Molecular detection of tick-borne bacteria and protozoa in cervids and wild boars from Portugal.
ABSTRACT: Wildlife can act as reservoir of different tick-borne pathogens, such as bacteria, parasites and viruses. The aim of the present study was to assess the presence of tick-borne bacteria and protozoa with veterinary and zoonotic importance in cervids and wild boars from the Centre and South of Portugal.One hundred and forty one blood samples from free-ranging ungulates including 73 red deer (Cervus elaphus), 65 wild boars (Sus scrofa) and three fallow deer (Dama dama) were tested for the presence of Anaplasma marginale/A. ovis, A. phagocytophilum, Anaplasma/Ehrlichia spp., Babesia/Theileria spp., Borrelia burgdorferi (sensu lato) (s.l.), and Rickettsia spp. DNA by PCR.Anaplasma spp. DNA was detected in 33 (43.4 %) cervids (31 red deer and two fallow deer) and in two (3.1 %) wild boars while Theileria spp. were found in 34 (44.7 %) cervids (32 red deer and two fallow deer) and in three (4.6 %) wild boar blood samples. Sequence analysis of msp4 sequences identified A. marginale, A. ovis, while the analysis of rDNA sequence data disclosed the presence of A. platys and A. phagocytophilum and T. capreoli and Theileria sp. OT3. Anaplasma spp./Theileria spp. mixed infections were found in 17 cervids (22.4 %) and in two wild boars (3.1 %). All samples were negative for Babesia sp., B. burgdorferi (s.l.), Ehrlichia sp. or Rickettsia sp.This is the first detection of Anaplasma marginale, A. ovis, A. phagocytophilum, A. platys, Theileria capreoli and Theileria sp. OT3 in cervids and wild boars from Portugal. Further studies concerning the potential pathogenicity of the different species of Anaplasma and Theileria infecting wild ungulates, the identification of their vector range, and their putative infectivity to domestic livestock and humans should be undertaken.
Project description:Data on the prevalence of piroplasms in buffaloes and large game animal species are lacking from several central European countries. Therefore, to investigate the presence of Babesia/Theileria DNA in these hosts, 239 blood and 270 spleen samples were taken from cervids (red, fallow, and roe deer), as well as from water buffaloes, mouflons, and wild boars in southwestern Hungary, followed by DNA extraction and molecular analysis for piroplasms. All samples from buffaloes and wild boars were PCR negative. Based on spleen samples, the prevalence of piroplasms was significantly higher in red deer (41.7%) than in fallow deer (23.5%). Two genotypes of Theileria capreoli were identified, which showed significant association with their host species (i.e. genotype "capreoli-CE1" was exclusively found in roe deer, whereas red and fallow deer harbored only genotype "elaphi-CE1"). Genotype "elaphi-CE1" of T. capreoli was also detected in one mouflon. No Babesia spp. were identified. In conclusion, in the evaluated region, genotypes of T. capreoli show host-associations among cervids, and at least one of these genotypes may infect mouflons.
Project description:BACKGROUND:Free-living ungulates are hosts of ixodid ticks and reservoirs of tick-borne microorganisms in central Europe and many regions around the world. Tissue samples and engorged ticks were obtained from roe deer, red deer, fallow deer, mouflon, and wild boar hunted in deciduous forests of south-western Slovakia. DNA isolated from these samples was screened for the presence of tick-borne microorganisms by PCR-based methods. RESULTS:Ticks were found to infest all examined ungulate species. The principal infesting tick was Ixodes ricinus, identified on 90.4% of wildlife, and included all developmental stages. Larvae and nymphs of Haemaphysalis concinna were feeding on 9.6% of wildlife. Two specimens of Dermacentor reticulatus were also identified. Ungulates were positive for A. phagocytophilum and Theileria spp. Anaplasma phagocytophilum was found to infect 96.1% of cervids, 88.9% of mouflon, and 28.2% of wild boar, whereas Theileria spp. was detected only in cervids (94.6%). Importantly, a high rate of cervids (89%) showed mixed infections with both these microorganisms. In addition to A. phagocytophilum and Theileria spp., Rickettsia helvetica, R. monacensis, unidentified Rickettsia sp., Coxiella burnetii, "Candidatus Neoehrlichia mikurensis", Borrelia burgdorferi (s.l.) and Babesia venatorum were identified in engorged I. ricinus. Furthermore, A. phagocytophilum, Babesia spp. and Theileria spp. were detected in engorged H. concinna. Analysis of 16S rRNA and groEL gene sequences revealed the presence of five and two A. phagocytophilum variants, respectively, among which sequences identified in wild boar showed identity to the sequence of the causative agent of human granulocytic anaplasmosis (HGA). Phylogenetic analysis of Theileria 18S rRNA gene sequences amplified from cervids and engorged I. ricinus ticks segregated jointly with sequences of T. capreoli isolates into a moderately supported monophyletic clade. CONCLUSIONS:The findings indicate that free-living ungulates are reservoirs for A. phagocytophilum and Theileria spp. and engorged ixodid ticks attached to ungulates are good sentinels for the presence of agents of public and veterinary concern. Further analyses of the A. phagocytophilum genetic variants and Theileria species and their associations with vector ticks and free-living ungulates are required.
