Secondary zoonotic dog-to-human transmission of SARS-CoV-2 suggested by timeline but refuted by viral genome sequencing.

PURPOSE: The risk of secondary zoonotic transmission of SARS-CoV-2 from pet animals remains unclear. Here, we report on a 44 year old Caucasian male presenting to our clinic with COVID-19 pneumonia, who reported that his dog displayed respiratory signs shortly prior to his infection. The dog tested real-time-PCR (RT-PCR) positive for SARS-CoV-2 RNA and the timeline of events suggested a transmission from the dog to the patient. METHODS: RT-PCR and serological assays were used to confirm SARS-CoV-2 infection in the nasopharyngeal tract in the dog and the patient. We performed SARS-CoV-2-targeted amplicon-based next generation sequencing of respiratory samples from the dog and patient for sequence comparisons. RESULTS: SARS-CoV-2 infection of the dog was confirmed by three independent PCR-positive pharyngeal swabs and subsequent seroconversion. Sequence analysis identified two separate SARS-CoV-2 lineages in the canine and the patient's respiratory samples. The timeline strongly suggested dog-to-human transmission, yet due to the genetic distance of the canine and the patient's samples paired-transmission was highly unlikely. CONCLUSION: The results of this case support current knowledge about the low risk of secondary zoonotic dog-to-human transmissions of SARS-CoV-2 and emphasizes the strength of genomic sequencing in deciphering viral transmission chains.

Clinical Burden of Respiratory Syncytial Virus in Hospitalized Children Aged ≤5 Years (INSPIRE Study).

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of hospitalizations in children (≤5 years of age); limited data compare burden by age. METHODS: This single-center retrospective study included children (≤5 years of age) hospitalized for >24 hours with reverse-transcription polymerase chain reaction (RT-PCR)-confirmed RSV infection (2015-2018). Hospital length of stay (LOS), intensive care unit (ICU) admissions, ICU LOS, supplemental oxygen, and medication use were assessed. Multivariate logistic regression analyses identified predictors of hospital LOS >5 days. RESULTS: Three hundred twelve patients had RSV infection (ages 0 to <6 months [35%], 6 to <12 months [15%], 1 to <2 years [25%], and 2-5 years [25%]); 16.3% had predefined comorbidities (excludes preterm infants). Median hospital LOS was 5.0 days and similar across age; 5.1% (16/312) were admitted to ICU (ICU LOS, 5.0 days), with those aged 0 to <6 months admitted most frequently (10/108 [9.3%]). Supplemental oxygen was administered in 57.7% of patients, with similar need across ages. Antibiotics were administered frequently during hospitalization (43.6%). Predictors of prolonged LOS included pneumonia (odds ratio [OR], 2.33), supplemental oxygen need (OR, 5.09), and preterm births (OR, 3.37). High viral load (RT-PCR RSV cycle threshold value <25) was associated with greater need for supplemental oxygen. CONCLUSIONS: RSV causes substantial burden in hospitalized children (≤5 years), particularly preterm infants and those aged <6 months.

One Health: EAACI Position Paper on coronaviruses at the human-animal interface, with a specific focus on comparative and zoonotic aspects of SARS-CoV-2.

The latest outbreak of a coronavirus disease in 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), evolved into a worldwide pandemic with massive effects on health, quality of life, and economy. Given the short period of time since the outbreak, there are several knowledge gaps on the comparative and zoonotic aspects of this new virus. Within the One Health concept, the current EAACI position paper dwells into the current knowledge on SARS-CoV-2's receptors, symptoms, transmission routes for human and animals living in close vicinity to each other, usefulness of animal models to study this disease and management options to avoid intra- and interspecies transmission. Similar pandemics might appear unexpectedly and more frequently in the near future due to climate change, consumption of exotic foods and drinks, globe-trotter travel possibilities, the growing world population, the decreasing production space, declining room for wildlife and free-ranging animals, and the changed lifestyle including living very close to animals. Therefore, both the society and the health authorities need to be aware and well prepared for similar future situations, and research needs to focus on prevention and fast development of treatment options (medications, vaccines).

Leptospira infection and shedding in dogs in Thailand.

