Babesia in North America: An Update.

Canine babesiosis results from infection of 1 of 5 identified protozoal species in the United States (Babesia conradae, Babesia sp. "coco," Babesia gibsoni, Babesia vogeli, and Babesia vulpes). They are part of the Apicomplexa family of protozoa and are obligate intraerythrocytic parasites. Domestic and wild canids are suspected of being intermediate hosts. This updated article aims to provide practical guidance about the clinical manifestations of disease, treatment options, and outcomes. In addition, the authors hope to provide some clarity about the taxonomy and nomenclature of these organisms, as they have undergone multiple changes since their initial discovery.

Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti.

Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as ß-actin, ef-1ß, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

Transfusion-associated babesiosis in China: A case report.

Babesiosis, a novel zoonosis, is endemic in the Northeast and Midwest United States. This disease is primarily transmitted by ticks and less commonly transmitted through blood transfusion. Here, we present a case of human babesiosis of unknown etiology. The patient may have been infected through blood transfusion. This patient had fever for more than 1 month, accompanied by fatigue, anemia, jaundice, and other symptoms. Clinical improvement was unsatisfactory with antibiotics. Subsequently, peripheral blood smears showed many circulating forms of parasites,morphologically consistent with Babesia in red blood cells. Gene sequencing suggested Babesia microti. We treated the patient with azithromycin combined with other symptomatic supportive treatment. Finally, the patient recovered and was discharged. The intensity of babesiosis ranges from mild to severe and can be fatal, so early diagnosis and treatment are warranted.

Bovine abortion, stillbirth and neonatal death associated with Babesia bovis and Anaplasma sp. infections in southern Brazil.

Anaplasmosis and babesiosis are tick-borne diseases widely disseminated in cattle herds in many parts of the world. These diseases represent important causes of death and economic losses in several countries, including Brazil, and are characterized by hemolytic disease and anemia. Animals of all ages may be affected. Although transplacental infections are known to occur, abortion, stillbirth and neonatal death directly associated with Anaplasma marginale and especially Babesia spp. infections have rarely been documented in cattle. The objective of the present study is to describe the pathological and molecular findings of two cases of bovine abortion, two cases of stillbirth and two cases of neonatal death associated with intrauterine anaplasmosis and/or babesiosis in southern Brazil. All cases occurred in beef farms in the state of Rio Grande do Sul, between 2017 and 2019. Angus and crossbred calves were affected. At the necropsy, the main gross lesions observed included different degrees of splenomegaly, enlarged and yellow liver, thick and grumous bile, pallor or jaundice of mucous membranes and carcass, and dark kidneys. Four calves also presented cherry-pink discoloration of the central nervous system. Cytological slides enabled the observation of intraerythrocytic organisms consistent with Babesia bovis (3/6) and A. marginale (2/6). Through PCR assays, it was possible to detect three cases of Babesia sp. infection alone, and one case of Anaplasma sp. infection alone. Co-infections with Anaplasma sp. and Babesia sp. were detected in two cases. These findings reaffirm that anaplasmosis and babesiosis should be considered as an important differential diagnosis of fetal loss, stillbirth and neonatal death in cattle in areas where these diseases occur.

The pathology of the spleen in lethal canine babesiosis caused by Babesia rossi.

To provide useful information based on the macropathology, histopathology and immunohistochemical investigation in the spleens of dogs with Babesia rossi infection. Control spleens were collected from four healthy dogs euthanized for welfare reasons. Nine dogs that died naturally because of a mono-infection with Babesia rossi were selected for the diseased group. One haematoxylin-and-eosin-stained section of splenic tissue from each of the infected and control dogs was examined under the light microscope. Immunohistochemical markers were applied to characterize different immunocyte populations. The application of analytic software enabled semi-quantitative comparison of leucocyte subpopulations. Routine splenic histopathology revealed diffuse intermingling of white and red pulp from infected dogs with a clear loss of distinction between these zones. Immunohistochemistry revealed an increase in the proportion of tissue resident and bone marrow origin macrophages in the infected spleens. Apart from a few remnant lymphocytes within the peri-arteriolar lymphatic sheaths and follicles, the majority of the immunocytes redistributed to the red pulp, supporting the observation of white and red pulp intermingling. The majority of our findings are in agreement with histomorphological descriptions of the spleen in a variety of noncanid mammalian hosts with lethal malaria or babesiosis.

Evaluation of acute kidney injury in dogs with complicated or uncomplicated Babesia rossi infection.

