Canine Blood Samples Research Articles

Feasibility of implementing RPA coupled with CRISPR-Cas12a (RPA-Cas12a) for Hepatozoon canis detection in dogs

Hepatozoonosis, caused by the protozoan Hepatozoon canis, is a prevalent blood disease affecting owned and stray dogs and cats. The prevalence of these parasites among companion animals in Thailand remains poorly understood. Diagnosing the old-world form of the disease is challenging due to the wide range of nonspecific clinical signs and the reliance on finding low levels of Hepatozoon gamonts in blood smears for conventional diagnosis. PCR demonstrates high specificity and sensitivity but it requires sophisticated instrumentation. Therefore, we established recombinase polymerase amplification (RPA) coupled with Cas12a for H. canis detection based on 18S rRNA. Our findings showed that RPA-Cas12a using gRNA_H was highly specific to H. canis, without yielding positives for other pathogen species including Babesia species. Even in cases of co-infection, RPA-Cas12a only detected positives in samples containing H. canis. This approach detected minimal amounts of H. canis18S rRNA-harboring plasmid at 10 copies per reaction, whereas plasmid-spiked canine blood enabled detection at a minimal amount of 100 copies per reaction. The performance of RPA-Cas12a was validated by comparing it with quantitative PCR-high resolution melting analysis (qPCR-HRM) and sequencing based on 35 canine blood samples. RPA-Cas12a demonstrated precision and accuracy values of 94 % and 90 %, respectively comparable to qPCR-HRM. Overall, these results indicate that RPA-Cas12a serves as a promising tool for H. canis detection as indicated by comparable performance to qPCR-HRM and is suitable for implementation in small animal hospitals or clinics due to its minimal resource requirements, thereby contributing to effective diagnosis and treatment for infected dogs.

Development and evaluation of a TaqMan® real-time PCR assay for species-specific detection of Ehrlichia canis

The aim of this work was to develop a real-time PCR assay with a TaqMan® probe that detects a species-specific part of the 16S rDNA gene of Ehrlichia canis. Canine blood samples (n = 207), collected and tested by a conventional PCR assay within a study conducted by De Salvo et al., were simultaneously analyzed with the novel designed real-time PCR, and the results of both assays were compared. The agreement between the two methods was 97.6 % with a kappa value of 0.92186. Hereby, the standard error was 0.034416 and the 95 % confidence interval from 0.8544 to 0.98931. While the conventional PCR assay showed false negative results (2.42 %; 5/207), the real-time PCR assays showed a specificity of 100 %. The results of the current study showed that the developed assay presents sensitivity and specificity for the detection of E. canis in blood samples, adding a new tool for the diagnosis of this pathogen.

Evaluation of Seroprevalence and Risk Factors of Heartworm Infection for Dogs in Rio de Janeiro with Access to Veterinary Care

Heartworm infection is a chronic disease with clinical signs and effects ranging from an asymptomatic condition to severe disease and death. The prevalence of heartworm disease in the state of Rio de Janeiro has been reported to be high (21.3%). The present study was conducted to evaluate the seroprevalence and risk factors of heartworm infection for the canine population with access to veterinary services in different areas of the state of Rio de Janeiro, Brazil. A total of 1787 canine blood samples were obtained from 135 practices across 8 different areas of Rio de Janeiro state (Rio de Janeiro municipality, São Gonçalo municipality, Niterói municipality, Baixada Fluminense, and the northern, southern, eastern, and mountainous areas) and tested for the presence of Dirofilaria immitis antigens and antibodies against several tick-borne disease pathogens using a commercial immunochromatography technique (Vetscan® Flex 4 Rapid Test; Zoetis; NJ USA). Pet owners reported living conditions, physical characteristics, demographics, and clinical signs for evaluation of risk factors for heartworm infection. Only two evaluated risk factors were shown to enhance the risk for D. immitis infection, including having a short hair coat vs. having a medium or long hair coat (OR 2.62) or positive for antibodies to tick-borne disease parasites (OR 3.83). Clinical signs reported for dogs with heartworm disease were typical for that condition. The overall prevalence of heartworm disease in the state was 8.2%, ranging from 2.4% in the mountainous region to 29.4% in the eastern area. It could not be determined if veterinarians were not diligent about dispensing heartworm preventatives or if poor levels of compliance by dog owners were responsible for higher infection rates in some areas of the state.

