Feline Blood Samples Research Articles

Detection of Bartonella henselae in feline erythrocytes in Egypt by using Giemsa staining, transmission electron microscopy, and polymerase chain reaction.

Bartonella species (Bartonella spp.) have gained recognition as a significant human pathogen, implicated in a wide range of diseases. Among these, Bartonella henselae infection has been extensively studied for its primary occurrence in cats and its role in the development of cat-scratch disease in humans. While light microscopy and transmission electron microscopy (TEM) have traditionally played crucial roles in identifying causative agents of infectious diseases, including Bartonella spp., the accuracy of these methods in identifying Bartonella spp. remains undefined. Therefore, this study aims to bridge this gap by employing both light microscopy and TEM to detect Bartonella in feline blood samples and to confirm B. henselae with polymerase chain reaction (PCR). Examination of blood smears stained with Giemsa and toluidine blue semithin sections by using light microscopy revealed the presence of intraerythrocytic corpuscles, suggesting Bartonella infection in six out of 33 examined cat blood samples. TEM findings corroborated these observations, showcasing the engulfment of bacteria by the erythrocyte membrane, along with the presence of some Bartonella spp., adhering to the erythrocyte wall. PCR-based molecular detection confirmed the presence of B. henselae in these six samples. It is concluded that light microscopy and TEM are considered valuable in the screening of cats' blood for the potential presence of Bartonella. However, further molecular techniques are essential for precise identification and confirmation of specific Bartonella spp. RESEARCH HIGHLIGHTS: Giemsa-stained blood smear and semithin section showed potential intraerythrocytic Bartonella spp. corpuscles. TEM demonstrated the engulfment of Bartonella spp. by the erythrocyte membrane, along with the presence of some Bartonella spp. adhering to the erythrocyte wall. Molecular analysis of blood samples from cats by PCR unveiled that six out of 33 (18.18%) samples tested positive for B. henselae infection.

A veterinary-calibrated point-of-care glucometer accurately measures blood glucose concentration in dogs and cats.

To determine the clinical and analytical accuracy of a new veterinary-calibrated portable blood glucose monitor (PBGM) compared to a reference laboratory analyzer. Client-owned dogs (n = 77) and cats (n = 64). Peripheral and paired capillary whole-blood glucose concentrations measured via PBGM were compared to plasma glucose concentrations measured via a Cobas c501 reference analyzer (Roche). Analytical accuracy was evaluated with the Spearman rank correlation coefficient, Bland-Altman difference plot analysis, and Deming regression. Clinical accuracy was evaluated with Parkes error grid analysis. Paired peripheral and capillary blood samples were compared with the Wilcoxon matched-pairs signed-rank test. There was a high correlation between PBGM and reference analyzer readings in dogs and cats. Human quality assurance standards (International Organization for Standardization 15197:2013 guidelines) for analytical accuracy were met for 95% of feline peripheral blood samples and 89% of canine samples. Similar veterinary standards (American Society of Veterinary Clinical Pathology guidelines) were met for 89% of canine and 92% of feline peripheral blood glucose measurements. Error grid analysis showed that all peripheral canine and 97% of feline measurements were clinically accurate (zone A). Any altered clinical decision for the remaining feline measurements was expected to minimally impact outcome (zone B). No significant difference was found between peripheral and capillary blood glucose measurements in either species. The PBGM produced clinically accurate results and is suitable for use in veterinary and home settings to measure blood glucose.

Feline vector-borne haemopathogens in Türkiye: the first molecular detection of Mycoplasma wenyonii and ongoing Babesia ovis DNA presence in unspecific hosts

