Successful Transmammary Treatment of Babesia gibsoni Infection in Newborn Puppies after the Administration of Malarone®, Azithromycin, and Artesunate to a Lactating Dam.

Babesia gibsoni is a parasitic protozoan transmitted through tick bites and can cause severe disease in dogs. It can also be transmitted through direct contact with infected blood during dog fights, blood transfusions, and from dam to offspring during the perinatal period, resulting in stillborn or dead newborn puppies. This study aimed to determine the incidence of infection, the viability of newborn puppies, and the degree of B. gibsoni transmission from infected dam to offspring during pregnancy and lactation. Using PCR-based molecular methods, B. gibsoni infection in a pregnant American Pit Bull Terrier and her newborn puppies was confirmed. The incidence of B. gibsoni infection in the litter reached 75%. Out of eight puppies, six were infected with B. gibsoni, and one died. A therapeutic protocol comprising Malarone®, azithromycin, and artesunate was administered to a lactating B. gibsoni-positive bitch. By day 77 after birth, three out of five positive puppies showed negative PCR tests for B. gibsoni, indicating successful treatment through breast milk during nursing. In the two remaining positive puppies, therapy was started and parasitemia was successfully eliminated.

The Impact of Altitude on Tick-Borne Pathogens at Two Mountain Ranges in Central Slovakia.

Ticks are ectoparasites of a wide range of animals and are important vectors of numerous pathogens affecting humans, livestock, and pets. This study investigates possible correlations between selected factors, altitude, soil pH, and a factor called 'amount' (number of ticks examined in pooled samples) on the occurrence of I. ricinus ticks positive for selected tick-borne microorganisms. Questing I. ricinus ticks were collected in 2016 and 2017 across various altitudes, at two mountain ranges in central Slovakia. Tick pools were screened for the presence of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato (Bbsl), Babesia/Theileria spp., Rickettsia spp., and tick-borne encephalitis virus (TBEV) using molecular methods. Regression analysis was employed to evaluate relationships between selected factors and the occurrence of vector-borne microorganisms. This study revealed a statistically significant influence of altitude on the occurrence of A. phagocytophilum; increasing altitude of the sampling site was associated with increased probability of pathogen occurrence. For Babesia/Theileria spp., neither altitude nor soil pH significantly affected pathogen occurrence. The occurrence of Bbsl was notably impacted by both altitude and soil pH; higher altitudes were associated with a decreased probability of pathogen presence, whereas higher soil pH increased the likelihood of pathogen occurrence. The presence of Rickettsia in a pooled sample was not affected by altitude and soil pH, but the 'amount' factor was a significant predictor, increasing the probability of pathogen detection. Neither altitude nor soil pH had a significant impact on TBEV occurrence. The regression models showed moderate goodness-of-fit levels to the data, underscoring their utility in examining the role of altitude and soil pH on pathogen occurrence. However, they explained only a small portion of the overall variance in pathogen occurrence, indicating the presence of other significant factors not covered in this study.

Clinical Efficacy and Safety of Malarone®, Azithromycin and Artesunate Combination for Treatment of Babesia gibsoni in Naturally Infected Dogs.

Babesia gibsoni is a tick-borne protozoal blood parasite that may cause hemolytic anemia, thrombocytopenia, lethargy, and/or splenomegaly in dogs. Many drugs have been used in management of canine babesiosis such as monotherapy or combined treatment, including diminazene aceturate, imidocarb dipropionate, atovaquone, and antibiotics. This report examines the effectiveness and safety of Malarone®, azithromycin (AZM) and artesunate (ART) combination for the treatment of babesiosis in dogs naturally infected with Babesia gibsoni. Twelve American Pit Bull Terriers were included in the experiment. Examined dogs underwent clinical and laboratory analysis including hematology and biochemistry profile and serum protein electrophoresis. After diagnosis, the dogs received combined therapy with Malarone® (13.5 mg/kg PO q24 h), azithromycin (10 mg/kg PO q24 h) and artesunate (12.5 mg/kg PO q24 h) for 10 days. The combined treatment improved hematology and biochemical parameters to the reference range gradually during the first 14 days already, resulting in the stable values until day 56 after treatment. No clinically apparent adverse effects were reported during treatment and monitoring. No relapses of parasitemia were detected in control days 180, 360, 540 and 720 in all dogs. Results of the study indicate that the combined treatment leads to successful elimination of parasitemia in chronically infected dogs with B. gibsoni.

