Diversity of Anaplasma species and importance of mixed infections in roe deer from Spain.

Although wildlife can act as reservoirs of some Anaplasma species, studies on the presence and distribution of Anaplasma spp. in wild cervids are mainly limited and focused on zoonotic species. In order to identify the Anaplasma species in roe deer from Spain and to detect co-infections, 224 spleen samples were tested for Anaplasma spp. using a commercial qPCR; positive samples were further characterized using generic 16S rRNA primers and species-specific primers targeting the msp2 and groEL genes. Anaplasma DNA was detected in the 50.9% of samples, and four Anaplasma species were identified. Anaplasma phagocytophilum (43.8%) was predominant, followed by Anaplasma bovis (13.8%), Anaplasma capra (5.8%) and Anaplasma ovis (2.2%). In addition, strains similar to Anaplasma platys were found in nine animals. Most positive roe deer (71.9%) were infected with a single Anaplasma species, whereas co-infections with two (19.3%) or three (8.8%) Anaplasma species were also found. This study confirms the widespread occurrence of Anaplasma spp. in roe deer from Spain, being the first report of A. platys-like strains and A. capra in this cervid; it is also the first report of A. capra in Spain. The detection of Anaplasma species pathogenic for humans and/or domestic animals in roe deer suggests that this cervid may play a role in the sylvatic cycle of these bacteria contributing to the appearance of clinical anaplasmosis cases. In addition, co-infections are common in roe deer revealing that Anaplasma species specific PCR assays are essential for a reliable identification as well as for determining their real prevalence.

An Update on Cephenemyiosis in the European Roe Deer: Emergent Myiasis in Spain.

Cephenemyia stimulator is a Palearctic species developing in the nasal cavity and pharynx of roe deer (Capreolus capreolus). It is widely spread in the range of distribution of this ungulate in Europe. Since the first report of C. stimulator in Spain in 2001, a rapid geographic expansion has been observed, first in the north of the country, with high prevalence and intensities of infestation that caused some mortal cases, and, lately, also in Extremadura and Andalucía, the southernmost populations of European roe deer. These observations suggest an adaptation of this parasite to different ecosystems of the Iberian Peninsula. Almost simultaneously, C. stimulator is also expanding its range to northern Europe, with the first cases being reported in Sweden. Thus, Cephenemyia stimulator may be an example of a parasite currently displaying distributional changes along its southernmost and northernmost range margins. Thus, it is of the utmost importance to unravel all the epidemiological and clinical aspects of this myiasis, as well as implementing surveillance measures including reliable and non-invasive diagnostic techniques to monitor its expansion and adaptation to different ecosystems and/or hosts and to reduce the negative impact on roe deer populations.

Efficacy of Two Commercial Ready-To-Use PCV2 and Mycoplasma hyopneumoniae Vaccines under Field Conditions.

Porcine Circovirus Type 2 (PCV2) and Mycoplasma hyopneumoniae are economically important pathogens in swine farms. Vaccination is the main preventive measure for both infections. In order to test two ready-to-use bivalent vaccines, 646 piglets from a herd actively infected with both pathogens were stratified according to the sow parity number and randomly assigned to three groups: A and B were vaccinated with two different vaccines, respectively, while C remained as the unvaccinated control. Vaccine efficacy was assessed based on the weight, average daily weight gain (ADWG), degree of lung lesions, presence of PCV2 viremia by qPCR and presence of PCV2 and M. hyopneumoniae antibody levels by ELISA. Our data revealed that the sow parity did not influence the vaccine outcomes. Good results for most of the analyzed parameters were observed in both vaccinated groups. ADGW and final weight were higher and lung lesions were less evident in both vaccinated groups than in the control one, but only Group A showed a significant improvement. PCV2 viremia was not detected in Group A, but it did appear in Group B coinciding with its peak in Group C. Finally, both the PCV2 and M. hyopneumoniae serological patterns differed depending on the employed vaccine.

The Age-Related Cryptosporidium Species Distribution in Asymptomatic Cattle from North-Western SPAIN.

An age-related distribution of Cryptosporidium species has been reported in cattle, with C. parvum being predominant in suckling calves, C. bovis and C. ryanae being predominant in post-weaned calves and C. andersoni being predominant in adults. However, variants to this pattern have recently been reported. Thus, fecal samples (n = 594) from asymptomatic cattle were collected in north-western Spain. Animals were classified as <1 month (G1), 1-2 months (G2), 2-12 months (G3), 12-24 months (G4) and >2 years (G5). Cryptosporidium detection and species identification were performed by SSU rRNA PCR. Individual Cryptosporidium prevalence was 16.7%; it significantly decreased with age. Cryptosporidium parvum was predominant in G1 and C. bovis was predominant in the rest of the age classes; C. bovis and C. ryanae were especially prevalent in G2 and G3. Cryptosporidium occultus was not found in suckling calves. Finally, C. andersoni and C. xiaoi were occasionally detected in G5. The presence of C. parvum in all age classes implies significant animal and public health concerns. The predominance of C. bovis in cattle older than 1 month supports the idea that the age-related pattern of Cryptosporidium species described in cattle is not fully consistent, and thus further studies are still needed to identify those factors determining the species distribution.