Development of a Colorimetric Loop-Mediated Isothermal Amplification Assay for the Detection of Trypanosoma cruzi in Low-Resource Settings.

Chagas disease is an inflammatory parasitic infection caused by Trypanosoma cruzi (T. cruzi). Early diagnosis is crucial in guiding treatment and slowing disease progression; however, current diagnostic methods have insufficient detection limits and often require skilled technicians. Molecular tests, especially isothermal nucleic acid assays, are advantageous due to their excellent sensitivity, specificity, speed, and simplicity. Here, we optimized a colorimetric loop-mediated isothermal amplification (LAMP) assay for T. cruzi. We can detect as few as 2 genomic copies/reaction using three different T. cruzi strains. We examined selectivity using other parasitic protozoans and successfully detected T. cruzi DNA extracted from parasites in human whole blood down to 1.2 parasite equivalents/reaction. We also performed a blinded study using canine blood samples and established a 100% sensitivity, specificity, and accuracy for the colorimetric LAMP assay. Finally, we used a heated 3D printer bed and an insulated thermos cup to demonstrate that the LAMP incubation step could be performed with accessible, low-cost materials. Altogether, we have developed a high-performing assay for T. cruzi with a simple colorimetric output that would be ideal for rapid, low-cost screening at the point of use.

Microfluidic PCR and network analysis reveals complex tick-borne pathogen interactions in the tropics

BackgroundIxodid ticks, particularly Rhipicephalus sanguineus s.l., are important vectors of various disease-causing agents in dogs and humans in Cuba. However, our understading of interactions among tick-borne pathogens (TBPs) in infected dogs or the vector R. sanguineus s.l. remains limited. This study integrates microfluidic-based high-throughput real-time PCR data, Yule's Q statistic, and network analysis to elucidate pathogen-pathogen interactions in dogs and ticks in tropical western Cuba.MethodsA cross-sectional study involving 46 client-owned dogs was conducted. Blood samples were collected from these dogs, and ticks infesting the same dogs were morphologically and molecularly identified. Nucleic acids were extracted from both canine blood and tick samples. Microfluidic-based high-throughput real-time PCR was employed to detect 25 bacterial species, 10 parasite species, 6 bacterial genera, and 4 parasite taxa, as well as to confirm the identity of the collected ticks. Validation was performed through end-point PCR assays and DNA sequencing analysis. Yule's Q statistic and network analysis were used to analyse the associations between different TBP species based on binary presence-absence data.ResultsThe study revealed a high prevalence of TBPs in both dogs and R. sanguineus s.l., the only tick species found on the dogs. Hepatozoon canis and Ehrlichia canis were among the most common pathogens detected. Co-infections were observed, notably between E. canis and H. canis. Significant correlations were found between the presence of Anaplasma platys and H. canis in both dogs and ticks. A complex co-occurrence network among haemoparasite species was identified, highlighting potential facilitative and inhibitory roles. Notably, H. canis was found as a highly interconnected node, exhibiting significant positive associations with various taxa, including A. platys, and E. canis, suggesting facilitative interactions among these pathogens. Phylogenetic analysis showed genetic diversity in the detected TBPs.ConclusionsOverall, this research enhances our understanding of TBPs in Cuba, providing insights into their prevalence, associations, and genetic diversity, with implications for disease surveillance and management.Graphical abstract

Exploring multiplex qPCR as a diagnostic tool for detecting microfilarial DNA in dogs infected with Dirofilaria immitis: A comparative analysis with the modified Knott’s test