Detection of pathogens in ixodid ticks collected from animals and vegetation in five regions of Ukraine.

The distribution and prevalence of zoonotic pathogens infecting ixodid ticks in Western Europe have been extensively examined. However, data on ticks and tick-borne pathogens in Eastern Europe, particularly Ukraine are scarce. The objective of the current study was, therefore, to investigate the prevalence of Anaplasma phagocytophilum, Anaplasmataceae, Rickettsia spp., Babesia spp., Bartonella spp., and Borrelia burgdorferi sensu lato in engorged and questing ixodid ticks collected from five administrative regions (oblasts) of Ukraine, namely Chernivtsi, Khmelnytskyi, Kyiv, Ternopil, and Vinnytsia. The ticks were collected from both wild and domestic animals and from vegetation. Of 524 ixodid ticks collected, 3, 99, and 422 ticks were identified as Ixodes hexagonus, Ixodes ricinus, and Dermacentor reticulatus, respectively. DNA samples individually extracted from 168 questing and 354 engorged adult ticks were subjected to pathogen-specific PCR analyses. The mean prevalence in I. ricinus and D. reticulatus were, respectively: 10 % (10/97) and 3 % (12/422) for A. phagocytophilum; 69 % (67/97) and 52 % (220/422) for members of the Anaplasmataceae family; 25 % (24/97) and 28 % (117/422) for Rickettsia spp.; 3 % (3/97) and 1 % (6/422) for Babesia spp.; and 9 % (9/97) and 5 % (20/422) for Bartonella spp. Overall, between the five cities, there was no significant difference in the prevalence of any of the pathogens for the respective ticks (p > 0.05). The prevalence of B. burgdorferi s. l. in the questing and engorged I. ricinus varied from 0 to 27 % and 14-44%, respectively, with no statistical significance identified between the five cities (p > 0.05). In addition to reporting the updated data for Kyiv and Ternopil, this study is the first to provide the prevalences of the tick-borne pathogens for Chernivtsi, Khmelnytskyi, and Vinnytsia. This investigation is also the first to detect Neoehrlichia mikurensis in ixodid ticks from Ukraine. These new data will be useful for medical and veterinary practitioners as well as public health officials when diagnosing infections and when implementing measures to combat tick-borne diseases in Ukraine.

Differences in serum protein electrophoretic pattern in dogs naturally infected with Babesia gibsoni and Babesia canis.

Canine babesiosis may cause several hematological and biochemical changes, but only limited studies are available regarding the possible differences of changes in animals infected by different Babesia parasites. The study focused on the evaluation of the differences in serum protein electrophoretic pattern between dogs naturally infected with B. gibsoni (17 dogs) and B. canis (40 dogs). The mean values of total proteins, ß1-, ß2- and γ-globulins were in dogs infected with B. gibsoni significantly higher (P < 0.05 and P < 0.001) than in dogs infected with B. canis. The relative concentrations of albumin, α1-, α2-globulins and the A/G ratios were in the B. gibsoni infected dogs significantly lower (P < 0.001), no significant differences were found in the relative concentrations of ß1- and ß2-globulins. Significant differences were found in most of the evaluated parameters when comparing the results in relation to the form of B. canis infection to B. gibsoni infection. Hematological indices showed significant differences between dogs infected with B. gibsoni and the complicated form of B. canis infection. In conclusion, the obtained results suggest differences in the changes of serum protein electrophoretic pattern between dogs infected with both Babesia species and thus, in the response to the infection caused by various Babesia parasites.

Effect of Climate and Land Use on the Spatio-Temporal Variability of Tick-Borne Bacteria in Europe.

The incidence of tick-borne diseases caused by Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. has been rising in Europe in recent decades. Early pre-assessment of acarological hazard still represents a complex challenge. The aim of this study was to model Ixodes ricinus questing nymph density and its infection rate with B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in five European countries (Italy, Germany, Czech Republic, Slovakia, Hungary) in various land cover types differing in use and anthropisation (agricultural, urban and natural) with climatic and environmental factors (Normalized Difference Vegetation Index (NDVI), Normalized Difference Water Index (NDWI), Land Surface Temperature (LST) and precipitation). We show that the relative abundance of questing nymphs was significantly associated with climatic conditions, such as higher values of NDVI recorded in the sampling period, while no differences were observed among land use categories. However, the density of infected nymphs (DIN) also depended on the pathogen considered and land use. These results contribute to a better understanding of the variation in acarological hazard for Ixodes ricinus transmitted pathogens in Central Europe and provide the basis for more focused ecological studies aimed at assessing the effect of land use in different sites on tick-host pathogens interaction.

