Occurrence of Babesia Species and Co-Infection with Hepatozoon canis in Symptomatic Dogs and in Their Ticks in Eastern Romania

Although the distribution of Babesia spp. and Hepatozoon canis is well known in Romania, there is still a marked lack of information in many places of the country. This study aimed to investigate the occurrence of these haemoparasites in symptomatic dogs and in their ticks in Iasi, eastern Romania. Ninety owned dogs were subjected to clinical examination at the Faculty of Veterinary Medicine of Iasi and all detectable ticks (58 ticks from 15 dogs) were collected. Additionally, 124 ticks collected from the coat of other dogs (no. = 23) were included. Three Babesia species were found in dogs: Babesia canis (94.4%), Babesia vogeli (3.3%), and Babesia rossi (2.2%). All the dogs resulted negative for H. canis. The ticks were identified as follows: Ixodes ricinus (64%), Dermacentor reticulatus (33%), and Rhipicephalus sanguineus group (3%). B. canis (Minimum Infection Rate; MIR = 81%), B. vogeli (MIR = 3%), and Babesia microti-like piroplasm (MIR = 1%) were found in ticks. Moreover, 15 ticks were positive for H. canis, 6 were co-infected with B. canis, and 1 with B. microti-like piroplasm. This is the first molecular identification of B. rossi in two symptomatic dogs from Romania, although further studies are needed to investigate the vector competence of other ticks from Europe.

Genetic Detection and Phylogenetic Relationship of Babesia Species Infecting Domestic Dogs from Select Regions in Kenya

The genus Babesia has more than 100 species that are transmitted by ticks with some being zoonotic. They can infect humans, livestock, and wildlife. Although canine babesiosis occurs locally, published studies on the species involved are limited. Babesia parasites cause severe disease in dogs which can be fatal. Drawbacks of the current control methods necessitate vaccine development. The study objective was to identify the Babesia species infecting dogs from three Kenyan counties; Nairobi, Mombasa, Nakuru and determine their phylogenetic relationship. This will enable improved control and rule out zoonotic potential. The study period was October 2018 to November 2019.The study design was descriptive and sampling opportunistic. One hundred and forty-three dogs were sampled. From whole blood, total DNA was extracted using the TanBead extractor followed by PCR amplification targeting Babesia 18S rRNA. Positive samples were purified and sequenced using the Sanger Dideoxy method.CLC Genomics Workbench, GenBank™ and BLASTn™ on NCBI were used for sequence processing and analysis. Geneious prime™ was used for multiple sequence alignment and phylogenetic analysis. The overall prevalence of Babesia canis was 9.0% (95% CI: 4.37–13.81). Two out of 13 positive samples (2/13) were identified as Babesia canis vogeli, with a prevalence of 1.4% (95% CI: 1.38–14.2, n = 143) while 11/13 were identified as Babesia canis rossi, with a prevalence of 7.69% (95% CI: 3.3–12, n = 143). The Babesia rossi sequences identified were closely related to sequences from black-backed jackals, while the Babesia vogeli ones were related to sequences from a pet cat in China. Babesia rossi which causes severe canine babesiosis was identified in 84.6% of the positive samples, immediate and aggressive clinical intervention is necessary. The possible sylvatic cycle of Babesia rossi and low levels of infections by Babesia vogeli should inform pertinent control measures.

Kinetics of the inflammatory response during experimental Babesia rossi infection of beagle dogs

