The Diverse Pathogenicity of Various Babesia Parasite Species That Infect Dogs.

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.

Regenerative response in dogs naturally and experimentally infected with Babesia rossi.

BACKGROUND: The regenerative response following Babesia rossi infection in dogs is mild, despite severe hemolytic anemia. OBJECTIVE: We aimed to compare the admission absolute reticulocyte count (ARC) and reticulocyte indices in 103 dogs naturally infected with B. rossi with 10 dogs suffering from immune-mediated hemolytic anemia (IMHA) and 14 healthy control dogs. The regenerative response was also evaluated in five dogs experimentally infected with B. rossi. METHODS: This is a retrospective observational study of records generated on the ADVIA 2120 hematology analyzer. RESULTS: The median hematocrits (HCT) of the B. rossi and IMHA groups were significantly lower than the control group (p < .001 for both); however, no differences were seen between the B. rossi and IMHA groups. Compared with the control group, the median ARC was significantly higher in the B. rossi (p = .006) and IMHA (p = .019) groups but significantly lower in the B. rossi group than the IMHA group (p = .041). In the experimentally infected dogs, there was a sudden decrease in the ARC approximately 48 h after the detection of peripheral parasitemia, which was followed by an increase after treatment. Reticulocytes of naturally infected B. rossi dogs were larger, with more variation in cellular volume. The reticulocytes of the experimentally infected dogs decreased in size with decreasing hemoglobin concentrations as the study progressed. CONCLUSIONS: The regenerative response in dogs naturally infected with B. rossi is inadequate, given the severity of the anemia observed, and it might be a result of direct suppressive action by the parasite or host response on the bone marrow.

Serum proteome profiling of naturally acquired Babesia rossi infection in dogs.

Babesiosis is a disease of significant medically and veterinary importance with worldwide distribution. It is caused by intra-erythrocyte protozoal parasites, with Babesia rossi causing the most severe clinical signs of all the large Babesia parasites infecting dogs. The disease can be clinically classified into uncomplicated and complicated forms with a wide range of clinical presentations from a mild, subclinical illness to complicated forms and death. The aim of this study was to assess serum proteomic profiles from dogs with babesiosis and healthy dogs using a label-based proteomics approach. Altogether 32 dogs naturally infected with B. rossi (subdivided into 18 uncomplicated cases and 14 complicated cases of babesiosis) and 20 healthy dogs were included. There were 78 proteins with significantly different abundances between the three groups of dogs. Elucidation of proteins and pathways involved in canine babesiosis caused by B. rossi have revealed key differences associated with haemostasis, innate immune system, lipid metabolism and inflammation. Shotgun proteomic profiling allowed identification of potential serum biomarkers for differentiation of disease severity in canine babesiosis caused by B. rossi. These findings may be applicable to the study of host-parasite interactions and the development of novel therapeutic targets.

The seroprevalence of African horse sickness virus, and risk factors to exposure, in domestic dogs in Tshwane, South Africa.

Dogs are the only non-equid species to develop the fatal form of African horse sickness (AHS). Research conducted in 2013 questioned the long-held belief that naturally occurring cases of AHS in dogs were contracted exclusively through the ingestion of contaminated horse meat. Culicoides midges, the vector of AHS virus (AHSV) for horses, have an aversion to dog blood meals and dogs were believed to be dead-end or incidental hosts. More recently, dog mortalities have occurred in the absence of horse meat consumption and vector transmission has been suspected. The current study is a retrospective serological survey of AHSV exposure in dogs from an endemic area. Dog sera collected from dogs (n = 366) living in the city of Tshwane, Gauteng Province, South Africa, were randomly selected from a biobank at a veterinary teaching hospital, corresponding to the years 2014-2019. The study used a laboratory in-house indirect recombinant VP7 antigen-based enzyme-linked immunosorbent assay (iELISA) with a test cut-off calculated from AHSV exposure-free dog sera (n = 32). Study AHSV seroprevalence was 6 % (22/366) with an estimated true prevalence of 4.1 % (95 % confidence interval (CI) = 1.3-8.1 %). Incidence was estimated for dogs with multiple serological results with seroconversion occurring at a rate of 2.3 seroconversions per 10 dog years at risk (95 % CI = 0.6-6.2). A subsection of the study sera was tested with AHSV viral neutralisation test (VN) (n = 42) for serotype determination. Antibodies to AHSV serotype 6 were most prevalent (90 %) in VN seropositive dogs (n = 20) with most dogs seemingly subclinically infected (>95 %). Seroprevalence descriptively varied by year and identified risk factors were annual rainfall > 754 mm (odds ratio (OR) = 5.76; 95 % CI = 2.22 - 14.95; p < 0.001), medium human population densities, 783-1663 people/km2 (OR = 7.14; 95 % CI = 1.39 - 36.73; p = 0.019) and 1664-2029 people/km2 (OR = 6.74; 95 % CI = 1.40 - 32.56; p = 0.018), and the month of March (OR = 5.12; 95 % CI = 1.41 - 18.61; p = 0.013). All identified risk factors were consistent with midge-borne transmission to dogs. The relatively high seroprevalence and seroconversion rates suggest frequent exposure of dogs to AHSV and indicates the need to investigate the role dogs might play in the overall epidemiology and transmission of AHSV.

