Plasma leptin concentrations in cats: reference range, effect of weight gain and relationship with adiposity as measured by dual energy X-ray absorptiometry

The aims of our study were to determine a reference range for plasma leptin in healthy, normal-weight cats and to measure the effect of weight gain on plasma leptin levels. To increase our understanding of the association between leptin and feline obesity, we investigated the relationship between plasma leptin and measures of adiposity in cats. Twenty-six normal-weight cats were used to determine the reference range for feline leptin using a multispecies radioimmunoassay. In the second part of the study, plasma leptin concentrations were determined in 16 cats before and after approximately 10 months of spontaneous weight gain. Dual energy X-ray absorptiometry scans (DEXA) were performed after weight gain. The tolerance interval for plasma leptin concentrations was 0.92-11.9 ng/ml Human Equivalent (HE) with a mean concentration of 6.41+/-2.19 ng/ml HE. In part two of the study, 16 cats gained on average 44.2% bodyweight over 10 months. The percentage of body fat in obese cats ranged from 34.2 to 48.7%. Mean plasma leptin concentrations increased from 7.88+/-4.02 ng/ml HE before weight gain to 24.5+/-12.1 ng/ml HE after weight gain, (P<0.001). Total body fat and body fat per cent were the strongest predictors of plasma leptin in obese cats (r=0.8 and r=0.78, P<0.001, respectively). In conclusion, plasma leptin concentrations increased three-fold in cats as a result of weight gain and were strongly correlated with the amount of adipose tissue present. Despite elevated leptin levels, cats continued to eat and gain weight, suggesting decreased sensitivity to leptin. This investigation into the biology of leptin in cats may aid the overall understanding of the role of leptin and the development of future treatments to help prevent and manage feline obesity.     

Sandwich enzyme-linked immunosorbent assay of canine leptin

Leptin is the ob gene product secreted from adipocytes in mammals, and thereby its plasma level reflects body fat content. To establish an assay method for leptin in the dog, rabbit anti-canine leptin antibody was obtained using canine recombinant leptin as an antigen. This antibody reacted to canine leptin much stronger than mouse, rat and human leptins. Sandwich enzyme-linked immunosorbent assay (ELISA) using this antibody was developed. The serum leptin levels of 13 healthy dogs were in a range from 1.4 to 5.6 ng/ml with the mean +/- SEM of 3.0 +/- 0.3 ng/ml.     

Relationship between serum leptin immunoreactivity and body fat mass as estimated by use of a novel gas-phase Fourier transform infrared spectroscopy deuterium dilution method in cats

OBJECTIVE: To validate a recently developed commercially available leptin radioimmunoassay (RIA) for use with feline serum and evaluate the relationship between serum leptin concentrations and body fat mass in domestic cats. ANIMALS: 19 sexually intact male specific-pathogen-free domestic cats that weighed 3.8 to 7.1 kg and were 1.1 to 3.5 years old. PROCEDURE: Specificity for feline leptin was evaluated by use of gel filtration chromatography and reverse-phase high-performance liquid chromatography fractionation of serum. Body fat mass was determined by use of the deuterium oxide (D2O) dilution method. Serum water D2O enrichment was measured by use of gas-phase Fourier transform infrared spectroscopy. RESULTS: Body fat mass and percentage body fat ranged from 0.3 to 2.3 kg and 7.5 to 34.9%, respectively. Serum leptin concentrations were lower in the unfed versus the fed state and ranged between 1.6 and 4.9 ng/ml human equivalent (HE); mean +/- SD value was 2.9 +/- 0.2 ng/ml HE. Leptin concentrations increased with increasing body fat mass and percentage of body fat. CONCLUSIONS: Leptin is in the serum of domestic cats in free (> 78%) and apparently bound forms. The relationship between body fat and serum leptin concentration was similar to that observed in humans and rodents and indicative of a lipostatic role for leptin in cats. Cats that have an overabundance of body fat appear to be less sensitive to the weight-normalizing action of leptin than cats of ideal body condition.     

Detection of feline coronavirus antibody, feline immunodeficiency virus antibody, and feline leukemia virus antigen in ascites from cats with effusive feline infectious peritonitis

To investigate the usefulness of ascites as a material for viral tests in cats with effusive feline infectious peritonitis (FIP), we attempted to detect anti-feline coronavirus antibody, anti-feline immunodeficiency virus antibody, and feline leukemia virus antigen in ascites from 88 cats clinically suspected with effusive FIP. In each of these three viral tests, all cats positive for serum antibody/antigen were also positive for ascitic antibody/antigen, while cats negative for serum antibody/antigen were also negative for ascitic antibody/antigen. This finding indicates that ascites is useful for these viral tests.     

