Search

Introduction Rhipicephalus bursa is a common tick parasite of small-to-medium size ungulates, principally in warm, temperate, and subtropical areas. Although common in livestock and showing a wide geographic distribution, its epidemiological role in tick-borne bacterial disease is barely known. This study addressed the knowledge gap and aimed to screen for the presence of Anaplasmataceae and spotted fever group (SFG) Rickettsia species in R. bursa ticks collected from domestic animals in Romania, Eastern Europe. Material and Methods A total of 64 pools of R. bursa ticks collected from small ungulates were tested by PCR for Anaplasmataceae DNA presence using group-specific primers. Specific testing was performed for Anaplasma marginale/A. centrale/A. ovis, A. platys, A. phagocytophilum, Ehrlichia canis, and SFG Rickettsia. The positive samples were purified and sequenced, and sequences analysis was used to identify the species and to confirm the PCR results. Results The only pathogen identified in this study was E. canis. The obtained sequences confirmed the PCR results. The presence of E. canis in R. bursa in Romania and in ticks from sheep was shown for the first time in this study. Conclusion Based on these findings, it may be presumed that the E. canis DNA originated from ticks; however, the vectorial role of R. bursa (and other arthropod species) in the transmission of E. canis should be proved experimentally.

Tickborne bacterial pathogens have been described worldwide as risk factors for both animal and human health. Spotted fevers caused by Rickettsiae may cause non-specific symptoms, which make clinical diagnosis difficult. The aim of the current study was to evaluate and review the diversity of SFG Rickettsiae in ticks collected in 41 counties in Romania. A total of 2028 questing and engorged ticks collected in Romania belonging to five species were tested by PCR amplification of

Introduction Bovine mastitis is an inflammatory disease of the udder that causes important economic losses in the animal breeding and dairy product industries. Nowadays, the conventional livestock antibiotic treatments are slowly being replaced by alternative treatments. In this context, the main aim of this study was to evaluate the efficacy of natural products in alternative treatment of bovine mastitis. Material and Methods Two natural formulations with previously suggested in vitro antimicrobial effect were tested in vivo on mastitic cows. Animals with a positive diagnosis for mastitis (n = 20) were divided into three treatment groups: two groups (n = 8) were administered formulations of propolis, alcoholic extracts of Brewers Gold and Perle hops, plum lichen, common mallow, marigold, absinthe wormwood, black poplar buds, lemon balm, and essential oils of oregano, lavender, and rosemary designated R4 and R7 (differing only in the latter being more concentrated) and one group (n = 4) a conventional antibiotic mixture. In vivo efficacy of treatments was evaluated by somatic cell and standard plate counts, the treatment being considered efficacious when both parameters were under the maximum limit. Results R7 was effective in the most cases, being therapeutically bactericidal in six out of eight cows, while R4 gave good results in three out of eight cows, and conventional antibiotics cured one out of four. Conclusion These results suggest the possible therapeutic potential of these natural products in bovine mastitis.

Honey bees (Apis mellifera) perform pollination service for many agricultural crops and contribute to the global economy in agriculture and bee products. However, honey bee health is an ongoing concern, as illustrated by persistent local population decline, caused by some severe bee diseases (e.g., nosemosis, AFB, EFB, chalkbrood). Three natural recipes are in development based on the bioactive compounds of different plants extract (Agastache foeniculum, Artemisia absinthium, Evernia prunastri, Humulus lupulus, Laurus nobilis, Origanum vulgare and Vaccinium myrtillus), characterised by HPLC-PDA. The antimicrobial activity of these recipes was tested in vitro against Paenibacillus larvae, Paenibacillus alvei, Brevibacillus laterosporus, Enterococcus faecalis, Ascosphaera apis and in vivo against Nosema ceranae. A mix of 20% blueberry, 40% absinthium, 10% oakmoss, 10% oregano, 10% Brewers Gold hops, 5% bay laurel and 5% anise hyssop extract showed the strongest antibacterial and antifungal activity. Combing several highly active plant extracts might be an alternative treatment against bee-disease-associated parasites and pathogens, in particular to replace synthetic antibiotics.

An amendment to this paper has been published and can be accessed via the original article.