BackgroundCats are hosts and reservoirs for many haemopathogens such as piroplasms, Rickettsia, hemotropic Mycoplasma, Bartonella, Ehrlichia, and Anaplasma, which are transmitted by various vector arthropods and some of which have a zoonotic concern. Although it is noteworthy that the rate of ownership of companion animals has increased in Türkiye in recent years and that cats account for a large proportion of these animals, there is limited research on the vector-borne infectious agents carried by them. The present study aimed to provide a comprehensive molecular epidemiological data and molecular characterization of feline vector-borne haemopathogens (FVBHs), including piroplasms, anaplasmataceae, rickettsias, haemoplasmas, and Bartonella species in Türkiye. In total, 250 feline blood samples were collected from client-owned cats (n = 203) and shelter cats (n = 47) brought to the Small Animal Hospital of Selcuk University, Veterinary Faculty.ResultsOverall, 40 (16%) cats were found to be infected with at least one of the investigated haemopathogens and piroplasm, Mycoplasma spp. and Bartonella spp. prevalence was 1.6%, 11.2%, and 4.8%, respectively. No Anaplasma/Ehrlichia spp. and Rickettsia spp. DNA was detected in the investigated feline samples. Sequence analysis revealed that all four piroplasms belonged to Babesia ovis with a 97.93–99.82% nucleotide sequence identity to 18S rRNA gene sequences from Spain and Türkiye, while some sequenced hemoplasmas were Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm) and Mycoplasma wenyonii, and Bartonella spp. were Bartonella henselae and Bartonella koehlerae species. Co-infections with Mycoplasma spp. and Bartonella spp. were also detected in 4 cats (1.6%) in this study, where single infections were predominant.ConclusionThis study provides valuable information on zoonotically important feline vector-borne hemopathogens in Türkiye, some of which have received attention under the One Health perspective, and is the first molecular epidemiological study to demonstrate the presence of Babesia ovis, the causative agent of ovine babesiosis, and Mycoplasma wenyonii DNA, the causative agent of bovine haemotropic mycoplasmosis, in cats. Further studies on the roles of such pathogens detected in unspecific hosts and the host specificity of the vectors that transmit them will contribute to the elucidation of this situation.

Adapting the SMART tube technology for flow cytometry in feline full blood samples.

Flow cytometry of blood samples is a very valuable clinical and research tool to monitor the immune response in human patients. Furthermore, it has been successfully applied in cats, such as for infections with feline immune deficiency virus (FIV). However, if cells are not isolated and frozen, analysis of anticoagulated blood samples requires mostly prompt processing following blood collection, making later analysis of stored full blood samples obtained in clinical studies often impossible. The SMART Tube system (SMART TUBE Inc., California, United States; SMT) allows fixation and long-term preservation of whole blood samples at -80°C. However, this system has so far only been applied to human biological samples. In the present study, a new flow cytometry SMART Tube protocol adapted for feline whole blood samples was successfully established allowing quantification of T-helper cells, cytotoxic T-cells, B-cells, monocytes, and neutrophils up to 2 years post sampling. Results obtained from frozen stabilized and fresh blood samples were compared for validation purposes and correlated to differential blood counts from a conventional hematology analyzer. Clinical applicability of the new technique was verified by using samples from a treatment study for feline infectious peritonitis (FIP). Using the new SMT protocol on retained samples, it could be demonstrated that long-term storage of these SMT tubes is also possible. In summary, the newly adapted SMT protocol proved suitable for performing flow cytometry analysis on stored feline whole blood samples, thus opening up new avenues for veterinary research on a variety of aspects of clinical interest.

Alterations in some hematological parameters of feline blood samples preserved at different temperatures for a duration of up to 48 hours

Hematological parameters serve as vital diagnostic tools in veterinary medicine, aiding in the identification of various diseases and metabolic conditions in animals. However, their reliability can be influenced by a multitude of factors, including storage conditions and duration. Pre-analytical errors, which account for up to 70% of laboratory diagnostic errors, primarily stem from issues such as sample collection, transportation, and storage. To address this, the European Federation of Clinical Chemistry and Laboratory Medicine-Working Group for the Preanalytical Phase conducted a survey to understand pre-analytical practices among human laboratories. Despite significant interest, diversity in monitoring approaches was noted among European laboratories. The complete blood count (CBC) stands as a frequently requested test by veterinarians, but its accuracy can be affected by pre-analytical variables like storage temperature and delay in analysis. This study aimed to assess the stability of hematological parameters in feline blood samples stored at different temperatures (4°C and 20°C) for up to 48 hours. Ten clinically healthy cats were included in the study, with blood samples collected and analyzed immediately and at various time points over 48 hours. Results showed stability in hemoglobin concentration for 48 hours at both temperatures. However, RBC and PCV values increased significantly after 24 hours at 20°C. MCV, MCH, and MCHC values remained relatively stable over time, with minor fluctuations. Notably, WBC counts declined significantly after 48 hours at 20°C, while no significant changes were observed at 4°C. These findings underscore the importance of prompt analysis and appropriate storage conditions in maintaining the integrity of hematological parameters in veterinary practice. In conclusion, blood samples from cats stored at 4°C remain reliable for up to 48 hours for most hematological parameters, while samples stored at 20°C are suitable for up to 24 hours. These findings provide valuable insights for veterinarians and laboratory professionals, emphasizing the need for standardized pre-analytical practices to ensure accurate diagnostic results in veterinary hematology.

