First Molecular Characterization of Hypoderma actaeon in Cattle and Red Deer (Cervus elaphus) in Portugal.
ABSTRACT: Hypoderma spp. larvae cause subcutaneous myiasis in several animal species. The objective of the present investigation was to identify and characterize morphologically and molecularly the larvae of Hypoderma spp. collected from cattle (Bos taurus taurus) and red deer (Cervus elaphus) in the district of Castelo Branco, Portugal. For this purpose, a total of 8 larvae were collected from cattle (n=2) and red deer (n=6). After morphological identification of Hypoderma spp. larvae, molecular characterization was based on PCR-RFLP and mitochondrial CO1 gene sequence analysis. All larvae were morphologically characterized as the third instar larvae (L3) of H. actaeon. Two restriction enzymes were used for molecular identification of the larvae. TaqI restriction enzyme was not able to cut H. actaeon. However, MboII restriction enzyme differentiated Hypoderma species showing 210 and 450 bp bands in H. actaeon. Furthermore, according to the alignment of the mt-CO1 gene sequences of Hypoderma species and to PCR-RFLP findings, all the identified Hypoderma larvae were confirmed as H. actaeon. This is the first report of identification of Hypoderma spp. (Diptera; Oestridae) from cattle and red deer in Portugal, based on morphological and molecular analyses.
Project description:Nematodes of the genera Elaphostrongylus and Dictyocaulus are associated with disease in semi-domesticated tundra reindeer and farmed red deer whereas less knowledge exists in the wild. Their first stage larvae (L1) develop to the infective third stage (L3) in the environment; Elaphostrongylus spp. within intermediate gastropod hosts and Dictyocaulus spp. as free-living larvae. Larval development of Elaphostrongylus is highly temperature dependent with a developmental minimum of 9-10?°C. Larval development of Dictyocaulus spp. may occur at low temperatures (5?°C) but the larvae are sensitive to desiccation. We examined the prevalence and intensity of Elaphostrongylus spp. and Dictyocaulus spp. infections in six wild reindeer and two wild red deer populations in relation to altitude, temperature and rainfall in their respective main summer pasture area over the 5 summers prior to sampling. The parasitological examination was based upon morphological identification of L1 in the faeces of hunted animals. Altitude was calculated from animal position data and temperature and precipitation by means of a nationwide gridded data set. Temperature decreased with increasing altitude, from 13.3?°C for the lowest located red deer population (300?m) to 6.1?°C for the highest located reindeer population (1400?m). No significant relationship between altitude and rainfall was identified. Elaphostrongylus spp. infection decreased in prevalence with increasing altitude, being identified in 89% of investigated samples from the lowest located population and in 3% of samples from the highest. The prevalence of Dictyocaulus spp. infection varied between 28 and 80% and no relationship with altitude was found. The intensity of Elaphostrongylus spp. infection was low in reindeer and moderate in red deer whereas the intensity of Dictyocaulus spp. infection was moderate in both species. Our results indicated that the climatic conditions in all areas studied were suitable for Dictyocaulus spp., whereas summer temperature was a restrictive factor for Elaphostrongylus sp. in reindeer.
Project description: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: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: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:The aim of the study was to identify genes which are differentially expressed in the peripheral blood nuclear cells of two breeds of cattle (Holstein-Friesian and Polish Red) and cervine in different points in their physiological states (dry-off period, peak of lactation) RNA from peripheral blood nuclear cells taken from cattle and cervine in peak lactation and dry period were hybridized to Agilent two color microarrays with a common reference. There were four Holstein-Friesian cattle, four Polish Red cattle and four deer investigated. The whole blood was drawn in two time point from each animal – during dry period and peak lactation. This means that there were six research groups (Holstein-Friesian cattle in dry period and Holstein-Friesian cattle in peak lactation; Polish Red cattle in dry period and Polish Red cattle in peak lactation; Deer in dry period and Deer in peak lactation). Using Gene Spring Software (one-way ANOVA and Tukey's HSD Post-hoc test) three lists of differentially expressed transcripts were obtained: a list of 576 transcripts which differ deer in dry period and in peak lactation, a list of 437 transcripts which differ Holstein-Friesian cattle in dry period and in peak lactation and a list of 158 transcripts which differ Polish Red cattle in dry period and in peak lactation.
Project description:PURPOSE:Sarcocystis spp. are protozoan parasites of livestock which also infect birds, lower vertebrates and mammals, including man. Wild and domestic ruminants such as red deer, roe deer, fallow deer, cattle, sheep and goats may act as intermediate hosts for many Sarcocystis species, some of which are significant pathogens causing sarcocystosis in livestock and humans. The purpose of the present study was to determine the prevalence of Sarcocystis species in fallow deer farmed in an open pasture system. METHODS:Samples of heart and oesophagus tissue taken from five fallow deer were examined by light microscope for the presence of sarcocysts. Genomic DNA was extracted from individual sarcocysts. ssu rRNA was successfully amplified using their DNA as templates. RESULTS:Analysis of the ssu rRNA identified the presence of two S. morae sarcocysts in the heart tissue; similarly, S. gracilis sarcocysts were identified in the heart and oesophagus, and Sarcocystis sp. most closely related to S. linearis and S. taeniata were detected in oseophagus. CONCLUSIONS:These findings confirm the presence of Sarcocystis spp. in farmed fallow deer in Poland; however, more molecular studies are needed.