Efficacy of Oxyclozanide and Closantel against Rumen Flukes (Paramphistomidae) in Naturally Infected Sheep.

Paramphistomosis is considered an emergent disease of ruminants in Europe. Some drugs have been found effective for treating paramphistomid infections in cattle, but data in sheep are currently limited. Thus, faecal samples from 25 adult sheep naturally infected with paramphistomids were collected weekly to test the efficacy of oxyclozanide and closantel. Three groups were performed: nine animals orally treated with a single dose of oxyclozanide (15 mg/kg bodyweight (BW) integrated the G-OXI group, whereas eight sheep orally treated with a single dose of closantel (10 mg/kg BW) were placed in a group called G-CLS. Eight untreated controls constituted the group G-CON. Oxyclozanide showed efficacies up to 90% until week 11 post-treatment, with a maximum efficacy of 98.3%, and significant differences were found between G-OXI and G-CON until the 26th week post-treatment. Closantel was insufficiently active (0-81%) throughout the study and differences compared to G-CON were never found. The present study reveals that oxyclozanide given at a single oral dose of 15 mg/kg BW is highly effective against adult rumen flukes in sheep. In addition, the use of a single oral dose of closantel at 10 mg/kg BW is not recommended for treating paramphistomid infections in sheep.

Environmental distribution of Porcine Circovirus Type 2 (PCV2) in swine herds with natural infection.

Porcine circovirus type 2 (PCV2) is the aetiological agent of PCV2-Systemic Disease (PCV2-SD) and PCV2-Subclinical Infection (PCV2-SI). PCV2 is highly resistant to environmental conditions, being able to remain in the farm environment and thus represent a risk for infection maintenance. The aim of this study was to identify, under field conditions, the possible critical points in the environment of non-vaccinated farrow-to-weaning swine farms where PCV2 could accumulate and persist. For that, environmental samples from five swine farms with PCV2-SD or PCV2-SI were taken and analysed by qPCR, including different farm areas, farm personnel and management implements. PCV2 DNA was detected in the environment of all farms (42.9% of positive samples). Overall, the PCV2-SD herd seemed to present more positive samples and higher viral loads than the PCV2-SI herds. At individual farm level, weaning areas appeared to be the most contaminated facilities. In addition, PCV2 was found at high levels in most samples from farm workers, especially work boots, suggesting that they may play a role in within-farm transmission. In addition, PCV2 was detected in areas without animals the like warehouses, offices and farm perimeter. Therefore, this study is helpful to improve measures to reduce within-farm PCV2 dissemination.

Longitudinal Study of Infection with Borrelia spp. in Questing Ticks from North-Western Spain.

Introduction:Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme borreliosis, is the most prevalent tick-borne pathogen in Europe, where it is mainly transmitted by Ixodes ricinus. This tick also circulates Borrelia miyamotoi, a member of the relapsing fever group of species. Objectives: A longitudinal study was performed to assess the prevalence of Borrelia spp. in questing ticks. Relationships between Borrelia prevalence in ticks and some variables such as tick development stage, sampling area, and questing tick density were assessed; in addition, the distribution of B. burgdorferi s.l. was analyzed to establish the periods of acarological risk. Methods: Ticks were collected monthly by flagging in three different ecological areas (coast, plateau, and mountain) in north-western Spain during a 2-year study. Borrelia DNA was detected by PCR, targeting the flagellin (fla) gene. Positive samples were also characterized at the rrfA-rrlB intergenic spacer region and the glycerophosphodiester phosphodiesterase (GlpQ) gene. Results:B. burgdorferi s.l. DNA was detected in 11.84% of I. ricinus. Five Lyme Borrelia species were identified (Borrelia afzelii, B. burgdorferi sensu stricto, Borrelia garinii, Borrelia lusitaniae, and Borrelia valaisiana). One single relapsing fever species (B. miyamotoi) was detected (0.85%). Questing Ixodes frontalis, Ixodes acuminatus, Dermacentor reticulatus, and Dermacentor marginatus yield negative results. The prevalence of B. burgdorferi s.l. was significantly higher in female ticks and in the mountain area. In addition, a seasonal pattern in the B. burgdorferi s.l. prevalence distribution throughout the study was not detected. Conclusions: The detection of a noticeable prevalence of B. burgdorferi s.l. in questing I. ricinus suggests a high acarological risk, especially in mountain area. There is no evidence of a relationship between the prevalence of B. burgdorferi s.l. and the density of questing ticks, nor traces of a seasonal pattern in the values of prevalence in ticks.

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.

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

BACKGROUND: Ixodes 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. METHODS: In 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. RESULTS: Borrelia 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. CONCLUSIONS: The 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.

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.