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.

Validation of storage and shipping of feline blood samples for analysis on the Platelet Function Analyzer-200 for determining the effect of clopidogrel.

The Platelet function analyzer-200 (PFA-200) can determine the effect of clopidogrel in cats, but analysis traditionally must be performed at point-of-care (POC). The ability to ship samples of blood to a laboratory would allow widespread access. We aimed to validate the shipping of blood samples for PFA-200 analysis in cats to determine the effect of clopidogrel. Twenty healthy cats and 10 cats receiving clopidogrel were recruited. Blood was collected from cats and aliquoted into two samples, one was analyzed at POC within 2 hours using the PFA-200, and the other was packaged and transported to a location 4 km away, stored, and transported back to the lab for analysis the following day. Median closure times (CTs) with the collagen/adenosine diphosphate (COL/ADP) cartridge in healthy cats were 51.5 seconds (POC) and 78.8 seconds (shipped), which were significantly different (P < 0.001), and for cats on clopidogrel, median CTs were 147.5 seconds (POC) and 190 seconds (shipped), which were not significantly different (P = 0.131). Median CTs with the P2Y cartridge in healthy cats were 50.5 seconds (POC) and 64.9 seconds (shipped), which were significantly different (P = 0.03), and in cats receiving clopidogrel, median CTs were 300 seconds (POC) and 300 seconds (shipped) which were not significantly different (P = 1.000). Reference intervals for CTs differed for COL/ADP at POC (19.8-89.7 seconds) and shipped (50.9-161.6 seconds) and for P2Y at POC (35.5-118.8 seconds) and shipped (35.1-108.9 seconds). Receiver operating characteristics showed similar areas under the curve (AUCROCs) regarding the effect of clopidogrel for COL/ADP at POC (0.994 seconds) and shipped (0.932) and for P2Y at POC (0.904 seconds) and shipped (0.975 seconds). When classifying for the presence of clopidogrel effects, Cohen's Kappa was 0.62 for COL/ADP and 1.00 for P2Y. Shipping blood samples for PFA analysis are feasible with similar performance to POC analyses for determining the effect of clopidogrel in cats.

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.

A Serodiagnostic IgM ELISA to Detect Acute Cytauxzoonosis.

Cytauxzoonosis is a tick-borne infectious disease affecting domestic cats with high mortality and limited treatment modalities. Because early diagnosis and therapeutic intervention are crucial to survival of infected cats, the objective of this study was to develop an ELISA capable of detecting cytauxzoonosis and differentiating acute vs. chronic infection in clinical feline blood samples. A microsphere immunoassay (MIA) was developed to evaluate the production of Cytauxzoon felis-specific IgM and IgG antibodies in serial plasma samples from cats with experimental C. felis infection by targeting a C. felis-specific transmembrane protein (c88). Recombinant c88 protein was utilized to develop indirect ELISAs to detect IgM and IgG antibodies in clinical plasma samples from: PCR-positive cats with acute C. felis infection (n = 36), C. felis-negative cats with pyrexia (n = 10), healthy C. felis-negative cats (n = 22), and chronic C. felis carriers (n = 4). Anti-c88 IgM antibodies were detectable at day 12 post-tick infestation in cats with experimental C. felis infection (within 24 hours of developing clinical signs), while anti-c88 IgG was detectable at day 15 post-tick infestation – indicating IgM could be used to detect early infection. Using a cut-off value of 19.85 percent positive, the C. felis IgM ELISA detected acute cytauxzoonosis in 94.44% (34/36) of cats presented with clinical signs of acute cytauxzoonosis with 100% specificity (indicating a “Strong Positive” result). When a lower cutoff of 8.60 percent positive was used, cytauxzoonosis was detected in the 2 remaining PCR-positive cats with 87.88% specificity (indicating of a “Weak Positive” result). One C. felis-negative, febrile cat had high IgG, and chronic carriers had variable IgM and IgG results. Combined interpretation of IgM and IgG ELISAs did not reliably differentiate acute vs. chronic infection. While further validation on assay performance is needed, the C. felis IgM ELISA is a promising test to detect acute cytauxzoonosis and can be utilized to develop a point-of-care test for clinical use.