Experimental and clinical studies on plasma leptin in obese dogs

Leptin is a protein synthesized and secreted primarily by adipocytes, and the circulating leptin concentration is elevated in obese humans and rodents. Recently, we have established a sandwich enzyme-linked immunosorbent assay for canine leptin. In the present study, plasma leptin concentrations were measured in experimentally developed obese beagles and in clinically obese dogs. When 5 male beagles were given a high-energy diet for 3 months, all of them became obese and the plasma leptin concentration significantly increased from 2.4+/-1.2 to 4.9+/-0.9 ng/ml, positively correlating with body fat content estimated by the deuterium oxide dilution method (r=0.87). The leptin concentrations of plasma samples collected from 59 dogs in veterinary practices were compared with their body condition scores (BCS). The plasma leptin concentrations of obese dogs were 9.7+/-0.7 and 12.3+/-1.5 ng/ml at BCS=4 and BCS=5, respectively, which were significantly higher than those of optimal (BCS=3) dogs (2.7+/-0.3 ng/ml). There was no significant effect of sex and breed. A weak positive correlation (r=0.37) was found between the plasma leptin concentration and age, probably due to the lesser content of visceral fat in puppies younger than 1 year old. These results indicate that plasma leptin is a good index of adiposity in dogs regardless of breed, age and sex, and may be useful for quantitative assessment of obesity in small animal practice.     

Production of polyclonal antisera against feline immunoglobulin E and its use in an ELISA in cats with experimentally induced asthma

Serum samples from six cats with experimentally induced asthma were used to purify feline IgE using gel filtration and affinity chromatography. The resultant IgE, evaluated for purity by immunoelectrophoresis (IEP) and reactivity by Prausnitz-Kustner (PK) testing, was used to develop polyclonal rabbit anti-feline IgE antisera. Using reverse cutaneous anaphylaxis (RCA), the antisera were determined to be specific for feline IgE. The polyclonal rabbit anti-feline IgE antiserum was then validated in an allergen-specific ELISA. Serum samples from an additional five asthmatic cats sensitized with Bermuda grass allergen (BGA) were evaluated prior to sensitization, after parenteral sensitization, and after aerosol sensitization and challenge. A significant increase in serum BGA-specific IgE was noted over time.     

Obesity increases initial rate of fibrin formation during blood coagulation in domestic shorthaired cats

Obesity predisposes to a prothrombotic state in humans, but whether a similar state occurs in obese animals is unknown. The objective of the current study was to examine the effect of body fat percentage (BF) on haemostatic parameters including thromboelastography with tissue factor as activator (TF-TEG) in client owned indoor-confined physically inactive cats. Seventy-two cats were included following an initial thorough health examination, and a complete blood count, biochemistry panel, conventional coagulation panel and a TF-TEG analysis were performed with tissue factor (1:50,000) as activator. The cats were anaesthetized, and BF was measured using Dual-energy X-ray absorptiometry. Significant difference between lean (BF < 35%, n = 26), overweight (35% < BF < 45%, n = 28) and obese (BF > 45%, n = 18) cats was identified using ANOVA. The correlation between BF, serum leptin and total adiponectin, respectively, with individual TEG and conventional coagulation parameters was evaluated. Obese cats showed a faster rate of fibrin formation (TF-TEG(R), p < 0.05), and TF-TEG(R) was positively correlated with plasma leptin levels. Increasing BF did not affect other conventional coagulation or TF-TEG parameters. In conclusion, this study indicates a connection between body fat content and altered haemostasis, also in cats. Whether feline obesity causes a hypercoagulable state of clinical relevance should be further investigated.     

Correlation between plasma leptin concentration and body fat content in dogs

OBJECTIVE: To evaluate the relationship between plasma leptin concentration and body fat content in dogs. ANIMALS: 20 spayed female Beagles that were 10 months old at the start of the experiment. PROCEDURE: Dogs were kept under regulated feeding and exercise conditions for 21 weeks, resulting in a wide range of body weights, body condition scores (BCS), and subcutaneous thicknesses. Plasma leptin concentration was measured by use of a canine leptin-specific ELISA test to evaluate its correlation to body fat content estimated by the deuterium oxide dilution method. Plasma concentrations of glucose, cholesterol, triglycerides (TG), and nonesterified fatty acids (NEFA) were also measured. RESULTS: Body fat content (9 to 60% of body weight) was positively and closely correlated (r = 0.920; n = 20; P < 0.001) to plasma leptin concentration (0.67 to 8.06 ng/ml), compared with other variables (ie, glucose, cholesterol, TG, and NEFA; r = 0.142, 0.412, 0.074, and 0.182, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: The positive relationship between plasma leptin concentration and body fat content in dogs was similar to correlations reported for humans and rodents, suggesting that plasma leptin is a quantitative marker of adiposity in dogs.     