Neglected intravascular pathogens, Babesia vulpes and haemotropic Mycoplasma spp. in European red fox (Vulpes vulpes) population.

Wild animals, especially canids, are important reservoirs of vector-borne pathogens, that are transmitted by the ticks and other bloodsucking arthropods. In total, 300 red foxes (Vulpes vulpes), shot by the hunters in eastern and northern Slovakia, were screened for the presence of vector-borne pathogens by PCR-based methods Blood samples were obtained from nine red foxes and tissue samples originated from 291 animals (the liver tissue samples from 49 foxes and spleen samples from 242 red foxes). Babesia vulpes and haemotropic Mycoplasma species were identified by amplification and sequencing of 18S rRNA and 16S rRNA gene fragments, respectively. Overall, the presence of these pathogens was recorded in 12.3% of screened DNA samples. Altogether 9.7% (29/300) of investigated foxes carried DNA of Babesia spp. In total, 12 out of 29 Babesia spp. PCR - positive amplicons were further sequenced and identified as B. vulpes (41.4%; 12/29), remaining 17 samples are referred as Babesia sp. (58.6%; 17/29). Overall prevalence of B. vulpes reached 4.0% (n=300). Thirteen (4.3%) samples tested positive for distinct Mycoplasma species. To the best of our knowledge, this study brings the first information on B. vulpes infection in red foxes in Slovakia, and the first data on the prevalence and diversity of haemotropic Mycoplasma spp. in European red fox population. Moreover, co-infections with B. vulpes and Mycoplasma spp. were confirmed in 1.7% of tested DNA samples. The relatively high rates of blood pathogen' prevalence and species diversity in wild foxes indicate the role of the fox population in the maintenance of the parasites in sylvatic cycles and strengthen the assumption that foxes play an important role in spreading of infectious microorganisms within and outside the natural foci.

Emergence of tick-borne pathogens (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Ricketsia raoultii and Babesia microti) in the Kyiv urban parks, Ukraine.

To date, only limited data about the presence of ticks and circulation of tick-borne pathogens in urban parks of Kyiv in northern Ukraine are available. In total, 767 ticks (696 Ixodes ricinus and 69 Dermacentor reticulatus) collected in seven urban parks and one suburban oak wood park in Kyiv were individually analyzed by the PCR assays. Tick-borne pathogens, namely spirochetes from Borrelia burgdorferi sensu lato complex, Anaplasma phagocytophilum, and Babesia microti, were detected in 11.1% of tested I. ricinus ticks. In total, 4% of I. ricinus ticks tested positive for the presence of B. burdorferi s.l. (Borrelia afzelii and Borrelia garinii), 5.2% for A. phagocytophilum, and Ba. microti was confirmed in 1.9% of examined ticks. Mixed infections were recorded in four DNA samples, representing the prevalence of 0.6%. One female and two I. ricinus nymphs were simultaneously infected with B. afzelii and A. phagocytophilum, and one female carried B. afzelii and Ba. microti. In addition, 10.1% of D. reticulatus ticks tested positive for Rickettsia raoultii. Identification of infectious agents and their diversity, assessment of the relative epidemiological importance and determination of the prevalence in questing ticks from central parts of the cities are crucial steps towards the tick-borne diseases surveillance in urban environment.

Molecular screening for bacteria and protozoa in great cormorants (Phalacrocorax carbo sinensis) nesting in Slovakia, central Europe.

This study brings the data about the occurrence of bacterial and protozoan pathogens in 32 great cormorants (Phalacrocorax carbo sinensis), representing approximately 20% of the population nesting in the surroundings of water basin Liptovská Mara (northern part of Central Slovakia). A survey revealed the presence of tick-borne bacteria Anaplasma phagocytophilum (6.25%) and parasitic protozoa Toxoplasma gondii (3.1%). These data indicate an infectious status of the great cormorant population nesting in Slovakia; they might suggest a degree of environmental contamination by infectious agents and demonstrate the role of migratory seabirds in the circulation and dispersal of pathogens with zoonotic potential.