Benefits, companion animal zoonotic disease prevalence and public perceptions of pet ownership among people experiencing homelessness in northern California.

California has the highest proportion of unhoused individuals in the country, and up to 25% of unhoused individuals own pets, providing substantial benefits but unique challenges including access to housing, transportation and unfounded grounds for social stigmatization. Unhoused individuals and pets may also be at risk for diseases due to impaired access to sanitation facilities. The purpose of this cross-sectional survey was to evaluate differences in perceived benefits, challenges and public perceptions among pet owners of varying housing security and the prevalence of diseases among their pets. Questionnaires were administered to housed and unhoused pet owners and pet blood screened for rickettsiosis, bartonellosis, ehrlichiosis, anaplasmosis, borreliosis, West Nile fever and heartworm. Among 147 canine and 16 feline blood samples, seropositivity of ectoparasitic diseases did not vary by housing status. Among 45 housed and 56 unhoused owners, unhoused owners were significantly more likely to report protective benefits, challenges obtaining housing, finding a flea on their pet, using bottled water for their pet and their pet sleeping in their bed. Housed owners were significantly more likely to report companionship and entertainment benefits, challenges with pet sitting and consistently administering parasite preventatives. Similar (96-98%) percentages stated they would not give up their pet for better housing and 31% of housed pet owners believed that people should not own pets if they do not have secure housing. Social stigma against unhoused pet owners is present within the community, requiring education to change public perception and guide policy regarding housing for pet owners experiencing homelessness.

Detection of frequent neutrophil misclassification by the ProCyte Dx in sick dogs and how to avoid it.

ObjectivesA common and severe error in identifying neutrophils in feline blood samples by the IDEXX ProCyte Dx haematology analyser (ProCyte) has been reported. The hypothesis was that the same or similar error would be identified during analysis of canine blood samples and that white blood cell dot plot evaluation would be critical to detect and avoid erroneous results.Materials and MethodsEighty‐six canine blood samples collected for clinical diagnosis of hospital patients were evaluated. Differential leukocyte counts were determined by the ProCyte Dx, ADVIA 2120 and manual methods. ProCyte neutrophil percentage results were considered unacceptable if the result was 15% different than percentage results from both ADVIA 2120 and manual counts. ProCyte WBC dot plots and instrument flags were evaluated for correctness.ResultsThe ProCyte neutrophil counts were unacceptably lower than the ADVIA 2120 and manual neutrophil counts in 13 samples (15% of 86 samples). Neutrophils misclassified by the instrument were erroneously classified as monocytes and/or lymphocytes. All these samples were from patients with systemic inflammation. The error could be eliminated by rejecting results from samples with incorrect separation of cell clusters in the ProCyte WBC dot plots.Clinical SignificanceThe ProCyte neutrophil count error with canine blood samples is common, severe and might affect clinical decisions. Operators of the instrument must evaluate white blood cell dot plots for correctness to avoid the error.

Amikacin prevents platelet aggregation in feline venous blood samples.

Physiologically, feline platelets are more reactive and prone to aggregation, which interferes with platelet counts using automated counters and manual methods. The use of aminoglycoside amikacin in association with EDTA has proven to be efficient in preventing platelet aggregates in cases of pseudo thrombocytopenia (PTP) in people. This study evaluated the efficacy of amikacin in preventing platelet aggregation in EDTA-containing feline blood samples and investigated the possible effects on hematologic measurands. Blood samples (1.0mL) collected from 100 healthy cats were stored in two EDTA tubes: 0.5mL in a microtube containing 10μL of 250mg/mL amikacin (EDTA-AMK group) and 0.5mL in a microtube containing only K2 EDTA 10% (EDTA group). A CBC was executed with an automated impedance blood analyzer, and a microscopic examination of the blood smears was performed. Platelet clumps were observed in 56% of samples from the EDTA group and 5% of samples from the EDTA-AMK group. Platelet counts (PLT), plateletcrit (PCT), and WBC counts were significantly higher (P<.001) in the EDTA-AMK group compared withi the EDTA group. Amikacin prevents platelet aggregation in feline venous blood samples and does not cause clinically relevant changes in other hematologic measurands. To our knowledge, this is the first report showing the use of amikacin in preventing platelet aggregation in feline blood samples. Based on this study, amikacin could be added to EDTA collection tubes for complete blood counts in cats.

