Efficacy of combined atovaquone and azithromycin for therapy of chronic Babesia gibsoni (Asian genotype) infections in dogs

Babesiosis caused by Babesia gibsoni (Asian genotype) is an emerging disease in dogs in the United States. To date, no drugs have been shown to eliminate B. gibsoni (Asian genotype) infections from dogs. Twenty-two dogs that remained persistently infected with B. gibsoni (Asian genotype) after either imidocarb diproprionate and or diminazine aceturate therapy were identified and randomly and evenly distributed into 2 groups. One group was treated with atovaquone and azithromycin combination therapy, and the other group received a placebo. Eight of 10 dogs in the treatment group had no detectable B. gibsoni (Asian genotype) DNA, as determined by a sensitive and specific polymerase chain reaction (PCR) assay, in any of their posttreatment samples. In contrast, B. gibsoni (Asian genotype) DNA was detectable by PCR in the posttreatment samples from 11 of 11 of the placebo-treated dogs. One dog in the treatment group was excluded from the treatment outcome analysis. This dog had 2 consecutive negative PCR assay results and was euthanized because of ongoing degenerative joint disease prior to completion of the study. No adverse effects of treatment were reported in any dog during the study period. A combination of atovaquone and azithromycin is the 1st described treatment that will either eliminate B. gibsoni (Asian genotype) infections or suppress the parasitemia below the limit of detection in the majority of treated dogs.     

Azotemia and mortality among Babesia microti-like infected dogs

Babesia microti-like piroplasms are a recently recognized cause of illness in dogs in northwest Spain. Our objective was to describe the clinical characteristics and investigate the risk factors for azotemia and death among 58 B microti-like infected dogs. Twenty-one of the 58 (36%) dogs were azotemic at the time that the infection was diagnosed. The case fatality rate during the following week was 22%. Dogs with azotemia at the time of diagnosis were 10 times (95% CI, 3.26-28.8) more likely to die during the following week. Azotemia was the main cause of death for B microti-like infected dogs (attributable fraction = 90%). Severe anemia was present in 45 of the 58 (78%) dogs. Azotemic dogs also presented with hyperphosphatemia, hypoalbuminemia, hypercholesterolemia, proteinuria, and high urine protein: creatinine ratios, suggesting a glomerular component to the disease. Age was the only factor significantly associated with the risk of azotemia (P = .042): on average, a 4-year age increase doubled the risk of an infected dog being azotemic. The only factor significantly associated with mortality was azotemia (P = .001). We concluded that B microti-like infection is associated with a high risk of azotemia and mortality.     

Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs

The morphologically small Babesia species isolated from naturally infected dogs in Europe, Japan, and US are described as Babesia gibsoni despite the fact that molecular techniques show that they should be assigned to two or three separate taxons. The morphologically large Babesia isolated from dogs in Europe, Africa, and US were generally classified as B. canis until it was proposed to distinguish three related, albeit genetically distinct subspecies of this genus, namely B. canis canis, B. canis rossi, and B. canis vogeli. The insight into the molecular taxonomy of canine piroplasms is, however, limited because only partial small subunit ribosomal RNA (ssrRNA) sequence data exist for two species from the B. canis group. In this work, we molecularly characterised natural Babesia infections in 11 dogs from Croatia, France, Italy, and Poland. These infections were diagnosed as caused by B. canis canis and B. canis vogeli based on the analysis of the complete sequence of the ssrRNA genes. Phylogenetic analysis confirmed that the large Babesia species of dogs belong the to the Babesia sensu stricto clade, which includes species characterised by transovarial transmission in the tick vectors and by exclusive development inside the mammalian host erythrocytes. The new data facilitate the reliable molecular diagnosis of the subspecies of B. canis.     