BACKGROUND: Leptospirosis is a widespread zoonosis and has been recognized as a re-emerging infectious disease in humans and dogs, but prevalence of Leptospira shedding in dogs in Thailand is unknown. The aim of this study was to determine urinary shedding of Leptospira in dogs in Thailand, to evaluate antibody prevalence by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA), and to assess risk factors for Leptospira infection. In Northern, Northeastern, and Central Thailand, 273 stray (n = 119) or client-owned (n = 154) dogs from rural (n = 139) or urban (n = 134) areas were randomly included. Dogs that had received antibiotics within 4 weeks prior to sampling were excluded. No dog had received vaccination against Leptospira. Urine was evaluated by real-time polymerase chain reaction (PCR) specific for lipL32 gene of pathogenic Leptospira. Additionally, urine was cultured for 6 months in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. Antibodies were measured by ELISA and MAT against 24 serovars belonging to 15 serogroups and 1 undesignated serogroup. Risk factor analysis was performed with backwards stepwise selection based on Wald. RESULTS: Twelve of 273 (4.4%; 95% confidence interval (CI): 2.0-6.8%) urine samples were PCR-positive. In 1/273 dogs (0.4%; 95% CI: 0.01-1.1%) Leptospira could be cultured from urine. MAT detected antibodies in 33/273 dogs (12.1%; 95% CI: 8.2-16.0%) against 19 different serovars (Anhoa, Australis, Ballum, Bataviae, Bratislava, Broomi, Canicola, Copenhageni, Coxi, Grippotyphosa, Haemolytica, Icterohaemorrhagiae, Khorat, Paidjan, Patoc, Pyrogenes, Rachmati, Saxkoebing, Sejroe). In 111/252 dogs (44.0%; 95% CI: 37.9-50.2%) immunoglobulin M (IgM) and/or immunoglobulin G (IgG) antibodies were found by ELISA. Female dogs had a significantly higher risk for Leptospira infection (p = 0.023). CONCLUSIONS: Leptospira shedding occurs in randomly sampled dogs in Thailand, with infection rates comparable to those of Europe and the USA. Therefore, the potential zoonotic risk should not be underestimated and use of Leptospira vaccines are recommended.

Utility of an immunocytochemical assay using aqueous humor in the diagnosis of feline infectious peritonitis.

OBJECTIVE: In cats suffering from feline infectious peritonitis (FIP) without effusion, antemortem diagnosis is challenging. Uveitis is common in these cats. It was the aim of this study to evaluate sensitivity and specificity of an immunocytochemical assay (ICC) in aqueous humor of cats suspected of having FIP. ANIMALS STUDIED: The study included 26 cats with immunohistochemically confirmed FIP and 12 control cats for which FIP was suspected due to similar clinical or laboratory changes, but which suffered from other diseases confirmed via histopathology. PROCEDURES: All aqueous humor samples were collected postmortem by paracentesis. ICC was carried out as avidin-biotin complex method. Sensitivity, specificity, and the overall accuracy including 95% confidence intervals (95% CI) were calculated. RESULTS: Immunocytochemistry was positive in 16 of 25 cats with FIP and 2 of 11 control cats (one cat with lymphoma, one with pulmonary adenocarcinoma). Aqueous humor samples of one cat with FIP and of one control cat were excluded from statistical analysis. Sensitivity was 64.0% (95% CI: 42.5-82.0); specificity 81.8% (95% CI: 48.2-97.7); and overall accuracy 69.4% (95% CI: 51.9-83.7). CONCLUSIONS: As false-positive results occurred and specificity is most important in the diagnosis of FIP, the diagnostic utility of ICC in aqueous humor is limited. Further studies are required to clarify the origin of false-positive ICC results.

Risk factors of different hemoplasma species infections in cats.