Effects of handling and storage on potassium concentration in plasma and serum samples obtained from cats.

To compare potassium concentrations in feline plasma and serum samples analyzed promptly after collection or after 20 to 28 hours of refrigerated storage. 41 cats. A venous blood sample was obtained from each cat. Aliquots were placed in 2 tubes without anticoagulant (blood was allowed to clot to derive serum) and 2 tubes with heparin (to derive plasma). One serum and 1 plasma sample were kept at room temperature and analyzed within 60 minutes after collection (baseline); the other serum and plasma samples were analyzed after 20 to 28 hours of refrigerated storage. At both time points, serum and plasma potassium concentrations were measured. Median baseline serum potassium concentration (4.3 mmol/L) was significantly higher than median baseline plasma potassium concentration (4.1 mmol/L). The median difference between those values was 0.4 mmol/L (95% CI, 0.2 to 0.5 mmol/L). Compared with their respective baseline measurements, the median serum plasma concentration (4.8 mmol/L) and median plasma potassium concentration (4.6 mmol/L) were higher after 20 to 28 hours of refrigeration. Results indicated that with regard to potassium concentration in feline blood samples, clotting or refrigerated storage for 20 to 28 hours results in a significant artifactual increase. Detection of an unexpectedly high potassium concentration in a cat may represent pseudohyperkalemia, especially if the blood sample was placed in a no-additive tube, was stored for 20 to 28 hours prior to analysis, or both.

Development of a real-time recombinase-aided amplification (RT-RAA) molecular diagnosis assay for sensitive and rapid detection of Toxoplasma gondii

Toxoplasma gondii, a protozoan intracellular parasite, is present in a wide range of hosts, including virtually all species of warm-blooded vertebrates. Toxoplasmosis spreads to humans through a variety of pathways, including contaminated food or water, and close contact with various types of domestic animals. It poses a severe threat to human health, and contributes to important economic losses, not only in cost-of-illness but also in surveillance programs. It is thus necessary to develop a rapid point-of-care field diagnostic technology to control or prevent pathogen transmission to economically important livestock animals, domestic animals, and human beings. In this study, we develop a real-time isothermal amplification method capable of detecting the T. gondii genome in swine and feline blood samples. This method can detect toxoplasma genome with a lowest detection limit of 102 copies of per reaction under optimal reaction conditions of 36 °C for 25 min. The assay displayed advantages in sensitivity and specificity in comparison to traditional real-time PCR, and can be performed in a portable instrument.

Molecular Characterization of a Reemergent Brugia malayi Parasite in Sri Lanka, Suggestive of a Novel Strain.

Sri Lanka achieved elimination status for lymphatic filariasis in 2016; still, the disease remains a potential public health issue. The present study is aimed at identifying a subperiodic Brugia sp. parasite which has reemerged in Sri Lanka after four decades via molecular-based analysis. Polymerase chain reaction performed with pan-filarial primers specific for the internal transcribed spacer region-2 (ITS-2) of the rDNA of Brugia filarial parasites isolated from human, canine, and feline blood samples yielded a 615 bp band establishing the species identity as Brugia malayi. Comparison of the ITS2 sequences of the reemerged B. malayi isolates with GenBank sequences revealed a higher sequence homology with B. pahangi than B. malayi with similar phylogenetic evidence. However, the mean interspecies Kimura-2-parameter pairwise divergence between the generated Brugia sequences with B. malayi and B. pahangi was less than 3%. During the analysis of parsimony sites of the new ITS2 sequences, substitutions at A36T, A296G, T373A, and G482A made the sequences different from both B. pahangi and B. malayi suggesting the possibility of a new genetic variant or a hybrid strain of B. malayi and B. pahangi. Mosquito dissections and xenomonitoring identified M. uniformis and M. annulifera as vectors of this novel strain of B. malayi circulating among cats, dogs, and humans in Sri Lanka.