Project description:Cervids are known to be reservoirs of zoonotic bacteria transmitted by ticks. This study aimed to identify the Anaplasma species carried by captive red deer and swamp deer in a wild fauna reserve in France. Blood from 59 red deer and 7 swamp deer was collected and analyzed over a period of two years. A semi-nested PCR targeting the 23S rRNA was performed to detect and characterize Anaplasma spp. and determine the presence of zoonotic species. Anaplasma phagocytophilum was identified in 14/59 red deer (23.7%) but it was not identified in any of the swamp deer (7 animals). Three sequences could not be assigned to any particular species based on the 23S rRNA sequences. Complementary nested PCR targeting 16S rRNA, gltA and groEL genes and sequencing analysis then identified these sequences as a recently reported zoonotic species, Anaplasma capra; this species was found in 2 red deer (Cervus elaphus) and 1 swamp deer (Rucervus duvaucelii). This is the first report of the tick-borne zoonotic bacterium A. capra in France, a species otherwise described only in China, Japan, Malaysia and South Korea in goats, sheep, deer, cattle and Japanese serows (Capricornis crispus). While this bacterium may have been introduced into the reserve by infected imported animals, its local epidemiological cycle via tick transmission seems possible as locally born deer were found infected. Diagnostic methods, especially molecular ones, should take into account the potential infection of animals and humans with this species.
Project description:BACKGROUND:Cryptosporidium spp. are causative agents of gastrointestinal diseases in a wide variety of vertebrate hosts. Mortality resulting from the disease is low in livestock, although severe cryptosporidiosis has been associated with fatality in young animals. METHODS:The goal of this systematic review and meta-analysis was to review the prevalence and molecular data on Cryptosporidium infections in selected terrestrial domestic and wild ungulates of the families Bovidae (bison, buffalo, cattle, goat, impala, mouflon sheep, sheep, yak), Cervidae (red deer, roe deer, white-tailed deer), Camelidae (alpaca, camel), Suidae (boar, pig), Giraffidae (giraffes) and Equidae (horses). Data collection was carried out using PubMed, Scopus, Science Direct and Cochran databases, with 429 papers being included in this systematic analysis. RESULTS:The results show that overall 18.9% of ungulates from the investigated species were infected with Cryptosporidium spp. Considering livestock species (cattle, sheep, goats, pigs, horses and buffaloes), analysis revealed higher Cryptosporidium infection prevalence in ungulates of the Cetartiodactyla than in those of the Perissodactyla, with cattle (29%) being the most commonly infected farm animal. CONCLUSIONS:Overall, the investigated domestic ungulates are considered potential sources of Cryptosporidium contamination in the environment. Control measures should be developed to reduce the occurrence of Cryptosporidium infection in these animals. Furthermore, literature on wild populations of the named ungulate species revealed a widespread presence and potential reservoir function of wildlife.
Project description:In the northern hemisphere, Caribou (Rangifer spp.) populations are known to be infested with the skin-penetrating ectoparasite, Hypoderma tarandi (Diptera; Oestridae). Although regarded as host specific, H. tarandi has been reported from other species, and has become of increasing concern as a zoonosis infecting humans. In February 2012, concurrent with the hunting of muskoxen, we examined carcasses for muscle and tissue parasites, and recorded warble larvae infestations. DNA extracted from samples of larvae was amplified targeting 579 bp of the COI gene, and subsequently sequenced, to be confirmed as H. tarandi. Infestation by oestrid flies has not previously been reported in muskoxen in West Greenland.
Project description:Anaplasma phagocytophilum and Babesia spp. are causative agents of tick-borne infections that are increasingly considered as a threat to animal and public health. To assess the role of cervids in the maintenance of zoonotic pathogens in Norway, we investigated the prevalence of A. phagocytophilum and Babesia spp. in free-ranging roe deer and red deer. Initial screening of spleen samples of 104 animals by multiplex real-time PCR targeting the major surface protein (msp2) gene and 18S rRNA revealed the presence of A. phagocytophilum infection in 81.1% red deer (Cervus elaphus) and 88.1% roe deer (Capreolus capreolus), and Babesia spp. parasites in 64.9% red deer and 83.6% roe deer, respectively. Co-infections were found in 62.2% red deer and 79.9% roe deer. Nested PCR and sequence analysis of partial msp4 and 18S rRNA genes were performed for molecular characterization of A. phagocytophilum strains and Babesia species. A total of eleven A. phagocytophilum msp4 gene sequence variants were identified: five different variants were 100% identical to corresponding A. phagocytophilum sequences deposited in the GenBank database, while other six sequence variants had unique nucleotide polymorphisms. Sequence analysis of the 18S rRNA gene demonstrated the presence of multiple Babesia species, including Babesia capreoli, Babesia divergens, Babesia venatorum and Babesia odocoilei/Babesia cf. odocoilei. This study is the first report demonstrating the prevalence and molecular characterization of A. phagocytophilum strains and Babesia species in roe deer and red deer in Norway. The high infection and co-infection rates with A. phagocytophilum and Babesia spp. in red deer and roe deer suggest that these cervids may play an important role in the transmission of single and multiple pathogens.