Proteomic analysis reveals pathogen-derived biomarkers of acute babesiosis in erythrocytes, plasma, and urine of infected hamsters.

Babesiosis among humans is on the rise in North America. Current diagnostic assays for the screening of babesiosis require blood collection by venipuncture, which is an invasive method. Urine on the other hand is a desirable biospecimen for biomarker analysis of Babesia microti infections because it can be collected periodically and non-invasively. Our group uses a new class of biomarker harvesting nanocage technology, which, when combined with mass spectrometry (MS), can determine the presence of parasite proteins shed in different bodily fluids of mammalian hosts, including urine. Using the hamster model of babesiosis, our nanoparticle-MS approach identified several B. microti proteins in erythrocytes, plasma, and urine samples. Surface and secreted antigens previously shown to elicit host immune responses against the parasite were particularly abundant in erythrocytes and plasma compared to other proteins. Two of these antigens, BmSA1 and BMR1_03g00947, showed different localization patterns by immunofluorescence of infected erythrocytes. Hamster urine samples from parasitemic animals harbored lower numbers of B. microti proteins compared to erythrocytes and plasma, with glycolytic enzymes, cytoskeletal components, and chaperones being the most frequently detected proteins. By applying novel nanoparticle-MS methods, a high level of analytical sensitivity can be achieved to detect multiple B. microti proteins in blood and urine. This is generally difficult to obtain with other techniques due to the masking of parasite biomarkers by the complex biomolecular matrix of bodily fluids from the host.

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.

Glomerular and tubular kidney damage markers in canine babesiosis caused by Babesia canis.

Canine babesiosis is a tick-borne disease caused by the haemoprotozoan parasites of the genus Babesia. The aim of this study was to assess renal dysfunction in dogs with babesiosis caused by B. canis, using serum and urinary markers for both glomerular and tubular dysfunction. Assays previously not validated for use in canine samples were validated and the potential interference of haemoglobin, lipids and bilirubin with these analyses was additionally considered. In this study 42 dogs naturally infected with B. canis and 14 healthy dogs were included. Dogs with babesiosis were divided into 3 groups: group A consisted of 9 non-azotemic dogs with normal urine protein to creatinine ratio (UPC < 0.5), group B of 27 non-azotemic dogs with UPC > 0.5 and group C of 6 azotemic dogs with UPC > 2. The concentrations of urinary immunoglobin G (IgG), retinol binding protein (RBP), uromodulin, kidney injury molecule - 1 (KIM-1), and serum symmetric dimethylarginine were measured by ELISA assays, while urinary albumin and N-acetyl-b-D-glucosaminidase (NAG) were evaluated by an immunoturbidimetric and enzymatic colorimetric assay, respectively. Urinary markers were normalized to urine creatinine concentration. All tested markers, with exception of uromodulin, showed significant differences between dogs with babesiosis and healthy dogs, and also showed strong or very strong positive correlation with UPC. Increases of urinary albumin and IgG suggested glomerular damage, and increases of KIM-1, RBP and NAG proximal tubular damage in dogs with babesiosis. They demonstrated clear advantages compared to conventional parameters by showing earlier changes in detecting renal damage.

Stability of glomerular and tubular renal injury biomarkers in canine urine after 4 years of storage.

Urine biomarkers are sensitive indicators of early-stage renal injury, consequently, research in this area is expanding in both human and veterinary medicine. However, studies investigating the impact of preanalytical factors, such as storage conditions, on urine biomarker concentrations are largely lacking in veterinary medicine. Therefore, we evaluated the stability of several renal injury biomarkers in canine urine after storage for 4 y at -72°C. Urine samples were collected from 26 dogs: 18 dogs with babesiosis and 8 healthy dogs. Concentrations of urine immunoglobulin G (uIgG), urine C-reactive protein (uCRP), and urine retinol-binding protein (uRBP) were measured, using validated commercial immunoassays, at the start of the study and 4 y later. To investigate the effect of long-term storage, absolute and relative differences between both measurements were compared. Additionally, dogs with babesiosis were compared with the healthy controls at both time points. Storage caused significant absolute and relative decreases in concentrations of all 3 biomarkers. Significant differences between dogs with babesiosis and healthy dogs were found in uIgG and uRBP at both times; however, the difference in uCRP between both groups lost significance after storage. Because the main goal of these urine biomarkers is to detect early-stage renal injury, the statistically significant decrease in their concentrations will be clinically relevant when a mild degree of renal injury is present. Our data indicate that the investigated urine biomarkers show significant decay after 4 y of storage at -72°C, adversely affecting their diagnostic utility.

Utility of urinary markers in the assessment of renal dysfunction in canine babesiosis.