Sex-differential non-specific effects of adjuvanted and non-adjuvanted rabies vaccines versus placebo on all-cause mortality in dogs (NERVE-Dog study): a study protocol for a randomized controlled trial with a nested case-control study.

BACKGROUND: It has been proposed that childhood vaccines in high-mortality populations may have substantial impacts on mortality rates that are not explained by the prevention of targeted diseases, nor conversely by typical expected adverse reactions to the vaccines, and that these non-specific effects (NSEs) are generally more pronounced in females. The existence of these effects, and any implications for the development of vaccines and the design of vaccination programs to enhance safety, remain controversial. One area of controversy is the reported association of non-live vaccines with increased female mortality. In a previous randomized controlled trial (RCT), we observed that non-live alum-adjuvanted animal rabies vaccine (ARV) was associated with increased female but not male mortality in young, free-roaming dogs. Conversely, non-live non-adjuvanted human rabies vaccine (NRV) has been associated with beneficial non-specific effects in children. Alum adjuvant has been shown to suppress Th1 responses to pathogens, leading us to hypothesize that alum-adjuvanted rabies vaccine in young dogs has a detrimental effect on female survival by modulating the immune response to infectious and/or parasitic diseases. In this paper, we present the protocol of a 3-arm RCT comparing the effect of alum-adjuvanted rabies vaccine, non-adjuvanted rabies vaccine and placebo on all-cause mortality in an owned, free-roaming dog population, with causal mediation analysis of the RCT and a nested case-control study to test this hypothesis. METHODS: Randomised controlled trial with a nested case-control study. DISCUSSION: We expect that, among the placebo group, males will have higher mortality caused by higher pathogen loads and more severe disease, as determined by haematological parameters and inflammatory biomarkers. Among females, we expect that there will be no difference in mortality between the NRV and placebo groups, but that the ARV group will have higher mortality, again mediated by higher pathogen loads and more severe disease. We anticipate that these changes are preceded by shifts in key serum cytokine concentrations towards an anti-inflammatory immune response in females. If confirmed, these results will provide a rational basis for mitigation of detrimental NSEs of non-live vaccines in high-mortality populations.

The dataset for the inflammatory response during experimental infection and treatment of dogs with Babesia rossi.

Babesia rossi causes severe morbidity and mortality in dogs in sub-Saharan Africa. This was an experimental study designed to observe systemic changes caused by Babesia rossi infection within a canine disease model as well as investigate the influence of inoculum dose and treatment on the progression of inflammation and clinical disease. Six healthy male beagle dogs formed the study population, one dog was splenectomised and used to raise the infectious inoculum, three were administered a high B. rossi infectious dose and two a low infectious dose. Clinical examination, complete blood count (CBC) and C-reactive protein (CRP) were determined daily. Cytokines were quantified on stored plasma collected during the study, using a canine specific cytokine magnetic bead panel (Milliplex©). The experiment was terminated, and treatment administered once predetermined experimental or humane endpoints were reached. The data and information provided in the following article is the summary of all data points collected over the course of the eight-day experimental infection.

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

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

Temporal analysis of the bovine lymph node transcriptome during cattle tick (Rhipicephalus microplus) infestation.

