Evaluation of chronic stress status and quality of life in cats suffering from chronic kidney disease and suspected feline infectious peritonitis based on hair cortisol concentration analysis and a questionnaire.

Hair cortisol concentration (HCC) and a questionnaire were used as indicators of chronic stress status and quality of life (QoL), respectively, in cats. To date, there has been limited research on the simultaneous application of both indicators in unwell cats. Our aim was to evaluate HCC and questionnaire data obtained from a healthy cat cohort (n = 61) and cat cohorts with either chronic kidney disease (CKD) (n = 78) or suspected feline infectious peritonitis (FIP) (n = 24). Furthermore, we also investigated the correlation between HCC and clinical pathological data. For this study, hair from the abdomen of cats was collected and analyzed for HCC using a commercial ELISA kit. Owners also completed a questionnaire, from which average-item-weighted-impact-scores (AWISs) were calculated. Cats with late-stage-CKD (median, HCC = 330.15 pg/mg, AWIS = -0.43) presented with a significantly higher HCC (p < 0.01) and a significantly lower AWIS (p < 0.01) than cats with early-stage-CKD (HCC = 183.56 pg/mg, AWIS = 1.08). Similarly, there were significant differences in both HCC (p < 0.001) and AWIS (p < 0.001) between cats with suspected FIP (HCC = 896.27 pg/mg, AWIS = -1.97) and healthy cats (HCC = 181.24 pg/mg, AWIS = 1.24). The degree of consistency between the HCC results and the questionnaire results reminds us that the severity of a chronic disease or the presence of a life-threatening disease can significantly increase stress and thus can affect the QoL of cats.

Urinary angiotensin-converting enzyme 2 and its activity in cats with chronic kidney disease.

Introduction: Angiotensin-converting enzyme 2 (ACE2) played an important role in the renin-angiotensin-aldosterone system (RAAS) and it was proved to be renoprotective in renal disease. Urinary angiotensin-converting enzyme 2 (uACE2) has been shown to reflect renal injury in human and experimental studies, but its role in feline kidney disease remains unknown. Aims: Our objectives involve comparing uACE2 concentrations and activities in cats across CKD stages with healthy controls, investigating the relationship between uACE2 concentrations, activities, and clinicopathological data in feline CKD patients, and assessing the predictive abilities of both for CKD progression. Methods: A retrospective, case-control study. The concentration and activity of uACE2 were measured by commercial ELISA and fluorometric assay kits, respectively. The concentration was adjusted to give uACE2 concentration-to-creatinine ratios (UACCRs). Results: In total, 67 cats consisting of 24 control and 43 chronic kidney disease (CKD), including 24 early-stage CKD and 19 late-stage CKD, were enrolled in this study. UACCR values were significantly higher in both early-stage (2.100 [1.142-4.242] x 10-6) and late-stage feline CKD (4.343 [2.992-5.0.71] x 10-6) compared to healthy controls (0.894 [0.610-1.076] x 10-6; p < 0.001), and there was also significant difference between-early stage group and late-stage group (p = 0.026). Urinary ACE2 activity (UAA) was significantly lower in CKD cats (1.338 [0.644-2.755] x pmol/min/ml) compared to the healthy cats (7.989 [3.711-15.903] x pmol/min/ml; p < 0.001). UACCR demonstrated an independent, positive correlation with BUN (p < 0.001), and UAA exhibited an independent, negative correlation with plasma creatinine (p < 0.001). Both UACCR and UAA did not yield significant results in predicting CKD progression based on the ROC curve analysis. Conclusion and clinical importance: uACE2 concentration and activity exhibit varying changes as renal function declines, particularly in advanced CKD cats.

Investigating the Efficacy of Kidney-Protective Lactobacillus Mixture-Containing Pet Treats in Feline Chronic Kidney Disease and Its Possible Mechanism.