Dogs with babesiosis can present with multiple complications, including acute kidney injury (AKI). The objective of this study was to characterize AKI in dogs with babesiosis caused by Babesia rossi at presentation and after treatment. Thirty-five client-owned dogs with B. rossi infection and 10 control dogs were included in this prospective observational study. Blood and urine were collected in Babesia-infected dogs at presentation (T0, n = 35), after 24 h (T24h, n = 11), and after 1 month (T1m, n = 9). The following urinary kidney injury biomarkers were assessed: urinary protein to creatinine ratio (UPC), urinary glomerular injury biomarkers (immunoglobulin G (uIgG) and C-reactive protein (uCRP)), and urinary tubular injury biomarkers (retinol-binding protein (uRBP) and neutrophil gelatinase-associated lipocalin (uNGAL)). Serum functional renal biomarkers were creatinine (sCr) and symmetric dimethylarginine (sSDMA). Post-mortem kidney biopsies were analyzed by light and transmission electron microscopy. At T0, all kidney injury biomarkers were significantly higher in Babesia-infected dogs compared to healthy controls (P < 0.001), while functional renal biomarkers were not significantly different (P > 0.05). At T24h, all urinary tubular injury biomarkers and UPC decreased significantly (P < 0.01), while glomerular injury biomarkers did not (P = 0.084). At T1m, all urinary kidney injury biomarkers decreased to values not significantly different from healthy controls (P > 0.5). Significant changes in functional renal biomarkers were not seen after treatment (P > 0.05). Dogs with complicated babesiosis had significantly higher glomerular injury biomarkers, UPC, and sSDMA compared to uncomplicated cases (P < 0.05), while all tubular injury biomarkers and sCr were not significantly different (P > 0.1). Dogs with babesiosis caused by B. rossi showed transient kidney injury, which was detected by all kidney injury biomarkers, but remained undetected by functional biomarkers. All infected dogs, irrespective of disease severity, suffered comparable kidney injury based on tubular injury biomarker concentrations, while loss of function was seen more often in dogs with complicated babesiosis based on sSDMA results.

Urinary proteome of dogs with kidney injury during babesiosis.

BACKGROUND: Acute kidney injury is the most frequent complication of babesiosis in dogs and may provide a natural model for identifying early and specific markers of kidney injury in this species. There are limited data on urine proteomics in dogs, and none of the effect of babesiosis on the urine proteome. This study aimed to identify urinary proteins of dogs with kidney injury during the natural course of babesiosis caused by Babesia canis, and to compare them with proteins in a control group to reveal any potential biomarkers predicting renal injury before the presence of azotemia. Urine samples were collected from 10 dogs of various breeds and sex with naturally occurring babesiosis, and 10 healthy dogs. Pooled urine samples from both groups were separated by 2D (two-dimensional) electrophoresis, followed by protein identification using MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometry. RESULTS: In total, 176 proteins were identified in the urine samples from healthy dogs, and 403 proteins were identified in the urine samples from dogs with babesiosis. Of the 176 proteins, 146 were assigned exclusively to healthy dogs, and 373 of the 403 proteins were assigned exclusively to dogs with babesiosis; 30 proteins were common for both groups. Characteristic analysis of 373 proteins found in dogs with babesiosis led to the isolation of 8 proteins associated with 10 metabolic pathways involved in immune and inflammatory responses. CONCLUSIONS: It was hypothesized that epithelial-mesenchymal transition might play an important role in the mechanisms underlying pathological changes in renal tissue during babesiosis, as indicated by a causal relationship network built by combining 5 of the 10 selected metabolic pathways, and 4 of the 8 proteins associated with these pathways; this network included cadherins, gonadotropin releasing hormone receptors, inflammatory responses mediated by chemokine and cytokine signalling pathways, integrins, interleukins, and TGF-ß (transforming growth factor ß) pathways. Those pathways were linked by interleukin-13, bone morphogenetic protein 7, α2(1) collagen, and tyrosine protein kinase Fer, which are potential biomarkers of damage during babesiosis in dogs, that might indicate early renal injury.

A clinical and pathological description of 320 cases of naturally acquired Babesia rossi infection in dogs.

