Canine Blood Samples Research Articles

Effects of calcitriol on apoptosis, toll-like receptor 4 expression, and cytokine production of endotoxin-primed canine leukocytes.

OBJECTIVE To determine the in vitro effect of calcitriol on indicators of immune system function in endotoxin-primed blood samples from healthy dogs. SAMPLE Blood samples from 6 healthy adult dogs. PROCEDURES Leukocytes were primed by incubation of blood samples with lipopolysaccharide (LPS; endotoxin) or PBS solution (unprimed control group) for 1 hour. Following priming, blood samples were incubated with calcitriol (2 × 10-7M) or ethanol (control substance) for 24 hours. After sample incubation, LPS-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) was measured with a canine-specific multiplex assay, and apoptosis and toll-like receptor 4 (TLR4) expression were evaluated via flow cytometry. RESULTS LPS stimulation of unprimed leukocytes but not endotoxin-primed leukocytes resulted in a significant increase in TNF and IL10 production, confirming the presence of endotoxin tolerance in dogs in vitro. Endotoxin priming significantly increased neutrophil viability with no effect on lymphocyte viability or TLR4 expression by neutrophils and monocytes. Calcitriol exposure significantly decreased LPS-stimulated production of TNF by unprimed and endotoxin-primed leukocytes. Conversely, calcitriol exposure had no effect on IL10 production by unprimed leukocytes but did significantly increase IL10 production by endotoxin-primed leukocytes. Calcitriol had no significant effect on the degree of neutrophil or lymphocyte apoptosis, nor was neutrophil and monocyte TLR4 expression affected in unprimed or endotoxin-primed leukocytes. CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in unprimed and endotoxin-primed canine leukocytes in vitro, without compromising neutrophil and monocyte TLR4 expression or altering the viability of neutrophils and lymphocytes in canine blood samples.

Effects of calcitriol on phagocytic function, toll-like receptor 4 expression, and cytokine production of canine leukocytes.

OBJECTIVE To determine the in vitro effects of calcitriol on indicators of immune system function in blood samples collected from healthy dogs. SAMPLE Blood samples from 8 healthy adult dogs. PROCEDURES Blood samples were incubated with calcitriol (10-7M) or control substance for 24 hours. Afterward, lipopolysaccharide (LPS)-, lipoteichoic acid (LTA)-, and N-acetylmuramyl-l-alanyl-d-isoglutamine hydrate (MDP)-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) were measured with a canine-specific multiplex assay. Phagocytosis of opsonized Escherichia coli and leukocyte expression of constitutive toll-like receptor 4 (TLR4) were evaluated via flow cytometry. Blood samples from 3 dogs were used to create a concentration-response curve to evaluate whether the observed cytokine modulation was concentration dependent. RESULTS Incubation of canine blood samples with calcitriol resulted in significant decreases in LPS-, LTA-, and MDP-stimulated leukocyte production of TNF but not IL10. Blunting of TNF production was concentration dependent. Leukocyte calcitriol exposure had no significant effect on phagocytosis and TLR4 expression. CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in canine leukocytes exposed to LPS, LTA, and MDP in vitro, without altering phagocytosis or TLR4 expression. Thus, calcitriol could represent a novel candidate immunomodulatory treatment for dogs.

Canine filariasis in the Amazon: Species diversity and epidemiology of these emergent and neglected zoonoses

Dirofilaria immitis and Acanthocheilonema reconditum are common parasites in dogs but have also been reported parasitizing humans. The differential diagnosis and epidemiological evaluation of these zoonoses are important to the development of efficient public health policies and control strategies. The purpose of this study was to carry out an epidemiological survey by using molecular methods for the specific identification of filarid parasites of domestic dogs in the Marajó mesoregion, State of Pará (PA), Brazil. A total of 418 canine blood samples from Marajó mesoregion (Northern Brazil) were collected, submitted to DNA extraction, polymerase chain reaction (PCR) with “pan filarial” primer, subsequent sequencing and sequence analysis using BLASTn software comparison with previously deposited sequences in GenBank. After that, a phylogenetic analysis by Maximum Parsimony was performed to aid the specific diagnosis. The obtained sequences showed the occurrence of 9 (2.15%) dogs infected with D. immitis and 30 (7.18%) by A. reconditum, with a confidence interval of 95%, there were no cases of co-infection. We observed that male dogs were more likely to D. immits and A. reconditum infection. However, age was not significant to both infections. This study reports for the first time the occurrence of A. reconditum in the northern region of Brazil and confirmed the presence of D. immitis in the Marajó mesoregion.