Current recommendations to diagnose cardiopulmonary dirofilariosis in dogs caused by Dirofilaria immitis involves tandem antigen and circulating microfilariae tests. The modified Knott’s test is an important tool in heartworm diagnosis, allowing identification of circulating microfilariae. However, the subjective nature of the modified Knott’s test affects its accuracy and diagnostic laboratories usually do not provide a quantitative outcome. Quantitative enumeration of microfilariae enables clinicians to track treatment progress and acts as a proxy for detecting emerging macrocyclic lactone resistance. There is a need for better diagnostic tools suitable for routine use to efficiently and accurately quantify the presence of D. immitis microfilaremia. The aim of this study was to determine whether the quantitative modified Knott's test can be substituted by multiplex quantitative polymerase chain reaction (qPCR) targeting D. immitis and associated Wolbachia endosymbiont DNA in canine blood samples. To do this, genomic DNA samples (n = 161) from Australian dogs, collected as part of a previous 2021 study, were assessed in a TaqMan qPCR targeting DNA of D. immitis, Wolbachia sp. and Canis lupus familiaris. Of the 161 genomic DNA samples, eight were considered positive for D. immitis microfilariae. The qPCR assay demonstrated good efficiency (E = 90 to 110%, R2 > 0.94). Considering the performance and efficient use of bench time, this TaqMan qPCR assay is a suitable alternative to the modified Knott’s test for quantitative enumeration of microfilariae (Cohen’s kappa coefficient [κ]: κ = 1 using D. immitis qPCR marker, κ = 0.93 using Wolbachia qPCR marker). The qPCR data demonstrated a comparable result to that of the quantitative modified Knott’s test in a 2022 survey of D. immitis in Australian dogs (n = 23) before and after macrocyclic lactone (ML) administration. Improving the detection and diagnosis of canine heartworm infections will assist veterinarians in better managing and controlling disease outcomes and will be valuable for tracking the spread of ML resistance in Australia.

Comparison of visual assessments of anisocytosis in canine blood smears and analyzer-calculated red blood cell distribution width.

Red blood cell distribution width (RDW) and visual assessments of anisocytosis assess variability in erythrocyte size. Veterinary studies on the correlation between the two methods and on observer agreement are scarce. The objectives were to assess the correlation of the grading of anisocytosis by means of conventional microscopy of canine blood smears to RDW, and to assess intra- and inter-observer variation in assessing the degree of anisocytosis. The study included 100 canine blood samples on which blood smear examination and RDW measurement were performed. RDW was measured on the Advia 2120i analyzer. The degree of anisocytosis was based on a human grading scheme assessing the ratio between the size of the representative largest red blood cell and that of the representative smallest red blood cell (1+ if <2x, 2+ if 2-3x, 3+ if 3-4x, and 4+ if >4x). Three observers participated and assessed the blood smears by conventional microscopy twice, 3 weeks apart by each observer. The correlation was assessed for each observer on each occasion using Kendahl-tau-b analysis. Intra-observer agreement was assessed using quadratically weighted kappa. Inter-observer agreement was assessed using free-marginal multi-rater kappa. Anisocytosis graded on blood smears correlated significantly with RDW values as assessed by Kendahl-tau-b ranging between 0.37 and 0.51 (p < 0.0001). Intra-observer agreement ranged from weak to moderate with resulting kappa-coefficients being 0.58, 0.68, and 0.75, respectively. Inter-observer agreement was weak (Kappa-values 0.44). The weak to moderate observer agreement in the visual assessment of anisocytosis indicates that the more precise and more repeatable RDW measurement should be used for clinical decision-making.

Evaluation of canine and feline leukocyte differential counts obtained with the scil vCell 5 compared to the Advia 2120 hematology analyzer and a manual method.

