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.

Babesia species infect a very wide range of mammal hosts across the globe, and zoonotic infections are of growing concern. Several species of the Babesia genus infect dogs, and some of these cause significant morbidity and mortality. The Apicomplexan parasite resides within the red cell and infections result in direct damage to the host through intra- and extravascular hemolysis. An exuberant inflammatory response by the host to some species of Babesia parasites also results in significant collateral damage to the host. Canine infections have been the subject of many studies as the well-being of these companion animals is increasingly threatened by the spread of tick vectors and an increasingly mobile dog population. There are currently no widely available and effective vaccines, and effective treatment can be challenging. Understanding disease pathogenesis underlies the development of new treatments. The varying pathogenicity of the various Babesia parasite species that infect dogs offers an opportunity to explore the molecular basis for the wide range of diseases caused by infection with this parasite genus. In this review, we focus on what has been reported about the clinical presentation of Babesia-infected dogs in an attempt to compare the severity of disease caused by different Babesia species.

BACKGROUND: Babesia gibsoni, the causative agent of canine babesiosis, belongs to the phylum Apicomplexa. The development of in vitro culture technology has driven research progress in various kinds of omics studies, including transcriptomic analysis of Plasmodium spp. between in vitro and in vivo environments, which has prompted the observation of diagnostic antigens and vaccine development. Nevertheless, no information on Babesia spp. could be obtained in this respect, which greatly hinders the further understanding of parasite growth and development in the blood stage.
METHODS: In this study, considerable changes in the morphology and infectivity of continuous in vitro cultured B. gibsoni (Wuhan isolate) were observed compared to in vivo parasites. Based on these changes, B. gibsoni (Wuhan isolate) was collected from both in vivo and in vitro cultures, followed by total RNA extraction and Illumina transcriptome sequencing. The acquired differentially expressed genes (DEGs) were validated using qRT-PCR, and then functionally annotated through several databases. The gene with the greatest upregulation after in vitro culture was cloned from the genome of B. gibsoni (Wuhan isolate) and characterized by western blotting and indirect immunofluorescence assay for detecting the native form and cellular localization.
RESULTS: Through laboratory cultivation, multiple forms of parasites were observed, and the infectivity of in vitro cultured parasites in dogs was found to be lower. Based on these changes, Illumina transcriptome sequencing was conducted, showing that 377 unigenes were upregulated and 334 unigenes were downregulated. Notably, an AP2 transcription factor family, essential for all developmental stages of parasites, was screened, and the transcriptional changes in these family members were tested. Thus, the novel AP2 transcription factor gene (BgAP2-M) with the highest upregulated expression after in vitro adaptation was selected. This gene comprises an open reading frame (ORF) of 1989 base pairs encoding a full-length protein of 662 amino acids. BgAP2-M contains one AP2 domain and one ACDC conserved domain, which may be involved in the nuclear biology of parasites. The prepared polyclonal antibodies against the BgAP2-M peptides further detected a native size of ~ 73 kDa and were localized to the nuclei of B. gibsoni.
CONCLUSIONS: This study presents a thorough transcriptome analysis of B. gibsoni in vivo and in vitro for the first time, contributing to a detailed understanding of the effects of environmental changes on the growth and development of parasites in the blood stage. Moreover, it also provides a deeper investigation for the different members of the ApiAP2 transcription factor family as various life stage regulators in Babesia spp.

Tick-borne haemoparasite Babesia gibsoni has been detected rarely in cats, in surveys of apparently healthy animals. In stored blood from a 6-year-old male-neutered domestic shorthair cat in Hong Kong, B. gibsoni DNA was detected retrospectively using PCR for Babesia spp. 18S rRNA and mitochondrial cytochrome B genes, followed by sequencing and basic local alignment search tool (BLAST) analysis. The cat presented with severe haemolytic anaemia and thrombocytopenia. The cat responded to supportive care and glucocorticoids and was clinically normal despite persistent subclinical thrombocytopenia until six months after presentation, when it succumbed to a fatal haemorrhagic episode. Necropsy revealed severe intestinal and pulmonary haemorrhage and hypocellular bone marrow with megakaryocytosis but no other causes of immune-mediated thrombocytopenia (IMTP) or immune-mediated haemolytic anaemia (IMHA). Blood stored on days 158 and 180 tested PCR negative for Babesia spp. This report demonstrates that geographic range of B. gibsoni detection in cats includes Hong Kong. The exclusion of other causes suggests that B. gibsoni might have potentially played a role in triggering immune-mediated disease in this case.