Microbiota-based strategies are a novel auxiliary therapeutic and preventative way of moderating chronic kidney disease (CKD). Lactobacillus mixture (Lm) was previously demonstrated to exert a renal-protective function in the CKD mice model. The efficacy of probiotics in pet foods is a relatively new area of study, and thus verifying the potential health benefits is necessary. This study evaluated the efficacy of Lm treats in feline CKD and elucidated the mechanisms underlying host-microbe interactions. CKD cats (2 and 3 stages) were administrated probiotic pet treats daily (10 g) for 8 weeks. The results demonstrated that during the eight weeks of Lm administration, creatinine was reduced or maintained in all cats with CKD. Similarly, gut-derived uremic toxin (GDUT), indoxyl sulfate (IS), were potential clinical significance in IS after Lm treatment (confidence intervals = 90%). The life quality of the cats also improved. Feline gut microbiome data, metabolic functional pathway, and renal function indicator analyses revealed the possible mechanisms involved in modulating CKD feline microbial composition. Further regulation of the microbial functions in amino acid metabolism after Lm administration contributed to downregulating deleterious GDUTs. The current study provides potential adjuvant therapeutic insights into probiotic pet foods or treats for pets with CKD.

Epidemiology of Severe Fever with Thrombocytopenia Syndrome in Dogs and Cats in Taiwan.

Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by the SFTS Virus (SFTSV), is a global health threat. SFTSV in Taiwan has only been reported in ruminants and wild animals. Thus, we aimed to investigate the infection statuses of dogs and cats, the animals with closer human interactions. Overall, the SFTSV RNA prevalence was 23% (170/735), with dogs showing a 25.9% (111/429) prevalence and cats at 19.3% (59/306) prevalence. Noticeably, the prevalence in stray animals (39.8% 77/193) was significantly higher than in domesticated ones (17.2%, 93/542). Among the four categories analyzed, the highest SFTSV prevalence was found in the stray dogs at 53.9% (120/193), significantly higher than the 24.2% prevalence noted in stray cats. In contrast, domesticated animals exhibited similar prevalence rates, with 17.1% for dogs and 17.2% for cats. It is noteworthy that in the domesticated animal groups, a significantly elevated prevalence (45%, 9/20) was observed among cats exhibiting thrombocytopenia compared to those platelet counts in the reference range (4.8%, 1/21). The high infection rate in stray animals, especially stray dogs, indicated that exposure to various outdoor environments influences the prevalence of infections. Given the higher human interaction with dogs and cats, there is a need for proactive measures to reduce the risk associated with the infection of SFTSV in both animals and humans.

A simple electrochemical immunosensor based on a gold nanoparticle monolayer electrode for neutrophil gelatinase-associated lipocalin detection.

An electrochemical immunosensor for the accurate detection of cat neutrophil gelatinase-associated lipocalin (NGAL) in urine samples based on an electrode with a monolayer of gold nanoparticles (AuNPs) was proposed in this study. To fabricate the sensing electrode, a nickel mold with concave micron hemisphere array was prepared and then used to transfer the micron hemispherical structure onto a polyethylene terephthalate (PET) film using the hot embossing technique. A gold thin film was sputtered onto the micron hemispherical structure array, after which 1,6-hexanedithol and AuNPs were uniformly deposited on the PET membrane to form a sensing electrode. The NGAL concentrations were measured by electrochemical impedance spectroscopy after attaching the anti-NGAL. Results revealed that the proposed sensing scheme exhibited a wide dynamic detection range from 1 to 100 ng/mL, which is far enough to distinguish the healthy (NGAL concentration <10 ng/mL) from the damaged kidney. A low limit of detection and high sensitivity of 0.47 ng/mL and 10261.8 Ω ng-1mL, respectively, were also measured. After performing real sample detection using urine samples from cats collected at a veterinary hospital, the results confirmed that the proposed NGAL detection approach in this research could accurately detect the concentration of NGAL in cat urine samples.

Evaluation of and the prognostic factors for cats with big kidney-little kidney syndrome.

