolus) hunted in Galicia (Northwestern Spain) were examined, by the compression method, for the presence of Sarcocystis spp. infection. The structure of the cyst wall was examined by light (LM) and transmission electron microscopy (TEM). The overall prevalence of infection was very high (99%), with a density of 404 cysts/sample (SD 812). The prevalence was very similar in the different examined muscle types (99% heart and diaphragm, and 98.9% oesophagus). A significantly higher intensity of infection was found in the heart (831; SD 1281), followed by the diaphragm (197; SD 190) and the oesophagus (180; SD 205). Macrocysts (>1500 μm long) were only detected in the oesophagus of 48.5% of the examined roe deer; their mean size was 2055.4 μm (SD 632.46). Cysts localised in the myocardium were significantly shorter (371.5 μm; SD 160.47) than those found in the diaphragm (678.2; SD 546) and the oesophagus (973.4 μm; SD 667.87). By LM, most of the cysts (98.8%) displayed a thin wall, which was consistent with those of Sarcocystis sp., S. gracilis and S. capreolicanis; only 1.2% of the cysts had a thick striated wall, consistent with Sarcocystis silva. Three morphological distinct sarcocysts were observed by TEM: the unnamed species Sarcocystis sp., S. capreolicanis and S. gracilis. The wall ultrastructure of the examined macrocysts was consistent with S. gracilis. This study has revealed that Spanish roe deer harbours 4 morphologically distinct types of sarcocysts; being the first record of S. gracilis in roe deer from Spain.

Seroprevalence of Toxoplasma gondii and Neospora caninum in goats from north-western Spain

INTRODUCTION AND OBJECTIVE Toxoplasma gondii and Neospora caninum are protozoans involved in reproductive failure especially in ruminant livestock. The objective was to estimate the seroprevalence of both parasites in goats from north-western Spain and to study the influence of some factors on seropositivity. MATERIALS AND METHOD Blood samples from 638 goats were collected in 50 farms. Presence of T. gondii and N. caninum antibodies were detected by direct agglutination and competitiveELISA techniques, respectively. The risk factor analysis was performed using a mixed-effects logistic regression. RESULTS Individual (48%) and herd-level (74%) T. gondii seroprevalence values were high; the within-herd prevalence was 53%. In contrast, 6% of animals tested positive to N. caninum and 38% of the herds had at least one positive animal, with a true within-herd prevalence of 10%. Mixed infections were limited; 91% of N. caninum seropositive goats were also positive to T. gondii. The risk factor analysis showed that T. gondii seroprevalence is influenced by the presence of sheep in the farm (OR=4.9) and the seropositivity to N. caninum (OR=16.5); goats from the Central-coastal area, more humid and warm, had a 15.7-fold probability of being seropositive to T. gondii than those from the Mountainous area. Cross-breed goats (OR=4.5) and the seropositivity to T. gondii (OR= 9.5) were factors associated with N. caninum seropositivity. CONCLUSIONS The high T. gondii seroprevalence in goats constitute a noticeable zoonotic risk. The consideration of the risk factors identified in designing T. gondii and N. caninum control programs in goat herds should allow the implementation of more efficient measures, avoiding the appearance of outbreaks of reproductive disorders by both protozoans in goats.

Cryptosporidium species in post-weaned and adult sheep and goats from N.W. Spain: Public and animal health significance

Application of molecular approaches has led to a significant progress on the knowledge of the epidemiology of Cryptosporidium spp. Nevertheless, molecular information on the occurrence of cryptosporidiosis in domestic small ruminants, especially in goats, are limited and restricted to the study of a modest number of isolates, mainly from diarrhoeic neonates. In order to determine the Cryptosporidium species present in healthy post-weaned and adult small ruminants from north-western Spain and to analyse a possible age-related distribution of species, faecal specimens were collected in sheep and goat farms without neonatal diarrhoea outbreaks the year before the sampling. Cryptosporidium spp. DNA was detected by SSU-rRNA PCR-RFLP, using restriction enzymes SspI, VspI and MboII. C. parvum and C. ubiquitum isolates were further characterized at the GP60 locus. Our results reveal that Cryptosporidium spp. is widely distributed in small ruminant farms (47.4-50.0%), although its prevalence is low in both hosts (5.9-6.0%). No significant differences in individual prevalence were detected between age groups. C. xiaoi and the zoonotic C. parvum and C. ubiquitum were identified. In sheep, C. parvum was the predominant species and its prevalence increased with age, in contrast to C. xiaoi; C. ubiquitum was an occasional finding in adults. In goats, C. xiaoi and C. ubiquitum were the most frequent species and slightly more prevalent in adults than in post-weaned kids, in contrast to C. parvum. Subtyping analysis of C. parvum isolates revealed the presence of IIaA15G2R1 and IIaA14G2R1 in sheep, whereas IIaA13G1R1 and IIdA17G1 were restricted to goats; only the C. ubiquitum XIIa subtype 3 was found. Although the prevalences detected are low, these values are probably underestimated due to, amongst others, the cross-sectional design of the study and the intermittent oocyst-excretion of post-weaned and adult small ruminants. Thus, these animals may play an important role in the appearance of cryptosporidiosis outbreaks in humans and domestic ruminant neonates and therefore should be considered as a potential threat to animal production and human health.