Background Spotted fever group rickettsiae represent one of the most diverse groups of vector-borne bacteria, with several human pathogenic species showing an emerging trend worldwide. Most species are vectored by ticks (Ixodidae), with many zoonotic reservoir species among most terrestrial vertebrate groups. While the reservoir competence of many different vertebrate species is well known (e.g. birds, rodents and dogs), studies on insectivorous bats have been rarely performed despite their high species diversity, ubiquitous urban presence and importance in harboring zoonotic disease agents. Romania has a high diversity and ubiquity of bats. Moreover, seven out of eight SFG rickettsiae species with zoonotic potential were previously reported in Romania. Based on this, the aim of this study was to detect Rickettsia species in tissue samples in bats. Methods Here we report a large-scale study (322 bats belonging to 20 species) on the presence of Rickettsia spp. in Romanian bat species. Tissue samples from insectivorous bats were tested for the presence of Rickettsia DNA using PCR detection amplifying a 381 bp fragment of the gltA gene. Positive results were sequenced to confirm the results. The obtained results were statistically analyzed by chi-squared independence test. Results Positive results were obtained in 14.6% of bat samples. Sequence analysis confirmed the presence of R. monacensis in two bat species (Nyctalus noctula and Pipistrellus pipistrellus) in two locations. Conclusion This study provides the first evidence of a possible involvement of these bat species in the epidemiology of Rickettsia spp., highlighting the importance of bats in natural cycles of these vector-borne pathogens. Graphical Abstract

Wild vertebrate hosts can serve as reservoirs or amplification hosts for tick-borne pathogens (TBPs). Wild carnivores due to their large size have an increased risk for harboring large numbers of ticks. Moreover, their large home ranges and long lives may increase the risk of exposure to ticks and TBPs. Wild carnivores therefore may be good sentinel species with which to monitor the distribution of TBPs. We aimed to evaluate the presence of rickettsial DNA in wild carnivores and to compare its presence in different types of samples. In total, 95 wild carnivores from nine species, originating from 17 counties of Romania collected during 2014-18, were included in the study. From each animal, DNA was extracted from multiple tissue samples, including blood clot, heart, liver, lungs, spleen, kidney, lymph node, and bone marrow, and screened for the presence of rickettsial pathogen DNA (Anaplasma phagocytophilum, Ehrlichia canis, and Rickettsia spp.). Samples from 10 animals from six species (Canis aureus, Ursus arctos, Canis lupus, Felis sylvestris, Lutra lutra, and Martes foina) were found to be positive for A. phagocytophilum. The most frequently positive sample was the spleen. No animal was positive for Ehrlichia spp. or Rickettsia spp. Wild carnivores may be involved in the ecoepidemiology of A. phagocytophilum by maintaining the infection in synanthropic environments.

Ticks are medically important vectors of infectious diseases that are able to transmit pathogens to humans and animals. Tick-borne diseases represent a major health concern, posing an increasing risk to the public health during the last century and affecting millions of people. The aim of the current study was to provide epidemiological data regarding the presence of certain tick-borne pathogens in ticks feeding on humans in Romania. Overall, 522 Ixodes ricinus ticks collected from humans were screened for six pathogens: Borrelia spp., Neoehrlichia mikurensis, Babesia spp., Coxiella spp., Bartonella spp., and Francisella tularensis. Ticks attached to humans were collected between 2013&ndash, 2015 in Cluj County, Romania. Conventional, nested and quantitative PCR were used to detect specific genetic sequences of each pathogen. For identifying the infectious agents, positive samples were sequenced. The infection prevalence was 21.07% from which 8.18% were mixed infections. The detected agents were Borrelia spp., N. mikurensis and Babesia spp. The present data reveal the endemic occurrence of potentially zoonotic pathogens in Romania. Revealing the current distribution of tick-borne pathogens in ticks collected from humans may provide new insights in understanding the complex ecology of tick-borne diseases and enlightens current knowledge about the infection prevalence at local, regional and national levels.

The objective of this study was to determine the occurrence of Staphylococcus spp., Escherichia coli, somatic coliphages, F-specific RNA bacteriophages, hepatitis E virus (HEV), and bovine enterovirus (BEV) in fecal and water samples. The occurrence of Staphylococcus spp. was investigated in treated wastewater samples collected from slaughterhouse of Tunisia. Results showed that Staphylococcus spp. were detected in the totality of collected samples with an average mean of 5.44 Log10 (CFU/100 ml). Regarding fecal indicator bacteria, E. coli was detected in the totality of water samples and was more abundant in Tunisian samples than in samples collected from Romania (P