Babesia rossi causes severe morbidity and mortality in dogs in sub-Saharan Africa, and the complications associated with this disease are likely caused by an unfocused, excessive inflammatory response. During this experimental B. rossi study we investigated inflammatory marker and cytokine kinetics during infection and after treatment. We aimed to determine whether infectious dose and treatment would influence the progression of the inflammatory response and clinical disease. Five healthy male beagle dogs were infected with B. rossi , three with a high infectious dose (HD group) and two with a low infectious dose (LD group). Clinical examination, complete blood count (CBC) and C-reactive protein (CRP) were determined daily. Cytokines were quantified on stored plasma collected during the study, using a canine specific cytokine magnetic bead panel (Milliplex©). The experiment was terminated when predetermined endpoints were reached. Parasitemia occurred on day 1 and 3 in the HD group and LD group respectively. The rate of increase in parasitemia in the HD group was significantly faster than that seen in the LD group. Significant differences were found in heart rate, blood pressure, interferon gamma (INFγ), keratinocyte chemoattractant (KC), INFγ-induced protein 10 (IP10), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP1), tumor necrosis factor alpha (TNFα), interleukin 2 (IL-2), IL-6, IL-7, IL-8, IL-10 IL-15, IL-18, CRP, neutrophils and monocytes between groups at multiple time points during the course of the infection. Our findings suggest that the initiation of inflammation occurs before the onset of clinical disease in B. rossi infection and infectious dose influences the onset of the inflammatory response. Treatment not only fails to curb the inflammatory response but may enhance it. Finally, we found that there is an imbalance in pro/anti-inflammatory cytokine concentrations during infection which may promote parasite replication.

Molecular and Biological Characterization of Haemaphysalis Leachi (Acari: Ixodidae) in Nigeria West Africa

Ticks constitute a serious threat to the wellbeing of humans and other animals. The accurate identification of ticks is paramount in epidemiological investigations. Genetic markers have been identified and used to overcome the limitations of phenotypic identification of ticks. In this study, the cytochrome c oxidase 1 (Cox1) gene was amplified and sequenced for the identification of Haemaphysalis leachi, the putative vector of Babesia rossi in Nigeria. Amplification was successful in 34 out of 39 (87 %) ticks collected from dogs in three Nigerian states with sequence homology of 99 % to H. leachi in Genbank (GenBank: MN663156.1). Maximum Likelihood phylogenetic analysis showed significant grouping of H. leachi sequences in independent monophyletic subclade with a bootstrap value of 100 %. Genetic distance analysis of H. leachi identified in this study indicated a very low level of intraspecific diversity (0.016 %) compared to 0.150—0.190 % interspecific distance to other Haemaphysalis species. The number of eggs laid by engorged female ticks maintained in the laboratory ranged from 885 to 2190 and was proportional to the ticks’ initial weight. The mean value of other biological parameters; female engorgement weight, pre-oviposition period, oviposition period, total egg mass, egg size, efficiency rates of female ticks in converting their food reservoir to eggs and incubation period are, 147.5 mg, 7.8 days, 13.2 days, 59.5 mg, 485.5 × 348.7 µm, 41.2 % and 26.2 days, respectively. This study reports the first molecular identification of H. leachi in Nigeria.

Genetic Detection and Phylogenetic Relationship of Babesia Species Infecting Domestic Dogs from Select Regions in Kenya

The Babesia genus has more than 100 species which are transmitted by ticks and infects humans, livestock and wildlife, some of which are zoonotic. New species continue being discovered which are poorly characterized. Locally, Babesia species occur in wildlife and livestock. Published literature on the species infecting dogs is limited.Local management practices enable close interaction between wildlife, livestock and humans. The societal role of dogs enable them serve as conduits for pathogens.Canine babesiosis causes a severe disease in dogs which can be fatal. Treatment required is lengthy and expensive. Current control methods rely on acaricide use. A vaccine against the disease is needed. Genetic characterization of local canine Babesia species would lay foundations for such development and assess any zoonotic potential.Molecular and bioinformatic methods i.e. DNA extraction (143 dogs sampled), PCR, sequencing and bioinformatic analysis were used in the study.13 samples were positive for Babesia canis; prevalence 9.0%, 95% confidence interval, (0.0437 to 0.1381).From the 13 positive samples, 2 were identified as Babesia canis vogeli; prevalence 1.4%, 95% confidence interval, (0.0138 to 0.142).While 11 were identified as Babesia canis rossi; prevalence 7.69%, 95% confidence interval, (0.033 to 0.12).Babesia rossi and Babesia vogeli were 84.6% and 15.4% of cases respectively. Phylogenetic analysis revealed the Kenyan B.rossi sequences to be closely related to B.rossi sequences from black-backed jackals. The B.vogeli sequences were closely related to a B.vogeli sequence obtained from a pet cat in China.Babesia rossi is known to cause the most severe form of canine babesiosis, 84.6% of the cases were positive for this parasite which requires immediate and aggressive medical intervention. The role of wildlife in the maintenance of the parasites especially B.rossi was noted, control measures would of necessity have to incorporate this component of the parasite lifecycle.

