A novel time-saving multiplex PCR assay for detecting and discriminating the most common canine Babesia species in Europe.

In Europe, most cases of canine babesiosis are caused by Babesia canis, Babesia vogeli (large piroplasms) and Babesia vulpes (small piroplasm). Molecular diagnosis is recommended due to its high sensitivity. Species identification after sequencing allows applying a rapid and efficient treatment, leading to a better prognosis; however, it is expensive and time-consuming. Thus, the objective of the present study was to develop a time-saving multiplex polymerase chain reaction (PCR) for simultaneously detecting and discriminating between large and small forms without sequence analysis. A new multiplex PCR was designed and tested using blood samples from 79 dogs showing clinical signs compatible with babesiosis which were previously analysed using blood smears and molecular methods. Multiplex PCR successfully discriminated between both Babesia groups showing bands of 700 and 890 bp for B. canis/B. vogeli and B. vulpes, respectively. No significant differences in the results of both PCR were detected and a substantial agreement between protocols (κ = 0.64) was found. Our multiplex PCR represents a reliable tool for detecting infections by the major Babesia spp. in dogs from Europe. Since no sequence analysis is required for identifying the species involved, this PCR allows the rapid administration of an appropriate treatment, thus improving the survival rate of the infected animals. In addition, it will represent a helpful tool for unravelling the real prevalence and distribution of B. vulpes and its implication in clinical cases.

Tick-borne pathogens in ticks from urban and suburban areas of north-western Spain: Importance of Ixodes frontalis harbouring zoonotic pathogens.

To identify the questing tick populations in urban and suburban areas from the city of Lugo (NW Spain), ticks were collected monthly by flagging. The presence of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum also was determined by polymerase chain reaction (PCR) and sequence analysis. Overall, 342 questing ticks were collected; the tick abundance was higher in suburban (95.9%) than in urban areas (4.1%). Ixodes frontalis was the most abundant (86.5%); 88.5% were larvae, 11.1% nymphs and 0.3% adults. All development stages of I. ricinus (7.3%) and adults of Rhipicephalus sanguineus sensu lato (5.8%) and Dermacentor reticulatus (0.3%) were found. Rickettsia spp. (31.9%) was more prevalent than Borrelia spp. (2.7%); no ticks were positive to A. phagocytophilum. Six Rickettsia species were identified (R. slovaca, R. monacensis, R. massiliae, R. raoultii, R. sibirica subsp. mongolitimonae and R. aeschielmanii); Candidatus Rickettsia rioja and two novel Rickettsia species also were detected. In addition, Borrelia turdi (1.8%) and B. valaisiana (0.9%) were identified in Ixodes ticks. This is the first report of R. slovaca in R. sanguineus s.l. and of R. monacensis, R. raoultii, R. slovaca, R. sibirica subsp. mongolitimonae and Ca. R. rioja in I. frontalis. Since most of the pathogens detected are zoonotic, their presence in these areas may have implications for public health.

Specificity of Stenurus (Metastrongyloidea: Pseudaliidae) infections in odontocetes stranded along the north-west Spanish coast.

Parasites extracted from the lungs and the pterygoid sinus complex of 6 species of odontocetes stranded along the north-west Spanish coast (Northeast Atlantic) between 2009 and 2019 were morphologically identified. The samples belonged to 14 specimens, including 3 harbour porpoises, Phocoena phocoena, 6 short-finned pilot whales, Globicephala macrorhynchus, 1 long-finned pilot whale, Globicephala melas, 1 Risso's dolphin, Grampus griseus, 1 striped dolphin, Stenella coeruleoalba and 2 bottlenose dolphins, Tursiops truncatus. All animals (14/14) were infected by nematodes of the genus Stenurus spp.; moreover, two of them presented a mixed lung nematode infection by Stenurus spp. and Halocercus spp., and another two a mixed infection by Stenurus spp. and the trematode Nasitrema spp. in the pterygoid sinuses. The morphological characterization of the Stenurus specimens revealed the existence of three different species: Stenurus minor, present in the pterygoid sinuses of harbour porpoises with a mean intensity of 43.0 ± 9.0; Stenurus globicephalae, in the pterygoid sinuses of pilot whales and the Risso's dolphin (370.3 ± 579.4); and Stenurus ovatus infecting bottlenose and striped dolphins' lungs (47.7 ± 76.5). This is the first citation of S. minor and S. ovatus in odontoceti from the Galician coast. Nematodes of the genus Stenurus are frequent in odontocetes stranded along the north-west Spanish coast. A clear host-parasite association was observed between S. minor and the Phocoenidae family, between S. globicephalae and the subfamily Globicephalinae and between S. ovatus and subfamily Delphininae. Different trophic position and niche segregation may lead to different patterns of specificity.

Traumatic myiasis by Wohlfahrtia magnifica in sheep flocks from southeastern Spain: prevalence and risk factors.

Wohlfahrtia magnifica (Diptera: Sarcophagidae) is an obligate myiasis-causing fly of livestock that is widely distributed throughout the Mediterranean basin. A total of 73 683 sheep from 122 flocks in Albacete Province (southeastern [SE] Spain) were examined to detect active traumatic myiasis. The influence of several individual and flock factors on the prevalence was also assessed. The overall flock prevalence of traumatic myiasis was 95.9%, with an individual prevalence of 7.1%. Wohlfahrtia magnifica was identified in all of the positive animals except one that was infested by Lucilia sericata (Diptera: Calliphoridae). Adults of both genders showed a higher risk of infestation than juveniles, whereas males of all ages displayed an increased risk of infestation than females of all ages; the existence of a lambing building on the farm, poor husbandry facilities and the location area were also identified as risk factors. Prevalence was highest in the summer, and the fly season lasted from March to November. Genitalia were the main location of infestation in males (94.4%) and females (76.3%). Our results reveal that traumatic myiasis by W. magnifica is widespread in southeastern Spain, hence preventive measures minimizing the attractiveness of sheep to gravid flies are necessary to avoid health and welfare problems and significant production losses in sheep flocks.