Thelazia callipaeda (Spirurida, Thelaziidae) is a vector-borne zoonotic nematode parasite of the conjunctival sac of a large spectrum of hosts, currently having an expanding distribution range throughout Europe. In Romania, the parasite has a relatively large distribution range and host spectrum. However, the presence of T. callipaeda in red foxes ( Vulpes vulpes) has not yet been confirmed. The aim of the present study was to assess the prevalence and geographical distribution of T. callipaeda in red foxes from Romania. A total of 514 animals originating from 10 counties of western Romania were evaluated for the presence of T. callipaeda. Overall, 151 red foxes (29.38%) were positive. No significant differences were noticed between sex and age groups of positive foxes. In total, 3,350 adult nematodes were recovered. The intensity of infection had a mean value of 23.2, while the sex ratio was 2.2:1 in favor of females. In 91 foxes, both eyes were affected, while 60 harbored unilateral infection. Sequence analysis of the cox 1 gene of one nematode from each positive fox ( n=151) revealed the exclusive presence of haplotype h1. We report a relatively high prevalence and distribution range of T. callipaeda infection in red foxes in Romania, highlighting their potential role as a wildlife reservoir.

Contagious sheep footrot is an infectious disease with chronic evolution, usually enzootic, characterized by necrotic or necrotic-purulent inflammation of the soft tissues of the hoof, which causes partial or total detachment of the horn. Cu-based products represented by (I) 1-hydroxyquinoline + Cu on 5% hydroxyapatite, (II) 2 Hydroxyquinoline + Cu + NO3 on 5% Hydroxyapatite and (III) 3 Nitrohydroxyquinoline + Cu on 5% Hydroxyapatite were conditioned in aqueous suspension in a volume of 100 ml, pH 6,0 – 6,4. The products were placed into wells, 20 μL/well, along with 0.5 % copper sulphate solution (Cu control) and Enroxil and Oxitetracycline (control for antibiotics).The results demonstrated that 7 of the samples (58.33 %) are sensitive to the product I, 6 samples (50%) to the product III, 4 samples (33.33 %) to the product 2 and the 0.5% Copper sulphate solution only has a bacteriostatic effect. In comparison to the two antibiotics, 9 samples (75 %) are sensitive to enrofloxacin and only 5 samples (41.66 %) to oxytetracycline. Regarding the associations of microorganisms in the samples with resistance, we have found that these are represented by the most common germs of genus Bacillus, Corynebacterium and Gram negative bacteria, and rarer associations with Micrococcus and Staphylococcus.

Background Tick-borne diseases pose a major threat in public health. The epidemiological dynamics of these diseases depends on the tick vector species and their hosts, as well as the geographical distribution and ecology of both. Among many possible hosts for ticks, small mammals have a major role in the development of immature stages of several tick species. Small mammals are also important reservoir hosts for several pathogenic agents and possible reservoirs for Anaplasma phagocytophilum. In this context, the aim of our study was to evaluate the prevalence of A. phagocytophilum in small mammal species in Romania. Results A total of 791 small mammals of 31 species were tested by PCR, targeting the rrs gene for detection of A. phagocytophilum DNA. Positive results were obtained in 20 small mammals: five Apodemus flavicollis (6.49%), three Sorex araneus (9.09%), three A. uralensis (4.84%), two A. sylvaticus (3.92%), and one of each Spermophilus cittelus (7.14%), Microtus agrestis (3.85%), Sorex minutus (3.85%), Muscardinus avellanarius (3.13%), Crocidura suaveolens (2.44%), Mus spicilegus (2%) and M. arvalis (1.75%). Conclusions Eleven small mammal species were found to be carriers of A. phagocytophilum, suggesting a possible involvement of these species in its epidemiology. To our knowledge, this is the first report of A. phagocytophilum in S. minutus, C. suaveolens, M. spicilegus, M. avellanarius and S. citellus. Electronic supplementary material The online version of this article (10.1186/s13071-018-2791-y) contains supplementary material, which is available to authorized users.