Canine seroprevalence to Orientia species in southern Chile: A cross-sectional survey on the Chiloé Island.

BackgroundScrub typhus is a potentially life-threatening vector-borne infection caused by Orientia species. It occurs mainly in the Asian-Pacific region, where it causes significant morbidity and mortality. Recently, an endemic focus of scrub typhus has been described in South America, on Chiloé Island in southern Chile. Dogs have been used as sentinel hosts to determine the presence and spatial distribution of various vector-borne infections. Their suitability to gain insight into human exposure to Orientia tsutsugamushi has been suggested in studies from Asia.MethodologyIn January 2016, we conducted a cross-sectional study, which included the two main cities on Chiloé Island. Canine blood samples were obtained in households, chosen by double stratified random sampling in urban and by convenience in rural locations. Specimens were tested by ELISA for IgG antibodies against whole-cell antigen preparations from three strains of O. tsutsugamushi. Data were further analyzed for factors associated with seropositivity including spatial clustering.ResultsSerum samples from 202 dogs (104 urban, 98 rural) were tested for IgG against O. tsutsugamushi, of which 43 (21.3%) were positive. Seroprevalence rates were higher in rural than in urban settings (p<0.01) and in older compared to younger dogs (p<0.01). Spatial analysis by LISA indicated the presence of four localities of highly grouped cases.ConclusionsThe detected seroprevalence supports the endemicity of scrub typhus in southern Chile and suggests a wide exposure of household dogs to the infected, yet unknown vector(s). The spatial data will be used for future research identifying further human cases as well as the local vector(s)/reservoirs for scrub typhus in southern Chile. The study reinforces that dogs are useful sentinels for Orientia spp. in regions of uncertain endemicity and distribution.

Tick-borne haemoparasites and Anaplasmataceae in domestic dogs in Zambia

Tick-borne diseases (TBDs), including emerging and re-emerging infectious diseases, are important threats to human and animal health worldwide. Indeed, the number of reported human and animal infectious cases of novel TBD agents has increased in recent decades. However, TBDs tend to be neglected, especially in resource-limited countries that often have limited diagnostic capacity. The aim of this molecular survey was to detect and characterise tick-borne pathogens (Babesia, Theileria, and Hepatozoon parasites and Anaplasmataceae bacteria) in domestic dogs in Zambia. In total, 247 canine peripheral blood samples were collected in Lusaka, Mazabuka, Monze, and Shangombo. Conventional PCR to detect the selected pathogens was performed using DNA extracted from canine blood. One hundred eleven samples were positive for protozoa and 5 were positive for Anaplasmataceae. Sequencing of thirty-five randomly selected protozoa-positive samples revealed the presence of Babesia rossi, Babesia vogeli, and Hepatozoon canis 18S rDNA. Based on these sequences, a multiplex PCR system was developed to yield PCR products with different amplicons, the size of which depended on the parasite species; thus, each species could be identified without the need for sequence analysis. Approximately 40% of dogs were positive for H. canis. In particular, the positive rate (75.2%) of H. canis infection was significantly higher in Shangombo than in other sampling sites. Multiplex PCR assay detected B. rossi and B. vogeli infections in five and seven dogs, respectively, indicating that this approach is useful for detecting parasites with low prevalence. Sequencing analysis of gltA and groEL genes of Anaplasmataceae revealed that two and one dogs in Lusaka were infected with Anaplasma platys and Ehrlichia canis, respectively. The data indicated that Zambian dogs were infected with multiple tick-borne pathogens such as H. canis, B. rossi, B. vogeli, A. platys, E. canis and uncharacterized Ehrlichia sp. Since some of these parasites are zoonotic, concerted efforts are needed to raise awareness of, and control, these tick-borne pathogens.