Livestock production is a fundamental source of revenue and nutrition, wherein cattle-farming constitutes one of the major agricultural industries. Vectors and vector-borne diseases constitute one of the major factors that decrease the livelihood of all farming communities, more so in resource-poor communities and developing countries. Understanding the immunological responses during tick infestation in cattle is instrumental in the development of novel and improved tick control strategies, such as vaccines. In this study, gene expression patterns were compared within the lymph nodes of three cattle breeds at different life stages of the cattle tick, Rhipicephalus microplus. For Bonsmara (5/8Bos taurus indicus × 3/8B. t. taurus) cattle specifically, some 183 genes were found to be differentially expressed within the lymph nodes during larval and adult tick feeding, relative to uninfested cattle. Overall, the data provides evidence for a transcriptional regulatory network that is activated during immature tick infestation, but is down-regulated towards basal transcriptional levels when adult ticks are feeding. Specific processes in the lymph nodes of Bonsmara cattle were found to be differentially regulated on a transcriptional level. These include: (1) Leukocyte recruitment to the lymph node via chemokines and chemotaxis, (2) Trans-endothelial and intranodal movement on the reticular network, (3) Active regulation of cellular transcription and translation in the lymph node (including leukocyte associated cellular regulatory networks) and (4) Chemokine receptors regulating the movement of cells out of the lymph node. This work provides a first transcriptome analysis of bovine lymph node responses in tick-infested cattle. Findings show a dynamic immune response to tick infestation for the Bonsmara cattle breed, and that suppression of the maturation of the cattle hosts' immunity is especially evident during the larval feeding stages.

Role of electrolyte abnormalities and unmeasured anions in the metabolic acid-base abnormalities in dogs with parvoviral enteritis.

BACKGROUND: The strong ion model (SIM) is an alternative paradigm in the characterization of acid-base disturbances particularly in complex disorders. HYPOTHESIS/OBJECTIVES: To compare the acid-base changes in dogs with parvoviral enteritis (PE) using the Henderson-Hasselbalch (HH) approach, with 2 strong ion approaches. ANIMALS: Forty-four dogs with PE, and 16 age-matched control dogs. METHODS: Prospective controlled observational study. Acid-base status was evaluated using the HH model, Fencl-Stewart (FS) approach and a validated strong ion model (VDM). The acid-base changes according to each model were classified and compared. Statistical correlations between pH, CO2 , and various SIM variables were performed, as well as between the sum of effects (SOE) of the SIM and the individual variables comprising the SOE. RESULTS: The HH model identified acid-base disorders in 31/44 cases of which 16/31 were mixed with metabolic acidosis and concurrent respiratory alkalosis the most common (10/31). Using the FS approach, metabolic changes were present 36/42 cases, with changes in free water (FW), chloride, and unmeasured anions (UA) being the most prevalent. Both FW and UA correlated well with pH; however, UA were most consistently abnormal in severe acidemia. Similarly to the HH, the VDM detected acid-base disturbances in 28/44 cases. Major contributors to the acid-base changes were hyponatremia, hypochloremia, and Atot acidosis because of elevated globulins and increased UA. CONCLUSIONS AND CLINICAL IMPORTANCE: Acid-base changes are common and complex in dogs with PE, and were easier to understand using a SIM paradigm. Increases in UA have not been documented in PE in dogs.

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.

Rabies Vaccination of 6-Week-Old Puppies Born to Immunized Mothers: A Randomized Controlled Trial in a High-Mortality Population of Owned, Free-Roaming Dogs.

To achieve global elimination of human rabies from dogs by 2030, evidence-based strategies for effective dog vaccination are needed. Current guidelines recommend inclusion of dogs younger than 3 months in mass rabies vaccination campaigns, although available vaccines are only recommended for use by manufacturers in older dogs, ostensibly due to concerns over interference of maternally-acquired immunity with immune response to the vaccine. Adverse effects of vaccination in this age group of dogs have also not been adequately assessed under field conditions. In a single-site, owner-blinded, randomized, placebo-controlled trial in puppies born to mothers vaccinated within the previous 18 months in a high-mortality population of owned, free-roaming dogs in South Africa, we assessed immunogenicity and effect on survival to all causes of mortality of a single dose of rabies vaccine administered at 6 weeks of age. We found that puppies did not have appreciable levels of maternally-derived antibodies at 6 weeks of age (geometric mean titer 0.065 IU/mL, 95% CI 0.061-0.069; n = 346), and that 88% (95% CI 80.7-93.3) of puppies vaccinated at 6 weeks had titers ≥0.5 IU/mL 21 days later (n = 117). Although the average effect of vaccination on survival was not statistically significant (hazard ratio [HR] 1.35, 95% CI 0.83-2.18), this effect was modified by sex (p = 0.02), with the HR in females 3.09 (95% CI 1.24-7.69) and the HR in males 0.79 (95% CI 0.41-1.53). We speculate that this effect is related to the observed survival advantage that females had over males in the unvaccinated group (HR 0.27; 95% CI 0.11-0.70), with vaccination eroding this advantage through as-yet-unknown mechanisms.