Serial haematology results in transfused and non-transfused dogs naturally infected with Babesia rossi

This prospective longitudinal study investigated the progression of haematological changes in 32 transfused and 54 non-transfused dogs naturally infected with Babesia rossi over the 1st 6 days following diagnosis and treatment. The effect of patient age on the results of complete blood counts was determined. Haematology data were analysed at presentation and at 24 hours, 3 days and 6 days after presentation. Dogs were treated with diminazene aceturate at diagnosis and a blood transfusion was given if deemed clinically required. Mildly to moderately regenerative normocytic normochromic anaemia was observed in all dogs throughout the study period. Transfused dogs more often had an inflammatory leukogram at presentation and at 24 hours, than dogs that were not transfused. In dogs with a left shift, a concurrent normal or decreased segmented neutrophil count was found more commonly than neutrophilia. Severe thrombocytopenia that resolved within a week was common. Blood transfusion alleviated the anaemia, but had no significant effect on white blood cell or platelet responses. Blood cell responses were not significantly influenced by age. In conclusion, the red blood cell and white blood cell responses were less than expected in dogs with babesiosis, given the degree of anaemia and inflammation present. The magnitude of thrombocytopenia and rapid return of the platelet count to normal suggested a possible immune-mediated mechanism for the thrombocytopenia.     

Clinicopathological changes and effect of imidocarb therapy in dogs experimentally infected with Babesia canis

In this study one spleen-intact dog (A) and two splenectomised dogs (BSE, CSE) were infected with Babesia canis. All animals developed an acute disease characterised by fever, haemoglobinuria and anaemia, the latter being more severe in the splenectomised dogs. Fever and parasitised red blood cells were detected for three days after imidocarb treatment in the splenectomised animals. Haematological abnormalities included regenerative anaemia, thrombocytopenia and leukopenia (due to neutropenia and lymphopenia) in the acute phase, soon followed by leukocytosis, neutrophilia and left shift a few days later. Acute hepatopathy was detected in all dogs with elevated ALT activity, which was more seriously altered in the splenectomised dogs. Diffuse changes in liver structure and hepatomegaly were seen by ultrasonography. Liver biopsy and histology revealed acute, non-purulent hepatitis in the splenectomised dogs. Both splenectomised dogs were successfully cured after collection of 400 ml highly parasitised blood, proving that large-amount antigen production is possible with rescuing the experimental animals. Whole blood transfusion, imidocarb and supportive care with infusions, antipyretics, glucocorticoids and diuretics were applied. The spleen-intact dog clinically recovered after receiving supportive treatment, with no imidocarb therapy. Microbial infections developed in both splenectomised animals (BSE: haemobartonellosis, CSE: osteomyelitis caused by Escherichia coli), probably as a consequence of immunosuppression after splenectomy and glucocorticoid therapy.     

Evidence of an acute phase response in dogs naturally infected with Babesia canis

The erythrocyte sedimentation rate (ESR), white blood cell count (WBC), haematocrit (HCT) and platelet number (PLT) were quantified and compared with the acute phase proteins (APPs) in dogs naturally infected with Babesia canis and healthy dogs. Both groups were treated with imidocarb dipropionate on the day of admission and both groups were monitored for all parameters on the admission day and on the first, second, third, fourth and seventh days in order to determine the presence of an acute phase reaction, to assess the diagnostic value of these markers in uncomplicated canine babesiosis and to evaluate the use of APPs in treatment monitoring. It was demonstrated that an acute phase response occurs in dogs naturally infected with Babesia canis, with significant increases in the concentration of major acute phase proteins. The serum concentration of C-reactive protein (CRP), serum amyloid A (SAA) and the erythrocyte sedimentation rate (ESR) decreased daily after treatment and approached reference range values by the eighth day. PLT and haematocrit (HCT) increased daily after treatment and approached reference range values by the fourth day. WBC and haptoglobin increased after treatment and then decreased from the third and fourth days, respectively, to the eighth day. The diagnostic sensitivity of CRP, SAA and PLT was significantly higher compared to haptoglobin, ESR, HCT and the WBC count. CRP and SAA were of clinical use in monitoring the response to antibabesial treatment.     

Efficacy of atovaquone and azithromycin or imidocarb dipropionate in cats with acute cytauxzoonosis