Determination of reference intervals and comparison of venous blood gas parameters using a standard and nonstandard collection method in 51 dogs.

The aim of this study was to determine reference intervals (RI) for venous blood parameters determined with the RAPIDPoint 500 (RP500) blood gas analyzer using blood gas syringes (BGS) and to determine whether immediate analysis of venous blood collected into lithium heparin (LH) tubes can replace anaerobic blood sampling into BGS. The null hypothesis was that canine venous blood samples collected in BGS and in LH tubes are comparable. Jugular blood was collected from 51 healthy dogs into a BGS and a LH tube. The BGS was immediately analyzed followed by the LH tube. The RI were calculated from BGS results. The BGS and LH tubes results were compared using paired t-test or Wilcoxon matched-pairs signed-rank test and Bland-Altman analysis. To assess clinical relevance, the bias between BGS and LH tubes was compared with the allowable total error (TEa). Values derived from LH tubes showed no significant difference for standard bicarbonate (HCO3std), whole blood base excess (BE B), Na, K, Cl, glucose and hemoglobin (tHb). The pH, partial pressure of carbon dioxide and oxygen, actual bicarbonate, extracellular base excess, ionized Ca, anion gap and lactate were significantly (p.

Evaluation of tissue trauma and healing on the basis of tumour necrosis factor-alpha, interleukin-6, and C-reactive protein in peripheral blood during and after pyometra in bitches

In this study, inflammation in the blood of bitches with pyometra (PG) was compared before and 15 days after ovariohysterectomy (OVH). The results were compared with those for control dioestrus bitches (CG) to reveal the evidence of inflammation in blood after a routine surgery. Inflammation was tracked by tumour necrosis factor-alpha (TNF-α), interleukine-6 (IL-6) and C-reactive protein (CRP), using immunocytochemistry (ICC) and immunofluorescence capture of blood cells in cell blocks. ICC is performed mainly during routine cytological examinations, whereas the use of cell blocks in blood examination is uncommon. Insofar as we know, this is the first study using cell block techniques on canine blood samples. Three commercially available antibodies against TNF-α, IL-6 and CRP, forming two panels, were evaluated. A standard streptoavidin-biotin complex technique was used for ICC. TNF-α and IL-6 labelling was scored for colour and intensity, and CRP for immunofluorescence capture. TNF-α and IL-6 colour and intensity scores differed significantly between the PG and CG groups, and were higher in PG before OVH (P&amp;lt;0.01, P&amp;lt;0.01, P&amp;lt;0.001, P&amp;lt;0.01, respectively). IL-6 intensity was significantly greater in PG 15 days after OVH (P&amp;lt;0.05). CRP capture in PG was strong before OVH and high in both groups 15 days later. Low-dose anti-inflammatory agents or an anti-cytokine therapy may be useful in pyometra treatment in the future because these treatments may offer protection from systemic inflammatory response syndrome before and after OVH. .

Frequency of Babesia vogeli in domestic dogs in the metropolitan area of Piura, Peru

The aim of this study was to assess the presence of piroplasms in dogs in the metropolitan region of the city of Piura, Peru. Two hundred and twelve canine blood samples were randomly collected. The deoxyribonucleic acid was extracted from each blood sample and was tested using the polymerase chain reaction, restriction fragment length polymorphism and sequence analyses. The study showed the occurrence of Babesia vogeli. For the first time, this approach revealed the presence of canine babesiosis caused by B. vogeli in Peru. This highlights the need to test for pathogens that might be responsible for causing canine babesiosis, through using proper molecular tools.

In vitro effect of blood cell counts on multiple-electrode impedance aggregometry in dogs.