Background Filarioids are vector-borne parasitic nematodes of vertebrates. In Europe, eight species of filarioids, including zoonotic species, have been reported mainly in domestic dogs, and occasionally in wild carnivores. In Romania, infections with Dirofilaria spp. and Acanthocheilonema reconditum are endemic in domestic dogs. Despite the abundant populations of wild carnivores in the country, their role in the epidemiology of filarioid parasites remains largely unknown. The aim of the present study was to assess the host range, prevalence and distribution of filarioid infections in wild carnivores present in Romania. Methods Between May 2014 and February 2016, 432 spleen samples originating from 14 species of wild carnivores have been tested for the presence of DNA of three species of filarioids (D. immitis, D. repens and A. reconditum). Results Overall 14 samples (3.24%) were molecularly positive. The most prevalent species was D. immitis (1.62%), accounting for 50% (n = 7) of the positive animals. The prevalence of D. repens was 1.39%, while that of A. reconditum was 0.23%. No co-infections were detected. Dirofilaria immitis DNA was detected in five golden jackals, Canis aureus (7.58%), one red fox, Vulpes vulpes (0.33%), and one wildcat, Felis silvestris (10%). The presence of D. repens DNA was detected in two red foxes (0.66%), two golden jackals (3.03%), one grey wolf (7.14%), and one least weasel, Mustela nivalis (33.33%). Acanthocheilonema reconditum DNA was found only in one red fox (0.33%). Conclusion The present study provides molecular evidence of filarial infections in wild carnivore species in Romania, suggesting their potential epidemiological role and reports a new host species for D. repens.

Background Jackals are medium-sized canids from the wolf-like clade, exhibiting a unique combination of ancestral morphotypes, broad trophic niches, and close phylogenetic relationships with the wolf and dog. Thus, they represent a potential host of several pathogens with diverse transmission routes. Recently, populations of the Eurasian golden jackal Canis aureus have expanded into the Western Palaearctic, including most of Europe. The aim of our study was to examine Eurasian golden jackals from Romania, Czech Republic and Austria for a wide spectrum of vector-borne protists and to evaluate the role of this species as a reservoir of disease for domestic dogs and/or humans. Results Diagnostic polymerase chain reaction (PCR) DNA amplifications revealed 70% of jackals to be positive for Hepatozoon, 12.5% positive for piroplasms, and one individual positive for Leishmania infantum. Phylogenetic analyses of partial 18S rDNA sequences invariably placed sequenced isolates of Hepatozoon into the H. canis clade. For piroplasms, both the 18S and cox1 sequences obtained confirmed the presence of Babesia canis and “Theileria annae” in 5 and 2 individuals, respectively, providing the first records of these two piroplasmids in Eurasian golden jackals. A single animal from Dolj County (Romania) was PCR-positive for L. infantum, as confirmed also by sequencing of ITS1-5.8S. Conclusions Apparently, expanding populations of jackals can play a significant role in spreading and maintaining new Babesia canis foci in Central Europe. The role of jackals in the epidemiology of “Theileria annae” and H. canis is probably similar to that of red foxes and should be taken into account in further research on these parasites. Also the presence of L. infantum deserves attention. Our study confirms that once established, the populations of Eurasian golden jackals constitute natural reservoirs for many canine vector-borne diseases, analogous to the role of the coyotes in North America.

Red foxes (Vulpes vulpes (L.)) are widespread across Europe, tolerant of synanthropic ecosystems, and susceptible to diseases potentially shared with humans and other animals. We describe flea fauna on red foxes in Romania, a large, ecologically diverse country, in part because fleas may serve as an indicator of the risk of spillover of vector-borne disease. We found 912 individual fleas of seven species on the 305 foxes assessed, for an infestation prevalence of 49.5%. Mean flea load per fox was 5.8 (range 0-44 fleas), and flea detections were most abundant in fall and early spring. Fleas included generalists (Ctenocephalides canis (Curtis), 32.6% of all fleas), Ct. felis (Bouché, 0.1%), and Pulex irritans L. (29.9%), the fox specialist Chaetopsylla globiceps (Taschenberg, 32.5%), mesocarnivore fleas Paraceras melis Walker (3.2%) and Ch. trichosa Kohaut (1.5%), and the small mammal flea Ctenophthalmus assimilis (Taschenberg, 0.1%), which is rarely or never reported from carnivores. There were significantly more female than male Ch. globiceps, Ct. canis, and Pu. irritans, and these three species were the most broadly distributed geographically. Diversity indices suggested reduced diversity in mountainous areas above 700 m. When compared to other flea studies on foxes in Europe, Romania had flea diversity near the median of reports, which was unexpected given Romania's high ecological diversity. Notably absent prey specialists, compared to other studies, include Archaeopsylla erinacei (Bouché) and Spilopsyllus cuniculi (Dale). Further studies of possible disease agents in fox fleas could help elucidate possible risks of vector-borne disease in foxes, domestic animals, and humans as well.