Experimental Babesia rossi infection induces hemolytic, metabolic, and viral response pathways in the canine host

Background Babesia rossi is a leading cause of morbidity and mortality among the canine population of sub-Saharan Africa, but pathogenesis remains poorly understood. Previous studies of B. rossi infection were derived from clinical cases, in which neither the onset of infection nor the infectious inoculum was known. Here, we performed controlled B. rossi inoculations in canines and evaluated disease progression through clinical tests and whole blood transcriptomic profiling. Results Two subjects were administered a low inoculum (104 parasites) while three received a high (108 parasites). Subjects were monitored for 8 consecutive days; anti-parasite treatment with diminazene aceturate was administered on day 4. Blood was drawn prior to inoculation as well as every experimental day for assessment of clinical parameters and transcriptomic profiles. The model recapitulated natural disease manifestations including anemia, acidosis, inflammation and behavioral changes. Rate of disease onset and clinical severity were proportional to the inoculum. To analyze the temporal dynamics of the transcriptomic host response, we sequenced mRNA extracted from whole blood drawn on days 0, 1, 3, 4, 6, and 8. Differential gene expression, hierarchical clustering, and pathway enrichment analyses identified genes and pathways involved in response to hemolysis, metabolic changes, and several arms of the immune response including innate immunity, adaptive immunity, and response to viral infection. Conclusions This work comprehensively characterizes the clinical and transcriptomic progression of B. rossi infection in canines, thus establishing a large mammalian model of severe hemoprotozoal disease to facilitate the study of host-parasite biology and in which to test novel anti-disease therapeutics. The knowledge gained from the study of B. rossi in canines will not only improve our understanding of this emerging infectious disease threat in domestic dogs, but also provide insight into the pathobiology of human diseases caused by Babesia and Plasmodium species.

Experimental Babesia Rossi Infection Induces Hemolytic, Metabolic, and Viral Response Pathways in the Canine Host

Background: Babesia rossi is a leading cause of morbidity and mortality among the canine population of sub-Saharan Africa, but pathogenesis remains poorly understood. Previous studies of B. rossi infection were derived from clinical cases, in which neither the onset of infection nor the infectious inoculum was known. Here, we performed controlled B. rossi inoculations in canines and evaluated disease progression through clinical tests and whole blood transcriptomic profiling.Results: Three subjects were administered a low inoculum (104 parasites) while two received a high (109 parasites). Subjects were monitored for 8 consecutive days; anti-parasite treatment with diminazene aceturate was administered on day 4. Blood was drawn prior to inoculation as well as every experimental day for assessment of clinical parameters and transcriptomic profiles. The model recapitulated natural disease manifestations including anemia, acidosis, inflammation and behavioral changes. Rate of disease onset and clinical severity were proportional to the inoculum. To analyze the temporal dynamics of the transcriptomic host response, we sequenced mRNA extracted from whole blood drawn on days 0, 1, 3, 4, 6, and 8. Differential gene expression, hierarchical clustering, and pathway enrichment analyses identified genes and pathways involved in response to hemolysis, metabolic changes, and several arms of the immune response including innate immunity, adaptive immunity, and response to viral infection. Conclusions: This work comprehensively characterizies the clinical and transcriptomic progression of B. rossi infection in canines, thus establishing a large mammalian model of severe hemoprotozoal disease to facilitate the study of host-parasite biology and in which to test novel anti-disease therapeutics. The knowledge gained from the study of B. rossi in canines will not only improve our understanding of this emerging infectious disease threat in domestic dogs, but also provide insight into the pathobiology of human diseases caused by Babesia and Plasmodium species.