Babesia rossi causes the most severe clinical disease in dogs of all the babesia parasites. We included 320 naturally-infected dogs that presented for care at the Onderstepoort Veterinary Academic Hospital between 2006 and 2016. All dogs had mono-infections confirmed by multiplex PCR. The data allowed more accurate clinical classification of the disease and identified parameters that were associated with disease severity and death. Odds ratios for dying were significant (P < 0.05) for increased band neutrophil count, collapse at presentation; presence of cerebral signs; hypoglycaemia; hyperlactatemia; high urea, high creatinine; hyperbilirubinaemia; hypercortisolaemia; and hypothyroxinaemia. Joint component analysis confirmed that the variables with significant odds ratios grouped together with death. Yet, multivariate logistic regression was unable to identify a group of significant independent predictors of death. Receiver Operator Characteristic curves indicated that low total thyroid hormone, high bilirubin, high serum urea and high cortisol concentrations were the variables with the highest sensitivity and specificity for death. These data provide both the clinician and researcher with a set of easily-measured laboratory and clinical assessments to classify cases into those that are uncomplicated and those that are complicated. The disease is complex and multisystemic and probably involves mechanisms more proximal in the pathogenesis than those that have been evaluated.

Canine babesiosis among working dogs of organised kennels in India: A comprehensive haematological, biochemical, clinicopathological and molecular epidemiological multiregional study.

Canine babesiosis is a serious disease among tick-borne haemoprotozoan diseases, globally. The present study was envisaged for carrying out thorough investigation of the disease among working dogs of organised kennels situated in different agro-climatic zones of India as comprehensive understanding of the disease from this country was pertinently lacking. During the study period of three years (2012-2014), 330 dogs suspected for babesiosis were examined for clinicopathology by their physical examination, haematological and biochemical parameters estimation, while the detection of apicomplexan parasites was confirmed by using various diagnostic techniques i.e. by conventional microscopy, by two different Babesia specific 18S rRNA based PCR protocols (conventional/simple PCR and nested PCR assays) followed by sequencing of obtained PCR amplicons for Babsesia spp. identification. Out of 330 clinical cases screened 5.15% (17/330), 9.09% (30/330) and 15.45% (51/330) were found to be positive in microscopic examination, simple- and nested- PCR assay, respectively. Comparative statistical analyses of these diagnostic assay results revealed that significant difference exists among the three diagnostic methodologies and thus it is recommended that the nested PCR technique be relied upon as a screening molecular assay and also for epidemiological studies of the disease in this country. Phylogenetic analysis based on 18S rRNA depicted the monophyletic nature and clonal expansion among all the B. gibsoni, under study. Sequencing results of PCR amplicons revealed that B. gibsoni has predominantly established itself over B. vogeli as former was incriminated in 47 cases while latter was confirmed in only four animals. Based on the clinical severity, these 51 affected animals were classified into three main groups' of 17 animals each viz., apparently healthy-, simple or uncomplicated babesiosis- and atypical or complicated babesiosis- group. Haematological and biochemical profiling of these dogs confirmed the characteristics findings of infection by both the Babesia spp. It was observed that the infection by small form of Babesia (B. gibsoni) is posing a significant therapeutic challenge and chemosterilization by commonly prescribed anti-protozoal drugs was not achieved as clinical relapses were often observed. The clinical signs, sequence based confirmation and severity of the infection suggested that there is a positive selection of B. gibsoni (smaller form) over B. vogeli (larger form) in this country and raises serious concerns as prognosis in former is considered to be poor compared to latter. Thus, these findings have opened new paradigms for planning of pragmatic control strategies against this emerging canine health problem.

Unravelling the cellular and molecular pathogenesis of bovine babesiosis: is the sky the limit?