OBJECTIVE To assess the effect of decreased platelet and WBC counts on platelet aggregation as measured by a multiple-electrode impedance aggregometer in dogs. ANIMALS 24 healthy dogs. PROCEDURES From each dog, 9 mL of blood was collected into a 10-mL syringe that contained 1 mL of 4% sodium citrate solution to yield a 10-mL sample with a 1:9 citrate-to-blood ratio. Each sample was then divided into unmanipulated and manipulated aliquots with progressively depleted buffy-coat fractions such that 2 to 3 blood samples were evaluated per dog. The Hct for manipulated aliquots was adjusted with autologous plasma so that it was within 2% of the Hct for the unmanipulated aliquot for each dog. All samples were analyzed in duplicate with a multiple-electrode impedance aggregometer following the addition of ADP as a platelet agonist. The respective effects of platelet count, plateletcrit, Hct, and WBC count on platelet aggregation area under the curve (AUC), aggregation, and velocity were analyzed with linear mixed models. RESULTS WBC count was positively associated with platelet AUC, aggregation, and velocity; blood samples with leukopenia had a lower AUC, aggregation, and velocity than samples with WBC counts within the reference range. Platelet count, plateletcrit, and Hct did not have an independent effect on AUC, aggregation, or velocity. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that WBC count was positively associated with platelet aggregation when ADP was used to activate canine blood samples for impedance aggregometry. That finding may be clinically relevant and needs to be confirmed by in vivo studies.

Comparison of three methods for recovery of Brucella canis DNA from canine blood samples

Brucella canis, a gram-negative, facultative intracellular and zoonotic bacterium causes canine brucellosis. Direct methods are the most appropriate for the detection of canine brucellosis and bacterial isolation from blood samples has been employed as gold-standard method. However, due to the delay in obtaining results and the biological risk of the bacterial culturing, the polymerase chain reaction (PCR) has been successfully used as an alternative method for the diagnosis of the infection. Sample preparation is a key step for successful PCR and protocols that provide high DNA yield and purity are recommended to ensure high diagnostic sensitivity. The objective of this study was to evaluate the performance of PCR for the diagnosis of B. canis infection in 36 dogs by testing DNA of whole blood obtained through different extraction and purification protocols. Methods 1 and 2 were based on a commercial kit, using protocols recommended for DNA purification of whole blood and tissue samples, respectively. Method 3 was an in-house method based on enzymatic lysis and purification using organic solvents. The results of the PCR on samples obtained through three different DNA extraction protocols were compared to the blood culture. Of the 36 dogs, 13 (36.1%) were positive by blood culturing, while nine (25.0%), 14 (38.8%), and 15 (41.6%) were positive by PCR after DNA extraction using methods 1, 2 and 3, respectively. PCR performed on DNA purified by Method 2 was as efficient as blood culturing and PCR performed on DNA purified with in-house method, but had the advantage of being less laborious and, therefore, a suitable alternative for the direct B. canis detection in dogs.

Cytokine Concentrations Measured by Multiplex Assays in Canine Peripheral Blood Samples.

Cytokines are known to play important roles in a wide range of pathologic conditions spanning all organ systems in every species studied. As our knowledge of the physiology of individual cytokines expands and our ability to measure multiple cytokines in smaller biological samples increases, we gain more insight into the significance and function of each cytokine and the importance of cytokine networks. Previous studies that reported measurements of cytokine concentrations from serum or plasma in dogs with infectious, autoimmune, metabolic, endocrine, and neoplastic diseases yield an appreciation for the complexity of cytokine control and potential applications for cytokine measurements in the diagnosis, prognosis, and therapy of a variety of disease conditions. In this review, we highlight the benefits of multiplex cytokine analysis, summarize clinical and experimental reports that have used this technology in dogs, and discuss the strengths and limitations of data analysis for the interpretation of results in these studies. We describe how differences in technical acuity, data reporting tactics, statistical analysis, study population selection criteria, and cross-sectional experimental design methods may affect interpretation of results from this technology. We also suggest methods for analysis in future studies, such as reporting median fluorescence intensity values, analyzing the proportion of patients above population medians, and performing longitudinal studies.

