Alberto Prieto

Seroprevalence and risk factors associated with Visna/Maedi virus in semi-intensive lamb-producing flocks in northwestern Spain

A cross-sectional study was carried out to determine Visna/Maedi virus (VMV) seroprevalence and risk factors in semi-intensive lamb-producing flocks as a prelude to establishing a monitoring program in northwestern (NW) Spain. A total of 15,155 serum samples were taken from 78 commercial flocks and were submitted to an indirect VMV ELISA. Association between potential risk factors and seroprevalence at the flock level was assessed using a multivariable logistic regression model. A Generalized Estimating Equations (GEE) model and Exhaustive Chi-squared Automatic Interaction Detector (CHAID) were used to determine the seropositivity against VMV at the individual animal level. Individual seropositivity was 24.8% while 52.6% of the flocks examined had a true seroprevalence ≥1%. Flock size and introduction of new animals in the flock were significantly associated with seropositivity at the flock level. Flock size, sheep-goat contact, type of housing of lambs prior to weaning and age were significantly associated with individual VMV seropositivity. Confinement of lambs in preweaning lamb groups and high sheep-goat contact, regardless of the low number of goats per flock, were risk factors associated with individual VMV seropositivity, suggesting that these two factors are crucial for VMV control in semi-intensive lamb-producing flocks. These factors should be considered for developing more efficient strategies that will reduce the rate of VMV transmission.

Application of real-time PCR to detect Aleutian Mink Disease Virus on environmental farm sources

The Aleutian Mink Virus (AMDV) causes the Aleutian Mink Disease (AMD) or Mink Plasmacytosis, a disease responsible of high economic losses for industry worldwide. Despite there is evidence of the environmental persistence of the virus, there is not literature on the detection of this virus in environmental samples in farms and this fact would have great importance in the control programs of the disease. In order to detect contamination caused by AMDV on farms, several environmental samples were taken and examined using qPCR. 93.9% of samples taken from farms confirmed to be infected tested positive. The virus was also detected on a farm which, despite having no previous positive results, was sharing personnel with an infected farm. All samples taken from AMD-free farms tested negative, including a farm where an eradication procedure by stamping out had been performed during the preceding months. Higher contamination levels were observed in samples from those surfaces in direct contact with animals. These results are the first demonstration of environmental contamination in farms, hitherto suggested by epidemiological evidences, caused by AMDV on surfaces, furniture and equipments inside mink farms. qPCR is an useful tool for evaluating the spread of AMDV into the environment, and it may have important applications within the disease control programs.

Fetal gender determination by first-trimester ultrasound in dairy cows under routine herd management in Northwest Spain

Ultrasonography (US) provides detailed visualization of the fetus in early pregnancy in cows, thus allowing for fetal sex determination. The objective of this prospective observational study was to determine the feasibility and accuracy of a single US examination to diagnose fetal sex in dairy cattle under routine reproductive management conditions. For this purpose, 953 Holstein cows at 7-16 weeks of gestation were examined. Gender assignment was performed in 822 cows, while the genitalia could not be clearly visualized in 131 (13.7%) of the fetuses. After calving, it was verified that 99.3% of the diagnoses were accurate. Fetal sex was correctly determined by US in 99.5% of male fetuses and 98.8% of female fetuses. Fetal sex determination was less accurate when conducted before d 55 of gestation. Likewise, it was verified that fetal sex, cow age and ultrasonographic diagnosis section did not have a significant influence (P>0.05) on diagnostic accuracy. With respect to the plane used for diagnosis, the sagittal view was poorly used for early pregnancy diagnosis, whereas the longitudinal and cross-sectional planes were used most frequently. These results demonstrate that US can be routinely applied under farm conditions to accurately determine the fetal sex in cattle between days 51 and 111 of gestation without apparent influence of cow age, US scanning plane or fetal sex. Conversely, days of gestation affected the accuracy and feasibility of US gender determination, showing poorer results when the diagnosis was made before day 55 of gestation.

High spread of Schmallenberg virus among roe deer (Capreolus capreolus) in Spain.

Schmallenberg virus (SBV) is an emergent virus in northwestern Europe since 2011. We conducted this study to evaluate the spread of this virus in a wild ruminant community. During 2013 and 2014, 75 serum samples of roe deer were collected from different locations of the Spanish geography and analysed for the presence of antibodies. The results revealed a widespread exposure to SBV, as well as a high seroprevalence (53.3%, CI95% 42.16–64.18). These findings demand more research with regard to the impact of SBV on roe deer health and the interactions with livestock. Results also show this species as potentially suitable for monitoring the spread of the virus through sylvatic areas.

Distribution of Aleutian mink disease virus contamination in the environment of infected mink farms

Control and eradication of Aleutian Mink Disease Virus (AMDV) are a major concern for fur-bearing animal production. Despite notably reducing disease prevalence, current control programs are unable to prevent the reinfection of farms, and environmental AMDV persistence seems to play a major role regarding this issue. In this study 114 samples from different areas and elements of seven infected mink farms were analyzed by qPCR in order to evaluate the environmental distribution of AMDV load. Samples were classified into nine categories, depending on the type of sample and degree of proximity to the animals, the main source of infection. Two different commercial DNA extraction kits were employed in parallel for all samples. qPCR analysis showed 69.3% positive samples with one kit and 81.6% with the other, and significant differences between the two DNA extraction methods were found regarding AMDV DNA recovery. Regarding sample categorization, all categories showed a high percentage of AMDV positive samples (31%-100%). Quantification of positive samples showed a decrease in AMDV load from animal barns to the periphery of the farm. In addition, those elements in direct contact with animals, the street clothes and vehicles of farm workers and personal protective equipment used for sampling showed a high viral load, and statistical analysis revealed significant differences in AMDV load between the first and last categories. These results indicate high environmental contamination of positive farms, which is helpful for future considerations about cleaning and disinfection procedures and biosecurity protocols.

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.

Epidemiology of reproductive pathogens in semi-intensive lamb-producing flocks in North-West Spain: a comparative serological study.

The aim of this study was to better understand the epidemiology of five major reproductive pathogens in semi-intensive lamb-producing flocks in North-West Spain. Two thousand four hundred serum samples were collected from 44 flocks, and several factors were studied to assess their influence on seropositivity. Farms that tested seropositive for more than one pathogen were common (84.1%), but seroprevalence was high only for Toxoplasma gondii (38.1%), and low for the other four pathogens, namely, Neospora caninum (5.5%), Coxiella burnetii (4.0%), Chlamydia abortus (3.9%) and Pestivirus (2.3%). Farm level exposure was highest for T. gondii and N. caninum (100% and 72.7%, respectively). For both of these pathogens, and for C. burnetii, seropositivity was associated with age and area. Farm level exposure was lowest for Pestivirus and C. abortus (13.6% and 18.2%, respectively), and, for the latter, seropositivity was principally related to flock size.

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.

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.