BACKGROUND: The term big kidney-little kidney syndrome in cats has been used for many years, but the definitions are not consistent and relevant research is limited. OBJECTIVE: To determine the factors that differ between normal and BKLK cats, as well as to develop models for predicting the 30-day survival of cats with ureteral obstruction (UO). ANIMALS: Sixteen healthy cats and 64 cats with BKLK. METHODS: Retrospective study. To define BKLK by reference to data from clinically healthy cats. The demographic and clinicopathological data among groups were statistically analyzed. RESULTS: Big kidney-little kidney syndrome cats had higher blood urea nitrogen (BUN) (median [interquartile range] 69 [28-162] vs 21 [19-24] mg/dL, P < .001), creatinine (5.6 [1.9-13.3] vs 1.3 [1.05-1.40] mg/dL, P < .001), and white blood cells (10 800 [7700-17 500] vs 6500 [4875-9350] /µL, P < .001) and lower hematocrit (32.8 [27.1-38.4] vs 39.1 [38.1-40.4]%, P < .001), urine specific gravity (1.011 [1.009-1.016] vs 1.049 [1.044-1.057], P < .001) and pH (5.88 [5.49-6.44] vs 6.68 [6.00-7.18], P = .001) compared to the control cats. A lower body temperature (BT; 38.1 [37.9-38.2] vs 38.7 [38.3-39.2]°C, P = .009), higher BUN (189 [150-252] vs 91 [36-170] mg/dL, P = .04), and creatinine (15.4 [13.3-17.4] vs 9.0 [3.1-14.2] mg/dL, P = .03) were found among the UO cats that were not 30-day survivors. A combination of BUN, phosphorus, and BT can predict 30-day survival among UO cats with an area under receiver operating characteristic curve of 0.863. (P = .01). CONCLUSION: An increase in the length difference between kidneys can indicate UO, but cannot predict outcome for BKLK cats.

Hypochlorite-induced porcine model of peritoneal fibrosis through the activation of IL1ß-CX3CL1-TGFß1 signal axis.

Patients with kidney failure rely on life-saving peritoneal dialysis to facilitate waste exchange and maintain homeostasis of physical conditions. However, peritoneal dialysis often results in peritoneal fibrosis and organ adhesion that subsequently compromise the efficiency of peritoneal dialysis and normal functions of visceral organs. Despite rodent models provide clues on the pathogenesis of peritoneal fibrosis, no current large animal model which shares high degree of physiological and anatomical similarities to human is available, limiting their applications on the evaluation of pre-clinical therapeutic efficacy. Here we established for the first time, hypochlorite-induced porcine model of peritoneal fibrosis in 5-week-old piglets. We showed that administration 15-30 mM hypochlorite, a dose- and time-dependent severity of peritoneal fibrosis characterized by mesothelium fragmentation, αSMA+ myofibroblasts accumulation, organ surface thickening and type I collagen deposition were observed. We also demonstrated in vitro using human mesothelial cells that hypochlorite-induced fibrosis was likely due to necrosis, but not programmed apoptosis; besides, overexpression of IL1ß, CX3CL1 and TGFß on the peritoneal mesothelium in current model was detected, similar to observations from peritoneal dialysis-induced peritoneal fibrosis in human patients and earlier reported mouse model. Moreover, our novel antemortem evaluation using laparoscopy provided instant feedback on the progression of organ fibrosis/adhesion which allows immediate adjustments on treatment protocols and strategies in alive individuals that can not and never be performed in other animal models.

Urine hemojuvelin in cats with naturally occurring kidney disease.

BACKGROUND: Soluble-type hemojuvelin in serum and urine has been shown to be a biomarker in humans for chronic kidney disease (CKD) and acute kidney injury (AKI). No similar research has been conducted on cats. OBJECTIVE: Urine hemojuvelin (u-hemojuvelin) can be used as a clinical indicator for cats with various renal diseases. ANIMALS: Eighteen healthy cats, 10 cats with AKI, 21 cats with acute-on-chronic kidney injury (ACKI), and 45 cats with CKD were enrolled. METHODS: The expression profile of u-hemojuvelin was assessed by Western blot analysis, whereas the u-hemojuvelin concentration was measured using an in-house sandwich ELISA. Each cat's u-hemojuvelin-to-creatinine ratio (UHCR) also was determined. RESULTS: Significant differences were found in both u-hemojuvelin concentration and UHCR between the control cats and the other cats (AKI, CKD, ACKI). Both u-hemojuvelin and UHCR had high areas under the receiver operator curve (AUROC) for diagnoses of AKI (u-hemojuvelin, 0.885; UHCR, 0.982), CKD (hemojuvelin, 0.869; UHCR, 0.959), and ACKI (hemojuvelin, 0.910; UHCR, 1). Late stage (International Renal Interest Society, IRIS stages 3 and 4) CKD cats had significantly higher u-hemojuvelin concentration and UHCR than did early stage cats (IRIS stages 1 and 2). Both u-hemojuvelin and UHCR were significantly correlated with high blood urea nitrogen, plasma creatinine, and plasma phosphate concentrations and with low hematocrit (Hct), red blood cell (RBC) count, and plasma albumin concentration. The UHCR values were also significantly correlated with white blood cell count in blood. CONCLUSION: Both u-hemojuvelin and UHCR potentially can serve as diagnostic indicators for a range of renal diseases in cats.