The global impact of bovine babesiosis caused by the tick-borne apicomplexan parasites Babesia bovis, Babesia bigemina and Babesia divergens is vastly underappreciated. These parasites invade and multiply asexually in bovine red blood cells (RBCs), undergo sexual reproduction in their tick vectors (Rhipicephalus spp. for B. bovis and B. bigemina, and Ixodes ricinus for B. divergens) and have a trans-ovarial mode of transmission. Babesia parasites can cause acute and persistent infections to adult naïve cattle that can occur without evident clinical signs, but infections caused by B. bovis are associated with more severe disease and increased mortality, and are considered to be the most virulent agent of bovine babesiosis. In addition, babesiosis caused by B. divergens has an important zoonotic potential. The disease caused by B. bovis and B. bigemina can be controlled, at least in part, using therapeutic agents or vaccines comprising live-attenuated parasites, but these methods are limited in terms of their safety, ease of deployability and long-term efficacy, and improved control measures are urgently needed. In addition, expansion of tick habitats due to climate change and other rapidly changing environmental factors complicate efficient control of these parasites. While the ability to cause persistent infections facilitates transmission and persistence of the parasite in endemic regions, it also highlights their capacity to evade the host immune responses. Currently, the mechanisms of immune responses used by infected bovines to survive acute and chronic infections remain poorly understood, warranting further research. Similarly, molecular details on the processes leading to sexual reproduction and the development of tick-stage parasites are lacking, and such tick-specific molecules can be targets for control using alternative transmission blocking vaccines. In this review, we identify and examine key phases in the life-cycle of Babesia parasites, including dependence on a tick vector for transmission, sexual reproduction of the parasite in the midgut of the tick, parasite-dependent invasion and egression of bovine RBCs, the role of the spleen in the clearance of infected RBCs (IRBCs), and age-related disease resistance in cattle, as opportunities for developing improved control measures. The availability of integrated novel research approaches including "omics" (such as genomics, transcriptomics, and proteomics), gene modification, cytoadhesion assays, RBC invasion assays and methods for in vitro induction of sexual-stage parasites will accelerate our understanding of parasite vulnerabilities. Further, producing new knowledge on these vulnerabilities, as well as taking full advantage of existing knowledge, by filling important research gaps should result in the development of next-generation vaccines to control acute disease and parasite transmission. Creative and effective use of current and future technical and computational resources are needed, in the face of the numerous challenges imposed by these highly evolved parasites, for improving the control of this disease. Overall, bovine babesiosis is recognised as a global disease that imposes a serious burden on livestock production and human livelihood, but it largely remains a poorly controlled disease in many areas of the world. Recently, important progress has been made in our understanding of the basic biology and host-parasite interactions of Babesia parasites, yet a good deal of basic and translational research is still needed to achieve effective control of this important disease and to improve animal and human health.

Investigating disease severity in an animal model of concurrent babesiosis and Lyme disease.

The incidence of babesiosis, Lyme disease and other tick-borne diseases has increased steadily in Europe and North America during the last five decades. Babesia microti is transmitted by species of Ixodes, the same ticks that transmit the Lyme disease-causing spirochete, Borrelia burgdorferi. B. microti can also be transmitted through transfusion of blood products and is the most common transfusion-transmitted infection in the U.S.A. Ixodes ticks are commonly infected with both B. microti and B. burgdorferi, and are competent vectors for transmitting them together into hosts. Few studies have examined the effects of coinfections on humans and they had somewhat contradictory results. One study linked coinfection with B. microti to a greater number of symptoms of overall disease in patients, while another report indicated that B. burgdorferi infection either did not affect babesiosis symptoms or decreased its severity. Mouse models of infection that manifest pathological effects similar to those observed in human babesiosis and Lyme disease offer a unique opportunity to thoroughly investigate the effects of coinfection on the host. Lyme disease has been studied using the susceptible C3H mouse infection model, which can also be used to examine B. microti infection to understand pathological mechanisms of human diseases, both during a single infection and during coinfections. We observed that high B. microti parasitaemia leads to low haemoglobin levels in infected mice, reflecting the anaemia observed in human babesiosis. Similar to humans, B. microti coinfection appears to enhance the severity of Lyme disease-like symptoms in mice. Coinfected mice have lower peak B. microti parasitaemia compared to mice infected with B. microti alone, which may reflect attenuation of babesiosis symptoms reported in some human coinfections. These findings suggest that B. burgdorferi coinfection attenuates parasite growth while B. microti presence exacerbates Lyme disease-like symptoms in mice.

Robust adaptive immune response against Babesia microti infection marked by low parasitemia in a murine model of sickle cell disease.

The intraerythrocytic parasite Babesia microti is the number 1 cause of transfusion-transmitted infection and can induce serious, often life-threatening complications in immunocompromised individuals including transfusion-dependent patients with sickle cell disease (SCD). Despite the existence of strong long-lasting immunological protection against a second infection in mouse models, little is known about the cell types or the kinetics of protective adaptive immunity mounted following Babesia infection, especially in infection-prone SCD that are thought to have an impaired immune system. Here, we show, using a mouse B microti infection model, that infected wild-type (WT) mice mount a very strong adaptive immune response, characterized by (1) coordinated induction of a robust germinal center (GC) reaction; (2) development of follicular helper T (TFH) cells that comprise ∼30% of splenic CD4+ T cells at peak expansion by 10 days postinfection; and (3) high levels of effector T-cell cytokines, including interleukin 21 and interferon γ, with an increase in the secretion of antigen (Ag)-specific antibodies (Abs). Strikingly, the Townes SCD mouse model had significantly lower levels of parasitemia. Despite a highly disorganized splenic architecture before infection, these mice elicited a surprisingly robust adaptive immune response (including comparable levels of GC B cells, TFH cells, and effector cytokines as control and sickle trait mice), but higher immunoglobulin G responses against 2 Babesia-specific proteins, which may contain potential immunogenic epitopes. Together, these studies establish the robust emergence of adaptive immunity to Babesia even in immunologically compromised SCD mice. Identification of potentially immunogenic epitopes has implications to identify long-term carriers, and aid Ag-specific vaccine development.