Project description:BACKGROUND:Small ruminants are important hosts for various tick species and tick-associated organisms, many of which are zoonotic. The aim of the present study was to determine the presence of tick-borne protozoans and bacteria of public health and veterinary significance in goats and wild Siberian roe deer (Capreolus pygargus) from Heilongjiang Province, northeastern China. METHODS:The occurrence of piroplasms, Anaplasma phagocytophilum, A. bovis, A. marginale, A. capra, A. ovis, Ehrlichia spp. and spotted fever group rickettsiae was molecularly investigated and analyzed in 134 goats and 9 free ranging C. pygargus living in close proximity. RESULTS:Piroplasm DNA was detected in 16 (11.9%) goats and 5 C. pygargus. Sequence analysis of 18S rRNA sequences identified 3 Theileria species (T. luwenshuni, T. capreoli and T. cervi). Four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) were identified in goats and C. pygargus. Anaplasma ovis and A. bovis were detected in 11 (8.2%) and 6 (4.5%) goats, respectively; A. phagocytophilum, A. bovis and A. capra were found in 3, 7 and 3 C. pygargus, respectively. Sequence analysis of 16S rRNA sequences revealed the presence of 5 different genetic variants of A. bovis in goats and C. pygargus, while the analysis of 16S rRNA and gltA sequence data showed that A. capra isolates identified from C. pygargus were closely related to the genotype identified from sheep and Haemaphysalis qinghaiensis, but differed with the genotype from humans. Anaplasma/Theileria mixed infection was observed in 2 (1.5%) goats and 5 C. pygargus, and co-existence involving potential zoonotic organisms (A. phagocytophilum and A. capra) was found in 2 C. pygargus. All samples were negative for A. marginale, Ehrlichia spp. and SFG rickettsiae. CONCLUSIONS:These findings report the tick-borne pathogens in goats and C. pygargus, and a greater diversity of these pathogens were observed in wild animals. Three Theileria (T. luwenshuni, T. capreoli and T. cervi) and four Anaplasma species (A. ovis, A. phagocytophilum, A. bovis and A. capra) with veterinary and medical significance were identified in small domestic and wild ruminants. The contact between wild and domestic animals may increase the potential risk of spread and transmission of tick-borne diseases.
Project description:Hunting constitutes an important industry in Europe. However, data on the prevalence of vector-borne bacteria in large game animal species are lacking from several countries. Blood or spleen samples (239 and 270, respectively) were taken from red, fallow and roe deer, as well as from water buffaloes, mouflons and wild boars in Hungary, followed by DNA extraction and molecular analyses for Anaplasma phagocytophilum, haemoplasmas and rickettsiae.Based on blood samples, the prevalence rate of A. phagocytophilum infection was significantly higher in red deer (97.9%) than in fallow deer (72.7%) and roe deer (60%), and in all these compared to mouflons (6.3%). In addition, 39.2% of the spleen samples from wild boars were PCR positive for A. phagocytophilum, but none of the buffalos. Based on blood samples, the prevalence rates of both Mycoplasma wenyonii (Mw) and 'Candidatus M. haemobos' (CMh) infections were significantly higher in buffaloes (Mw: 91.2%; CMh: 73.3%) than in red deer (Mw: 64.6%; CMh: 45.8%), and in both of them compared to fallow deer (Mw: 30.3%; CMh: 9.1%) and roe deer (Mw: 20%; CMh: 1.5%). The prevalence of Mw and CMh infection significantly correlated with the body sizes of these hosts. Furthermore, Mw was significantly more prevalent than CMh in buffaloes, red and roe deer. Mycoplasma ovis was detected in mouflons, M. suis in wild boars, R. helvetica in one fallow deer and one mouflon, and an unidentified Rickettsia sp. in a fallow deer.Forest-dwelling game animal species were found to be important carriers of A. phagocytophilum. In contrast, animals grazing grassland (i.e. buffaloes) were less likely to get infected with this Ixodes ricinus-borne pathogen. Water buffaloes, deer species, mouflons and wild boars harbored haemoplasmas that may affect domestic ungulates. Evaluated animals with larger body size had significantly higher prevalence of infection with haemoplasmas compared to smaller deer species. The above host species rarely carried rickettsiae.