The intracellular protozoan parasite Babesia gibsoni infects canine erythrocytes and causes babesiosis. The hazards to animal health have increased due to the rise of B. gibsoni infections and medication resistance. However, the lack of high-quality full-genome sequencing sets has expanded the obstacles to the development of pathogeneses, drugs, and vaccines. In this study, the whole genome of B. gibsoni was sequenced, assembled, and annotated. The genomic size of B. gibsoni was 7.94 Mbp in total. Four chromosomes with the size of 0.69 Mb, 2.10 Mb, 2.77 Mb, and 2.38 Mb, respectively, 1 apicoplast (28.4 Kb), and 1 mitochondrion (5.9 Kb) were confirmed. KEGG analysis revealed 2,641 putative proteins enriched on 316 pathways, and GO analysis showed 7,571 annotations of the nuclear genome in total. Synteny analysis showed a high correlation between B. gibsoni and B. bovis. A new divergent point of B. gibsoni occurred around 297.7 million years ago, which was earlier than that of B. bovis, B. ovata, and B. bigemina. Orthology analysis revealed 22 and 32 unique genes compared to several Babesia spp. and apicomplexan species. The metabolic pathways of B.gibsoni were characterized, pointing to a minimal size of the genome. A species-specific secretory protein SA1 and 19 homologous genes were identified. Selected specific proteins, including apetala 2 (AP2) factor, invasion-related proteins BgAMA-1 and BgRON2, and rhoptry function proteins BgWH_04g00700 were predicted, visualized, and modeled. Overall, whole-genome sequencing provided molecular-level support for the diagnosis, prevention, clinical treatment, and further research of B. gibsoni. IMPORTANCE The whole genome of B. gibsoni was first sequenced, annotated, and disclosed. The key part of genome composition, four chromosomes, was comparatively analyzed for the first time. A full-scale phylogeny evolution analysis based on the whole-genome-wide data of B. gibsoni was performed, and a new divergent point on the evolutionary path was revealed. In previous reports, molecular studies were often limited by incomplete genomic data, especially in key areas like life cycle regulation, metabolism, and host-pathogen interaction. With the whole-genome sequencing of B. gibsoni, we provide useful genetic data to encourage the exploration of new terrain and make it feasible to resolve the theoretical and practical problems of babesiosis.

Implementing hemoprotozoan control strategies in dogs has become difficult because of the co-infections. A multiplex polymerase chain reaction (PCR) was carried out for simultaneous detection of the co-infections of Babesia gibsoni, B. vogeli, Hepatozoon canis and Ehrlichia canis from dogs (N = 442) in Andhra Pradesh, South India. The co-infection combinations were classified as (i) B. gibsoni + B. vogeli + E. canis + H. canis (BEH), (ii) B. gibsoni + B. vogeli + E. canis (BE), (iii) B. gibsoni + B. vogeli + H. canis (BH) and (iv) E. canis + H. canis (EH) groups. The parasite-specific multiplex PCR amplified 18S rRNA gene of B. gibsoni, B. vogeli and H. canis and VirB9 gene of E. canis. The age, gender, breed, medium, living condition and region of dogs were studied as risk factors for co-infections using logistic regression model. Among the co-infections, the incidence was 1.81%, 9.28%, 0.69% and 0.90% for BEH, BE, BH and EH infections, respectively. Young age (< one year), females, mongrels, rural dogs, kennel dogs and presence of ticks were the identified risk factors for overall prevalence of tick-borne pathogens. The incidence of infection was less in rainy season, especially in dogs with a previous acaricidal treatment. The study concludes that the multiplex PCR assay could simultaneously detect natural co-infections in dogs, emphasizing the need for the assay in epidemiological studies to reveal the real pattern of pathogens and select pathogen-specific treatment protocols.

The establishment of in vitro culture methods has greatly facilitated the research of Babesia. However, the current Babesia gibsoni in vitro culture medium requires high concentrations of canine serum, which intensively limits the culture and is unable to satisfy the demands of long-term studies. In this study, AlbuMAX I (2 mg/mL) and 2.5% dog serum (vol/vol) were added to VP-SFM medium to develop a low-concentration serum culture medium named VP-SFMAD (2.5%), and the effectiveness of this medium was assessed by the growth of B. gibsoni. The results showed that VP-SFMAD (2.5%) could support the continuous growth of the parasite, and the parasitemia has no difference with the cultivation in RPMI 1640 with 20% dog serum. In contrast, either a low concentration of dog serum or absence of AlbuMAX I will significantly lower the parasite growth or fail to maintain B. gibsoni growth in the long term. The strategy of reducing the hematocrit was also evaluated, and VP-SFMAD (2.5%) improved the parasitemia to over 50% within 5 days. The high parasitemia is helpful for larger numbers of parasite collection, which is valuable for studying the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites. In addition, VP-SFMAD (2.5%) medium was successfully used for monoclonal parasite screening, which obtained monoclonal strains with parasitized erythrocytes about 3%, which is similar to RPMI-1640D (20%) medium that obtains monoclonal strains on the 18th day. Those results showed that VP-SFMAD can be applied to B. gibsoni continuous long-term, expansion culture, and subclone culture. IMPORTANCE The VP-SFM as a base medium supplemented with AlbuMAX I and a low concentration of canine serum (2.5%) allowed the continuous in vitro culture of Babesia gibsoni at both small and large volumes, which was to meet different experimental needs, such as long-term culture and obtaining high parasitemia and subclone culture. The establishment of in vitro culture systems allows researchers to better understand the metabolism and growth patterns of Babesia. Importantly, several technical problems impeding such studies have been overcome.