OBJECTIVE: Canine babesiosis is a common and clinically significant tick-borne disease caused by haemoprotozoan parasites of the genus Babesia. Acute renal failure is considered to be one of the most prevalent complications of canine babesiosis. This complication leads to a decrease in the glomerular filtration rate and in consequence causes azotemia and uremia. The objective of this study was to assess the localization and extent of renal damage in dogs infected with Babesia canis using an urinary marker for glomerular (urinary immunoglobulin G, uIgG), proximal tubular dysfunction (urinary retinol binding protein, uRBP) and distal tubular dysfunction (urinary Tamm-Horsfal protein, uTHP). Material und methods: In 10 dogs naturally infected with B. canis and 10 healthy control dogs the levels of urinary biomarkers were measured using commercially available ELISA tests. RESULTS: Higher concentrations of uIgG, uRBP and uTHP were found in the urine of all dogs with babesiosis than in those from the control group. This indicates that in the course of the disease, the glomeruli as well as the renal tubules become damaged. CONCLUSION AND CLINICAL RELEVANCE: The study results allow a better understanding of the pathogenesis of canine babesiosis. However, in order to fully determine the extent and the nature of the damage to the kidneys of the infected dogs, it is advisable to conduct additional histopathological examinations of these organs.

Assessment of renal dysfunction using urinary markers in canine babesiosis caused by Babesia rossi.

Renal damage is deemed a common, yet poorly documented, complication in canine babesiosis. Serum urea and creatinine are insensitive and non-specific markers of early renal dysfunction and their measurements are influenced by hemolysis caused by babesiosis. Therefore, the aim of this study was to use urinary markers to assess the localization and degree of renal dysfunction in dogs with Babesia rossi infection. Urinary immunoglobulin G (uIgG) and urinary C-reactive protein (uCRP) were measured as markers for glomerular dysfunction, while urinary retinol-binding protein (uRBP) was used as a marker for tubular dysfunction. Eighteen dogs presenting with uncomplicated babesiosis were included and compared with eight clinically healthy dogs. Previously validated commercial ELISA kits were used for the measurement of uIgG, uCRP, and uRBP. Results were related to urinary creatinine concentrations (c). Dogs with babesiosis had significantly higher concentrations of all three measured urinary markers compared to healthy dogs. Except for urinary protein/c ratio (UPC), routine urinary and serum markers for renal function (urine specific gravity (USG), serum urea and creatinine (sCr)) were not significantly different between dogs with babesiosis and healthy dogs. All three urinary markers were positively correlated with each other and with UPC. The data supports the presence of both glomerular and tubular dysfunction in dogs suffering from uncomplicated B. rossi infection. Urinary markers were superior to USG, serum urea and creatinine concentrations for the early detection of renal dysfunction in dogs with babesiosis.

Changes in the SUSPPUP ratio and fractional excretion of strong monovalent electrolytes in hospitalized dogs with canine babesiosis.

In this study an increased SUSPPUP ratio and fractional excretion of potassium in dogs infected with Babesia canis suggested mineralocorticoid excess in canine babesiosis. A significant increase in strong monovalent electrolyte fractional excretions in azotaemic dogs infected with B. canis probably resulted from acute tubular necrosis.

Free light-chain proteinuria and normal renal histopathology and function in 11 dogs exposed to Lleishmania infantum, Ehrlichia canis, and Bbabesia canis.

The purpose of this retrospective study was to investigate the relationship among proteinuria consisting of immunoglobulin free light chains (FLCs), renal histopathologic findings, and routine markers of renal function in 11 dogs exposed to Leishmania infantum (n = 8), Ehrlichia canis (n = 2), and Babesia canis (n = 1). FLC proteinuria was suspected based on identification of a 22- to 27-kDa band by sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) and later confirmed by immunofixation electrophoresis. SDS-AGE identified an isolated band of 22-27 kDa in 8 dogs, whereas the remaining 3 had a 22- to 27-kDa band and an additional band of 67-72 kDa. The median urine protein-to-urine creatinine ratio was 0.37 (range, 0.11-2.24) and increased ratios were found in 6 dogs (54.5%) (reference value, <0.7). All dogs underwent histologic examination of renal percutaneous biopsy specimens and determination of serum creatinine and urea concentrations. Tissue samples for light microscopy were stained with hematoxylin-eosin, periodic acid-Schiff, Goldners trichrome, and methenamine silver. In the study group, the glomerular tufts, mesangium, tubulointerstitium, and vessels appeared unaffected. The median serum creatinine concentration in these 11 dogs was 1.3 mg/dL (range, 0.8-1.5 mg/dL; reference range, 0.6-1.5 mg/dL), whereas the concentration for urea was 28 mg/dL (range, 22-52 mg/dL; reference range, 20-50 mg/dL). All dogs had normal renal morphology and had normal serum creatinine and urea concentrations, suggesting that immunoglobulin FLC may be detected in the urine of dogs exposed to L. infantum, E. canis, and B. canis without any apparent structural or functional renal derangement.

Nitric oxide metabolites in naturally occurring canine babesiosis.