BACKGROUND: Hemoplasma species (spp.) commonly cause infections in cats worldwide. However, data on risk factors for infections are limited. The aim of this study was to determine the prevalence of hemoplasma spp. infections in cats in Southern Germany and to assess risk factors associated with infection. RESULTS: DNA was extracted from blood samples of 479 cats presented to different veterinary hospitals for various reasons. DNA of feline hemoplasmas was amplified by use of a previously reported PCR assay. Direct sequencing was used to confirm all purified amplicons and compared to hemoplasma sequences reported in GenBank. Results were evaluated in relation to the age, sex, housing conditions, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) status of the cats. The overall hemoplasma prevalence rate was 9.4% (45/479; 95% CI: 7.08-12.36). 'Candidatus Mycoplasma (M.) haemominutum' (Mhm) DNA was amplified from 42 samples, M. haemofelis from 2, and M. haemocanis from 1 sample. There was a significantly higher risk of hemoplasma infection in cats from multi-cat households, in outdoor cats, as well as in cats with FIVinfection and in cats with abortive FeLV infection, but not in cats with progressive or regressive FeLV infection. CONCLUSIONS: Mhm infection is common in cats in Southern Germany. Higher prevalence in multi-cat households and associations with FeLV infection likely reflect the potential for direct transmission amongst cats. Outdoor access, male gender, and FIV infection are additional risk factors that might relate to aggressive interactions and exposure to vectors.

Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis.

BACKGROUND: Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) specifically designed to detect FCoV spike gene mutations at two nucleotide positions. It was hypothesized that this test would correctly discriminate feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). METHODS: The study included 63 cats with signs consistent with FIP. FIP was confirmed in 38 cats. Twenty-five control cats were definitively diagnosed with a disease other than FIP. Effusion and/or serum/plasma samples were examined by real-time RT-PCR targeting the two FCoV spike gene fusion peptide mutations M1058 L and S1060A using an allelic discrimination approach. Sensitivity, specificity, negative and positive predictive values including 95% confidence intervals (95% CI) were calculated. RESULTS: FIPV was detected in the effusion of 25/59 cats, one of them being a control cat with chronic kidney disease. A mixed population of FIPV/FECV was detected in the effusion of 2/59 cats; all of them had FIP. RT-PCR was negative or the pathotype could not be determined in 34/59 effusion samples. In effusion, sensitivity was 68.6% (95% CI 50.7-83.2), specificity was 95.8% (95% CI 78.9-99.9). No serum/plasma samples were positive for FIPV. CONCLUSIONS: Although specificity of the test in effusions was high, one false positive result occurred. The use of serum/plasma cannot be recommended due to a low viral load in blood.

Patient's and physician's awareness of kidney disease in coronary heart disease patients - a cross-sectional analysis of the German subset of the EUROASPIRE IV survey.

BACKGROUND: Chronic kidney disease (CKD) is a common comorbid condition in coronary heart disease (CHD). CKD predisposes the patient to acute kidney injury (AKI) during hospitalization. Data on awareness of kidney dysfunction among CHD patients and their treating physicians are lacking. In the current cross-sectional analysis of the German EUROASPIRE IV sample we aimed to investigate the physician's awareness of kidney disease of patients hospitalized for CHD and also the patient's awareness of CKD in a study visit following hospital discharge. METHODS: All serum creatinine (SCr) values measured during the hospital stay were used to describe impaired kidney function (eGFRCKD-EPI < 60 ml/min/1.73m2) at admission, discharge and episodes of AKI (KDIGO definition). Information extracted from hospital discharge letters and correct ICD coding for kidney disease was studied as a surrogate of physician's awareness of kidney disease. All patients were interrogated 0.5 to 3 years after hospital discharge, whether they had ever been told about kidney disease by a physician. RESULTS: Of the 536 patients, 32% had evidence for acute or chronic kidney disease during the index hospital stay. Either condition was mentioned in the discharge letter in 22%, and 72% were correctly coded according to ICD-10. At the study visit in the outpatient setting 35% had impaired kidney function. Of 158 patients with kidney disease, 54 (34%) were aware of CKD. Determinants of patient's awareness were severity of CKD (OReGFR 0.94; 95%CI 0.92-0.96), obesity (OR 1.97; 1.07-3.64), history of heart failure (OR 1.99; 1.00-3.97), and mentioning of kidney disease in the index event's hospital discharge letter (OR 5.51; 2.35-12.9). CONCLUSIONS: Although CKD is frequent in CHD, only one third of patients is aware of this condition. Patient's awareness was associated with kidney disease being mentioned in the hospital discharge letter. Future studies should examine how raising physician's awareness for kidney dysfunction may improve patient's awareness of CKD.

Supportive Treatment of a Dog with Leishmaniosis and Severe Glomerulopathy with Immunoadsorption.