Alleviating chronic kidney disease progression through modulating the critical genus of gut microbiota in a cisplatin-induced Lanyu pig model.

In the present study, we investigated the effects of Probiotic mix 1 (Pm1) with Lactobacillus plantarum subsp. plantarum, Lactobacillusparacasei subsp. paracasei, and Streptococcus salivarius subsp. thermophilus on preventing renal injury using a chronic kidney disease (CKD) minipig model previously developed in our lab using cisplatin-induced CKD in Lanyu pigs. The results indicated that the high dosage Pm1 (H.Pm1) group demonstrated lower incidence of lesions, including atrophy, mononuclear inflammation, cell infiltration, and interstitial fibrosis in renal tubules in hematoxylin and eosin (H&E) and Masson's trichrome stain. We further systematically investigated the preventing effect of Pm1. The H.Pm1 group decreased inflammatory cytokines production and increased the level of superoxide dismutase activity in plasma. The pigs fed with high dosage of Pm1 group also showed reduced both creatinine and blood urea nitrogen (BUN) when compared with the cisplatin group. Microbiota results indicated that Pm1-intervention not only reduced the abundance of Gram-negative bacteria but also affected the abundance of specific genera biomarkers, Anaerovibrio, possible_genus_SK018, Holdemanella, and Lachnospiraceae_UCG_010 in gut microbiota, leading to decreased inflammation and apoptosis in the kidney and further prevention/alleviation of the symptoms of CKD.

Identification of urine neutrophil gelatinase-associated lipocalin molecular forms and their association with different urinary diseases in cats.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL), a promising renal biomarker, can exists as a monomer, a dimer and/or in a NGAL/matrix metalloproteinase-9 (MMP-9) complex form when associated with different urinary diseases in humans and dogs. In this study, the presence of the various different molecular forms of NGAL in cat urine (uNGAL) was examined and whether these forms are correlated with different urinary diseases was explored. RESULTS: One hundred and fifty-nine urine samples from cats with various different diseases, including acute kidney injury (AKI, 22 cats), chronic kidney disease (CKD, 55 cats), pyuria (44 cats) and other non-renal and non-pyuria diseases (non-RP, 26 cats), as well as healthy animals (12 cats), were collected. The molecular forms of and concentrations of urinary NGAL in these cats were analyzed, and their uNGAL-to-creatinine ratio (UNCR) were determined. The cats with AKI had the highest UNCR (median: 2.92 × 10- 6), which was followed by pyuria (median: 1.43 × 10- 6) and CKD (median: 0.56 × 10- 6); all of the above were significantly higher than the healthy controls (median: 0.17 × 10- 6) (p < 0.05). Three different NGAL molecular forms as well as the MMP-9 monomer were able to be detected in the cat urine samples. Moreover, the cases where urine NGAL monomer were present also had significantly higher levels of BUN (median: 18.9 vs 9.6 mmol/L) and creatinine (327.1 vs 168 umol/L). The presence of dimeric NGAL was found to be associated with urinary tract infections. Most cats in the present study (126/159, 79.2%) and more than half of healthy cats (7/12, 58.3%) had detectable NGAL/MMP-9 complex present in their urine. CONCLUSIONS: The monomeric and dimeric molecular forms of uNGAL suggest upper and lower urinary tract origins of disease, respectively, whereas the presence of the uNGAL/MMP-9 complex is able to be detected in most cats, including seemingly healthy ones.

Expression and the molecular forms of neutrophil gelatinase-associated lipocalin and matrix metalloproteinase 9 in canine mammary tumours.