Hypoderma actaeon: an emerging myiasis in roe deer (Capreolus capreolus)

Subcutaneous larvae of Hypoderma spp. (Diptera: Oestridae) were detected in the dorsal region in 10 roe deer, Capreolus capreolus (Artiodactyla: Cervidae), hunted in central Spain between January and March 2016. All larvae were found in the inner side of the hide during the skinning of the animals. The study of the morphological features of eight larvae of different stages collected from two animals allowed the identification of Hypoderma actaeon Brauer. The small size (4–5 mm) of some of the first instars suggests that the internal lifecycle of H. actaeon may be exclusively subcutaneous. This is the first confirmation of H. actaeon in roe deer; however, further studies to assess the spread of the parasite and to follow the evolution of this myiasis in roe deer are needed.

Prevalence and risk factors associated to Eimeria spp. infection in unweaned alpacas (Vicugna pacos) from Southern Peru.

A total of 350 faecal samples from unweaned alpacas over 3 months of age were collected from 23 herds in order to determine the prevalence of Eimeria spp. in Southern Peru and to identify the risk factors associated to Eimeria infection in young alpacas. Samples were examined by a flotation technique and the identification of risk factors was assessed by a logistic regression analysis. Sixty four percent of the examined animals shed Eimeria oocysts; herd prevalence was 96%, with an intra-herd prevalence of 60% (range 5.9-100%). Five different Eimeria species were identified, being E. lamae (91%), E. alpacae (87%) and E. punoensis (78%) the most prevalent; E. macusaniensis (35%) and E. ivitaensis (13%) were less common. Mixed-species infections were more frequent (78%) than single infections (22%). E. lamae was the most common monospecific infection and E. lamae/E. alpacae the most frequent association. The geographical area has a significant effect on Eimeria infection rates (74.9% wet Puna vs 37.4% dry Puna) as well as the breeding system (65.1% traditional vs 63.8% modern). In contrast, the sex of the animals (64.6% males vs 64.0% females) showed no influence on the prevalence of infection by Eimeria. The high prevalence found at both individual and herd level and the common presence of highly pathogenic Eimeria species may lead to important economic losses for alpaca breeders and could require the implementation of suitable control measures.

Molecular identification of Borrelia spirochetes in questing Ixodes ricinus from northwestern Spain

BackgroundIxodes ricinus, the predominant tick species in Europe, can transmit the causative agents of important human diseases such as Lyme borreliosis (LB), caused by Borrelia spirochetes. In northern Spain, LB is considered endemic; recently, a significant increase of the annual incidence of LB was reported in the northwestern (NW) region.MethodsIn order to provide information on the prevalence of Borrelia spp., pooled and individually free-living I. ricinus from NW Spain were molecularly analyzed. Positive samples were characterized at the fla and Glpq genes and the rrfA-rrlB intergenic spacer region to identify Borrelia species/genospecies.ResultsBorrelia burgdorferi (sensu lato) (s.l.) individual prevalence and MIR were significantly higher in adult females (32.3 and 16%) than in nymphs (18.8 and 6.2%) and adult males (15.6 and 8.4%). Five Borrelia genospecies belonging to the B. burgdorferi (s.l.) group were identified: B. garinii was predominant, followed by B. valaisiana, B. lusitaniae, B. afzelii and B. burgdorferi (sensu stricto) (s.s.). One species belonging to the tick-borne relapsing fever group (B. miyamotoi) was also found, showing low individual prevalence (1%), positive pool (0.7%) and MIR (0.1%) values. To our knowledge, this is the first citation of B. miyamotoi in free-living ticks from Spain.ConclusionsThe significant prevalences of B. burgdorferi (s.l.) genospecies detected in questing ticks from NW Spain are similar to those detected in northern and central European countries and higher to those previously found in Spain. These results together with the high incidence of LB in humans and the high seroprevalence of B. burgdorferi (s.l.) in roe deer shown in other studies reveal that the northwest area is one of the most risky regions for acquiring LB in Spain.