Molecular detection and identification of piroplasms (Babesia spp. and Theileria spp.) and Anaplasma phagocytophilum in questing ticks from northwest Spain.

Anaplasma phagocytophilum and some piroplasm species are pathogens mainly transmitted by Ixodes ricinus. Considering that this tick species is predominant in north-western Spain, individual specimens (652 nymphs, 202 females and 202 males) and 23 larval pools were processed to determine the prevalence of these pathogens in questing I. ricinus from that region. Additionally, Dermacentor marginatus, Dermacentor reticulatus, Ixodes frontalis and Ixodes acuminatus were individually analysed. The groESL operon as well as the 16S rRNA and msp2 genes of Anaplasma were analysed. Similarly, piroplasms were identified at the 18S rRNA gene and the ITS1 of Babesia spp. and Theileria spp. Babesia venatorum (1.5%), A. phagocytophilum (0.7%), Babesia microti (0.3%) and Theileria sp. OT3 (0.2%) were detected in I. ricinus. A single I. frontalis (8.3%) tested positive to A. phagocytophilum. Although a low percentage of I. ricinus were infected with A. phagocytophilum and piroplasms, a potentially human pathogenic variant of A. phagocytophilum was detected, and both Babesia species found were zoonotic. Since the vector of Theileria sp. OT3 remains unknown, further investigations are needed to unravel the role of I. ricinus in the transmission of this piroplasm.

Cytokine expression in bovine PBMC cultures stimulated with Hypoderma lineatum antigens.

Hypoderma antigens are involved in host inflammation and immune response, conditioning larvae survival. In this study, peripheral blood mononuclear cell (PBMC) cultures from Hypoderma sensitized and unsensitized cattle were performed to determine the effect of H. lineatum antigens and incubation time (18, 24, 48 h) on IFN-γ, TNF-α, IL-10 and IL-4 mRNA gene expression determined by RT-qPCR. TNF-α and IL-4 gene expression were higher in Hypoderma previously sensitized PBMCs, suggesting that a mixed Th1/Th2 response may play a significant role in host defence reactions against Hypoderma exhibited by previously infested cattle. Incubation time had a significant effect on IL-10 and TNF-α gene expression, which decreased over time. Regarding to H. lineatum antigens, the crude larval extract and the purified fraction hypodermin B (HB) produced a significant reduction of the mRNA expression levels of the proinflammatory cytokine, IFN-γ; moreover, the HB had a stimulating effect on the mRNA gene expression of the anti-inflammatory cytokine IL-10, demonstrating that the parasite would modulate the host defence mechanisms by avoiding harmful immune responses that would limit its survival into the host tissues.

Temporal and spatial spread of Hypoderma actaeon infection in roe deer from peninsular Spain determined by an indirect enzyme-linked immunosorbent assay.

The host switching of Hypoderma actaeon (Diptera: Oestridae), a specific parasite of red deer (Cervus elaphus), towards roe deer (Capreolus capreolus) has been recently reported in Spain. To provide information about the temporal and spatial spreading of H. actaeon infection in roe deer, 244 serum samples from animals hunted in Spain between 2013 and 2018 were analysed by an indirect enzyme-linked immunosorbent assay. The overall seropositivity was 13.9%. Seropositivity was higher in continental (27.7%) and mountainous (12%) areas from central Spain, followed by southern-Mediterranean (11.2%) and northern-oceanic regions (3.5%). Differences were significant between central-continental and northern-oceanic regions (P = 0.003). No differences were found according to the sex and age of roe deer (P > 0.05). In 2013, all seropositive animals were concentrated in two distant areas in central and southern Spain, suggesting that the host switch could have occurred independently in both regions. Changes in the pattern of distribution of red deer and roe deer could have favoured the spreading of this myiasis towards roe deer, indicating that roe deer may become infested by H. actaeon in areas where both cervids coexist at high densities.

Occurrence of Borrelia and Borreliella species in Ixodes ricinus collected from roe deer in northwestern Spain.

Ixodes ricinus, comprising the predominant tick species in Europe, can transmit important human pathogens, including Borreliella spp., the causal agent of Lyme borreliosis. One hundred and seventy five roe deer hunted in two areas (plateau and mountain) of Galicia (northwest Spain) were examined for the presence of ticks; all roe deer were infested by I. ricinus. Nymphs (n = 1000), males (n = 1449) and females (n = 1000) of I. ricinus were analysed in pools of up to 10 ticks to detect both Borreliella and Borrelia DNA. The average number of I. ricinus per roe deer was similar in both areas, regardless of the life stage; although the percentage of Borreliella and Borrelia positive pools was higher in ticks collected from roe deer hunted in the plateau area, no significant differences were detected. Sequence analysis at the flagellin gene allowed the identification of four Borreliella species (Borreliella afzelii, Borreliella garinii, Borreliella lusitaniae and Borreliella valaisiana) and Borrelia miyamotoi in adult males; only B. valaisiana and B. miyamotoi were detected in nymphs and all females were negative. All Borreliella and Borrelia species found in roe deer were previously identified in questing I. ricinus collected in the same study area, although the prevalence was lower in the present study. The analysis of male I. ricinus ticks collected from roe deer gives a good estimation of Borreliella diversity in questing ticks.