Anaplasma phagocytophilum and spotted fever group Rickettsia are obligate intracellular Gram-negative tick-borne bacteria, among which several may cause clinical infections in humans. Several Rickettsia spp. and A. phagocytophilum are transmitted in Europe by Ixodes ricinus, the most common tick species feeding on humans in this area. The aim of this study was to evaluate the annual prevalence of Rickettsia spp. and A. phagocytophilum in I. ricinus collected from humans during three consecutive years. The mean prevalences of the infection with the investigated pathogens in I. ricinus ticks collected from human patients were as follows: A. phagocytophilum (5.56%), R. helvetica (4.79%) and R. monacensis (1.53%). In the present study, no significant differences of pathogens prevalence between the three years study period were observed, except the prevalence of R. helvetica, which had a significant increase in 2015, suggesting an increasing risk for humans to be exposed to this zoonotic pathogen.

Canine granulocytic anaplasmosis (CGA) is an important tick-borne disease with worldwide distribution. The importance of this disease resides in the ability of Anaplasma phagocytophilum to infect humans and several animal species. The aim of the study was to evaluate the prevalence rate of CGA in different altitudinal areas of Romania. A total of 357 canine blood samples were collected during 2010-2013 from eight counties. To assess the influence of the altitude on A. phagocytophilum prevalence, the samples were collected from four different altitude areas (coastal 0-5 meters; lowland 6-100 meters; hilly areas 200-300 meters; low mountain areas >500 meters). These samples were evaluated for the presence of A. phagocytophilum DNA by amplifying part of the Ankyrin repeat protein (AnkA) gene. A higher prevalence was obtained for coastal compared with remaining areas, suggesting an influence of altitude on the CGA. Moreover, the results suggest an influence of climate and rainfall. In the present research work, we highlight the risk of granulocytic anaplasmosis in Central and Southern Romania, with a greater risk associated to Southern lowland region, especially in coastal areas. The importance of these results resides in the zoonotic potential of the canine A. phagocytophilum strains. In conclusion, the altitude and precipitation level may be risk factors for A. phagocytophilum infection in dogs and other hosts, including humans.

An extensive survey of parasites in red foxes ( Vulpes vulpes ) was independently conducted in Romania and the Czech Republic. Carcasses were examined by necropsy, and small, dark nodules apparently containing ticks were noticed in the subcutaneous tissue of several foxes. Histopathological examination was performed using hematoxylin and eosin (HE) staining. Of the 91 foxes examined from the Czech Republic, 14 (15.4%) were harboring ticks in the subcutaneous tissue. In the majority of these cases, 1–3 nodules/fox were found, with a maximum of 31 nodules/fox. In Romania a single examined fox had subcutaneous ticks. All ticks collected from subcutaneous tissue were partially engorged adults. Based on morphological features, Ixodes ricinus , I. hexagonus , I. crenulatus and Dermacentor reticulatus were identified. The histopathological examination revealed chronic granulomatous panniculitis with peripheral fibrosis and intralesional presence of the ticks. Only few data are available regarding ticks localized in the subcutaneous tissue of any host. All the ticks were dead or already decomposed and it is evident that subcutaneous location does not represent an evolutionary advantage, as the detachment and finishing the life cycle is impossible.