The vCell 5 (scil Animal Care), a point-of-care hematology analyzer (POCA), was recently introduced to veterinary laboratories. This laser- and impedance-based analyzer is capable of providing a CBC with 5-part WBC differential count (Diff) along with WBC cytograms and flags serving as interpretation aids for numerical results. We compared the scil POCA-Diff to reference methods (i.e., manual differential count, Advia 2120 hematology analyzer [Siemens]) for canine and feline blood samples and considered WBC cytograms and flags. Total observed error (TEo), calculated from CV and bias%, was compared to total allowable error (TEa). Data were analyzed before and after a review process (exclusion of flagged and samples with invalid cytograms). For both species, correlation was good-to-excellent (rs = 0.81-0.97) between both analyzers for all variables, except for feline monocytes (rs = 0.21-0.63) and canine monocyte% (rs = 0.50). Smallest biases were seen for neutrophils (dog: -5.7 to 0.8%; cat: 1.5-9.4%) with both reference methods. Quality requirements (TEo < TEa) were fulfilled for canine and feline neutrophils (TEo = 5.3-10.6%, TEa = 15%) and eosinophils (TEo = 67.1-83%, TEa = (90)-50%) considering at least one reference method. Our review process led to mildly higher rs-values for most variables. Although not completely satisfactory, the scil POCA provides reliable results in compliance with ASVCP quality goals for canine and feline neutrophils and eosinophils. Analyzer flag and cytogram analysis served as useful tools for QA, indicating the necessity for manual review of blood smears, and contributed to improvement of scil POCA performance.

Performance of the Sysmex XN-V hematology analyzer in determining the immature platelet fraction in dogs: A preliminary study and reference values.

Immature platelets (IPs) are newly formed platelets released into circulation that have been demonstrated as good markers of thrombopoiesis. Although many flow cytometric and fully automated-based methods are available, the latest Sysmex XN-V hematology analyzer for veterinary use is equipped with a specific fluorescent platelet channel (PLT-F) that detects platelets using a platelet-specific dye. The aims of this study were to evaluate the performance of the Sysmex XSN-1000 V in determining the IPF (immature platelet fraction) and other selected PLT-F channel parameters and to propose IPF reference intervals (RIs) for canine blood samples. Canine EDTA blood samples were analyzed on the Sysmex XN-1000 V to assess linearity, imprecision, carryover, stability, and the effect of platelet clumping on selected platelet parameters from the PLT-F channel. We also reported the de novo generated RIs for the IPF in dogs. Imprecision was acceptable (CV <10%) for all parameters except for the absolute IPF values (IPF#), in which the reproducibility was 12.15% for the normal-low concentration samples. Linearity and carryover were excellent for all variables. Relative IPF values (IPF %) and IPF# remained stable for both storage conditions for up to 48 hours; however, a nonsignificant progressive increase in these parameters was observed from 12 hours at 4°C. We observed a statistical increase in IPF% and IPF# and a statistically significant decrease in PLT-F counts after intentional in vitro platelet aggregation. RIs were generated for all reference samples (n = 69) and for samples with (n = 25) or without (n = 44) platelet clumps. The performance of the new PLT-F channel-derived variables for dogs was excellent. Specific RIs for IPF should be used when platelet aggregates are present.

Seroepidemiological Analysis of Canine Leptospira Species Infections in Changchun, China.

Leptospirosis is a significant worldwide zoonotic infectious disease that infects a wide range of animals and humans. Leptospira will colonize the animal's urinary and reproductive systems and be excreted with urine, potentially causing a wide range of infections. Dogs are an essential host for Leptospira, and epidemiological investigation studies of leptospirosis must be conducted to clarify the prevalence of leptospirosis and to reduce the risk of transmission to humans. This study aimed to investigate the seroepidemiology of leptospiral infection in dogs from Changchun, China, using Microscopic Agglutination Test (MAT). A total of 1053 canine blood samples were collected and tested by MAT. The positive rate of MAT was approximately 19.1%. The main prevalent Leptospira serogroups were L. Icterohaemorrhagiae (8.1%), L. Canicola (7.6%), L. Australis (5.3%), L. Ballum (4.7%) and L. Pyrogenes (4.2%). No statistically significant difference among different varieties, sexes and sampling seasons (p > 0.05), except the age (p < 0.05). The seropositive rate was much higher in adult and aged dogs than in juvenile dogs. Our results showed the seroprevalence and the prevalent serogroup of Canine leptospirosis in Changchun, China.