A new PCR assay for the detection and differentiation of Babesia canis and Babesia vogeli

Babesia spp. are globally distributed tick-borne protozoan parasites that infect the red blood cells of a wide range of vertebrate hosts, including humans. Diagnosis of babesiosis is often impeded by the transient presence of the parasites in peripheral blood, as well as by their pleomorphic nature. Given the reports of an expanding and, in some cases, sympatric geographical distribution of Babesia canis and Babesia vogeli in dogs and associated vectors, in Europe, the development of time-efficient and cost-effective diagnostic tools to detect and differentiate these two species is warranted. In this study, we designed and developed a novel polymerase chain reaction (PCR) assay targeting the parasite cytochrome c oxidase subunit 1 (cox1) gene, for the simultaneous detection and differentiation of B. canis and B. vogeli. The analytical sensitivity of the PCR was evaluated using serial dilutions of genomic DNA extracted from individual and artificially mixed canine blood samples infected by B. canis (3×102 infected erythrocytes/ml, ie/ml) and B. vogeli (2.1×101 ie/ml). The analytical specificity of the assay was assessed using blood samples positive for Hepatozoon canis, Ehrlichia canis, Anaplasma platys, Babesia microti, Babesia rossi and Theileria annae (syn. Babesia vulpes). The clinical specificity of the PCR assay was evaluated on 147 blood samples from dogs and 128 tick specimens (Dermacentor reticulatus and Rhipicephalus sanguineus sensu lato). Species-specific bands of the expected sizes (i.e., 750bp for B. canis and 450bp for B. vogeli), and two bands in the mixed blood samples were obtained. The PCR assay developed herein detected a low number of infected erythrocytes (i.e., 3×10−2B. canis, 2.1×10−2B. vogeli ie/ml). Of the 147 blood samples, nine (6.1%) were positive for B. canis and six (4.1%) for B. vogeli, whereas only one tick (D. reticulatus) was positive for B. canis. This PCR assay represents a rapid and reliable tool for the diagnosis of B. canis and B. vogeli in areas where these species occur in sympatry, and will assist future epidemiological investigations aimed to track the expanding range of these parasites.

Kai 1 and Kai 2: Characterization of these dog erythrocyte antigens by monoclonal antibodies.

Dog Erythrocyte Antigens (DEA) have thus far been found by sensitizing dogs with canine allogeneic blood and are clinically important regarding blood transfusion incompatibilities, but remain poorly defined. The goals of this study were to discover and characterize two DEAs, named as Kai 1 and Kai 2. The monoclonal antibodies were produced by mouse hybridoma techniques and examined by ELISA isotyping, immunoblotting, and affinity chromatography. Canine blood samples were typed and the development of alloantibodies was examined in transfused dogs. The monoclonal Kai 1 and Kai 2 antibodies were isotyped as IgM kappa and IgG3 lamda, respectively, and identified two different erythrocyte membrane proteins of 200 kDa and 80 kDa in molecular weights, respectively. Either Kai 1 or Kai 2 can be expressed but not both in an individual dog. There were no naturally occurring anti-Kai 1 or Kai 2 alloantibodies. In addition, Kai 1- and/or Kai 2- dogs developed Kai 1 and Kai 2 alloantibodies, respectively, when transfused with mismatched blood. This is the first discovery of canine blood types by screening monoclonal antibodies. Kai 1 and Kai 2 are novel blood types which can induce anti-Kai 1 or anti-Kai 2 alloantibodies when Kai 1- and/or Kai 2- dogs are transfused with Kai 1+ or Kai 2+ blood. These canine blood types may explain some of the blood incompatibilities and transfusion reactions observed in dogs in clinical practice.

Comparison of a full-spectrum multi-analyte clinical analyser with six reference instruments using canine and feline blood samples