In Romania, data regarding hard-tick diversity and tick-host associations in wild carnivores are scarce. We aimed to identify tick species in wild carnivores and to establish reliable data on tick-host associations. The study was conducted in various Romanian localities from all five ecoregions found in the country. Fourteen species of wild carnivores were examined. Immature and adult ticks were collected and identified using the morphological keys. The frequency and mean intensity of tick infestation, overall and differentiated by species, developmental stage and host were calculated. Of 202 wild carnivores, 68 were parasitized by seven tick species (predominantly Ixodes ricinus, I. hexagonus and Dermacentor reticulatus). The mean intensity of tick infestation was similar in males (6.97, BCa 95% CI 5.15-9.88) and females (5.76, BCa 95% CI 4.15-9.17). The highest prevalence of infested animals was recorded in the pannonian and steppic ecoregions, 66.7 and 52.7%, respectively. In the continental ecoregion the prevalence was 26.7%, whereas in the pontic ecoregion it was 28%. The lowest value, 16.7% was recorded in the alpine ecoregion. In total 430 ticks were collected, and 24.8% (n = 50) of the animals were infested with more than one tick species. Fourteen new tick-host associations were recorded. Our results suggest that anthropogenic changes of the environment lead to the diminishing of the boundaries, between wild and domestic animals, increasing the exposure for both animals and humans, to infective agents, including tick-borne pathogens.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) comprises obligate intracellular Gram-negative bacteria that are mainly transmitted by ticks, and currently includes six species: Anaplasma bovis, Anaplasma centrale, Anaplasma marginale, Anaplasma phagocytophilum, Anaplasma platys, and Anaplasma ovis. These have long been known as etiological agents of veterinary diseases that affect domestic and wild animals worldwide. A zoonotic role has been recognized for A. phagocytophilum, but other species can also be pathogenic for humans. Anaplasma infections are usually challenging to diagnose, clinically presenting with nonspecific symptoms that vary greatly depending on the agent involved, the affected host, and other factors such as immune status and coinfections. The substantial economic impact associated with livestock infection and the growing number of human cases along with the risk of transfusion-transmitted infections, determines the need for accurate laboratory tests. Because hosts are usually seronegative in the initial phase of infection and serological cross-reactions with several Anaplasma species are observed after seroconversion, direct tests are the best approach for both case definition and epidemiological studies. Blood samples are routinely used for Anaplasma spp. screening, but in persistently infected animals with intermittent or low-level bacteremia, other tissues might be useful. These guidelines have been developed as a direct outcome of the COST action TD1303 EURNEGVEC (>European Network of Neglected Vectors and Vector-Borne Diseases>). They review the direct laboratory tests (microscopy, nucleic acid-based detection and in vitro isolation) currently used for Anaplasma detection in ticks and vertebrates and their application., This work was done under the frame of COST action TD1303.

Three species of the genus Angiostrongylus are known to infect European carnivores: A. vasorum (mainly in canids but also in other carnivores), A. chabaudi (in felids) and A. daskalovi (in mustelids). A. vasorum is responsible for clinically severe disease in domestic dogs, most commonly diagnosed based on fecal examination and serological detection of circulating antigens. Considering the poorly known host range and the challenging larval differentiation in the feces between the three species of Angiostrongylus infecting European carnivores, our aim was to evaluate the cross-reactivity of A. chabaudi and A. daskalovi with A. vasorum using a commercial serologic test developed for domestic dogs. Badgers (Meles meles) (n = 10) and wildcats (Felis silvestris) (n = 8) were examined between 2015 and 2016 by full parasitological necropsy with subsequent morphological and molecular identification of nematodes and by serology, using IDEXX Angio Detect™ tests. Five out of the ten badgers and two out of the eight wildcats were harboring nematodes in the pulmonary arteries. All nematodes were identified morphologically as A. daskalovi in badgers and A. chabaudi in wildcats, respectively. Serological examination of the plasma samples revealed the positivity of the same animals as found in necropsy. None of the animals negative at necropsy was positive at serology. The 100% correlation between the necropsy results and the serologic positivity to IDEXX Angio Detect™ in badgers infected with A. daskalovi and wildcats infected with A. chabaudi suggest that these rapid tests are able to identify circulating antigens of all species of Angiostrongylus found in European carnivores: A. vasorum, A. daskalovi and A. chabaudi. The possibility for future in-clinic use of this test in domestic cats should be further investigated.

Anaplasma phagocytophilum is the agent of tick-borne fever, equine, canine and human granulocytic anaplasmosis. The common route of A. phagocytophilum transmission is through a tick bite, the main vector in Europe being Ixodes ricinus. Despite the apparently ubiquitous presence of the pathogen A. phagocytophilum in ticks and various wild and domestic animals from Europe, up to date published clinical cases of human granulocytic anaplasmosis (HGA) remain rare compared to the worldwide status. It is unclear if this reflects the epidemiological dynamics of the human infection in Europe or if the disease is underdiagnosed or underreported. Epidemiologic studies in Europe have suggested an increased occupational risk of infection for forestry workers, hunters, veterinarians, and farmers with a tick-bite history and living in endemic areas. Although the overall genetic diversity of A. phagocytophilum in Europe is higher than in the USA, the strains responsible for the human infections are related on both continents. However, the study of the genetic variability and assessment of the difference of pathogenicity and infectivity between strains to various hosts has been insufficiently explored to date. Most of the European HGA cases presented as a mild infection, common clinical signs being pyrexia, headache, myalgia and arthralgia. The diagnosis of HGA in the USA was recommended to be based on clinical signs and the patient’s history and later confirmed using specialized laboratory tests. However, in Europe since the majority of cases are presenting as mild infection, laboratory tests may be performed before the treatment in order to avoid antibiotic overuse. The drug of choice for HGA is doxycycline and because of potential for serious complication the treatment should be instituted on clinical suspicion alone.

