Applicability of leukocyte esterase test strip in detection of canine pyuria

A commercially available leukocyte esterase assay was evaluated for application in analyzing canine urine for the detection of pyuria. In 229 urine samples, the leukocyte esterase activity was compared with leukocyte concentrations, as assessed by microscopic sediment analysis and chamber cell counts. The leukocyte esterase assay was specific (93.2%) for canine pyuria, but was poorly sensitive (46.0%) and did not appear to be applicable to analysis of canine urine samples.     

Onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleucopenia, feline calicivirus and feline herpesvirus

The induction of a quick onset of immunity against feline parvovirus (FPV), feline herpesvirus (FHV) and feline calicivirus (FCV) is critical both in young kittens after the decline of maternal antibodies and in cats at high risk of exposure. The onset of immunity for the core components was evaluated in 8–9 week old specific pathogen free kittens by challenge 1 week after vaccination with a combined modified live (FPV, FHV) and inactivated (FCV) vaccine. The protection obtained 1 week after vaccination was compared to that obtained when the challenge was performed 3–4 weeks after vaccination. The protocol consisted of a single injection for vaccination against FPV and two injections 4 weeks apart for FHV and FCV.At 1 week after vaccination, the kittens showed no FPV-induced clinical signs or leukopenia following challenge, and after FCV and FHV challenges the clinical score was significantly lower in vaccinated animals than in controls. Interestingly, the relative efficacy of the vaccination was comparable whether the animals were challenged 1 week or 3–4 weeks after vaccination, indicating that the onset of protection occurred within 7 days of vaccination. Following the 1-week challenge, excretion of FPV, FHV and FCV was significantly reduced in vaccinated cats compared to control kittens, confirming the onset of immunity within 7 days of vaccination.     

Evaluation of serum galactomannan detection for diagnosis of feline upper respiratory tract aspergillosis

Measurement of serum galactomannan (GM), a polysaccharide fungal cell-wall component, is a non-invasive test for early diagnosis of invasive aspergillosis in humans. Feline upper respiratory tract (URT) aspergillosis is an emerging infectious disease in cats. Diagnosis requires biopsy for procurement of tissue specimens for cytological or histological detection of fungal hyphae and for fungal culture. The aim of this study was to evaluate serum GM measurement as a non-invasive diagnostic test for URT aspergillosis in cats. A one-stage, immunoenzymatic sandwich ELISA was used to detect serum GM in 4 groups of cats; Group 1 (URT aspergillosis) – confirmed URT aspergillosis (n = 13, sinonasal aspergillosis (SNA) n = 6 and sino-orbital aspergillosis (SOA) n = 7), Group 2 (URT other) – other URT diseases (n = 15), Group 3 (β-lactam) – cats treated with β-lactam antibiotics for non-respiratory tract disease (n = 14), Group 4a – healthy young cats (≤1 y of age, n = 28), Group 4b – healthy adult cats (>1 y of age, n = 16). One cat with SNA and two cats with SOA caused by an Aspergillus fumigatus-mimetic species, tested positive for serum GM. For a cut-off optical density index of 1.5, the overall sensitivity and specificity of the assay was 23% and 78% respectively. False positive results occurred in 29% of cats in Group 3 and 32% of cats in Group 4a. Specificity increased to 90% when Groups 3 and 4a were excluded from the analysis. Overall, serum GM measurement has a poor sensitivity but is a moderately specific, non-invasive screening test to rule out infection in patients with suspected feline upper respiratory tract aspergillosis.     