Leptin, body fat content and energy expenditure in intact and gonadectomized adult cats: a preliminary study

The present study was conducted to assess the body composition, leptin, and energy expenditure changes following gonadectomy in cats. Twenty-one females (12 intact and nine spayed) and 21 males (11 intact and 10 castrated) were used. Body weight was recorded. Serum plasma leptin was measured by radioimmunoassay and body composition and energy expenditure were assessed after injection of doubly labelled water. These results confirmed the gain in body weight and body fat following neutering and demonstrated a strong linear relationship between body fat and serum level of leptin. Energy expenditure decreased in castrated cats in comparison with intact ones. This study underlined the effect of gonadectomy as a major factor of obesity in cats and showed that the increase in circulating leptin reflected the amount of body fat. The present results provide further evidence that the regimen of gonadectomized cats should be carefully controlled to avoid excessive weight gain.     

Falciform fat:femur length ratio provides a novel method for objective postmortem estimation of total body fat in overweight and obese cats

Determination of the nutritional condition, including estimation of amounts of total body fat (tBF), at routine postmortem examination of cats is typically based on subjective visual assessment. Subjective assessment may result in uncertainties regarding degree of overweight, and objective methods that provide a numerical value reflecting the tBF could be valuable to accurately judge excess body fat. We investigated if the falciform fat pad weight (FFPW) was correlated to tBF and could be used to detect overweight and obesity in cats. The FFPW and the femur length (FL) were recorded at postmortem examination in 54 cats and the FFPW:FL ratio (FFR) calculated. Each cat was additionally assigned to a fat category (FC) according to subjective assessment. Computed tomography was used to determine tBF as the body fat percentage (%BF), the body fat volume (BFV), and BFV normalized to animal size (nBFV) in 39 cats. There was strong correlation between the FFPW and the BFV (r = 0.888) and between the FFR and the nBFV (r = 0.897). The correlation between the nBFV and %BF was very strong (r = 0.974). Using a lower FFR cutoff value of 3.5 for obesity and 1.6 for overweight, there was a discrepancy in FC between using the FFR and subjective assessment in 6 of 54 cats (11%). We conclude that the FFPW increases proportionally with tBF and that the FFR provides a method for objective tBF estimation. We suggest introducing the FFR to feline postmortem examination protocols as an objective estimate of tBF.     

A micro-enzyme-linked immunosorbent assay (ELISA) test for detection of feline leukemia virus in cats

The performance of a micro ELISA test for detection of feline leukemia virus (FeLV) infection was evaluated. The test was found specific for FeLV and feline sarcoma virus (FeSV) group-specific antigens in blood, plasma or serum of infected cats. Other common feline pathogens were negative to the test.Quantities as little as 7.8 ng of p-27 (the major group specific antigen of FeLV) per ml of sample gave positive results. The correlation between the micro ELISA test and the indirect immunofluorescent test commonly used for diagnosis of FeLV infection was 98% in 116 clinical cases and 184 samples from cats inoculated with FeLV and 100% in 100 specific pathogen-free cats.     

Feline plasma cortisol (hydrocortisone) measured by radioimmunoassay.

Plasma cortisol (hydrocortisone) was measured by radioimmunoassay in 6 normal cats. Blood was collected from the cats by venipuncture at intervals of 3 hours for 3 days. Resting plasma cortisol concentrations averaged 17.0 +/- 2.8 (SD) ng/ml and ranged from nondetectable (less than 3 ng/ml) to 82.8 ng/ml. Of 144 plasma samples, 95% contained less than 40 ng of cortisol/ml. Circadian rhythm of cortisol secretion was not detected, suggesting that adrenal function tests may be started in feline patients at any time of day. Intramuscular injection of 2.2 U of ACTH gel/kg of body weight caused detectable increase in plasma cortisol concentrations at 1 and 2 hours after injection. Maximal response to ACTH in the 6 cats ranged from 41.6 to 178.4 ng/ml. Oral administration of 0.1 mg of dexamethasone/kg suppressed plasma cortisol to nondetectable concentrations for 32 hours in 5 of the 6 cats.     