A Comparison Between Manual Count, Flow Cytometry and Quantitative Real-Time Polymerase Chain Reaction as a Means of Determining Babesia rossi Parasitaemia in Naturally Infected Dogs.

PURPOSE: Light microscopic manual count is the current gold standard for parasite quantification. The ability to determine parasite density in whole blood is crucial to understanding disease pathogenesis and finding a suitable automated method of Babesia rossi parasite quantification would facilitate higher throughput and provide results that are more objective. This study investigated both peripheral capillary and central venous whole blood to estimate the correlations between light microscopy, flow cytometry and quantitative real-time polymerase chain reaction (qPCR). METHODS: Peripheral capillary and central venous blood were sampled from 40 naturally B. rossi-infected dogs and 10 healthy control dogs. Samples were analysed by reverse line blot hybridization assay to confirm a mono-B. rossi infection. Capillary blood parasite density was detected using light microscopic manual counting and venous blood parasitaemia detected by manual counts, flow cytometry and qPCR. RESULTS: A significant correlation was found between the venous manual counts and flow cytometry (rs = 0.465; P < 0.001), as well as qPCR (rs = - 0.500; P < 0.001). A significant correlation was also observed between the capillary manual counts compared to venous manual counts (rs = 0.793; P < 0.001), flow cytometry (rs = 0.399; P = 0.004), and qPCR (rs = - 0.526; P < 0.001). CONCLUSIONS: The study results suggest that qPCR is of value as an alternative to the gold standard manual count for detecting B. rossi parasitaemia in canine whole blood and that flow cytometry may be useful with further refinement of issues such as background fluorescence and the influence of reticulocytes.

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.

Association between computed tomographic thoracic injury scores and blood gas and acid-base balance in dogs with blunt thoracic trauma.

OBJECTIVE: To determine the association between thoracic injuries evaluated by computed tomography (CT) and arterial blood gas and acid-base status in dogs with blunt thoracic trauma caused by motor vehicle accidents. DESIGN: Prospective observational clinical study. SETTING: University teaching hospital. ANIMALS: Thirty-one client owned traumatized dogs and 15 healthy dogs. PROCEDURES: All trauma group dogs underwent a CT scan and simultaneous arterial blood gas analysis within 24 hours, but not before 4 hours, after the traumatic incident within a 45-month enrollment period. MEASUREMENTS AND MAIN RESULTS: Thorax injuries were classified as pulmonary, pleural space, or rib cage and each of these components was scored for severity using a CT composite pulmonary, pleural, and rib score. The trauma group arterial blood gas and acid-base status were evaluated for statistical difference from the control group. The pulmonary-arterial oxygen pressure was significantly lower in the trauma group compared to the control group that was supported by significant differences in the calculated variables of arterial blood oxygenation as well. There was also a significant correlation between the composite lung score and pleural score and the variables of arterial oxygen status. The pulmonary-arterial carbon dioxide pressure was not significantly different to any of the thoracic injury variables indicating normal alveolar ventilation. Acid-base imbalances were generally mild, insignificant, and variable. CONCLUSIONS AND CLINICAL RELEVANCE: Blunt thoracic trauma causes significant pulmonary and pleural injury and the blood oxygen economy is significantly affected by this. The functional measures of arterial blood oxygenation were well correlated with thoracic CT pathology. Alveolar ventilation was mostly spared but a clinically significant ventilation perfusion mismatch was present.

Disease severity and blood cytokine concentrations in dogs with natural Babesia rossi infection.

AIMS: Babesia rossi causes severe disease in dogs. Here, we describe the association between serum cytokine concentrations and disease severity. METHODS: Seventeen controls and 55 PCR confirmed B rossi-infected dogs were included. Diseased dogs were subdivided into 23 critically ill and 32 relatively well cases. Serum concentrations of 11 cytokines and biochemical markers of disease severity were determined. RESULTS: Significant differences were detected for IL-6, IL-8, IL-10, MCP-1 and TNF-α between the groups. Generally, the more complicated the disease, the more pro-inflammatory the cytokine milieu. IL-8 showed a reverse trend and was negatively correlated with disease severity. IL-6, MCP-1 and TNF-α were also significantly higher in the dogs that died (n = 9) compared to the dogs that survived (n = 46). IL-8 showed the opposite. MCP-1 and TNF-α were negatively correlated with biochemical markers of severity. Glucose was negatively correlated with IL-6. Cortisol, peripheral parasite density and band neutrophil count were positively correlated, whilst thyroid hormone was negatively correlated with IL-6, MCP-1 and TNF-α. CONCLUSIONS: As in malaria and sepsis, B rossi infection induces a pro-inflammatory cytokine storm that correlates with disease severity and adverse outcome. The multiplicity of cytokines involved argues for redundancy in the system once the disease is established.