Background: Imidocarb or a combination of atovaquone and azithromycin (A&A) has been suggested for treatment of cats with cytauxzoonosis, but neither has been prospectively evaluated for efficacy. Hypothesis/Objectives: That survival to hospital discharge is improved by treatment with A&A as compared with imidocarb. Animals: Eighty acutely ill cats with Cytauxzoon felis infection treated at one of 18 veterinary clinics in 5 states. Methods: An open‐label, randomized prospective study compared survival in cats treated with atovaquone (15 mg/kg PO q8h) and azithromycin (10 mg/kg PO q24h) or imidocarb (3.5 mg/kg IM). All received heparin, fluids, and supportive care. Clinical and clinicopathologic data from initial presentation were collated. Parasitemia was quantified (n = 79) and pathogens genotyped (n = 60). Logistic regression was used to determine the impact of treatment group on the primary endpoint, survival to hospital discharge or death. Covariants were analyzed by rank‐sum testing. Results: Of 53 cats treated with A&A, 32 (60%) survived to discharge while only 7 of 27 cats (26%) treated with imidocarb survived (P= .0036; odds ratio 7.2, 95% CI 2.2, 24). Cats with a lower parasitemia were more likely to survive, as were cats with higher white blood cell counts and lower total bilirubin. Unique pathogen genotypes were identified from 15 cats, while genotype isolated from 21 cats had been described previously. There were multiple pathogen genotypes identified in 24 cats. Conclusions and Clinical Importance: Survival to discharge was more likely in cats treated with A&A as compared with imidocarb, although case fatality rate remained high.     

Histological and ultrastructural studies of renal lesions in Babesia canis infected dogs treated with imidocarb

Histological and electron microscopic examinations of the kidneys of 8 dogs suffering from fatal, naturally acquired Babesia canis infection and nephropathy are presented. Seven animals were treated with imidocarb dipropionate on average 4.5 days prior to death. Severe anaemia was present only in 2 cases. Degenerative histological changes observed mostly in the proximal convoluted tubules included vacuolar-hydropic degeneration, necrosis and detachment of renal tubular epithelial (RTE) cells from the basement membrane. Necrotic debris occasionally formed acidophilic casts within the tubules. In some cases, necrosis of the whole tubule was observed. Haemoglobin casts in the tubules and haemoglobin droplets in RTE cells seldom appeared. No significant histological changes were seen in the glomeruli. Ultrastructural lesions in RTE cells included nuclear membrane hyperchromatosis, karyopyknosis, karyolysis, swelling or collapse of mitochondria with fragmentation of cristae and vacuolar-hydropic degeneration in the endoplasmic reticulum and microvilli. Nuclear oedema was also observed. Many RTE cells exhibiting necrosis collapsed. Vacuolar-hydropic degeneration and necrosis were also observed in the glomerular and interstitial capillary endothelium. The severe acute tubular necrosis described in this study is probably the result of hypoxic renal injury. Systemic hypotension leading to vasoconstriction in the kidneys might be the most important cause of renal hypoxia in B. canis infections, but anaemia may also contribute to inadequate oxygenation. Imidocarb should be applied with caution in patients with possible renal involvement until further data become available on its potential nephrotoxicity in dogs.     

Changes in anti-erythrocyte membrane antibody level of dogs experimentally infected with Babesia gibsoni.

To account for the conflict between the excessive destruction of erythrocytes and the number of parasitized erythrocytes in dogs infected with Babesia gibsoni, we examined the correlation between anti-erythrocyte membrane antibody level (AEMAL) and the number of erythrocytes (RBC count) in dogs with experimentally induced babesiosis using hematological examination and an enzyme linked immunosorbent assay (ELISA). In the infected dogs without splenectomy, more prominent reduction in RBC count accompanied with the elevated AEMAL was presented than anticipated from parasitemia until the 21st day. Furthermore, autoagglutinated erythrocytes and spherocytes were demonstrated in blood films. These results suggest that a humoral immunologic mechanism may be involved in a decrease in RBC count in dogs infected with B. gibsoni.     

Anti-erythrocyte membrane antibodies detected in sera of dogs naturally infected with Babesia gibsoni.

Due to the potential for anti-erythrocyte membrane antibodies as possible enhancers of erythrocyte destruction, the presence of serum anti-erythrocyte membrane antibodies in 31 dogs with Babesia gibsoni infection admitted to a veterinary hospital was investigated by an enzyme linked immunosorbent assay (ELISA) and immunoblotting analyses. This infection resulted in an increase of anti-erythrocyte membrane antibodies in 84% (IgG) and 74% (IgM) of 31 infected dogs, respectively. This was confirmed by the similarity in the protein profiles of the erythrocyte membrane antigens immunoblotted with rabbit antiserum to dog erythrocyte membrane antigens and infected dog serum. These results suggest the production of anti-erythrocyte membrane antibodies was induced by B. gibsoni infection.     