In this study, we report the characterisation of a novel centrifugation and spectrum-integrated veterinary clinical analyser, the AmiShield&lt;sup&gt;TM&lt;/sup&gt;, which has been developed for the multiplex measurement of biochemical, electrolyte and immunoassay parameters in a point-of-care testing environment. The aims of this study were to evaluate the analytical performance of the AmiShield&lt;sup&gt;TM&lt;/sup&gt; and to compare it with six reference instruments using clinical blood samples. Two hundred and four canine and 120 feline blood samples collected from veterinary teaching hospitals were analysed in parallel using the AmiShield and appropriate reference instruments. All results were evaluated separately for canine and feline specimens. The instrument’s analytical performance was evaluated initially for short- and long-term precision, bias, and observed total error using quality control material. This was followed by comparison of clinical specimens on the AmiShield analyser in parallel with the Vitros and Hitachi for biochemical parameters, VetScan and SNAPshot for total bile acids, and VetLyte and Biolyte for electrolytes. Overall, the AmiShield analyser’s performance met the standards of the American Society for Veterinary Clinical Pathology for total allowable error for most analytes, and can be considered suitable for use in veterinary clinical practices. Using canine samples, excellent correlation coefficients (r ≧ 0.92) were identified for 14 analytes of various categories including glucose, total protein, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, amylase, blood urea nitrogen, creatinine, phosphorus, Na&lt;sup&gt;+&lt;/sup&gt;, K&lt;sup&gt;+&lt;/sup&gt;, Cl&lt;sup&gt;–&lt;/sup&gt; and total bile acid, while good correlations (0.91 ≧ r ≧ 0.80) were recorded for albumin (r = 0.91). Bland-Altman difference plots also showed agreement (greater than 95% within Limits of Agreement) for glucose, total protein, albumin, alanine aminotransferase, alkaline phosphatase, total bilirubin, amylase, blood urea nitrogen, creatinine, Na&lt;sup&gt;+&lt;/sup&gt;, K&lt;sup&gt;+&lt;/sup&gt;, Cl&lt;sup&gt;–&lt;/sup&gt; and total bile acid between AmiShield and the reference instruments. However, aspartate aminotransferase and phosphorus exhibited higher outliers, implying potential problems associated with matrix interferences such as lipemic samples, which warrant further study. This study demonstrates that the AmiShield compares favourably with standard reference instruments, and the new device generated data of high quality for most analytes in clinical canine and feline samples. The capability of reliably measuring multi-category analytes in one device using minute amounts (170 μl) of whole blood and short turn-around times (&amp;lt; 15 min) underlines the high potential of the device as a good alternative in-house diagnostic application.

Rickettsia amblyommatis infecting ticks and exposure of domestic dogs to Rickettsia spp. in an Amazon-Cerrado transition region of northeastern Brazil.

This study was performed in Maranhão state, a transition area two Brazilian biomes, Amazon and Cerrado. During 2011–2013, 1,560 domestic dogs were sampled for collection of serum blood samples and ticks in eight counties (3 within the Amazon and 5 within the Cerrado). A total of 959 ticks were collected on 150 dogs (9.6%). Rhipicephalus sanguineus sensu lato (s.l.) was the most abundant tick (68% of all collected specimens), followed by Amblyomma cajennense sensu lato (s.l.) (12.9%), Amblyomma parvum (9.2%), and Amblyomma ovale (5.2%). Other less abundant species (<1%) were Amblyomma oblongoguttatum, Rhipicephalus microplus, Haemaphysalis juxtakochi, and Amblyomma rotundatum. Females of A. cajennense s.l. ticks were morphologically identified as A. cajennense sensu stricto (s.s.) or A. sculptum. Molecular analyses of 779 canine ticks revealed three Rickettsia species: Rickettsia amblyommatis in 1% (1/100) A. cajennense s.l., ‘Candidatus Rickettsia andeanae’ in 20.7% (12/58) A. parvum, Rickettsia bellii in 6.8% (3/44) A. ovale and 100% (1/1) A. rotundatum ticks. An additional collection of A. sculptum from horses in a Cerrado area, and A. cajennense s.s. from pigs in an Amazon area revealed R. amblyommatis infecting only the A. cajennense s.s. ticks. Serological analysis of the 1,560 canine blood samples revealed 12.6% canine seroreactivity to Rickettsia spp., with the highest specific seroreactivity rate (10.2%) for R. amblyommatis. Endpoint titers to R. amblyommatis were significantly higher than those for the other Rickettsia antigens, suggesting that most of the seroreactive dogs were exposed to R. amblyommatis-infected ticks. Highest canine seroreactivity rates per locality (13.1–30.8%) were found in Amazon biome, where A. cajennense s.s. predominated. Lowest seroreactivity rates (1.9–6.5%) were found in Cerrado localities that were further from the Amazon, where A. sculptum predominated. Multivariate analyses revealed that canine seroreactivity to Rickettsia spp. or R. amblyommatis was statistically associated with rural dogs, exposed to Amblyomma ticks.