Neutrophil gelatinase-associated lipocalin (NGAL) is a new biomarker for renal injury. It is also involved in tumorigenesis of different human cancer types. The oncogenic role of NGAL is related to its molecular forms, and heterodimer formation with matrix metalloproteinase 9 (MMP9) promotes human breast cancer (HBC) invasion and metastasis. To date, the levels of NGAL and NGAL/MMP9 complex have not yet been explored in canine mammary tumours (CMTs). Hence, this study aimed to investigate whether NGAL and its molecular forms could be the biomarker for CMT diagnosis. To this end, expression profile of NGAL and MMP9 in mammary epithelial cells as well as in urine samples were detected. By immunohistochemistry staining, NGAL was expressed at variable levels. Unlike HBC, a significant reduction in NGAL expression was demonstrated in benign and malignant CMTs as compared with normal controls. Additionally, NGAL expression was significantly reduced in dogs with metastatic CMTs. By contrast, the mean score of MMP9 expression in ascending order was normal groups, benign, and malignant CMTs. Interestingly, analysis of the molecular form revealed the NGAL/MMP9 complex presents in most mammary tissues and urine of dogs with benign or malignant CMTs, whereas the complex was absent in samples from dogs without CMTs. In conclusion, NGAL and MMP9 are ubiquitously expressed in canine mammary epithelial cells in normal and cancerous status. However, the NGAL/MMP9 complex exclusively presents in mammary tissues and urine of dogs with tumours.

The association of indoxyl sulfate with fibroblast growth factor-23 in cats with chronic kidney disease.

BACKGROUND: Indoxyl sulfate (IS) has been reported not only to increase with the severity of impaired renal function, but also possibly to be a factor associated with bone abnormalities linked to fibroblast growth factor-23 (FGF-23) in humans with chronic kidney disease (CKD). It is not yet known whether this correlation between IS and FGF-23 holds true for cats with CKD. HYPOTHESIS: Accumulation of IS is related to FGF-23 secretion in cats with CKD. ANIMALS: Twenty clinically healthy cats and 73 cats with CKD cases were evaluated retrospectively. METHODS: The concentrations of IS and FGF-23 in plasma were determined by high-performance liquid chromatography and ELISA, respectively. Progression was defined as an increment of 0.5 mg/dL of serum creatinine concentration within 3 months. RESULTS: Plasma IS and FGF-23 concentrations were significantly increased concurrently with decreasing renal function. Higher concentration of FGF-23 was significantly associated with higher concentration of IS after adjusting for various confounding factors including creatinine and phosphate. Furthermore, the correlation between IS and phosphate was higher than that between FGF-23 and phosphate. When the renal progression group was compared with the non-progression group, both IS and FGF-23 were found to be significantly increased (P < .05). In addition, the area under receiver operator curve of the combination of IS and FGF-23 predicted renal progression at a level >0.9. CONCLUSIONS AND CLINICAL IMPORTANCE: Both FGF-23 and IS are associated with phosphate metabolism and CKD progression.

Honokiol, a Polyphenol Natural Compound, Attenuates Cisplatin-Induced Acute Cytotoxicity in Renal Epithelial Cells Through Cellular Oxidative Stress and Cytoskeleton Modulations.

Cisplatin is a potent anti-cancer drug that has been widely used in the treatment of various cancers; however, cisplatin administration results in severe nephrotoxicity and impedes its clinical applications. In this study, we showed that honokiol, a polyphenol constituent extracted from Magnolia officinalis exhibited a short-term protective effect against cisplatin-induced damages in renal epithelial cells in vitro. The protective effects of honokiol were resulted from the combination of (1) reduced cellular oxidative stress ranging from 53 to 32% reduction during a 24-h incubation, (2) the maintenance of cellular antioxidant capacity and (3) the stabilization of cytoskeletal structure of the kidney epithelial cells. By promoting the polymerization of actin (1.6-fold increase) and tubulin (1.8-fold increase) cytoskeleton, honokiol not only maintained epithelial cell morphology, but also stabilized cellular localizations of tight junction protein Occludin and adhesion junction protein E-Cadherin. With stabilized junction protein complexes and structural polymerized cytoskeleton network, honokiol preserved epithelial cell polarity and morphology and thus reduced cisplatin-induced cell disruption and damages. Our data demonstrated for the first time that honokiol could counteract with cisplatin-induced damages in renal epithelial cells in vitro, future in vivo studies would further validate the potential clinical application of honokiol in cisplatin-based cancer treatments with reduced nephrotoxicity.