Background Small mammals play an important role in the life-cycle of ticks and are reservoirs for several zoonotic pathogens. The aim of this study was to provide epidemiological data regarding the presence of Borrelia spp. in tissues of small mammals from Romania. Methods We examined 401 individuals belonging to 11 small mammal species collected in Romania. Collections cover the largest effort to survey these reservoirs in the country. Tissue samples were analyzed by multiplex qPCR targeting the ospA gene of Borrelia burgdorferi (s.l.) and a part of the flaB gene of B. miyamotoi. Positive samples were further analysed by conventional PCR and sequenced. Results The overall prevalence of infection with Borrelia spp. in small mammal tissues was 4.9%. The most commonly detected species were B. afzelii, followed by B. garinii/B. bavariensis, B. miyamotoi and B. burgdorferi (s.s.). To our knowledge, we report for the first time the detection of Borrelia spp. in Crocidura leucodon and C. suaveolens, and B. miyamotoi in the liver of Myodes glareolus. Conclusions To our knowledge, our study evaluates for the first time the occurrence of Borrelia spp. in small mammals in Romania, contributing to a better knowledge of the distribution of these bacteria. This survey upgrades previous data on the spatial distribution of the pathogens and reveals the importance of animal surveillance regarding Lyme borreliosis and relapsing fever caused by B. miyamotoi.

This study aimed to characterize raw honeys from different geographical origins in Romania, in respect of chemical composition, microbiological examination and evaluate their probiotic potential. The physico-chemical determinations were performed in APHIS-DIA Laboratory, Cluj-Napoca, Romania, using standard validated methods. Bacterial identification was performed for each sample and each colony type using Vitek® 2 Compact 15 system and PCR amplification using 16S rDNA bacterial universal primers (27F, 1492R), species being confirm by sequences analysis. In five raw honey samples, we have identified probiotic bacteria, such as: Bacillus mycoides, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus velezensis. Generally, all honey samples meet the standard values for chemical composition. However, one sample having 7.44% sucrose was found to have also probiotics bacteria from the genus Bacillus because sucrose is a substrate for probiotics development. In conclusion, the Romanian raw honey can be a potential reservoir of probiotics, which confer a health benefit for consumers.

Bovine mastitis is a cause of major financial losses in the dairy industry worldwide. Bovine mastitis is defined as the inflammatory response of the udder tissue due to either physical trauma or infections caused by different microorganisms, especially bacteria. More than 135 pathogenic agents have been identified from bovine mastitis. The aim of the study was to identify the bacterial genera involved in mastitis etiology in cattle and to test the antimicrobial susceptibility to antibiotics. The samples were collected before morning milking and evaluated by microscopic and cultural examination, and further identified using biochemical methods. The evaluation of antimicrobial susceptibility was performed using Kirby-Bauer disk diffusion test. Overall, Staphylococcus spp., Bacillus spp., Streptococcus spp., Micrococcus spp., Escherichia spp. and Trueperella spp. were identified. The most frequent bacterial associations were observed for Staphylococcus-Bacillus, Bacillus-Streptococcus and Staphylococcus-Micrococcus. Regarding the antimicrobial sensitivity profile, the majority of the bacterial strains we have isolated were classified as sensitive, with the exception of Escherichia coli, with one strain being resistant to all tested antibiotics. Among the antimicrobials, the Amoxicillin and clavulanic acid was the most efficient one and Trimethoprim/Sulfamethoxazole did not exhibit an inhibitory activity in 19 milk samples.

The conjunctival bacterial resident and opportunistic flora of dogs may represent a major source of dissemination of pathogens throughout the environment or to other animals and humans. Nevertheless, contamination with bacteria from external sources is common. In this context, the study of the antimicrobial resistance (AMR) pattern may represent an indicator of multidrug resistant (MDR) strains exchange. The present study was focused on a single predisposed breed—Saint Bernard. The evaluated animals were healthy, but about half had a history of ocular disease/treatment. The swabs collected from conjunctival sacs were evaluated by conventional microbiological cultivation and antimicrobial susceptibility testing (AST). The most prevalent Gram-positive was Staphylococcus spp., regardless of the history, while Gram-negative was Pseudomonas spp., exclusively from dogs with a history of ocular disease/treatment. Other identified genera were represented by Bacillus, Streptococcus, Trueperella, Aeromonas and Neisseria. The obtained results suggest a possible association between the presence of mixed flora and a history of ocular disease/treatment. A high AMR was generally observed (90%) in all isolates, especially for kanamycin, doxycycline, chloramphenicol and penicillin. MDR was recorded in Staphylococcus spp. and Pseudomonas spp. This result together with a well-known zoonotic potential may suggest an exchange of these strains within animal human populations and the environment.