A three-year-old, intact female mix-breed dog, weighing 30 kg, was presented due to vomitus and diarrhea. At presentation, the patient had a slightly reduced general condition and moderately enlarged mandibular and popliteal lymph nodes. The initial blood work showed severe azotemia and hypoalbuminemia. In the urinalysis, marked proteinuria with a urine protein/creatinine ratio (UPC) of 4.69 was found. Further workup showed a high leishmania antibody titer. The dog was diagnosed with leishmaniosis and glomerulonephritis. Initial treatment consisted of intravenous fluid therapy, allopurinol, miltefosine, amlodipine, clopidogrel, and a diet with a low purine content. Creatinine temporarily decreased but increased again after three days. For further supportive treatment, intermittent hemodialysis in combination with hemoperfusion with the cytosorb® adsorber was performed. A total blood volume of 17.7 L was processed within three hours. Thereafter, immunoadsorption (IA) was performed with the COM.TEC® and ADAsorb® platforms and a LIGASORB® adsorber to eliminate circulating immunocomplexes. Treatment time for IA was two hours with a blood flow of 50 mL/min. A total plasma volume of 2.4 L was processed. Over the following days, creatinine declined, and the patient improved significantly. UPC decreased to 1.74 on day 17 after IA. The patient was discharged after two and a half weeks. Two years after the initial event, the patient is still in excellent condition, with creatinine, UPC, and albumin levels in the reference range. Therefore, IA might be an additional therapeutic option for dogs with leishmaniosis-induced glomerulonephritis and subsequent severe azotemia to improve immunocomplex-mediated glomerulonephritis.

[Overview on utility of in-house tests for detection of systemic infectious diseases in dogs]. / Übersicht über Einsatzmöglichkeiten von In-house-Tests zum Nachweis von systemischen Infektionskrankheiten beim Hund.

For detection of infectious diseases, several point-of-care (POC) tests are on the market in addition to methods performed in commercial laboratories. These POC tests are based on enzyme-linked immunosorbent assay (ELISA) or other immunochromatographic technologies and present results within few minutes in veterinary practice. This article gives an overview of the utility of numerous POC tests of different manufacturers for detection of parvovirus antigen in feces, Dirofilaria (D.) immitis antigen in blood as well as antibodies against Borrelia (B.) burgdorferi, Anaplasma (A.) spp., Ehrlichia (E.) spp., Leptospira (L.) spp. and Leishmania (L.) infantum in blood (single or in different combinations). Sensitivity and specificity of these tests are important for their usefulness in veterinary practice. Furthermore, presence of antibodies or detection of antigen has to correlate with the presence of clinical signs. POC tests for detection of canine parvovirus antigen have a very high specificity, the sensitivity of all evaluated POC tests, however, is very low. POC tests for detection of D. immitis antigen have a very high sensitivity and specificity. As they detect antigen from the uterus of female adult parasites, test results are negative when only very few female or only male adults are present. POC tests for detection of antibodies against B. burgdorferi only indicate contact with Borrelia spp. and do not prove clinical Lyme disease, as the infection only extremely rarely causes clinical signs. POC tests for detection of antibodies against A. phagocytophilum are also not suitable for diagnosis of clinical anaplasmosis. Infections with A. phagocytophilum only lead to clinical disease in very rare cases and in these, clinical signs occur before the development of antibodies. POC tests for detection of antibodies against E. canis have a very high sensitivity as well as specificity. POC tests for detection of antibodies against L. infantum and Leptospira species (spp.) show a very high specificity and a high sensitivity. However, Leptospira spp. antibody-positive results may occur following vaccination, as the POC tests cannot distinguish between field and vaccination strains.

Anti-rabies humoral immune response in cats after concurrent vs separate vaccination against rabies and feline leukaemia virus using canarypox-vectored vaccines.