The different molecular forms of urine neutrophil gelatinase-associated lipocalin present in dogs with urinary diseases.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a useful biomarker for the early prediction of renal diseases. NGAL may exist as monomer, dimer and/or NGAL/MMP-9 complex forms in humans. In this study, the existence of various forms of NGAL in urine (uNGAL) was determined and whether these forms are related to the different urinary diseases found in dogs is further discussed. RESULTS: Eighty-one urine samples from dogs with different forms of renal disease (41), pyuria (19) and a number of non-renal related diseases (10), as well as healthy dogs (11), were collected. uNGAL concentrations and their molecular forms in dogs were measured by ELISA and Western blot analysis, respectively. The uNGAL concentrations of dogs with pyuria (median: 15.35 ng/mL) were significantly higher than those of the healthy control animals (median: 3.92 ng/mL) (p < 0.01), but lower than those of dogs with renal diseases (median: 23.77 ng/mL). Each NGAL molecular form could be detected in dog urine. In particular, monomer was detected more frequently in patients with renal disease than those with non-renal diseases; while the dimer form appeared in a significantly higher percentage of cases with pyuria compared to those without pyuria. The NGAL/MMP-9 complex was found to exist not only in the patients with cystitis, but also in the cases with renal injury. CONCLUSION: Different molecular forms of uNGAL can indicate different origins of the urinary abnormalities. Determining the molecular forms of uNGAL present in diseased dogs may provide clinical workers with a tool that will help the early and more precise detection of different urinary diseases.

Urine neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute canine kidney injury.

BACKGROUND: Biomarkers for the early prediction of canine acute kidney injury (AKI) are clinically important. Recently, neutrophil gelatinase-associated lipocalin (NGAL) was found to be a sensitive biomarker for the prediction of human AKI at a very early stage and the development of AKI after surgery. However, NGAL has not yet been studied with respect to dog kidney diseases. The application of NGAL canine AKI was investigated in this study. RESULTS: The canine NGAL gene was successfully cloned and expressed. Polyclonal antibodies against canine NGAL were generated and used to develop an ELISA for measuring NGAL protein in serum and urine samples that were collected from 39 dogs at different time points after surgery.AKI was defined by the standard method, namely a serum creatinine increase of greater than or equal to 26.5 µmol/L from baseline within 48 h. At 12 h after surgery, compared to the group without AKI (12 dogs), the NGAL level in the urine of seven dogs with AKI was significantly increased (median 178.4 pg/mL vs. 88.0 pg/mL), and this difference was sustained to 72 h. CONCLUSION: As the increase in NGAL occurred much earlier than the increase in serum creatinine, urine NGAL seems to be able to serve as a sensitive and specific biomarker for the prediction of AKI in dogs.

Molecular detection of autosomal-dominant feline polycystic kidney disease by multiplex amplification refractory mutation system polymerase chain reaction.

Feline autosomal-dominant polycystic kidney disease (ADPKD), with its characteristic growth of fluid-filled cysts of different sizes, is the most prevalent inherited genetic disease of cats. The point mutation (C-->A transversion) in exon 29 of the PKD1 gene is known to contribute to ADPKD development and can thus serve as a target for the molecular genetic diagnosis of ADPKD. To this end, a simple amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) was designed with 3 primers: 2 forward primers specifically targeting either the mutant or normal allele, and 1 universal reverse primer for amplification of both alleles. The new method was tested on the DNA from 35 feline blood samples, which included 15 mutant cats and 20 wild type cats. As verified by direct DNA sequencing, both sensitivity and specificity of this tri-primer ARMS PCR were 100%. As the multiplex ARMS PCR test can be performed in a single PCR reaction without other post-PCR procedures, it is a simple and accurate method for molecular studies of feline ADPKD.

Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus.

Canine distemper (CD) is a widely distributed disease of dogs, caused by the canine distemper virus (CDV). In the present study, the gene encoding the hemagglutinin (H) protein of a CDV isolate from central Taiwan was sequenced and compared with other strains. Sequence variations were noticed in the H gene from the field CDV strain that had previously been implicated in the increasing incidence of CD. To establish a serology-based diagnostic test, the full-length H protein, as well as five deletion mutants of a recombinant H protein of the local isolate, were produced using an E. coli expression system. Three truncated recombinant proteins with relatively high expression levels, designated HM3, HM4 and HM5, were used as antigens to examine their reactivity with canine sera. By using three negative sera and 17 CD-positive sera, the high specificity of recombinant H proteins was observed by ELISA. In addition, immunoblotting demonstrated that all three purified recombinant proteins exhibit an antigenic property recognized by the serum of a CD-suspected dog.