Histopathological changes and oxidative damage in hepatic tissue of rats experimentally infected with Babesia bigemina.

The present study was aimed to investigate oxidative stress, DNA damage, and histopatholog- ical alterations in hepatic tissues of splenectomized Wistar rats experimentally infected with Ba- besia bigemina. Rats were challenged with 5x106 infected erythrocytes. Babesia infection was con- firmed both with Giemsa's staining blood smears and nested-PCR amplified region of apical membrane antigen-1 (AMA-1) gene. Parasitemia reached approximately 10 % at day 5 post-in- fection. Livers of infected rats were enlarged and darker in color, became extremely brittle with marked congestion. Microscopic evaluation showed cytoplasmic clearing of hepatocytes and se- vere hydropic changes with significantly dilated sinusoids containing macrophages and also intra- sinosoidal parasitized erythrocytes. Severe infiltration of lymphoplasma cells was also present throughout the liver parenchyma. Furthermore, Kupffer cells were enlarged and, occasionally, containing Babesia-parasitized erythrocytes. The activity of Glutathione (GSH) and catalase (CAT), and total antioxidant capacity (TAC) were also significantly decreased (p ⟨ 0.05) after infection of rats with B. bigemina. B. bigemina infection also induced a significant increase (p ⟨ 0.05) in hepatic malondialdehyde (MDA) and nitric oxide-derived products (NOx) concentra- tions as well as amount of endogenous hepatocytes DNA damage. Hepatic damage was also re- flected through the measurement of lactic acid dehydrogenase (LDH) and protein carbonyl con- tent (PCO) in liver cells. These two indices of liver injury were also significantly elevated (p ⟨ 0.5) during B. bigemina infection. Evaluation of correlation between assayed variables in infected rats revealed that MDA levels were positively correlated with PCO, NOx, LDH and DNA damage in the infected group and negatively correlated with GSH, CAT and TAC. There was also an inverse relationship between the antioxidant enzymes activities of GSH, CAT and TAC with PCO, NOx and DNA damage in infected rats. However, NOx showed positive correlation with PCO and DNA damage in infected rats. On the basis of the above results it can be concluded that the Ba- besia infection increases oxidative stress markers, protein carbonyl content and DNA damage and decreases antioxidant enzymes activities in the liver. These results suggest that B. bigemina infec- tion could alter the liver histopathology and causes DNA damage following oxidative stress in hepatic tissue. Further studies are needed to precisely define how hepatic tissue damage takes place in B. bigemina infection.

Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka.

Bovine babesiosis is a serious threat to the cattle industry. We prepared blood DNA samples from 13 cattle with clinical babesiosis from the Badulla (n = 8), Jaffna (n = 3), and Kilinochchi (n = 2) districts in Sri Lanka. These DNA samples tested positive in PCR assays specific for Babesiabovis (n = 9), Babesia bigemina (n = 9), and Babesiaovata (n = 1). Twelve cattle were positive for B. bovis and/or B. bigemina One cow was negative for the tested Babesia species but was positive for Babesia on microscopic examination; the phylogenetic positions of 18S rRNA and cytochrome oxidase subunit III gene sequences suggested that the cow was infected with Babesia sp. Mymensingh, which was recently reported from a healthy cow in Bangladesh. We then developed a novel Babesia sp. Mymensingh-specific PCR assay and obtained positive results for one other sample. Analysis of gene sequences from the cow with positive B. ovata-specific PCR results demonstrated that the animal was infected not with B. ovata but with Babesia sp. Hue-1, which was recently reported from asymptomatic cattle in Vietnam. The virulence of Babesia sp. Hue-1 is unclear, as the cow was coinfected with B. bovis and B. bigemina However, Babesia sp. Mymensingh probably causes severe clinical babesiosis, as it was the sole Babesia species detected in a clinical case. The present study revealed the presence of two bovine Babesia species not previously reported in Sri Lanka, plus the first case of severe bovine babesiosis caused by a Babesia species other than B. bovis, B. bigemina, and Babesiadivergens.