Development and Validation of a Quantitative UHPLC–MS-MS Method for the Determination of Alpha-Chloralose in Feline Blood and Application on Blood Samples Collected from Cats with Symptoms of Alpha-Chloralose Poisoning

Alpha-chloralose (AC) is used as a rodenticide as well as an anesthetic agent in laboratory animals. It was previously also used as an avicide. Detection of AC in blood samples or in body tissues collected postmortem is key for the diagnosis of clinical cases and a requirement for surveillance of secondary toxicosis, including potential cases in wild animals. Reports on poisoning of humans and non-laboratory animals confirmed by the detection of AC or its metabolites are available, however poisoning of domestic animals are rarely available. Furthermore, reports on clinical cases in domestic animals rarely report quantifications of AC in blood or body tissues. The present study describes the validation of a quantitative ultra high performance liquid chromatography--tandem mass spectrometry (UHPLC--MS-MS) method that can be used in cases of suspected AC poisoning in cats. The validation study showed the method to be fit for purpose. In serum, the limit of quantification was 100 ng/mL and the limit of detection was 30 ng/mL. The new analytical method was applied on blood samples collected from 20 individual cats with a preliminary clinical diagnosis of acute AC poisoning. AC was confirmed in all 20 feline blood samples, and the concentration range of AC was 538–17,500 ng/mL. The quantitative method developed in this study was found to be a fast and selective method for confirmation of AC poisoning using blood samples from cats.

Erythrocyte phenotyping for feline AB system in domestic cats from the Ilhéus-Itabuna microregion, Bahia, Brazil

ABSTRACT: This study aimed to determine the erythrocyte phenotypes of the feline AB system and to check the presence of antigens other than those present in the feline AB system in domestic cats from Ilhéus-Itabuna microregion, Bahia, Brazil. Three-hundred feline blood samples were collected at the Veterinary Hospital of the “Universidade Estadual de Santa Cruz” (UESC) and in home visits to perform blood phenotyping using the tube-method testing. The reverse phenotyping was made between cats that tested phenotype B with blood samples of cats that tested phenotype A to confirm the blood phenotype B. The cross-tested among cats with phenotype A was made in order to verify the presence of different antigens of AB system in this blood phenotype. The results underwent macroscopic and microscopic analyses. Among the 300 animals tested, regarding breed, 290 were mixed-breed cats and among the remaining ten, five were Persians, four Siamese, and one Angora. 297 (99%) presented with phenotype A (including all the breeding cats) and three (1%) with phenotype B, and all this cats were mixed-breed cats. None (0%) of the cats showed the phenotype AB. All phenotype B bloods reacted to reverse phenotyping with phenotype A, confirming the phenotype B of these cats. All phenotype A bloods were compatible among each other, so no further erythrocyte antigens were detected through this test. The mother of one of the phenotype B cats was identified and had phenotype A, demonstrating phenotype A parents with phenotype B offspring. This finding indicates heterozygosis in the studied population. This data enable to conclude that the studied population presented different erythrocyte phenotypes, subsequently highlighting the importance of conducting phenotype analyses in these animals before performing blood transfusion to avoid serious hemolytic complications associated with incompatibility.

Comparison of Conventional Tube and Gel-Based Agglutination Tests for AB System Blood Typing in Cat.