Project description:(1) Background: Wild cervids play an important role in transmission cycles of tick-borne pathogens; however, investigations of tick-borne pathogens in sika deer in Germany are lacking. (2) Methods: Spleen tissue of 74 sympatric wild cervids (30 roe deer, 7 fallow deer, 22 sika deer, 15 red deer) and of 27 red deer from a farm from southeastern Germany were analyzed by molecular methods for the presence of <i>Anaplasma phagocytophilum</i> and <i>Babesia</i> species. (3) Results: <i>Anaplasma phagocytophilum</i> and <i>Babesia</i> DNA was demonstrated in 90.5% and 47.3% of the 74 combined wild cervids and 14.8% and 18.5% of the farmed deer, respectively. Twelve <i>16S rRNA</i> variants of <i>A. phagocytophilum</i> were delineated. While the infection rate for <i>A. phagocytophilum</i> among the four cervid species was similar (71.4% to 100%), it varied significantly for <i>Babesia</i> between roe deer (73.3%), fallow deer (14.3%), sika deer (27.3%) and red deer (40.0%). Deer ?2 years of age tested significantly more often positive than the older deer for both <i>A. phagocytophilum</i> and <i>Babesia</i> species. (4) Conclusions: This study confirms the widespread occurrence of <i>A. phagocytophilum</i> and <i>Babesia</i> species in wild cervids and farmed red deer in Germany and documents the co-occurrence of the two tick-borne pathogens in free-ranging sika deer.
Project description:Tick-borne diseases (TBDs) caused by Theileria, Babesia, Anaplasma and Ehrlichia species are common in tropical and subtropical regions. In this study, we investigated the presence and genetic diversity of Theileria spp., Anaplasma ovis, B. ovis, E. ruminantium and Anaplasma spp. in sheep from the Machakos and Homa Bay counties of Kenya. In order to improve the diagnosis and control of ovine TBDs, a total of 76 blood samples from apparently healthy sheep were screened using a polymerase chain reaction (PCR). The assays were conducted using primers based on Theileria spp. 18S rRNA, Anaplasma ovis Major surface protein-4 (AoMSP4), B. ovis 18S rRNA, E. ruminantium pCS20 and Anaplasma spp. 16S rRNA. The overall infection rates for Theileria spp., A. ovis, E. ruminantium and Anaplasma spp. were 39/76 (51.3%), 26/76 (34.2%), 6/76 (7.9%) and 31/76 (40.8%), respectively. The overall co-infection was 47/76 (61.8%). All Theileria spp. positive samples were confirmed to be of Theileria ovis on sequencing. A phylogenetic analysis of the 18S rRNA gene sequences of T. ovis revealed that all isolates of this study clustered with T. ovis sequences extracted from the GenBank suggesting this gene is highly conserved. E. ruminantium pCS20 sequences were in the same clade on the phylogenetic tree. However, three AoMSP4 sequences from this study appeared in the same clade, while one sequence formed a separate branch revealing genetic divergence. The 16S rRNA sequencing revealed uncharacterised Anaplasma spp. and A. ovis. The phylogenetic analyses of the uncharacterised Anaplasma spp. revealed that the two sequences from this study appear in an independent clade from other sequences extracted from the GenBank. This study provides important information regarding the occurrence of tick-borne pathogens and their degree of genetic diversity among sheep in Kenya, which is useful for the diagnosis and control of TBDs.
Project description:Anaplasma and Mycobacterium species are known to modify gene expression in ruminants. The objectives of this study were (a) to characterize global gene expression profiles in European red deer (Cervus elaphus) in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/M. avium sub. paratuberculosis (MAP) infections, (b) to compare the expression of immune response genes between A. ovis- and A. ovis/M. bovis/MAP-infected deer, and (c) to characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and A. marginale. The results of this study showed that global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer results in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes also showed pathogen-specific signatures and the effect of infection with multiple pathogens on red deer host immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera use similar mechanisms to infect and multiply within ruminant host cells while pathogen-specific mechanisms underline differences that could contribute to disease characterization and diagnosis in ruminants. Overall design: A gene expression pre analysis was made in deers naturally infected with Anaplasma ovis and Mycobacterium complex using Affymetrix Bos taurus microarray to detect differentialy expressed genes. The immune response genes with variation in expression were analyzed by real time RT-PCR in the same samples and a bigger group of deers. A real time RT-PCR analysis was also made in Bos taurus naturally infected with Anaplasma marignale.