Babesiosis, caused by the virulent haemoprotozoan parasite Babesia canis rossi, is an important disease of dogs in South Africa. The nitric oxide metabolites, nitrate and nitrite (collectively termed reactive nitrogen intermediates or RNIs) were measured in admission sera from dogs in a babesiosis-endemic area. Five groups were prospectively studied: mild uncomplicated (n=9), severe uncomplicated (severe anaemia) (n=10) and complicated babesiosis (n=11); and two groups of healthy aparasitaemic dogs: endemic controls from the study area (n=10) and experimental dogs kept in tick-free conditions (n=10). Four measures of RNI production were studied: (i) serum RNI; (ii) serum RNI/creatinine ratio; (iii) fractional clearance of RNI (FC(RNI)); (iv) fractional excretion of RNI (FE(RNI)). Marked elevations of serum RNI occurred in only two dogs, both in the severe uncomplicated group. The highest concentration (log value 5.29 micromol/l) was in a dog that died, but concentrations in the other four dogs that died were unremarkable (0, 0.34, 1.66 and 2.64 micromol/l). Age, appetite and free serum haemoglobin were significant covariates for measures of RNI production. There were no significant differences among the babesiosis groups for serum RNI. Adjustment for creatinine had minor effects on the results. All babesiosis groups had significantly higher serum RNI and RNI/creatinine than the tick-free control group, but did not differ from the endemic controls except for the severe uncomplicated group, which had higher RNI/creatinine. The complicated group had significantly lower FC(RNI) and FE(RNI) than all other groups, except for the tick-free control group, which had similar FE(RNI). The results indicate that, in an endemic area, measures of RNI production are unlikely to be useful indicators of severity or outcome in canine babesiosis.

Met-haemoglobinuria in naturally occurring Babesia canis infection.

Higher levels of urinary met-haemoglobin were found in dogs with naturally occurring Babesia canis infection (n = 6) than in control subjects (n = 5). The urinary haemoglobin in the affected cases ranged from 1 to 4 g/l of which 28-95% was met-haemoglobin. This was a significant finding in that met-haemoglobin has been shown to be nephrotoxic in the dog, especially in the presence of aciduria. Acidic urine was also demonstrated in those dogs with B. canis infection, which may aggravate the nephrotoxic effect of the met-haemoglobinuria. Thus naturally infected B. canis cases showing haemoglobinuria may in fact have significant met-haemoglobinaemia, which may predispose them to renal disease.

Rhabdomyolysis as a complication of canine babesiosis.

Rhabdomyolysis was diagnosed in two dogs with babesiosis. The first animal presented with muscle pain and caramel-coloured urine, and had markedly elevated serum myoglobin and muscle enzymes. Acute renal failure complicated the clinical picture. The second dog exhibited muscle pain and tremors, together with neurological signs and pulmonary oedema, and died soon after admission. Muscle necrosis and haemorrhage were found at necropsy. In human malaria, a disease clinically similar to canine babesiosis, rhabdomyolysis is unusual, but clinically silent muscle damage appears to be common. Likewise, biochemical evidence of muscle damage is readily found in experimental bovine babesiosis. Muscle enzymes were mildly elevated in three dogs with severe babesiosis and pigmenturia but there was no obvious muscle damage, indicating that this might also apply to canine babesiosis. The pathogenesis of infection-associated rhabdomyolysis and acute renal failure remains unclear, but inflammatory cytokines and nitric oxide could play an important role.

Studies on feline babesiosis. 4. Chemical pathology; macroscopic and microscopic post mortem findings.

Chemopathological changes were monitored in 20 experimentally infected and 70 clinical cases of feline babesiosis. Total serum proteins remained unchanged but there was a definite increase in gamma globulin and decrease in alpha and beta globulins. In most cases liver function was essentially normal although function tests occasionally indicated hepatic dysfunction. Renal function was unaffected. Venous blood pH remained normal throughout. Post mortem findings on the experimental cats included bile stasis and hepatic necrosis in some; marked internal icterus was only seen in 2 cases.

Disseminated intravascular coagulation: a complication of Babesia canis infection in the dog.

Disseminated intravascular coagulation is described as a complication of Babesia canis infection in the dog. B. canis infection in the dog is characterized as a mild (uncomplicated) or severe (complicated) disease. The clinical, coagulation and haematological, pathological and histopathological findings of the severe disease are described. Thrombocytopenia is reported as occurring in both the mild and severe forms of B. canis infection in the dog.

Acute Babesia bovis infections: renal involvement in the hypotensive syndrome.

Splenectomised calves in metabolism cages were infected with Babesia bovis. During the infection, urine samples were collected and analysed for electrolytes, proteins, kinin, and urinary kallikrein. During the later stages of the infection there were significant reductions in urinary volume, water intake, urinary kinin, kallikrein, and electrolytes. Proteinuria was detected from 3--8 days postinfection of which 15--20% was haemoglobin and most of the remainder was albumin (70--75%). Fibrin degradation products, fibrinogen-like products, and haptoglobin were not detected. Degeneration of cortical tubules was detected by histological studies. As these tubules produce urinary kallikrein it seems probable that diminished glomerular blood flow and hence glomerular filtration rate are due to decreased production of this enzyme.