Humoral immune response to a recombinant hemoplasma antigen in experimental 'Candidatus Mycoplasma turicensis' infection

'Candidatus Mycoplasma turicensis' is a feline hemoplasma species that was isolated in a cat with hemolytic anemia. PCR has been widely used to investigate and diagnose 'Candidatus Mycoplasma turicensis' infection, but so far, little is known about the humoral immune response in infected cats. Recently, enzyme-linked immunosorbent assays (ELISA) were developed to monitor anti-feline hemoplasma antibodies. The aim of the present study was to investigate the humoral immune response in cats experimentally infected with 'Candidatus Mycoplasma turicensis' and to monitor the influence of the pre-administration of methylprednisolone and subsequent antibiotic treatment. Serum and plasma samples from 15 specified pathogen-free cats infected with 'Candidatus Mycoplasma turicensis' were analyzed by ELISA. Seroconversion was demonstrated in all cats, and the antibodies remained detectable until the end of the study (up to 100 weeks post-exposure). In some cats, the ELISA seemed more sensitive and better able to demonstrate exposure to 'Candidatus Mycoplasma turicensis' than PCR. The peak antibody level occurred after the peak of the bacterial blood loads. The methylprednisolone administrations were associated with increased antibody levels, while antibiotic treatment, particularly with doxycycline, resulted in a decrease in antibody levels. Additionally, preliminary data indicated that three of four seropositive cats were protected from bacteremia after a subsequent challenge. In conclusion, the ELISA was found to be a useful tool to investigate the humoral immune response in hemoplasma-infected cats and a desirable addition to PCR to study the pathogenesis of hemoplasma infections.     

Development of a feline proinsulin immunoradiometric assay and a feline proinsulin enzyme-linked immunosorbent assay (ELISA): a novel application to examine beta cell function in cats

The prevalence of feline diabetes mellitus has increased several-fold over the last three decades. In humans, progression from obesity to diabetes is marked by changes in the release of proinsulin. A specific proinsulin (FPI) assay has not been available to examine similar changes in cats. The goal of this study was to develop a proinsulin assay for the analysis of beta cell function in cats. Monoclonal antibodies were developed against recombinant FPI and used in a two-site sandwich immunoradiometric assay (IRMA) and enzyme-linked immunosorbent Assay (ELISA). The antibody pair had negligible cross-reactivity with bovine insulin and feline C-peptide. The working range was 11-667pmol/L for the IRMA and 11-1111pmol/L for the ELISA. An intravenous glucose tolerance test was performed in six long-term obese and six lean adult healthy cats and serum glucose, insulin, and FPI concentrations were determined. The proinsulin and insulin secretion pattern in response to glucose was significantly different between lean and obese cats but the pattern was similar within a group. Both groups had similar baseline proinsulin/insulin ratios; however, obese cats showed a significantly higher proinsulin/insulin ratio during the first 15min of the IVGTT and a much lower ratio during the last 30min suggesting a time-delayed adjustment to the increased insulin demand. In conclusion, we report the development and validation of an IRMA and an ELISA for FPI. This novel assay is useful to elucidate FPI secretion and can be used similar to a C-petide assay to evaluate residual beta cell function in cats.     

Markers of feline leukaemia virus infection or exposure in cats from a region of low seroprevalence

Molecular techniques have demonstrated that cats may harbour feline leukaemia virus (FeLV) provirus in the absence of antigenaemia. Using quantitative real-time polymerase chain reaction (qPCR), p27 enzyme-linked immunosorbent assay (ELISA), anti-feline oncornavirus-associated cell-membrane-antigen (FOCMA) antibody testing and virus isolation (VI) we investigated three groups of cats. Among cats with cytopenias or lymphoma, 2/75 were transiently positive for provirus and anti-FOCMA antibodies were the only evidence of exposure in another. In 169 young, healthy cats, all tests were negative. In contrast, 3/4 cats from a closed household where FeLV was confirmed by isolation, had evidence of infection. Our results support a role for factors other than FeLV in the pathogenesis of cytopenias and lymphoma. There was no evidence of exposure in young cats. In regions of low prevalence, where the positive predictive value of antigen testing is low, qPCR may assist with diagnosis.     