Heat pretreatment of canine samples to evaluate efficacy of imidacloprid\u2009+\u2009moxidectin and doxycycline in heartworm treatment

BackgroundConsidering the recent information on the increase of Dirofilaria immitis antigen detection by rapid assays in canine blood samples after heat treatment, the proposal that immune complexes block D. immitis antigen detection and that macrocyclic lactone + doxycycline (alternative protocol) might lead to increased production of those immune complexes, resulting in the erroneous diagnosis of adult worm elimination, and that there is no recommended adulticide marketed in Brazil, a study was performed to evaluate the interference of moxidectin + doxycycline (moxi-doxy) on diagnostic procedures when heartworm positive dogs are treated with this alternative protocol. Twenty-two naturally infected pet dogs were treated monthly with topical 10% imidacloprid + 2.5% moxidectin and with oral doxycycline (10 mg/kg BID/30 days) (moxi-doxy). All the dogs had their microfilaremia level determined prior to the first day of treatment, and were tested every 6 months for microfilariae (Mf) detection prior to heating, and for antigen detection prior to and after heating, the sample.ResultsThe results indicate that the treatment protocol can eliminate adult heartworms as early as 6 months after the first dose, especially in low microfilaremic dogs (< 300 Mf/ml). In this study, all dogs were free of heartworm antigen after 18–24 months of treatment. In a comparison of pre-heated samples and non-heated samples, sample pre-heating increased antigen detection sensitivity, and non-heated samples tended to be antigen-negative earlier than the pre-heated samples, especially when dogs had low microfilaremia levels. These discrepancies were not present in a subsequent sample of the same dog 6 months later.ConclusionsTwo negative antigen test results 6 months apart can be recommended as the criterion to consider when a dog has been cleared of infection. The initial microfilaremia level of a dog can be used to estimate the necessary time frame to end the treatment period.

The genetics and expression of canine antigen receptors analysed with a novel RNA-seq strategy.

Abstract INTRODUCTION Dogs are an excellent model for human disease, e.g. the treatment of canine lymphoma is often predictive of the human response to that treatment. However, an incomplete picture of their antigen-receptor (AR) gene loci and a limited set of available experimental tools has restricted their use. This work advances the annotation of the canine AR loci, develops methods to interrogate their expression patterns, and looks into breed-specific features of the loci. METHODS A bioinformatic approach alongside unbiased RNA-seq was used to complete the annotation of the canine AR genes, and these sequences were used to query 107 whole genome sequences from 19 breeds. A 5’RACE strategy was developed to selectively amplify ARs for next generation sequencing, and this was applied to RNA isolated from canine peripheral blood samples. RESULTS &amp;gt;5,500 novel alleles were identified across the ~550 gene segments (of which 326 were newly annotated) of the AR loci, yielding insight into AR evolution as well as confirming the greater conservation between dog and human than mouse with either. The 5’RACE strategy was validated with a pilot study that cast light onto the baseline expression and mutation patterns of healthy dogs, finding they closely mirrored their human counterparts. CONCLUSION This work brings our understanding of the genetics and expression of ARs in dogs to the same high standard as mice and men. The large number of genome sequences serves as a reference for future studies, and has allowed statistically powerful conclusions to be drawn on the pressures that have shaped these loci. Finally, it confirms the dog as a superior immunological model, particularly in the context of the high conservation seen in both sequence and expression features.