Crows (Corvidae) are common city dwellers worldwide and are increasingly important subjects of epidemiology studies. Although their importance as hosts and transmitters of a number of zoonotic parasites and pathogens is well known, there are no studies on their importance as tick hosts. After mosquitoes, ticks are the most important vectors of zoonotic pathogens, especially for those causing emerging zoonotic diseases. Pathogenic bacteria, especially Borrelia spp., Rickettsia spp., and Anaplasma spp., vectored by ticks, are the cause for most vector-borne diseases in Europe. Here we report on ticks and tick-borne pathogens harbored by urban breeding crows. A total of 36 birds (33.33%, n = 108) hosted ticks, with 91 individual ticks belonging to 6 species (Haemaphysalis concinna, Haemaphysalis parva, Haemaphysalis punctata, Hyalomma marginatum, Ixodes arboricola, and Ixodes ricinus). Rickettsia spp. DNA was found in 6.6% of ticks and 1.9% of bird tissues, whereas Anaplasma phagocytophilum was found in 5.9% of ticks and 0.9% of birds. Two rickettsial genospecies were located, Rickettsia helvetica and Rickettsia monacensis. This is the first study to determine such a diverse tick spectrum feeding on urban corvids, while highlighting their importance as tick hosts and raising concerns about their potential risk to human health.

Background Babesia spp. are hemoparasites which infect the red blood cells of a large variety of mammals. In bats, the only known species of the genus is Babesia vesperuginis. However, except a few old reports, the host range and geographical distribution of this bat parasite have been poorly studied. This study aimed to investigate the presence of piroplasms in tissues of bats collected in four different countries from eastern and central Europe: Austria, Czech Republic, Hungary and Romania. Methods A total of 461 bat carcasses (24 species) were collected between 2001 and 2016 from caves, mines and buildings. PCR was performed using specific primers targeting a portion of the 18S rDNA nuclear gene and cytochrome c oxidase subunit 1 mitochondrial gene, followed by sequencing. Results The results of this study show for the first time the presence of B. vesperuginis in bats in central and eastern Europe. The phylogenetic analysis of the 18S rDNA nuclear gene revealed no variability between the sequences and the phylogenetic analysis of the cox1 mitochondrial gene proved that B. vesperuginis could be divided into two subclades. Conclusion Our study showed a broad geographical distribution of B. vesperuginis in European bats, reporting its presence in five new host species (M. cf. alcathoe, M. bechsteinii, M. myotis, Pi. nathusii and V. murinus) and three new countries. Electronic supplementary material The online version of this article (10.1186/s13071-017-2536-3) contains supplementary material, which is available to authorized users.

Red foxes, Vulpes vulpes are among the most widely spread carnivores in the world, invading also urban areas and are often parasitized by various ticks and directly exposed to several vector-borne pathogens, including the commonly present “Theileria annae”. Considering the paucity of data on the possible vectors of this pathogen and the presence of the infection in various locations across the globe, the aim of our study was to understand the potential role of various environmental factors on the distribution of “T. annae” in red foxes from a well-defined region within the Carpathians, Romania. Between July 2016 and April 2017, a total of 347 blood samples originating from red foxes from 13 counties were tested using a PCR specifically designed for “T. annae”. In order to assess the potential distribution of “T. annae” based on niche modelling, we used presence-only data and 15 ecological variables. The probability of presence models was built using MaxEnt software. Of all sampled foxes, 20.1% (66 unique locations in 8 counties) were positive for “T. annae” DNA. There was no significant difference between the prevalence in males and females, nor between juveniles and adults. The sequences were all identical to each other and showed 100% identity to other sequences deposited in GenBank. The highest contribution to the spatial model was represented by the agricultural land coverage. This is the first study to demonstrate the presence of “T. annae” in foxes in Romania and the first spatial analysis for “T. annae” highlighting the importance of the environmental factors on its distribution.