OBJECTIVES: Some expert groups recommend that cats should be vaccinated with non-adjuvanted feline leukaemia virus (FeLV) and rabies vector vaccines, which, in the European Union, are currently not licensed for concurrent use and have to be administered at least 14 days apart (different from the USA) and thus at separate visits, which is associated with more stress for cats and owners. The aim of this study was to assess the anti-rabies antibody response in cats after vaccination against rabies and FeLV at concurrent vs separate (4 weeks apart) visits using two canarypox-vectored vaccines (Purevax Rabies and Purevax FeLV; Boehringer Ingelheim) and to evaluate the occurrence of vaccine-associated adverse events (VAAEs). METHODS: Healthy FeLV antigen-negative client-owned kittens (n = 106) were prospectively included in this randomised study. All kittens received primary vaccinations against rabies (week 0) and FeLV (weeks 4 and 8). After 1 year, the study group (n = 52) received booster vaccinations against rabies and FeLV concurrently at the same visit (weeks 50-52). The control group (n = 54) received booster vaccinations against rabies (weeks 50-52) and FeLV (weeks 54-56) separately. Anti-rabies virus antibodies (anti-RAV Ab) were determined by fluorescent antibody virus neutralisation assay at weeks 4, 50-52 and 54-56, and compared between both groups using a Mann-Whitney U-test. RESULTS: Four weeks after the first rabies vaccination, 87/106 (82.1%) kittens had a titre ⩾0.5 IU/ml and 19/106 (17.9%) had a titre <0.5 IU/ml. Four weeks after the 1-year rabies booster, all cats had adequate anti-RAV Ab according to the World Organisation for Animal Health (⩾0.5 IU/ml), and the titres of the study group (median = 14.30 IU/ml) and the control group (median = 21.39 IU/ml) did not differ significantly (P = 0.141). VAAEs were observed in 7/106 (6.6%) cats. CONCLUSIONS AND RELEVANCE: Concurrent administration of Purevax FeLV and Purevax Rabies vector vaccines at the 1-year booster does not interfere with the development of anti-RAV Ab or cause more adverse effects and thus represents a better option than separate vaccination visits for cats and owners.

The Effect of Metronidazole versus a Synbiotic on Clinical Course and Core Intestinal Microbiota in Dogs with Acute Diarrhea.

The usefulness of antibiotics in dogs with acute diarrhea (AD) is controversial. It is also unclear what effect metronidazole has on potential enteropathogens such as Clostridium perfringens and Escherichia coli. Thus, the aim of this study was to evaluate the effect of metronidazole vs. a synbiotic on the clinical course and core intestinal bacteria of dogs with AD. Twenty-seven dogs with AD were enrolled in this prospective, randomized, blinded clinical trial and treated with either metronidazole (METg) or a synbiotic (SYNg; E. faecium DSM 10663; NCIMB 10415/4b170). The Canine Acute Diarrhea Severity (CADS) index was recorded daily for eleven days. Bacteria were quantified using qPCR. Data were analyzed using mixed models with repeated measures. A higher concentration of E. coli was observed in the METg group vs. the SYNg group on Day 6 (p < 0.0001) and Day 30 (p = 0.01). Metronidazole had no effect on C. perfringens. C. hiranonis was significantly lower in the METg group than in the SYNg group on Days 6 and 30 (p < 0.0001; p = 0.0015). No significant differences were observed in CADS index, fecal consistency, or defecation frequency between treatment groups (except for the CADS index on one single day). In conclusion, metronidazole negatively impacts the microbiome without affecting clinical outcomes. Thus, synbiotics might be a preferred treatment option for dogs with AD.

Molecular Detection of Anaplasma phagocytophilum in Cats in Europe and Associated Risk Factors.

Infections with Anaplasma (A.) phagocytophilum in cats seem to be rare. The study aimed to determine whether infections in cats are underestimated and to identify the risk factors for infection. Blood samples of 1015 cats across Europe (2017-2022), sent to IDEXX Laboratories, Germany, were tested for A. phagocytophilum DNA. The influence of the cats' origin on A. phagocytophilum infection was assessed by univariable analysis, while multivariable logistic regression evaluated associations with the cats' sex and age, and the years, and seasonality of the samples' submission. Furthermore, univariable linear regression was used to determine patterns in PCR orders. The number of submitted samples increased significantly during the 6 years (p = 0.042). Anaplasma phagocytophilum DNA was detected in 76/1015 of cats (7.5%, 95% CI 6.0-9.3%). Infections were significantly more common in Northern compared to Central (p < 0.001, OR: 8.70) and Southern Europe (p < 0.001, OR: 39.94). A significantly higher likelihood for infections during the summer compared with winter (p = 0.047, OR: 3.13) was found. Bacteremia with A. phagocytophilum in European cats is not uncommon. Anaplasma phagocytophilum infection should be considered an important risk, particularly in Northern Europe. Effective tick prevention is crucial for managing feline health across Europe, not just in the Mediterranean region.