Soluble parasite antigens from Babesia canis do not directly activate the kallikrein system in dogs infected with Babesia canis

Soluble parasite antigens (SPA) from Babesia canis have been shown to induce protective immunity when used as vaccine. In order to explain the immune mechanisms of vaccination, the precise role of SPA in the pathogenesis of canine babesiosis is under investigation. Earlier studies suggested that the plasma kallikrein system is central in the pathogenesis of babesiosis, malaria and trypanosomosis, and significant plasma kallikrein activation during acute B. bovis and P. knowlesi infections has been described. In the studies presented here dogs were experimentally infected with B. canis to investigate whether the plasma kallikrein system is activated during babesiosis infection. Results showed that prekallikrein levels decreased during episodes of peak parasitaemia. No effect was found on the kallikrein levels. In order to determine whether B. canis SPA could activate plasma kallikrein, dogs were infused with variable amounts of B. canis SPA and plasma samples were taken for (pre-) kallikrein determination. The results indicated that B. canis SPA did not affect plasma (pre-) kallikrein levels. In addition, the effect of B. canis SPA on (pre-) kallikrein levels in normal dog plasma was determined in vitro. Again, no effect on (pre-) kallikrein levels was found. The results suggest that, although the kallikrein pathway may be involved in B. canis-associated pathology, the system is not directly activated by B. canis SPA. Furthermore, infusion of B. canis SPA as well as stroma of normal dog erythrocytes triggered the production of the acute phase reactant, C-reactive protein. This suggests that the inflammatory response that is triggered during B. canis infection could be in part due to the release and exposure of self molecules. The implications of these findings are discussed.     

Efficacy of atovaquone against Babesia gibsoni in vivo and in vitro

The therapeutic efficacy of atovaquone against Babesia gibsoni was examined in three dogs experimentally infected with B. gibsoni isolated from naturally infected dogs in Aomori Prefecture, Japan. Once parasitemia reached 10%, atovaquone was administered orally (30 mg/kg twice daily for 7 days). Within 2 days of atovaquone treatment, the parasite disappeared from blood smears without any clinical side effects. Anemia and thrombocytopenia were significantly improved in all the dogs. However, a polymerase chain reaction assay revealed that a B. gibsoni marker gene was intermittently present in peripheral blood after atovaquone therapy, indicating that the organism had not been eliminated, and parasites reappeared in blood smears 33 days after the last treatment. To investigate the change in sensitivity against atovaquone, an in vitro sensitivity test was performed using peripheral blood obtained from an untreated dog that was infected with the original parasite isolate, and from two of the experimentally infected and atovaquone-treated animals (blood was collected at the time of the post-treatment recurrence of the B. gibsoni infection). Atovaquone was added to the culture medium to final concentrations of 0.1, 1, 10, 100, and 1000 nM. For the untreated parasites, complete growth inhibition occurred at 1000 nM of atovaquone, whereas the recurrent parasites were inhibited by only 39.52 +/- 8.34% and 31.31 +/- 8.14% at this concentration after 48 h of incubation. Thus, the recurring parasites were less sensitive to atovaquone than the untreated originally isolated parasites.     

Lymphocyte subsets and specific IgG antibody levels in clindamycin-treated and untreated dogs experimentally infected with Babesia gibsoni

This study was carried out to clarify the role of lymphocyte subpopulations and Babesia-specific antibody on the treatment of clindamycin in dogs infected with B. gibsoni. Ten beagle dogs were divided into two groups: an untreated group (5 dogs) and a clindamycin-treated group (5 dogs), which was administered clindamycin at 25 mg/ kg body weight, per os, q 12 hr from 7 days to 21 days post-infection (PI). On the acute stage of infection, clindamycin treatment resolved anaemia and other clinical findings. There were no significant differences between treated and untreated dogs either in parasitemia levels or Babesial IgG antibody levels. However, morphological changes that indicated degeneration in the majority of parasites were observed. The numbers of CD4(+) showed a significant increase in treated dogs, especially after treatment. On the chronic stage, CD4(+) cells maintained high level both of the treated and untreated dogs. Although parasitemia maintained low level, their relapses were occurred on the 49th day PI in treated dogs and on the 42nd and 63rd PI in untreated dogs. A rapid humoral antibody response was observed in treated dogs, however, lower humoral antibody responses in untreated dogs after relapses. The antibody levels of treated dogs were significantly higher than those of untreated dogs. These results suggested that clindamycin might not eliminate rapidly parasites from peripheral blood, but damage parasites, which might stimulate efficiently humoral and cellular immunity against Babesia infection, and result in an improvement of clinical conditions.     

Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland

Babesia canis has generally been considered the only large Babesia to infect dogs. In this study, we used PCR to detect and characterize B. canis canis isolated from naturally infected dogs in Poland by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from 76 Babesia-symptomatic dogs. A 559-bp fragment of the B. canis canis 18S rRNA gene was amplified by PCR. The PCR products were then digested with HincII restriction enzyme, and isolates were classified according to whether they were cut (group A) or not (group B) by this endonuclease. Sequencing of the PCR products from the isolates led to the identification of seven sequence variants (four in group A, and three in group B). Sequences were compared with GenBank sequences, and alignments showed that all B. canis canis isolates from Europe may be classified into groups A or B as defined in our study.     

Biochemical abnormalities observed in serum of dogs infected with large Babesia in Warsaw (Poland)

Biochemical abnormalities observed in canine babesiosis are related to the severity of the disease. The primary biochemical abnormalities found in affected dogs are: increase of the serum activity of transaminases and alkaline phosphatase, azotemia, and hypoglycemia. The purposes of this study were: 1) to estimate biochemical abnormalities in dogs infected with large Babesia in Warsaw and 2) to evaluate statistically changes observed during canine babesiosis in dogs from Warsaw. Samples of serum were collected from dogs naturally infected with large Babesia. Among 2023 positive samples, 202 were randomly selected. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine, blood urea nitrogen (BUN), total serum protein (TSP), albumin and blood glucose concentration were determined with a clinical chemistry analyser. Elevated activity of ALT, AST and ALP was detected accordingly in: 64.9, 92.6 and 31.7% of dogs. Elevated creatinine concentration and BUN were detected accordingly in 30.7 and 62.4% of dogs. Decrease of TSP, albumin, BUN, and hypoglycemia was detected accordingly in: 19.8, 32.7, 1.5 and 18.3% of dogs. The most common biochemical abnormalities found in affected dogs were: increase of activity of transaminases and ALP, elevated creatinine concentration, hypoalbuminemia and hypoglycemia. These abnormalities resulted from hepatopathy, renal failure and fasting.     

A review of the small canine piroplasms from California: Babesia conradae in the literature

Small piroplasms as a cause of canine babesiosis in southern California were first documented in 1990. Initially these piroplasms were considered to be Babesia gibsoni, the only small Babesia parasite known to infect dogs at that time. In the following decade, the use of molecular analysis made it clear that small canine Babesia in fact are comprised of at least three distinct species, and the isolates from dogs in southern California were not B. gibsoni. Molecular, antigenic, and morphological characteristics of the southern California species of canine piroplasm supported naming it as a distinct species, Babesia conradae. The renaming of this species prompted this literature review of small canine piroplasms in California in order to clarify clinical, diagnostic, epidemiological, and molecular characteristics of B. conradae in comparison to other small canine piroplasms. Clinical symptoms of B. conradae are similar to those of B. gibsoni; however, B. conradae infections may be more pathogenic, resulting in higher parasitaemia and more pronounced anaemia when compared with B. gibsoni-infected dogs. The immunofluorescent antibody test is the most commonly used test to diagnose B. conradae. It is important to specify which small Babesia species to test for since there is little serological cross reactivity between the small canine Babesia antigens or cross-detection in the newer molecular tests. Molecular characterization of B. conradae, based principally on the 18S small subunit rRNA gene, and recently the second internal transcribed spacer region, demonstrate that B. conradae is most closely related to piroplasms recovered from humans and animals in the western United States.     

Clindamycin in the treatment of Babesia gibsoni infections in dogs

This report examines the effectiveness of clindamycin for the treatment of babesiosis in dogs (n=10) experimentally infected with Babesia gibsoni (B. gibsoni). Clindamycin (25 mg/kg body weight, per os, q 12 hours for 14 days) gradually reduced parasitemia levels and induced morphological changes that indicated degeneration of parasites (e.g., segmentation; size reduction; localization in the cell limbic and/or torn state of the nucleus; and swelling, decrease, or disappearance of the cytoplasm) in the majority of dogs. Clindamycin treatment reduced the clinical symptoms characteristic of Babesia infection, including anemia, anorexia, and listlessness. Clindamycin might be useful as a medicine for treatment of B. gibsoni infection.