Development and validation of a multiplex, real-time PCR assay for Babesia rossi and Babesia vogeli.

Canine babesiosis is caused by tick-transmitted intraerythrocytic protozoan parasites occurring worldwide. In southern Africa, babesiosis is caused by Babesia rossi and B. vogeli and is one of the most common and important infectious diseases affecting dogs. There is no reliable, rapid and sensitive method for the detection of these parasites, especially when parasitaemia is low. The aim of this study was to develop a sensitive and specific multiplex TaqMan® MGB PCR assay for the diagnosis of canine babesiosis infections occurring in southern Africa, and to discriminate between Babesia rossi and B. vogeli. The fitness of purpose of the assay was to confirm diagnosis of suspect or clinical cases, and estimate prevalence of infection for research purposes. A total of 648 published sequences were used to design the assay. A set of group-specific canine Babesia spp. primers were designed to amplify a 117 nucleotide region of the 18S rRNA gene of all canine Babesia spp. Species-specific TaqMan® MGB probes were developed for B. rossi, B. vogeli, B. canis and B. gibsoni, but analytical validation was only performed for B. rossi and B. vogeli as a multiplex assay. The assay had a broad dynamic range and amplified B. rossi and B. vogeli efficiently (98.6% and 94.7% respectively). The assay was sensitive, with a 95% LOD of 10-2.67% parasitized erythrocytes (PE) for B. rossi and 10-2.03% PE for B. vogeli, and specific, with no cross reaction between B. rossi and B. vogeli and no detection of other haemoparasites that infect dogs, such as Ehrlichia canis and Anaplasma platys. Consistent repeatability within and between PCR runs was shown. This assay will be able to accurately and rapidly confirm babesiosis in canines and allow for treatment to be administered in the early stages of the disease, speeding up the recovery time in affected dogs.

Comparison of the differential regulation of T and B-lymphocyte subsets in the skin and lymph nodes amongst three cattle breeds as potential mediators of immune-resistance to Rhipicephalus microplus.

Although varying natural resistance to ticks between highly resistant Brahman (Bos taurus indicus), resistant Bonsmara (5/8 B. t. indicus x 3/8 B. t. taurus) and susceptible Holstein-Friesian (B. t. taurus) breeds is documented in skin and blood, little information is available describing draining lymph nodes. To elucidate the cellular dynamics during Rhipicephalus microplus induced immune responses, this study analysed immune factors from these cattle breeds using histology, immunohistochemistry and flow cytometry. Following the collection of skin and lymph node samples before artificial tick infestation, cattle were infested with R. microplus larvae. Subsequent sampling coincided with the tick larvae and adult developmental stages. A significant influx of CD20+ B-lymphocytes in the dermis all cattle breeds was observed while CD3+ T-lymphocytes were significantly increased for more tick resistant breeds. Eosinophil infiltration in germinal centres of lymph nodes was significant for all cattle breeds while tingible body macrophages were significantly increased for adult infested Brahman animals. A negligible fluctuation in CD20+ and CD79α+ B-lymphocyte numbers was present in the lymph node of more resistant cattle breeds, while susceptible animals showed a decrease in B-lymphocytes after infestation, followed by an increase between larvae to adult infested time points. Increased variability of γd T-lymphocyte populations in lymph nodes was correlated with tick susceptibility. In addition, a more stable T helper lymphocyte population was identified in the lymph nodes for the Brahman cattle breed. Results suggest the association of tick susceptibility with differential B-lymphocyte regulation in lymph node tissues, increased variability of WC1+ γδ T-lymphocyte populations in the lymph node as well as a decrease in T helper lymphocytes in the lymph node.

A flow cytometric assessment of the lymphocyte immunophenotypes in dogs naturally infected with Babesia rossi.