Gel technology is widely used for blood typing in human medicine. It has a number of advantages over routine tube testing, including standardization, stability, smaller sample volume, ease of performance and analysis, and speed. The aim of this study was to evaluate feline blood typing using the gel column technique. TUBE agglutination typing was performed in 143 feline blood samples from blood donors and recipients, healthy and sick patients, and whole-blood units anticoagulated with ethylenediamine tetraacetic acid or citrate phosphate dextrose adenine. Plasma from type B cats was used as anti-A reagent, Triticum vulgaris lectin as anti-B reagent, and the control was saline solution. Agglutination in backtyping of types B and AB samples with type A red blood cells (RBCs) was used to confirm whether the samples were type B (presence of alloantibodies) or type AB (absence of alloantibodies). Blood typing in a neutral gel column technique (GEL) using the same anti-A and anti-B reagents was performed on duplicate samples. Sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value, negative predictive value, overall accuracy, and Cohen κ coefficient (κ) for GEL were calculated, with TUBE considered the gold standard technique. Of 143 samples typed with TUBE, 98 (68.5%) were type A, 25 (17.5%,) type B, and 20 (14.0%) type AB. Backtyping confirmed the categorization of all types B and AB samples. Of these samples, gel testing produced 115 (80.4%) concordant results; a mixed-field agglutination pattern (layers of RBCs at both the top and at the bottom of the gel in either the A or B gel column) was seen in 27 samples, and one type B sample was misidentified as type AB. If the mixed-field pattern was interpreted as a negative result, 141/143 (98.6%) samples showed concordant results with an overall accuracy of the GEL of 100.0% for type A, 98.9% for type B, and 99.1% for type AB. Strength of agreement was very good (κ = 0.97). When the same anti-A and anti-B reagents are used, GEL is a sensitive and specific method for blood typing feline samples. Until additional studies have been performed, mixed-field patterns obtained in GEL testing should be classified as negative results.

Prospective Evaluation of Feline Sourced Platelet-Rich Plasma Using Centrifuge-Based Systems.

Objective: To evaluate the hematologic components of platelet-rich plasma (PRP) generated using feline blood with two commercially available centrifuge-based systems1,2.Materials and methods: Twenty healthy adult cats were enrolled in this prospective study from November 2018 to January 2019. Feline blood samples were obtained for analysis of whole blood (WB) cellular components and preparation of PRP product. PRP was prepared using two commercial systems and complete blood count (CBC) testing was performed on both WB and PRP samples. The cellular composition of the PRP product was compared to the WB sample for each patient.Results: Both systems showed significant decrease of median RBC concentration in PRP products compared to WB samples (P = 0.002 for both systems). System 1 significantly decreased median WBC concentration (P = 0.002). System 2 decreased WBC concentration, though statistical significance was not reached (P = 0.63). Median platelet concentration was decreased by 3% using System 1, and increased by 187% using System 2. Platelet aggregation presented a challenge with 8/20 (40%) of samples demonstrating platelet aggregation.Clinical relevance: Commercial systems available for generation of PRP may be useful for creating a feline sourced product and in this study showed promise in decreasing RBC and WBC concentration. Neither system tested achieved 2–5 times platelet concentration from baseline. Platelet aggregation presented a significant obstacle to reliable generation of PRP products using feline blood. This treatment modality may be particularly beneficial for feline patients with osteoarthritis and soft tissue injuries, though first characterizing the PRP product made using feline blood is critical to validate its use in further clinical studies.

Epidemiological investigation of feline infectious peritonitis in cats living in Harbin, Northeast China from 2017 to 2019 using a combination of an EvaGreen-based real-time RT-PCR and serum chemistry assays

Feline infectious peritonitis (FIP) is caused by the FIP virus (FIPV), a highly virulent mutant form of feline coronavirus (FCoV). This disease is one of the most important infectious diseases in cats, and it is associated with high mortality, particularly among younger cats. In this study, we isolated a wild-type FIPV HRB-17 epidemic strain from the blood sample of household pet cat exhibiting the characteristic wet-form FIP symptoms, which has been confirmed further by animal infection. Further, we developed an EvaGreen-based real-time RT-PCR assay for the accurate detection of FCoV based on the amplification of the highly conserved FIPV N gene. Then, using a combination of the real-time RT-PCR approach and a serum chemistry assay, we performed an epidemiological survey of FIPV infection in cats living in Harbin City, Northeast China. The results indicated that the EvaGreen-based real-time RT-PCR assay can be used for screening FCoV infection in the affected cats at an analytical detection limit of 8.2 × 101 viral genome copies/μL, but could not effectively distinguish FIPVs from FECVs. Additionally, the results of the epidemiological survey investigating feline blood samples (n = 1523) collected between July 2017 to July 2019 revealed an FIPV prevalence of approximately 12% (189/1523). Maybe, the prevalence would be less than 12% due to the real-time RT-PCR assay could not accurately differentiate FIPV and FECV. Nevertheless, it still highlighted the severity of the FIP epidemic in cats and reiterated the urgent need to develop effective anti-FIP therapeutic agents and anti-FIPV vaccines. As pet cats are household animals, risk communication and continuous region-extended surveillance cat programs are recommended.