Molecular detection of Bartonella and Borrelia in pet dogs in Metro Manila and Laguna, Philippines.

Bartonella and Borrelia are zoonotic vector-borne pathogens that can infect dogs and humans. Data on Bartonella and Borrelia in dogs in the Philippines are lacking. This study was conducted to validate previous reports and further investigate the occurrence of Bartonella and Borrelia spp. in cities of Metro Manila. A total of 182 canine blood samples were acquired with DNA using a commercial extraction kit from selected veterinary clinics in the cities of Metro Manila and Laguna, Philippines. The mammalian actin was amplified through polymerase chain reaction (PCR), followed by PCR assays targeting Bartonella gltA and Borrelia flaB. Further PCR assays targeting 16S of Borrelia and ospA and ospC of Borrelia burgdorferi were performed for those that showed flaB bands. A positive band for gltA of Bartonella was observed in 9 (4.95%) samples, whereas a positive band for flaB of Borrelia was observed in 15 (8.24%) samples. Subsequent PCR assays for other genes of Borrelia were negative. These results confirmed the presence of Bartonella and warranted further investigation for the possible presence of other Borrelia species.

Extended sample storage for platelet function testing in healthy dogs.

Platelet function testing is important for monitoring the effects of antiplatelet therapy but is not readily used due to time constraints for testing and the need for specialized equipment. This study evaluated the effects of various storage methods on selected platelet function tests to determine if delayed platelet function testing is feasible in canine blood samples. Our hypotheses were that platelet function would not decline during storage and, thus, no differences in test results would be found over time. Thirteen healthy dogs were studied. Citrated blood samples were tested with a Platelet Function Analyzer-200 (PFA), which mimics high-shear conditions, using P2Y and CADP cartridges, after being held at room temperature for 2 h and refrigerated for 24 and 48 h. Plateletworks (PW), which measures aggregation based on platelet counting, was performed on an optical hematology analyzer using 10-min-old native samples, citrated samples held at room temperature for 3-4 h and refrigerated for 24 and 48 h, and samples stored in the preservative solution, AGGFix, up to 7 days. PFA closure times increased with storage, especially with the P2Y cartridge. Median aggregation with fresh PW was 94%, and this was maintained at all time points (range of median values 88%-94%). Most samples showed decreased, yet still robust (>70%), aggregation with longer storage. Spontaneous aggregation in citrate was noted in most dogs. AGGFix stabilized platelet aggregates to allow for delayed testing. Delayed platelet function testing is feasible, but ranges of expected values may differ from tests using fresh samples.

Impact on canine neutrophil preservation with the addition of bovine serum albumin to K3 -EDTA whole blood samples.

Cellular deterioration occurs with blood sample aging, impacting white blood cell (WBC) identification and differential accuracy. This may be exacerbated in samples from patients experiencing inflammation. Previously, bovine serum albumin (BSA) has been shown to improve cellular preservation of blood and other samples, but the effect on cell preservation in canine blood has not been assessed. We aimed to determine the effects of BSA on neutrophil nuclear area when added to potassium ethylene diamine tetra-acetic acid (K3 -EDTA)-anticoagulated canine blood prior to blood smear preparation. We evaluated the impact of inflammatory leukograms, sample storage temperatures (4° and 20°C), and time on outcomes. Canine K3 -EDTA-anticoagulated blood samples stored at 4° and 20°C were used from unique patients, 10 with and 10 without inflammatory leukograms. Blood smears were prepared from aliquots with or without the addition of 22% BSA at 0, 4, 8, 24, 48, and 72 h. The nuclear area was measured for 25 randomly selected neutrophils per slide using Fiji software. Mixed-effect linear regression modeling was performed (significance: P < 0.05). Nuclear area increased over time with and without added BSA. Both sample storage temperatures and the presence or absence of an inflammatory leukogram significantly impacted neutrophil nuclear area. Samples with added BSA had slightly higher predicted nuclear areas than those without BSA, but this difference was not statistically significant. BSA did not significantly impact neutrophil nuclear area and did not improve neutrophil preservation in canine blood samples.