Babesia gibsoni is a tick-borne apicomplexan protozoan causing canine babesiosis. This parasite has diploid sexual reproduction in ticks, during which genetic exchanges can occur leading to increased genetic diversity, which is an important factor in adapting to environmental changes. Exploring the genetic variation of B. gibsoni population can provide a foundation for understanding the patterns of disease transmission and developing babesiosis control strategies. Partial 18S rRNA fragment sequences were obtained from 11 B. gibsoni isolates collected from different regions in China and 117 publicly available sequences were from 12 geographical areas including China. The genetic variation, demographic expansion and population structure were examined. A total of 34 haplotypes were identified among B. gibsoni populations. Analysis of molecular variance, pairwise Fst and structure analysis showed that high genetic variation within populations, low genetic differentiation and obvious mixture haplotype were apparent in a single continent, but higher genetic differentiation was detected across different continents. Neutrality tests implied that B. gibsoni populations had experienced population extension. These findings will contribute to understand the genetics and evolution of B. gibsoni and will be useful for formulating effective management strategies to prevent and control this parasite.

As there are few studies of Babesia spp. infection in cats in China, or anywhere in the world, the aim of this study was to explore the epidemic features of babesiosis in pet cats in China. In total, 429 blood samples were randomly collected in four different geographical regions. The 18S rRNA gene fragment of Babesia spp. was amplified by nest polymerase chain reaction (PCR), and haplotype and phylogenetic analysis of Babesia were performed to analyze the relationship of this protozoa. The total positive rate of infection was 2.8%. BLAST analysis indicated that Babesia gibsoni was detected in 12 cats. Among these, 4.3%, 3.1%, 0.8% and 2.0% were from Chongqing, Fujian, Hubei and Shandong, respectively. Haplotype and phylogenetic analysis showed that there were nine haplotypes and no obvious genetic variation among B. gibsoni populations. These findings will be helpful for understanding the epidemiology of Babesia spp. in China, and provide a foundation for developing effective preventative strategies.

OBJECTIVE: Babesiosis is one of the most important globally extended and quickly spreading tick-borne infections of dogs. Diagnosis of babesiosis in Sri Lanka is based on clinical signs followed by thin blood smears which could be error-prone due to undetected early infections, absence of clinical signs or low parasitemia. The present study investigated the prevalence of babesiosis in dogs presented to the Veterinary Teaching Hospital (VTH) at the Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, for treatments, vaccinations, and regular check-ups, and compared the diagnosis methods of microscopy and molecular analysis.
METHODS: Blood samples from dogs were collected from January to June 2019. First, Giemsa stained blood smears were prepared, and then the blood samples were subjected to PCR using genus-specific primers to amplify a 411-450 bp region in the 18S rRNA gene. Twenty samples from PCR amplified products were sequenced for species identification and phylogenetic analysis. Clinical signs of the dogs were noted down, and ticks were also collected from dogs if any.
RESULTS: Results show a very high prevalence of canine babesiosis (78.6%) among the dogs brought to the VTH. The parasite was identified microscopically and genetically as Babesia gibsoni. A large percentage (66.7%) of infections was asymptomatic. Out of 42 blood samples, 19 (45.2%) were microscopically positive for babesiosis while 33 (78.6%) were PCR positive, showing a significant difference in the two methods of diagnosis (chi-square test, χ2 = 9.462, p = 0.002). Three tick species: Rhipicephalus haemaphysaloides, Haemaphysalis bispinosa, and Rhipicephalus sanguineus were found attached to the dogs.
CONCLUSIONS: This study shows a very high prevalence of canine babesiosis among dogs in the Kandy area. Most of these infections might go undetected if only microscopy was used to diagnose. An improved, rapid diagnostic method such as the novel, PCR-based point-of-care diagnostic method that detects very low parasitemia within 30 min is needed. Moreover, as most infected dogs did not show clinical signs, they may act as reservoirs of infection. The ability of asymptomatic dogs to spread babesiosis should be investigated.