Antibody response after feline panleukopenia virus vaccination in kittens with and without intestinal parasites.

OBJECTIVES: Vaccinations should only be given to healthy cats, and deworming before vaccination is generally recommended; however, so far, no study has investigated the influence of intestinal parasitic infection on the immune response in kittens. The aim of this prospective study was to compare the antibody response to feline panleukopenia virus (FPV) vaccination in kittens with and without intestinal parasites. METHODS: Overall, 74 healthy kittens were included. Of these, 17 had intestinal parasites (12/17 Toxocara cati, 6/17 Cystoisospora felis, 1/17 Capillaria species). Both kittens with and without (n = 57) parasites received two primary kitten vaccinations with modified live FPV vaccines in a 4-week interval starting at the age of 8-12 weeks. Anti-FPV antibodies were determined at the beginning of the study (week 0) and at week 8 (4 weeks after the second vaccination) by haemagglutination inhibition. A ⩾four-fold titre increase (week 8 vs week 0) was defined as a response to vaccination. Comparison of the immune response in the kittens with and without intestinal parasites was performed using Pearson's χ2 test. RESULTS: Pre-vaccination antibodies were present in 4/17 (23.5%) kittens with intestinal parasites and in 24/57 (42.1%) without parasites. A ⩾four-fold titre increase was seen in 13/17 (76.5%) kittens with parasites compared with 32/57 (56.1%) kittens without parasites. There was neither a significant difference in pre-vaccination antibodies (P = 0.17), nor in vaccination response (P = 0.13) between kittens with and without parasites. CONCLUSIONS AND RELEVANCE: The results indicate that asymptomatic intestinal infections with endoparasites do not interfere with the immune response to kitten vaccination series. Parasitic infection (at least with T cati, C felis and Capillaria species) is therefore not a reason to postpone important vaccinations.

Anaplasma phagocytophilum infection associated with strong inflammatory response in 3 cats.

Anaplasmosis is a vector-borne disease caused by Anaplasma (A.) spp. which currently is still rarely diagnosed in cats. This article describes 3 independent cases of anaplasmosis in cats from different regions of Germany presented to veterinarians in 2021. All cats showed unspecific clinical signs, such as fever, reduced general condition, and decreased appetite. One cat additionally had generalized limb pain, another showed reluctance to move as well as vomiting. On complete blood cell count, only 1 of 3 cats showed mild thrombocytopenia. A. phagocytophilum was detected in blood samples of all 3 cats by polymerase chain reaction. Additionally, in 2 cats (in which blood smears were evaluated) morulae could be detected within neutrophilic granulocytes. Initially, all 3 cats had highly elevated serum amyloid A (SAA) concentrations. Treatment with doxycycline caused a rapid improvement of clinical signs, followed by a decrease of SAA concentrations to normal levels as well as negative PCR results after a treatment duration of at least 28 days. In cats with fever, otherwise unspecific clinical signs with only mild or no hematological changes, elevated SAA concentrations, and previous exposure to ticks, attending veterinarians should consider anaplasmosis as differential diagnosis.

Characterizing the blood microbiota in healthy and febrile domestic cats via 16s rRNA sequencing.

This study aimed to evaluate the blood bacterial microbiota in healthy and febrile cats. High-quality sequencing reads from the 16S rRNA gene variable region V3-V4 were obtained from genomic blood DNA belonging to 145 healthy cats, and 140 febrile cats. Comparisons between the blood microbiota of healthy and febrile cats revealed dominant presence of Actinobacteria, followed by Firmicutes and Proteobacteria, and a lower relative abundance of Bacteroidetes. Upon lower taxonomic levels, the bacterial composition was significantly different between healthy and febrile cats. The families Faecalibacterium and Kineothrix (Firmicutes), and Phyllobacterium (Proteobacteria) experienced increased abundance in febrile samples. Whereas Thioprofundum (Proteobacteria) demonstrated a significant decrease in abundance in febrile. The bacterial composition and beta diversity within febrile cats was different according to the affected body system (Oral/GI, systemic, skin, and respiratory) at both family and genus levels. Sex and age were not significant factors affecting the blood microbiota of febrile cats nor healthy ones. Age was different between young adult and mature adult healthy cats. Alpha diversity was unaffected by any factors. Overall, the findings suggest that age, health status and nature of disease are significant factors affecting blood microbiota diversity and composition in cats, but sex is not.