Analytical errors in nucleated red blood cell enumeration.

Enumeration of nucleated red blood cells (NRBCs) in peripheral blood of dogs and cats is performed by manual counting during blood film evaluation. Automated methods have increased precision and accuracy; however, most analyzers cannot distinguish leukocytes and NRBCs. The Sysmex XN-V Series may distinguish NRBCs and leukocytes; however, analytical errors occur. We aimed to investigate cases with discrepant automated and manual NRBC counts, and to evaluate reasons for analytical errors. Data from samples with increased NRBCs were collected retrospectively and compared with manual counts performed on blood films using Spearman's correlation, Passing-Bablok agreement analysis, and Bland-Altman comparisons. Precision of the automated method and interobserver agreement of manual counts were evaluated. Cases with discrepant results were investigated. Agreement between the methods was good at ≤1NRBC ×109 /L in dogs and cats, and inadequate at ≥1NRBC ×109 /L. The automated method demonstrated a negative proportional difference to the manual method. Precision was good for the automated method (overall CV 7.1%) and interobserver agreement for the manual method was poor overall (mean CV 27.3%, range 0%-106.1%). Inaccuracies in NRBC enumeration by the automated method occurred with high hematocrits, the mergence of the cell fragments and leukocyte clouds, and the presence of earlier erythroid precursors. NRBC enumeration by the WNR channel on the Sysmex XN-1000 V is precise and has good agreement with manual counts in canine and feline blood samples at ≤1NRBC ×109 /L. Manual film review is indicated for samples with ≥1NRBC ×109 /L, earlier erythroid precursors, samples from greyhounds and dehydrated patients, and if gating errors are noted.

Prevalence of Babesia and Ehrlichia in owned dogs with suspected tick-borne infection in Hong Kong, and risk factors associated with Babesia gibsoni.

Babesiosis and ehrlichiosis are the most clinically significant tick-borne infections in dogs. Although epidemiological investigations of these diseases have been performed in some Asian regions, little data is available in Hong Kong, where competent vector tick species are endemic. The objectives of this study were to determine the molecular prevalence of Ehrlichia canis and Babesia species (B. canis, B. gibsoni, B. vogeli) in owned dogs suspected of tick-borne infection in Hong Kong and to identify risk factors associated with B. gibsoni infection. Electronic records from the Veterinary Diagnostic Laboratory of City University of Hong Kong were searched to identify canine blood samples submitted for molecular testing of these pathogens by real time PCR between March 2018 and May 2021. Electronic patient records from the affiliated veterinary hospital were searched to identify a subset of tested dogs to investigate the potential risk factors for B. gibsoni infection using logistic regression models. Among 1508 tested dogs for all four pathogens of interest, Babesia spp. were detected in 435 (28.8%) and E. canis in 112 (7.4%). Babesia gibsoni was detected in 408 dogs while B. vogeli was detected in 27 dogs. Babesia canis was not detected in any dog. Co-infections of different combinations of B. gibsoni, B. vogeli and E. canis were present in 25 dogs. In multivariable logistic regression, mixed breed dogs were more likely to be infected with B. gibsoni than purebreds (P=0.005), while dogs >10 years of age were less likely to be infected than younger dogs (P=0.019). Hematological abnormalities significantly associated with B. gibsoni infection included thrombocytopenia, neutropenia, or pancytopenia. Babesiosis caused by B. gibsoni is a common infection in owned dogs suspected of tick-borne infection in Hong Kong. The risk factors reported should be considered in diagnosing dogs suspected of infection with this agent. Furthermore, consideration for testing for B. gibsoni infection should be given if the results of a complete blood count show thrombocytopenia even in the absence of anemia, neutropenia or pancytopenia.