Project description:Babesiosis and Theileriosis are important worldwide-distributed tick-borne diseases for human and animals. Their presence in a particular area depends on the presence of suitable tick-vector and host species as well as competent reservoirs such as roe deer, one of the most abundant wild cervids in Spain. Spleen samples from 174 roe deer hunted in Spain were analysed to determine the prevalence of Babesia and Theileria species. DNA of both piroplasms was firstly detected using a commercial qPCR. Then, positive samples were molecularly characterized at the 18S rRNA and ITS1 genes of Babesia spp. and Theileria spp. The possible influence of some factors such as ecological area, age and sex was also assessed. Overall, 89.7% of roe deer were positive to any of the two piroplasms. Theileria spp. was more prevalent (60.9%) than Babesia spp. (19.0%); species identification could not be achieved in 17.3% of positive samples. Babesia prevalence was significantly higher in young animals and in roe deer from Oceanic regions, in contrast to Theileria spp. Five species were identified: Theileria sp. OT3 (60.3%), Babesia capreoli (15.5%), Babesia venatorum (2.9%), Theileria sp. 3185/02 (0.6%) and Babesia bigemina (0.6%). The coinfection B. capreoli/T. sp. OT3 was the most common (4.6%) followed by B. venatorum/T. sp. OT3 (0.6%) and B. bigemina/T. sp. OT3 (0.6%). Our results reveal that Theileria spp. and Babesia spp. are prevalent piroplasms in roe deer from Spain. These cervids can act as reservoirs for several Babesia and Theileria species, including the zoonotic B. venatorum. This study represents the first description of B. venatorum and B. bigemina in roe deer from Spain.
Project description:Anaplasma and Mycobacterium species are known to modify gene expression in ruminants. The objectives of this study were (a) to characterize global gene expression profiles in European red deer (Cervus elaphus) in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/M. avium sub. paratuberculosis (MAP) infections, (b) to compare the expression of immune response genes between A. ovis- and A. ovis/M. bovis/MAP-infected deer, and (c) to characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and A. marginale. The results of this study showed that global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer results in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes also showed pathogen-specific signatures and the effect of infection with multiple pathogens on red deer host immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera use similar mechanisms to infect and multiply within ruminant host cells while pathogen-specific mechanisms underline differences that could contribute to disease characterization and diagnosis in ruminants. A gene expression pre analysis was made in deers naturally infected with Anaplasma ovis and Mycobacterium complex using Affymetrix Bos taurus microarray to detect differentialy expressed genes. The immune response genes with variation in expression were analyzed by real time RT-PCR in the same samples and a bigger group of deers. A real time RT-PCR analysis was also made in Bos taurus naturally infected with Anaplasma marignale.
Project description:Infections with Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale are endemic in Kenya yet there is a lack of adequate information on their genotypes. This study established the genetic diversities of the above tick-borne hemoparasites infecting cattle in Kenya.Nested PCR and sequencing were used to determine the prevalence and genetic diversity of the above parasites in 192 cattle blood samples collected from Ngong and Machakos farms. B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, A. marginale major surface protein 5, Theileria spp. 18S rRNA, T. parva p104 and T. orientalis major piroplasm surface protein were used as the marker genes.B. bovis, B. bigemina, T. parva, T. velifera, T. taurotragi, T. mutans and A. marginale were prevalent in both farms, whereas T. ovis, Theileria sp. (buffalo) and T. orientalis were found only in Ngong farm. Co-infections were observed in more than 50 % of positive samples in both farms. Babesia parasites and A. marginale sequences were highly conserved while T. parva and T. orientalis were polymorphic. Cattle-derived T. parva was detected in Machakos farm. However, cattle and buffalo-derived Theileria were detected in Ngong farm suggesting interactions between cattle and wild buffaloes. Generally, the pathogens detected in Kenya were genetically related to the other African isolates but different from the isolates in other continents.The current findings reaffirm the endemicity and co-infection of cattle with tick-borne hemoparasites, and the role of wildlife in pathogens transmission and population genetics in Kenya.