Simultaneous detection of the feline lungworms Troglostrongylus brevior and Aelurostrongylus abstrusus by a newly developed duplex-PCR

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to as the feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been identified as an agent of bronco-pulmonary infestations in cats. These two parasites have a similar biology, share ecological niches, potentially co-infesting cats, but are difficult to be differentiated due to the morphological similarities of their first-stage larvae (L1). This paper describes a molecular tool, based on single-step duplex polymerase chain reaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for the simultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both species were collected from faecal samples, morphologically identified, and single larval specimens isolated. An aliquot of faeces was used as a test sample for a case of mixed natural infestation. The duplex-PCR was performed using species-specific forward primer sets for the ITS-2 region (i.e., A. abstrusus: 220 bp; T. brevior: 370 bp). The detection limit of the molecular assay was also assessed by serial dilutions of DNA from single larvae of both species (from ～4.0 to 4.0 × 10^?5 μg/μl). The duplex-PCR carried out on individual DNA samples was able to detect as low as 5.2 × 10^?3 μg/μl of DNA for A. abstrusus, 4.9 × 10^?3 μg/μl for T. brevior, and as low as 4.0 × 10^?3 μg/μl for samples containing both species. Species-specific bands of the expected sizes and two bands were simultaneously amplified from the faecal sample containing both species. The phylogenetic analyses of the ITS-2 sequences here examined and those available for other metastrongyloids were concordant in clustering them with those of other Troglostrongylus brevior and A. abstrusus sequences available in GenBank database. This molecular approach proved to be effective and highly sensitive for the simultaneous detection of the two lungworms species and it might be used for molecular epidemiological studies and for monitoring therapeutic protocols.     

Gastric Helicobacter species as a cause of feline gastric lymphoma: a viable hypothesis

Gastric Helicobacter spp. are associated with chronic inflammation and neoplastic transformation in humans as well as domestic and laboratory species. The present study examined the association of Helicobacter heilmannii (Hhe) infection in pet cats with feline gastric mucosa associated lymphoid tissue (MALT) lymphoma. Tissues were collected via gastric biopsy or at necropsy from 47 pet cats with clinical signs of gastrointestinal disease, including vomiting and inappetance, and classified as gastritis (14/47), lymphoma (31/37), or normal (2/47). Tissues positive for argyrophilic organisms with Warthin–Starry stain (29/47) were assessed by fluorescent in situ hybridization (FISH) for the presence of Hhe strains 1–4 as well as with a fifth probe that detected Helicobacter salomonis, Helicobacter bizzozeronii, or Helicobacter felis. A significant association of positive Warthin–Starry status with Hhe infection was found in cases of sick cats (22/29; p < 0.05 by Chi-square; χ² = 7.034). Interestingly, a significant association between Hhe status and a diagnosis of lymphoblastic or lymphocytic lymphoma was observed as well in a subset of 24 Warthin–Starry positive lymphoma cases: of lymphoblastic lymphoma cases, 13/17 were positive for Hhe (p < 0.05; χ² = 4.854). Hhe strains 2 and 4 were most commonly found (18/29 and 17/29, respectively) among sick cats, although a higher than expected number of cats was also positive for Hhe1, which initial reports have described as rare in cats and common in humans. The association found between a positive Hhe status with the presence of feline gastric lymphoma, especially lymphoblastic lymphoma, argues for the need to conduct prospective studies to better identify the frequency and strain distribution of Hhe infection in both healthy and clinically ill cats, particularly those cats with gastric lymphoma.     

Clinical evolution and radiographic findings of feline heartworm infection in asymptomatic cats