Post-exposure treatment of cats with mouse-cat chimeric antibodies against feline herpesvirus type 1 and feline calicivirus

In order to confirm the in vivo effectiveness of anti- feline herpesvirus type 1 (FHV-1) mouse-cat chimeric antibody (FJH2), and anti-feline calicivirus (FCV) mouse-cat chimeric antibody (F1D7), cats that had been experimentally infected with FHV-1 or FCV were administered intravenously with the chimeric antibodies, and observed for clinical manifestations. The symptoms due to FHV-1 or FCV infection in the cats administered FJH2 or F1D7 were obviously decreased when compared with those of the non-administered control cats. From these results, it was confirmed that both FJH2 and F1D7 were effective at reducing the appearance of symptoms due to FHV-1 and FCV infection, respectively.     

Patterns of expression of feline cytokeratins in healthy epithelia and mammary carcinoma cells.

Expression of keratins (cytokeratins, CK) in healthy feline epithelia and 2 established feline mammary carcinoma cell lines was examined immunohistochemically and by use of immunoblotting analysis. A panel of specific anti-CK monoclonal antibodies (MAb) identifying epitopes unique to individual keratins or shared by 2 (or 3) CK polypeptides was used. Besides already available anti-human CK MAb, this panel of MAb consisted of 9 newly generated anti-human CK MAb and 1 newly generated anti-feline CK MAb. Immunohistochemical analysis on normal epithelia revealed that most of the anti-human CK MAb and the anti-feline CK MAb reacted with both feline and human epithelia, with a comparable tissue distribution pattern. However, slight differences in CK tissue distribution pattern between human beings and cats were detected by one MAb. Immunoblotting analysis revealed that all anti-human CK MAb that were immunohistochemically reactive with feline tissues detected analogous CK in cats, indicating the presence of a number of common epitopes on human and feline CK. Two continuous cell lines derived from 2 distinct feline mammary adenocarcinomas, K248C and K266, were analyzed with respect to their CK phenotype. Although no difference in CK expression between the 2 cell lines was detected in vitro, a difference in CK phenotype was detected on subcutaneous transplantation of the 2 cell lines into nude mice. Although the K248C-induced adenocarcinomas maintained the same CK phenotype as observed in vitro, the CK pattern of the K266 heterotransplants, growing as adenosquamous carcinomas, changed with squamous differentiation. Our findings confirm the high degree of homology between mammalian CK, and on the basis of those findings, we suggest that CK proteins provide a set of markers valuable for the characterization of normal and neoplastic feline tissues and for studies of squamous metaplasia.     

Identification of the feline CD63 homologue using retrovirus-mediated expression cloning

Recently, we combined a retrovirus-mediated expression cloning with a simple screening method using non-adherent cells and panning [Anal. Biochem. 315 (2003) 138]. In this study, we applied this method to identify the antigen recognized by an uncharacterized monoclonal antibody raised against a feline cell line, and identified it as the feline homologue of CD63. This simple method is useful for characterizing unknown antibodies that recognize cell surface molecules. Furthermore, the monoclonal antibody identified as an anti-feline CD63 antibody will be useful for studying feline molecular function(s).     

Development of a hyena immunology toolbox

Animals that hunt and scavenge are often exposed to a broad array of pathogens. Theory predicts the immune systems of animals specialized for scavenging should have been molded by selective pressures associated with surviving microbial assaults from their food. Spotted hyenas (Crocuta crocuta) are capable hunters that have recently descended from carrion feeding ancestors. Hyenas have been documented to survive anthrax and rabies infections, and outbreaks of several other viral diseases that decimated populations of sympatric carnivores. In light of the extreme disease resistance manifested by spotted hyenas, our objective was to identify tools available for studying immune function in spotted hyenas and use these tools to document the hyena antibody response to immunization. Domestic cats (Felis catus) are the closest phylogenetic relatives of hyenas that have been studied in detail immunologically, and we hypothesized that anti-cat isotype-specific antibodies would cross react with hyena immunoglobulin epitopes. We used ELISA and Western blots to test isotype-specific anti-feline antibodies for specific cross-reaction to hyena Ig epitopes. Molecular weights of heavy (IgA, IgG, IgM) and light chains of hyena immunoglobulins were determined by protein electrophoresis, and as expected, they were found to be similar to feline immunoglobulins. In order to further validate the cross-reactivity of the anti-feline antibodies and document the hyena humoral response, eight spotted hyenas were immunized with dinitrophenol conjugated keyhole limpet hemocyanin (DNP-KLH) and serum anti-DNP responses were monitored by enzyme-linked immunosorbent assay (ELISA) for one year. The full array of isotype-specific antibodies identified here will allow veterinarians and other researchers to thoroughly investigate the hyena antibody response, and can be used in future studies to test hypotheses about pathogen exposure and immune function in this species.