Re-emergence of canine Leishmania infantum infection in mountain areas of Beijing

Canine Leishmaniasis (CanL) is an endemic infectious disease in China, causing visceral Leishmaniasis (VL) and resulting in important public health problem. However, in the last 3 y, endemic trends have changed considerably and spatial–temporal aggregation areas have shifted from northwestern to central China. Although Beijing was an endemic area for CanL in the last century, this disease has not been reported in Beijing since control programs were implemented in the 1950s. In the present study, PCR and immunochromatographic (ICT) were used to estimate prevalence of Leishmania infection in domestic dogs living in Beijing, a VL re -emergencearea. In total, 4420 canine blood samples were collected at vet clinics in 14 districts of Beijing. Overall prevalence (percentage of dogs seropositive and/or PCR positive) of CanL infection in Beijing was 1.22% (54/4420). However, prevalence of CanL in the western mountain areas was 4.68% (45/961), significantly higher than that (0.26%, 9/3459) of the plains. In addition, multilocus sequence typing (MLST) of seven enzyme-coding genes was used to examine phylogenetic relationships of CanL strains. Forty-one Leishmania infantum isolates were well separated from the other strains and divided into five major clades (A to E) by MLST analysis. All clades were closely related to strains from Sichuan Province and Gansu Province. A phylogenetic tree, based on the MLST, revealed that L. infantum in Beijing was genetically related to strains from western endemic of Mountain type VL in China. In conclusion, CanL has re-emerged in Beijing, and almost 5% of dogs living in Beijing’s mountain areas were infected with L. infantum. The phylogenetic tree based on MLST effectively distinguished species of Leishmania and reflected geographical origins. Because dogs are considered a natural reservoir, comprehensive control measures including surveillance, phylogenetic analyses and management should be implemented to mitigate or eliminate Leishmaniasis.

Effects of time delay and blood storage methods on analysis of canine venous blood samples with an Element point-of-care analyzer.

Manufacturers of point-of-care (POC) analyzers recommend immediate processing and anaerobic collection of blood samples. However, it is not uncommon for clinical scenarios to result in delayed sample processing or room air exposure that could impact the test results. To investigate the effect of time delay and sample storage method on key POC analytes in canine venous blood samples processed with an Element POC analyzer. Blood gas analysis was performed on venous blood samples at times 0 (T0), 15, 30, and 60 minutes after sampling using three different storage methods: preheparinized plastic syringes and two different lithium heparin tubes. To determine clinical relevance, results were compared with allowable total error of the respective parameter. Significance was set at P < 0.05. Significant differences between the three storage methods at baseline were found for partial pressure of carbon dioxide (PCO2 ), partial pressure of oxygen (PO2 ), base excess, and total hemoglobin. No significant differences up to T60 were found within collection methods for actual bicarbonate (HCO3 - ), base excess, sodium, potassium, chloride, ionized calcium (iCa), glucose, and BUN. Significant differences within collection methods were found after T0 for creatinine, after 15 minutes for lactate, and after 30 minutes for pH and hematocrit. No significant differences were found for PO2 in samples stored in preheparinized plastic syringes at any time point. These results suggest that HCO3 - , sodium, potassium, chloride, iCa, glucose, and BUN are comparable within the three storage methods for up to 60 minutes after sampling without resulting in clinically relevant changes.

Assessment of the circulation of Dirofilaria immitis in dogs from northern Portugal through combined analysis of antigens, DNA and parasite forms in blood

Dirofilariasis is a vector-borne disease frequent in many countries. Not only infected dogs, but also cats and wild canids (including wolves and foxes), represent important sources of infection for mosquitoes, which are the pathogen vectors. The disease is endemic in Mediterranean countries with increasing incidence in Italy, France, Greece and Spain, but limited epidemiological data is available from Portugal regarding its distribution and impact. Aiming to clarify this, canine whole blood samples (n = 244) from the north of Portugal were tested for Dirofilaria spp. antigens by use of a commercial rapid immunomigration test. Polymerase chain reaction (PCR) and the modified Knott test were also used to assess the presence of microfilariae. Results were also compared to assess the performance of each test used. Of the 244 animals tested, 118 (48.4%) were positive for Dirofilaria immitis (heartworm) in the serological adult worm rapid antigen detection test, and 36 (14.8%) had circulating microfilariae, identified as D. immitis. A combined positivity of 51.6% (126/244) was found. Results indicate that the risk of exposure to D. immitis in dogs is high in this region of Portugal, and that prophylaxis against the parasite is advisable to decrease the occurrence of canine infection and disease. The present study highlights the diagnostic value of serological and molecular tests in determining the prevalence of D. immitis.