Anaplasma spp. in dogs and owners in north-western Morocco

BackgroundAnaplasma phagocytophilum is an emerging tick-borne zoonotic pathogen of increased interest worldwide which has been detected in northern Africa. Anaplasma platys is also present in this region and could possibly have a zoonotic potential. However, only one recent article reports on the human esposure to A. phagocytophilum in Morocco and no data are available on canine exposure to both bacteria. Therefore, we conducted a cross-sectional epidemiological study aiming to assess both canine and human exposure to Anaplasma spp. in Morocco. A total of 425 dogs (95 urban, 160 rural and 175 working dogs) and 11 dog owners were sampled from four cities of Morocco. Canine blood samples were screened for Anaplasma spp. antibodies by an enzyme-linked immunosorbent assay (ELISA) and for A. phagocytophilum and A. platys DNA by a real-time polymerase chain reaction (RT-PCR) targeting the msp2 gene. Human sera were tested for specific A. phagocytophilum immunoglobulin G (IgG) using a commercial immunofluorescence assay (IFA) kit.ResultsAnaplasma spp. antibodies and A. platys DNA were detected in 21.9 and 7.5% of the dogs, respectively. Anaplasma phagocytophilum DNA was not amplified. Anaplasma platys DNA was significantly more frequently amplified for working dogs. No statistically significant differences in the prevalence of Anaplasma spp. antibodies or A. platys DNA detection were observed between sexes, age classes or in relation to exposure to ticks. A total of 348 Rhipicephalus sanguineus (sensu lato) ticks were removed from 35 urban and working dogs. The majority of dog owners (7/10) were seroreactive to A. phagoyctophilum IgG (one sample was excluded because of hemolysis).ConclusionsThis study demonstrates the occurrence of Anaplasma spp. exposure and A. platys infection in dogs, and A. phagocytophilum exposure in humans in Morocco.

Genotyping Brucella canis isolates using a highly discriminatory multilocus variable-number tandem-repeat analysis (MLVA) assay

Differentiation of Brucella canis from other Brucella species are mainly performed through PCR-based methods and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) procedures. Both PCR-based and MLVA methods are limited in discriminating B. canis strains. A new MLVA-13Bc method for B. canis genotyping was established by combining eight newly-developed VNTRs with five published ones. During 2010 and 2016, 377 B. canis PCR-positives were identified from 6,844 canine blood samples from 22 U.S. states, resulting in 229 B. canis isolates. The MLVA-13Bc method was able to differentiate each of these 229 isolates. The Hunter-Gaston Discriminatory Index of the individual VNTR loci ranged from 0.516 to 0.934 and the combined discriminatory index reached 1.000. Three major clusters (A, B and C) and 10 genotype groups were identified from the 229 B. canis isolates. Cluster A mainly contains genotype groups 1 and 2, and a few group 3 isolates; nearly all Cluster B isolates were from group 6; other genotype groups were classified into Cluster C. Our newly developed MLVA-13Bc assay is a highly discriminatory assay for B. canis genotyping, and can serve as a useful molecular epidemiological tool, especially for tracing the source of contamination in an event of a B. canis outbreak.

Hematological and epidemiological characterization of Hepatozoon canis infection in dogs from Buenos Aires, Argentina.

Canine hepatozoonosis caused by Hepatozoon canis is widespread in America. In Argentina, since the first finding of the disease in Buenos Aires in 1999, several isolated cases were reported in other six provinces. However, there is no information regarding hematological and epidemiological characterization of the disease in the country. A total of 100,123 canine blood samples obtained during the period 2002-2013 from Southern Greater Buenos Aires were examined by light microscopy. Overall prevalence was 2.3%, with high parasitemia levels (>800gamonts/μl) in 680 samples, mild (100-800) in 1088, and low (<100) in 433 patients. Among parasitemic dogs, anemia (mostly non-regenerative) was present in 56.9%. Inflammatory leukogram, defined as neutrophilia with or without leukocytosis, with or without left shift and left shift with or without neutrophilia, was the main hematological abnormality and was present in 74.1% of positively tested dogs. A clear seasonal tendency was observed, with maximum values in summer and minimal in winter, and an increasing prevalence was recorded during the study decade. Young, male and mixed breed dogs showed higher prevalence values. Our findings strongly suggest that canine hepatozoonosis is endemic and expanding in the region.