Clinical manifestations of heartworm disease in cats are variable; most cats seem to tolerate the infection well for extended periods. Heartworm-infected cats may undergo spontaneous self-cure due to the natural death of parasites without any symptomatology, or they may suddenly show dramatic and acute symptoms. Sudden death in apparently healthy cats is not a rare event. Thoracic radiographs are important tool for the diagnosis of cardiopulmonary disease. However, thoracic abnormalities are often absent or transient and highly variable in heartworm-infected cats. Findings, such as enlargement of the peripheral branches of the pulmonary arteries, with a varying degree of pulmonary parenchymal disease and hyperinflation, are the most typical features consistent with infection. A field study was performed for cats referred to the Veterinary Hospital Città di Pavia from January 1998 to December 2001 for routine health examinations and procedures to evaluate the clinical evolution and radiographic findings of feline heartworm infection. Thirty-four asymptomatic cats diagnosed with feline heartworm infection by antibody and antigen tests together with an echocardiogram that allowed worm visualization were included in the follow-up study. Cats were routinely examined every 3 months from the time of heartworm diagnosis until the outcome (self-cure or death). Self-cure was defined as no positive serology for heartworm antigens and no visualization of worms by echocardiography. A final examination for antibodies was carried after 12 months as a final confirmation of self-cure. Twenty-eight cats (82.4%) self-cured; including 21 that showed no clinical signs of infection throughout the study. Six cats died. The most common clinical features observed were acute respiratory symptoms and sudden death. Infection lasted over 3 years in the majority of the cats enrolled in the study. Thoracic radiograph appearance was variable, and the most commonly observed findings were focal and diffuse pulmonary parenchymal abnormalities.     

Comparison of home monitoring methods for feline urine pH measurement

Background — Monitoring of urine pH, often done in the patient's home, is essential for proper clinical treatment and management of conditions such as urolithiasis. Objective — The purpose of this study was to assess the agreement in pH readings between a standard laboratory method and methods readily available for home monitoring. The influence of refrigerated storage on urine pH was also examined. Methods — Urine samples were obtained by cystocentesis from 40 clinically healthy cats, and pH was measured within 2 hours of collection. Each sample was evaluated using pH paper, urinalysis reagent strip, 2 brands of portable pH meters (Chek‐Mite, Corning, Corning, NY, USA; and Checker 1, Hanna Instruments, Woonsocket, RI, USA), and a standard laboratory benchtop pH meter. Urine samples were refrigerated, and a second pH reading was obtained with the laboratory benchtop meter after 24 hours. The degree of agreement was assessed among the different methods, with the laboratory benchtop pH meter as the reference method. Results — The closest agreement was obtained with the Chek‐Mite portable pH meter and least agreement with the Checker 1 portable pH meter, which had a constant negative bias of 0.31 units due to expiration of the electrode. As expected, pH paper and reagent strips had poor and intermediate agreement, respectively. The reagent strip method had a negative bias of 0.12 units when compared with the benchtop pH meter and wide disagreement at the low pH end. The reagent strip did not agree strongly with the reference method; only 50% of values were within 0.25 pH units of each other. The difference in pH between 0 hours (6.57 ± 0.54) and 24 hours of refrigeration (6.61 ± 0.53) was not considered clinically significant. Conclusion — Portable pH meters are excellent for monitoring urine pH at home as long as attention is given to electrode maintenance. Urine can be collected at home and kept refrigerated, and pH may be measured reliably within 24 hours using the reference method or a portable pH meter.     

A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues

For a proper determination of relative mRNA expression levels with real-time quantitative PCR (Q-PCR) internal standards, such as the expression of reference genes, are of utmost importance. For cats, in contrast to dogs, no validation of reference genes has been published. Our goal was to evaluate frequently used reference genes for the analysis of relative mRNA levels from feline tissues in a SYBR Green-based Q-PCR protocol. First, primers were optimized on mRNA-derived cDNA from liver and kidney tissues of randomly chosen (healthy and diseased) cats. Then, the expression variation and stability of each reference gene within a specific tissue was determined. Dental roots and crowns, heart (left ventricle), renal, liver, lung, and mammary gland tissues from 3 to 11 cats of different breeds, sexes, ages, and disease status were included in this study. Averaging relative stabilities over these six tissues revealed the usefulness of each tested gene as reference gene. In order to compensate for the expression variation of a reference gene within a specific tissue, as much as six reference genes (e.g. RPL17, RPL30, RPS7, YWHAZ, and HPRT) were required to obtain highly reliable data in cat tissues. The optimal set of reference genes depended on the tissue analyzed and should, ideally, be selected and evaluated at the start of each experimental condition. A comparison with a similar evaluation in dogs revealed three issues: (i) most ribosomal genes are suitable in both species; (ii) good non-ribosomal reference genes differ; (iii) more feline than canine reference genes are required for proper analysis.     