Diversity and geographic distribution of rickettsial agents identified in brown dog ticks from across the United States

Rhipicephalus sanguineus sensu lato, or brown dog ticks, transmit a variety of pathogens of veterinary and public health importance globally. Pathogens vectored by brown dog ticks and identified in the United States include Babesia vogeli, Ehrlichia canis, and several spotted fever group Rickettsia spp. (SFGR). Due to the challenge of collecting canine blood samples nationwide to screen for exposure to these pathogens, we took an indirect approach and tested brown dog ticks for molecular evidence of infection. Brown dog ticks (616 adults and 65 nymphs) collected from dogs and cats across the nation were tested by separate PCR assays detecting Babesia spp., E. canis, and SFGR. While no Babesia sp. was found, we identified rickettsial agents in 3.5% (24/681; 95% CI 2.4–5.2%) of the ticks. Pathogens and related organisms detected in ticks included E. canis (n = 1), Rickettsia amblyommatis (n = 3), Rickettsia massiliae (n = 11), Rickettsia monacensis (n = 3), Rickettsia montanensis (n = 5), and an undefined Rickettsia species (n = 1). These data demonstrate a wider geographic distribution of R. massiliae than previously known, and to the authors’ knowledge, reports R. monacensis in brown dog ticks for the first time. Due to the close association that brown dog ticks have with domestic dogs and humans, more research is needed to understand the full array of organisms, some of which are zoonotic, potentially transmitted by this widespread tick complex.

Comprehensive Map of Canine Angiostrongylosis in Dogs in Spain.

Simple SummaryAngiostrongylus vasorum (A. vasorum) is the causal agent of canine angiostrongylosis, an emerging disease in Europe. In Spain, A. vasorum mainly affects wild animals (red foxes and wolves), although studies in domestic dogs are scarce but show evidence of an expansion of the disease. The aim of this study was to analyze the presence of A. vasorum in domestic dogs throughout Spain, taking into account that there were still provinces where this canine infection has not been studied. Blood samples from 5544 domestic dogs were collected from January 2020 to March 2022. These samples were tested for the presence of circulating A. vasorum antigens. The overall prevalence of canine angiostrongylosis in Spain was 1.39%. No significant differences were found for sex and age, but significant differences were found for habitat. Infected domestic dogs were reported in most Spanish provinces, with lower prevalence observed for inland provinces and higher prevalence observed for provinces along the coast, where climatic factors seem to be determinant in the establishment of the parasite.Canine angiostrongylosis is an emerging disease caused by Angiostrongylus vasorum, mainly affecting wild carnivores and dogs. In Spain, there are studies reporting infections in foxes, wolves, and badgers in different regions of the country. However, there are hardly any publications on its prevalence in dogs. The aim of this study was to complete and update the epidemiologic map of A. vasorum in dogs in Spain. A total of 5619 canine blood samples from all autonomous cities and provinces of Spain were collected and tested for the presence of circulating A. vasorum antigens. The overall apparent prevalence of canine A. vasorum infection in Spain was 1.39%. No significant differences were found for sex or age, but significant differences between outdoor and indoor/outdoor dogs were found. A high prevalence was also observed in the northern third of the country, where an oceanic climate prevails, being humid and rainy and where abundant vegetation can be found, thus favoring the proliferation of intermediate hosts. The results suggest that A. vasorum canine infections are heterogeneously present in a large part of the territory, demonstrating its expansion throughout the country, and therefore, awareness and prevention campaigns for this disease should be promoted.