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.

Evaluation of a Revised Point-of-Care Test for the Detection of Feline Leukaemia p27 Antigen and Anti-p15E Antibodies in Cats.

The first point-of-care (PoC) test (v-RetroFel®; modified version 2021) determining the presence of FeLV p27 antigen and FeLV anti-p15E antibodies has become recently commercially available to identify different feline leukaemia virus (FeLV) infection outcomes. This study aimed to assess this PoC test's performance concerning FeLV p27 antigen and FeLV anti-p15E antibody detection. Sensitivity, specificity, positive and negative predictive values (PPV, NPV) were assessed after ten minutes (recommended) and 20 min (prolonged) incubation times. The test results were evaluated as either positive or negative. Serum samples from 934 cats were included, originating from Italy (n = 269), Portugal (n = 240), Germany (n = 318), and France (n = 107). FeLV p27 antigen and anti-p15E antibodies were measured by reference standard ELISAs and compared to the PoC test results. The PoC test was easy to perform and the results easy to interpret. Sensitivity and specificity for FeLV p27 antigen were 82.8% (PPV: 57.8%) and 96.0% (NPV: 98.8%) after both, ten and 20 minues of incubation time. Sensitivity and specificity for anti-p15E antibodies were 31.4% (PPV: 71.6%) and 96.9% (NPV: 85.1%) after ten minutes incubation time; sensitivity was improved by a prolonged incubation time (20 min) to 40.0% (PPV: 76.3%), while specificity remained the same (96.9%, NPV: 86.7%). Despite the improved sensitivity using the prolonged incubation time, lower than ideal sensitivities for both p27 antigen and especially anti-p15E antibodies were found, indicating that the PoC test in its current version needs further improvement prior to application in the field.

Alpha-1-Acid Glycoprotein Quantification via Spatial Proximity Analyte Reagent Capture Luminescence Assay: Application as Diagnostic and Prognostic Marker in Serum and Effusions of Cats with Feline Infectious Peritonitis Undergoing GS-441524 Therapy.

Until recently, the diagnosis of feline infectious peritonitis (FIP) in cats usually led to euthanasia, but recent research has revealed that antiviral drugs, including the nucleoside analog GS-441524, have the potential to effectively cure FIP. Alpha-1-acid glycoprotein (AGP) has been suggested as a diagnostic marker for FIP. However, AGP quantification methods are not easily accessible. This study aimed to establish a Spatial Proximity Analyte Reagent Capture Luminescence (SPARCLTM) assay on the VetBio-1 analyzer to determine the AGP concentrations in feline serum and effusion samples. Linearity was found in serial dilutions between 1:2000 and 1:32,000; the intra-run and inter-run precision was <5% and <15%, respectively; and AGP was stable in serum stored for at least 8 days at room temperature, at 4 °C and at -20 °C. Cats with confirmed FIP had significantly higher serum AGP concentrations (median: 2954 µg/mL (range: 200-5861 µg/mL)) than those with other inflammatory diseases (median: 1734 µg/mL (305-3449 µg/mL)) and clinically healthy cats (median 235 µg/mL (range: 78-616 µg/mL); pKW < 0.0001). The AGP concentrations were significantly higher in the effusions from cats with FIP than in those from diseased cats without FIP (pMWU < 0.0001). The AGP concentrations in the serum of cats with FIP undergoing GS-441524 treatment showed a significant drop within the first seven days of treatment and reached normal levels after ~14 days. In conclusion, the VetBio-1 SPARCLTM assay offers a precise, fast and cost-effective method to measure the AGP concentrations in serum and effusion samples of feline patients. The monitoring of the AGP concentration throughout FIP treatment provides a valuable marker to evaluate the treatment's effectiveness and identify potential relapses at an early stage.