Genetic diversity of Bartonella genotypes found in the striped field mouse (Apodemus agrarius) in Central Europe.

We investigated the diversity of Bartonella in Apodemus agrarius, an important rodent of peri-domestic habitats, which has spread into Europe in the past 1000 years. Spleen samples of 344 A. agrarius from Eastern Slovakia were screened for the presence of Bartonella spp. using 16S-23S rRNA internal transcribed spacer region and bacteria were detected in 9% of rodents. Based on sequencing of three housekeeping genes (gltA, rpoB and groEL) Bartonella genotypes were ascribed to the species typical for mice and voles: B. grahamii, B. taylorii and B. birtlesii. However, the study also confirmed presence of genotypes belonging to the B. clarridgeiae/B. rochalimae clade, and the B. elizabethae/B. tribocorum clade, which are not commonly found in woodland rodents. In addition, a potential recombination event between these two genotypes was noted, which highlights an important role of A. agrarius in shaping Bartonella diversity and evolution.

Wild boar (Sus scrofa) - reservoir host of Toxoplasma gondii, Neospora caninum and Anaplasma phagocytophilum in Slovakia.

In Central Europe the wild boar population is permanently growing and consequently Cf foodborne infections. In this study serological and molecular detection of Toxoplasma gondii and Neospora caninum in wild boars was evaluated. Moreover, same samples were screened for the presence and genetic variability of tick-borne bacterium Anaplasma phagocytophilum. Blood samples collected from 113 wild boars from Southern Slovakia were examined for antibodies to T. gondii by indirect and to N. caninum by competitive ELISA. The presence of parasitic DNA in blood samples was determined by standard or real time PCR techniques. Antibodies against T. gondii and N. caninum were detected in 45 (39.8%) and 38 (33.6%) animals, respectively. Females were more frequently infected for both pathogens than males. The high seropositivity against both coccidia indicates a permanent occurrence of these pathogens in the studied locality. T. gondii DNA was confirmed in five seropositive boars (4.4%) and N. caninum in 23 blood samples (20.4%). Three out of 23 N. caninum PCR positive animals did not show seropositivity. Three out of 113 blood samples of wild boars were positive for A. phagocytophilum (2.7%). The obtained A. phagocytophilum sequences were 100% identical with GenBankTM isolates from Slovak dog (KC985242); German horse (JF893938) or wild boar (EF143810) and red deer (EF143808) from Poland. Coinfections of T. gondii with N. caninum and N. caninum with A. phagocytophilum were detected in single cases. Results suggest a potential zoonotic risk of toxoplasmosis transmission to humans and the spread of neosporosis to farm animals.

First molecular identification of Babesia gibsoni in dogs from Slovakia, central Europe.

Canine babesiosis is a severe and potentially life threatening infection. In Europe, Babesia canis is considered to be the most common species responsible for the disease. We report two cases of babesiosis caused by Babesia gibsoni. The polymerase chain reaction, restriction fragment length polymorphism analysis and further sequencing of 18S rRNA gene fragments from blood samples of both dogs revealed the identity of isolates with B. gibsoni genotypes from other dogs worldwide. This species was previously not known to infect dogs in Slovakia. It is resistant to traditional anti-babesial therapy. Therefore, correct diagnosis is crucial for the successful treatment, especially in dogs with hemolytic anemia and febrile conditions.

Presence of Candidatus Neoehrlichia mikurensis and Babesia microti in rodents and two tick species (Ixodes ricinus and Ixodes trianguliceps) in Slovakia.