Clinical trial of a feline pheromone analogue for feline urine marking

Thirty-six cases of feline urine marking problem were collected through the cooperation of veterinary practitioners in the Kanto, Chubu, and Kansai areas in Japan, for an assessment of the clinical effect of treatment with a synthetic analogue of a feline cheek gland pheromone-like product. The mean frequency of urine marking was 14.2 times/week (median, 10; range, 1-77) at pre-treatment week (preW), and decreased significantly from the first week of treatment, dropping to 4.2 times/week (median, 2; range, 0-44) at the fourth week of treatment. This effect continued until the fourth week after cessation of treatment. These 36 cases were divided into 3 groups based on the effectiveness of treatment as demonstrated in the fourth week of treatment; 37% was categorized as the totally eliminated group (urine marking was not seen), 40% as the reduced group (the frequency of urine marking was equal to or less than 50% that of the preW), and 23% as the unchanged group (the frequency of urine marking was more than 50% that of the preW). Effectiveness of treatment in these groups was 38%, 24%, and 38% at the fourth week after the cessation of treatment, respectively. The decreasing rate of urine marking was compared between cats with and without intercat aggression, and it was revealed that the frequency of marking was sustained at high level in cats with intercat aggression. These results suggest that this pheromone treatment is as effective in Japan as has been reported in other countries for solving feline urine marking problems.     

Comparison of ultrasonography, radiography and a single computed tomography slice for fluid identification within the feline tympanic bulla

Evaluation of the tympanic bulla (TB) in cases of acute feline otitis media can be a diagnostic challenge, although a feature often associated with this condition is the accumulation of fluid or material within the middle ear cavity. A technique is reported allowing optimum imaging of the feline TB using ultrasound (US) and recording of the appearance of gas and fluid-filled TB. A random number of bullae in 42 feline cadavers were filled with lubricant and rostroventral-caudodorsal oblique radiographs, single slice computed tomography (CT) images and US images were created and interpreted by blinded operators. The content (fluid or gas) of each TB was determined using each technique and the cadavers were then frozen and sectioned for confirmation. CT remained the most accurate diagnostic method, but US produced better results than radiology. Given the advantages of US over other imaging techniques, these results suggest that further work is warranted to determine applications of this modality in the evaluation of clinical cases of feline otitis media.     

Acute myelomonocytic leukemia negative for alpha-naphthyl acetate esterase stain in a Holstein cow

A 4-year, 7-month-old Holstein cow presented with anorexia. Physical examination revealed masses in the interscapular region and vagina. Blast cells were detected in the masses and peripheral blood by fine needle aspiration cytology and hematological examination. By bone marrow aspiration, blast cells constituted up to 24.2% of all nucleated cells, and 22% and 2% of non-erythroid cells stained positive for myeloperoxidase and alpha-naphthyl acetate esterase (ANAE), respectively. Pathological examination revealed the mass lesions consisted of a proliferation of tumor cells, which were positive for monocytic markers (HLA-DR and Iba-1). The cow was diagnosed with acute myelomonocytic leukemia (AMML). Even when tumor cells are ANAE-negative, AMML cannot be completely ruled out and should be considered when diagnosing cattle with leukemia/lymphoma.     