Immunity to Babesia infection requires both innate and acquired responses, including cell mediated- and humoral responses. The aims of this study were to investigate the variation in selected peripheral blood lymphocyte phenotypes in dogs with virulent babesiosis at presentation and over time after treatment, and to determine whether these were correlated with the severity of clinical signs. Forty-four dogs naturally infected with B. rossi were studied and 5 healthy dogs were included as controls. Blood samples were collected from the jugular vein at admission, prior to any treatment, and at 24h and 48-72h. Leukocytes were incubated with canine specific, fluorochrome conjugated anti-CD3, anti-CD4, anti-CD8, and anti-B cell markers. Babesia-infected dogs were divided into complicated or uncomplicated groups on clinical grounds and in-house laboratory assays. The percentage CD3+ lymphocytes in the complicated group was lower compared to the controls (P=0.014) and uncomplicated group (P=0.007). The percentage CD4+ T lymphocytes in the complicated group was lower compared to the controls (P=0.027) and uncomplicated group (P=0.014). Both the complicated as well as the uncomplicated groups expressed a lower percentage CD8+ T lymphocytes compared to the control group (P<0.001 and P=0.005, respectively). The percentage B lymphocytes was higher in the complicated group at 48-72h. These findings could indicate the presence of a functional immune suppression secondary to increased apoptosis or redistribution of effector lymphocytes and/or a combination of other immune modulatory mechanisms induced by B. rossi infection.

Excessive Pro-Inflammatory Serum Cytokine Concentrations in Virulent Canine Babesiosis.

Babesia rossi infection causes a severe inflammatory response in the dog, which is the result of the balance between pro- and anti-inflammatory cytokine secretion. The aim of this study was to determine whether changes in cytokine concentrations were present in dogs with babesiosis and whether it was associated with disease outcome. Ninety-seven dogs naturally infected with B. rossi were studied and fifteen healthy dogs were included as controls. Diagnosis of babesiosis was confirmed by polymerase chain reaction and reverse line blot. Blood samples were collected from the jugular vein at admission, prior to any treatment. Cytokine concentrations were assessed using a canine-specific multiplex assay on an automated analyser. Serum concentrations of interleukin (IL)-2, IL-6, IL-8, IL-10, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF) and monocyte chemotactic protein-1 (MCP-1) were measured. Twelve of the Babesia-infected dogs died (12%) and 85 survived (88%). Babesia-infected dogs were also divided into those that presented within 48 hours from displaying clinical signs, and those that presented more than 48 hours after displaying clinical signs. Cytokine concentrations were compared between the different groups using the Mann-Whitney U test. IL-10 and MCP-1 concentrations were significantly elevated for the Babesia-infected dogs compared to the healthy controls. In contrast, the IL-8 concentration was significantly decreased in the Babesia-infected dogs compared to the controls. Concentrations of IL-6 and MCP-1 were significantly increased in the non-survivors compared to the survivors. Concentrations for IL-2, IL-6, IL-18 and GM-CSF were significantly higher in those cases that presented during the more acute stage of the disease. These findings suggest that a mixed cytokine response is present in dogs with babesiosis caused by B. rossi, and that an excessive pro-inflammatory response may result in a poor outcome.

Platelet indices in dogs with Babesia rossi infection.

BACKGROUND: Thrombocytopenia without clinical bleeding is a consistent finding in virulent canine babesiosis. OBJECTIVES: The purpose of the study was to investigate the platelet index phenotype in Babesia rossi-infected dogs and the association with disease outcome. We hypothesized that an increased proportion of large, activated platelets would be present. METHODS: Ninety-six infected and 15 control dogs were included. Babesia-infected dogs were further divided into survivors and nonsurvivors. Platelet count, mean platelet volume (MPV), platelet volume distribution width (PDW), plateletcrit (PCT), mean platelet mass (MPM), mean platelet component concentration (MPC), and platelet component distribution width (PCDW) were measured at presentation, and at 24 and 48 hours in admitted survivors. RESULTS: Mortality rate was 13% (12/96). At presentation, compared to controls, PLT and PCT were significantly decreased in survivors and nonsurvivors (P < .001 for both). Mean platelet volume was significantly increased in survivors and nonsurvivors compared to the controls (P < .001 for both); however, MPM was only significantly increased in the survivors (P < .001). There were no differences between the survivors and nonsurvivors for any of the indices at presentation. Platelet count and PCT were significantly increased at 24 and 48 hours, and MPC significantly increased at 24 hours for admitted survivors compared to the values at presentation. CONCLUSION: Large, activated platelets were significantly increased and may play a role in the lack of a bleeding phenotype, despite severe thrombocytopenia, in canine babesiosis.