Prevalence and distribution of infectious and parasitic agents in roe deer from Spain and their possible role as reservoirs

Abstract In order to provide up-to-date information about the prevalence of infectious and parasitic agents in Spanish roe deer (Capreolus capreolus), samples from 93 animals hunted from January 2013 to April 2015 were collected and analysed by parasitological, serological and molecular techniques. Sampled animals came from four roe deer populations corresponding to Oceanic, Continental, Mediterranean and Mountainous ecosystems of Spain. Data regarding sex, age and year were also considered. A high percentage of roe deer (95.7%) resulted positive for at least one agent. Sarcocystis spp. was the most frequently diagnosed genus (88.8%), followed by gastrointestinal nematodes (62.9%) and Schmallenberg virus (53.5%). Varestrongylus capreoli (38%), Anaplasma phagocitophylum (34.2%), Eimeria spp. (29.2%), Toxoplasma gondii (25%) and Cephenemyia stimulator (23.8%) displayed medium prevalences and, finally low percentages were registered for Moniezia spp. (6.7%), Dictyocaulus noerneri (2.4%) and Mycobacterium avium subsp. paratuberculosis (1.5%). No infections by Neospora caninum, Bovine herpesvirus, pestivirus or Coxiella burnetii were found. Climate was significantly associated with the prevalence of T. gondii, C. stimulator and A. phagocitophylum, with higher prevalences in animals from Oceanic and Mediterranean areas. Our results suggest that infections affecting Spanish roe deer, especially those of parasitic aetiology, represent one of the causes of the descent in the abundance of this ungulate in the last years. Moreover, the high prevalence of zoonotic agents such as T. gondii and A. phagocitophylum could also have a great relevance in the environmental and/or Public Health.

Lesser housefly (Fannia canicularis) as possible mechanical vector for Aleutian mink disease virus

Flies are known vectors for a variety of infectious diseases in animals. In fur mink farming, one of the most severe diseases is Aleutian disease, which is caused by the Aleutian mink disease virus (AMDV). The presence of large fly populations is a frequent issue in mink farms; however, no studies assessing their role as AMDV carrier vectors have been conducted to-date. In order to determine the presence of AMDV in aerial flies from an infected mink farm, flies (n = 254) and environmental swab samples (n = 4) were collected from two farm barns. Fannia canicularis (L.) (Diptera: Muscidae) represented more than 99% of the fly population. One hundred and fifty specimens of this species were divided into pools of ten flies and analysed by qPCR, and positive samples were further sequenced. All fly pools and environmental samples tested positive for AMDV, and sequence analysis revealed identical genotypes in both types of samples. This is the first report of AMDV contamination in flies from mink farms, suggesting that F. canicularis may act as an AMDV vector. These results may be of interest for epidemiological studies and also for the improvement of control measures against this virus in mink farms.

Tick species diversity and population dynamics of Ixodes ricinus in Galicia (north-western Spain)

Ticks are the major arthropod vectors of animal and human pathogens in Europe; thus, information of tick species and their phenology allows identifying those areas and periods involving a high risk of exposure to particular tick-borne pathogens. In order to assess the diversity of exophilic tick species in north-western Spain, questing ticks were collected by flagging in 17 different locations; ticks were microscopically and molecularly identified at the 16S rDNA loci. In addition, phenology of questing ticks in the same region was studied in representative sampling areas of three different ecological regions, where ticks were monthly collected in 300-m transects during two complete years. The possible influence of different climate variables on the normalized accumulated fraction of nymphal and adult Ixodes ricinus was assessed using a generalized linear mixed model (GLMN). I. ricinus was the most frequently and widely distributed tick species; Dermacentor marginatus, Dermacentor reticulatus, Ixodes frontalis and Ixodes acuminatus were sporadically found. Nymphs were the most commonly collected stage. Both nymphs and adults of I. ricinus presented a unimodal activity pattern; density showed a peak in late spring-early summer whereas the lowest levels were found at late summer-early autumn. GLMN showed that both nymphal and adult I. ricinus phenology was directly associated with the accumulated temperature, photoperiod and average daily maximum temperature and inversely associated with average daily maximum relative humidity. This information could be used to predict the annual periods associated with a higher exposure and risk of transmission of pathogens and to complete the global map of distribution of tick species.

Three-year monitoring of paratuberculosis in dairy cattle by pooled faecal culture and individual prevalence estimation

Abstract The objective of this study was to assess the implementation of a three-year Mycobacterium avium subsp. paratuberculosis monitoring programme using pooled faecal culture in small and mediumsized dairy herds to classify them as infected or non-infected and apply proper hygiene and biosecurity measures. Over a three-year period, 35 dairy herds were analysed annually by faecal culture of ten pooled samples. In addition, proper hygiene and biosecurity protocols were implemented in the farms after the first testing round. Considering a herd as infected with at least one culture positive in any of the three years, the accumulated percentage of infected herds was 25.7%, 40% and 45.7%, for each year respectively. Assuming that all infected herds had been detected at the end of the study, the percentage of infected herds detected each year was 56.25% and 87.5% for the first and second year, respectively. Using frequentist and Bayesian approaches, the estimated individual prevalence revealed a downward trend from 3.30-3.65% in the first year to 1.66-1.86% in the third year. The results of this study indicate that pooled faecal culture allowed for proper classification of the herds and can be a useful tool for monitoring dairy herds against paratuberculosis. In addition, statistical analysis of pooled faecal culture results can be used to evaluate the evolution of individual prevalence in the population and therefore the function of the implemented control programmes.