Evaluation of leukocyte counts and neutrophil‐to‐lymphocyte ratio as predictors of local recurrence of feline injection site sarcoma after curative intent surgery

Local recurrence (LR) is the major concern in the treatment of feline injection‐site sarcoma (FISS). Pretreatment leukocyte counts and ratios have been reported as diagnostic and/or prognostic markers in human and canine oncology. The aim of this retrospective study was to explore the prognostic impact on LR and overall survival time (OST) of pretreatment neutrophil‐to‐lymphocyte ratio (NLR), white blood cell count (WBCC), neutrophil count (NC) and lymphocyte count (LC) in cats with surgically excised FISS. Eighty‐two cats with histologically confirmed FISS at first presentation, without distant metastases, and with available pretreatment haematological analyses were retrospectively enrolled. The correlation of NLR, WBCC, NC, LC with tumour variables and patient variables was explored. NLR was correlated with tumour size (P = .004), histological pattern of tumour growth (P = .024) and histotype (P = .029), while WBCC and NC were associated with ulceration (P = .007, P = .011) and pattern of growth (P = .028, P = .004). No significant relationships emerged between LC and any of the considered variables. The impact of NLR, WBCC, NC, LC on LR and OST was then estimated in univariate and multivariate analysis. In univariate analysis, NLR, WBCC and NC were significant prognostic factors for both LR and OST. NLR, WBCC and NC remained prognostic in multivariate analysis for LR but not for OST. When NLR, WBCC and NC were jointly analysed, WBCC was the marker with the greater impact on LR. Preoperative NLR, WBCC and NC may aid in identifying cats at higher risk of LR.     

Comparison of a semiquantitative point-of-care assay for the detection of canine microalbuminuria with routine semiquantitative methods for proteinuria

Background: It has been speculated that renal disease can be identified through the detection and quantification of microalbuminuria, however, reliable measurement of albuminuria in any quantity can be challenging. Recently, a new point‐of‐care immunoassay was validated for the specific detection of microalbuminuria and early renal disease in dogs. Objectives: The goal of this study was to determine if measurement of microalbuminuria by the point‐of‐care immunoassay correlated with results from routine semiquantitative methods for detecting proteinuria in dogs. Methods: One hundred and thirty‐eight urine samples, from 133 different dogs, submitted for urinalysis to the Clinical Pathology Laboratory at the University of Missouri‐Columbia Veterinary Medical Teaching Hospital were eligible for the study. Samples that contained >20 RBC/high power field (hpf) or >20 WBC/hpf were excluded, as were samples with insufficient volume to complete all tests. All samples were evaluated with a urinary dipstick with or without a sulfosalicylic acid turbidimetric test, a urine protein:creatinine (UPC) ratio, and the immunoassay for microalbuminuria. Data were analyzed by the Spearman rank order correlation. Results: Microalbuminuria results correlated significantly with those of the dipstick (r= 0.715), sulfosalicylic acid test (r= 0.742), and UPC ratio (r= 0.830). Correlation between the immunoassay and UPC ratio was the same (r= 0.830) when only samples with trace or 1+ proteinuria by dipstick were analyzed (n = 51). Conclusions: The point‐of‐care immunoassay results for microalbuminuria correlated with the results of semiquantitative methods for detecting total proteinuria in dogs. Routine methods for canine proteinuria appear to be adequate for determining whether further testing for renal disease is warranted.     

In vivo expression of GFP transgene delivered via a replicating feline leukemia virus

We previously described replication-competent feline leukemia virus (FeLV) vectors with high-level and stable expression of a green fluorescent protein (GFP) or a suicide transgene in cell cultures in vitro. Considering that FeLV might potentially be used to deliver therapeutic genes in vivo, we first evaluated the expression of the GFP gene introduced in cats by the FeLV, Rickard subgroup A (FRA) construct. Eight newborn kittens were either inoculated with pFRA-GFP plasmid DNA intradermally, or challenged intraperitoneally with FRA-GFP-infected feline fibroblasts. During a 12-week observation period, five cats were shown to be progressively viremic. Quantitative PCR and RT-PCR analyses of plasma and tissue samples from these cats showed that GFP was retained in FeLV DNA or RNA to a variable degree, ranging from 0.002 to 27.890%. Tissue DNA samples were analyzed by PCR for the status of GFP and the env-transgene complex. While the proviruses carrying the GFP transgene were shown to be minor species, all tissues, however, retained the full-length GFP transgene. Despite the occurrence of predominant species with various deletions in the viral genome, approximately 1–3% of the total cell population was GFP-positive in the lymphoid tissues as visualized by laser confocal microscopy. Co-localization of immunofluorescent cells indicated that CD3-positive T cells, dendritic cells and macrophages were the major targets for GFP expression. These findings on the detectable in vivo expression of GFP for as long as a period of 3 months could be viewed positively for contemplating a therapeutic strategy for control of FeLV infection in the cats.     