Rodents are important reservoir hosts of many tick-borne pathogens. Their importance in the circulation of the emerging bacterial agent, Candidatus Neoehrlichia mikurensis and the intraerythrocytic protozoan parasite, Babesia microti has been recently proposed. The aim of the present study was to identify the presence and genetic diversity of Candidatus N. mikurensis and B. microti circulating in the natural foci among rodents and two species of ixodid ticks (Ixodes ricinus and Ixodes trianguliceps). In 2011-2013, rodents were captured at sampling sites in Eastern Slovakia. A total of 997 rodents (324 Apodemus agrarius, 350 Apodemus flavicollis, 271 Myodes glareolus, and 52 other rodent species), 788 feeding ticks from rodents, and 1375 questing ticks were investigated for the presence of pathogens by molecular methods followed by DNA sequencing. Candidatus N. mikurensis was detected in 2.4% of questing I. ricinus nymphs and 2.6% of questing adult I. ricinus ticks, spleens of rodents (1.6%), as well as in feeding larval I. ricinus (0.3%) and feeding larval I. trianguliceps ticks (3.3%). The 16S rRNA and gltA gene sequences of Candidatus N. mikurensis obtained in this study confirmed a high degree of genetic identity of this bacterium in Europe. DNA of B. microti was found in ear (0.6%) and spleen biopsies of rodents (1.9%), in rodent foetus (3.8%) and feeding larval (5.2%) and nymphal (8.7%) I. ricinus, in questing nymphal I. ricinus (0.5%) and questing adult I. ricinus ticks (0.3%). None of the 112 I. trianguliceps ticks were infected. B. microti was represented by two different genotypes: 92% of the positive samples belonged to the zoonotic type strain from Jena (Germany). The results of this study underline the importance of rodents in the circulation of both emerging pathogens in natural foci.

Distinct Anaplasma phagocytophilum genotypes associated with Ixodes trianguliceps ticks and rodents in Central Europe.

Rodents are important reservoir hosts of tick-borne pathogens. Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis of both medical and veterinary importance. In Europe, this pathogen is primarily transmitted by the Ixodes ricinus tick among a wide range of vertebrate hosts. However, to what degree A. phagocytophilum exhibits host specificity and vector association is poorly understood. To assess the extent of vector association of this pathogen and to clarify its ecology in Central Europe we have analyzed and compared the genetic variability of A. phagocytophilum strains from questing and feeding I. ricinus and Ixodes trianguliceps ticks, as well as from rodent' tissue samples. Tick collection and rodent trapping were performed during a 2-year study (2011-2012) in ecologically contrasting setting at four sites in Eastern Slovakia. Genetic variability of this pathogen was studied from the collected samples by DNA amplification and sequencing of four loci followed by Bayesian phylogenetic analyses. A. phagocytophilum was detected in questing I. ricinus ticks (0.7%) from all studied sites and in host feeding I. trianguliceps ticks (15.2%), as well as in rodent biopsies (ear - 1.6%, spleen - 2.2%), whereas A. phagocytophilum was not detected in rodents from those sites where I. trianguliceps ticks were absent. Moreover, Bayesian phylogenetic analyses have shown the presence of two distinct clades, and tree topologies were concordant for all four investigated loci. Importantly, the first clade contained A. phagocytophilum genotypes from questing I. ricinus and feeding I. ricinus from a broad array of hosts (i.e.,: humans, ungulates, birds and dogs). The second clade comprised solely genotypes found in rodents and feeding I. trianguliceps. In this study we have confirmed that A. phagocytophilum strains display specific host and vector associations also in Central Europe similarly to A. phagocytophilum' molecular ecology in United Kingdom. This study suggests that A. phagocytophilum genotypes associated with rodents are probably transmitted solely by I. trianguliceps ticks, thus implying that rodent-associated A. phagocytophilum strains may not pose a risk for humans.

Candidatus Neoehrlichia mikurensis and its co-circulation with Anaplasma phagocytophilum in Ixodes ricinus ticks across ecologically different habitats of Central Europe.

BACKGROUND: Candidatus Neoehrlichia mikurensis is a newly emerging tick-borne bacterium from the family Anaplasmataceae. Its presence in Ixodes ricinus ticks was reported from various European countries, however, it's ecology and co-circulation with another member of the same family, Anaplasma phagocytophilum has not been rigorously studied yet. FINDINGS: Candidatus N. mikurensis was detected in all sampling sites. In total, 4.5% of ticks were positive including larvae. The highest positivity was detected in Austria with a prevalence of 23.5%. The probability of Candidatus N. mikurensis occurrence increased with the proportion of ticks infected with Anaplasma phagocytophilum. CONCLUSION: A positive association between the occurrences of Candidatus N. mikurensis and A. phagocytophilum indicates that both bacteria share similar ecology for their natural foci in Central Europe.