Use of flow cytometry to develop and characterize a set of monoclonal antibodies specific for rabbit leukocyte differentiation molecules

Flow cytometry was used to identify and characterize monoclonal antibodies (mAbs) that react with rabbit leukocyte differentiation molecules (LDM). Screening sets of mAbs, developed against LDM in other species, for reactivity with rabbit LDM yielded 11 mAbs that recognize conserved epitopes on rabbit LDM orthologues and multiple mAbs that recognize epitopes expressed on the major histocompatibility class I or class II molecules. Screening of mAbs submitted to the Animal Homologues Section of the Eighth Human Leukocyte Differentiation Workshop yielded 7 additional mAbs. Screening of mAbs generated from mice immunized with leukocytes from rabbit thymus or spleen or concanavalin A activated peripheral blood and/or spleen lymphocytes has yielded 42 mAbs that recognize species restricted epitopes expressed on one or more lineages of leukocytes. Screening of the anti-rabbit mAbs against leukocytes from other species yielded one additional mAb. The studies show that screening of existing sets of mAbs for reactivity with rabbit LDM will not be productive and that a direct approach will be needed to develop mAbs for research in rabbits. The flow cytometric approach we developed to screen for mAbs of interest offers a way for individual laboratories to identify and characterize mAbs to LDM in rabbits and other species. A web-based program we developed provides a source of information that will facilitate analysis. It contains a searchable data base on known CD molecules and a data base on mAbs, known to react with LDM in one or more species of artiodactyla, equidae, carnivora, and or lagomorpha.     

Field efficacy and safety of a combination of moxidectin and imidacloprid for the prevention of feline heartworm (Dirofilaria immitis) infection

Throughout the end of March to beginning of May 2006, 212 owned cats and 608 owned dogs from a heavy endemic area for canine heartworm (HW) disease in northern Italy have been examined to assess HW infection prevalence. Both cats and dogs were clinically examined and blood samples were taken from each animal to be examined for HW antibody (Ab). Ab-positive cats were further examined for circulating microfilariae, HW antigens (Ag) and by echocardiography (ECHO) to assess the presence of adult worms. Dogs were clinically examined and blood samples taken from each animal were examined for circulating microfilariae and for HW Ag. Ten cats (4.7%) were found Ab positive. Of these, 6 cats were Ag positive (2.6%) and in 4 (1.8%) the worms were visualized by ECHO. HW prevalence in dogs was 36% (221/608). One hundred and seventy-six (29%) were both microfilaraemic and Ag positive, 40 (7%) had occult infections (no circulating microfilariae) and 7 (1%) were microfilaraemic but Ag negative. Upon owners’ consent, 132 cats (including cats Ab and/or Ag and ECHO positive) were prophylactically treated against HW disease with an imidacloprid/moxidectin spot-on combination (10% imidacloprid/1% moxidectin) monthly administered for 6 months. Cats were re-examined for HW infection in November, 1 month after the last drug administration, and in May–June 2007, 7–8 months after the last treatment. All 122 cats found HW negative before treatment, were found negative at the two examinations at the end of study. The 4 cats Ab positive, 2 cats Ab and Ag positive and 1 Ab, Ag and ECHO positive at the beginning of treatment were found negative. Throughout the treatment, transitory hypersalivation and generic signs of annoyance were reported by owners in 6 cats (4.5%). All signs regressed spontaneously.