TaqMan real-time PCR for detection of pathogenic Leptospira spp. in canine clinical samples.

Introduction: Canine leptospirosis has always been a differential diagnosis in dogs presenting with clinical signs and blood profiles associated with kidney and/or liver disease. The conventional polymerase chain reaction (PCR) provides diagnoses, but real-time PCR-based tests provide earlier confirmation and determine the severity of infection, especially in the acute stage, allowing early detection for immediate treatment decisions. To our knowledge, real-time PCR has not been routinely adopted for clinical investigation in Malaysia. This study evaluated TaqMan real-time PCR (qPCR) assays diagnosing leptospirosis and compared their applicability to clinical samples from dogs with kidney and/or liver disease against a conventional PCR reference. Material and Methods: The qPCR assays were validated using existing leptospiral isolates. Whole blood and urine samples were analysed using a conventional PCR, LipL32(1) and LipL32(2) qPCRs and a microscopic agglutination test. The sensitivity and specificity of the qPCRs were determined. Results: The LipL32(1) qPCR assay had more diagnostic value than the LipL32(2) qPCR assay. Further evaluation of this assay revealed that it could detect as low as five DNA copies per reaction with high specificity for the tested leptospiral strains. No cross-amplification was observed with other organisms. Analysing the clinical samples, the LipL32(1) qPCR assay had 100.0% sensitivity and >75.0% specificity. Conclusion: The LipL32(1) qPCR assay is sensitive, specific and has the potential to be applied in future studies.

A cross-sectional study of Angiostrongylus malaysiensis in rats and gastropod hosts from recreational parks in Kuala Lumpur, Malaysia: Detection, risk factors and pathology.

Angiostrongylus malaysiensis is a potential zoonotic parasite, which reported to co-occur with A. cantonensis in human cerebrospinal fluid. It is a heteroxenous nematode that primarily develops through the early larval stages in gastropods and attains sexual maturity within rats. This study was conducted to determine the host species responsible for the reservoir of A. malaysiensis and investigate the risk factor for transmission among the hosts in Kuala Lumpur, Malaysia. Sampling was conducted in six recreational parks. The rats were trapped alive using steel wire traps with bait, while the gastropods were collected by active searching. The rats were euthanized and dissected to collect any adult worms observed. The molecular detection of A. malaysiensis was performed by PCR on gastropod tissue samples. Biotic and landscape factors were recorded for risk factor analysis. In total, 82 rats and 330 gastropods were collected throughout the study. Overall, 3.64% of gastropods and 32.9% of rats were infected with A. malaysiensis. Rattus tiomanicus (Malayan wood rat) and Parmarion martensi (Yellow-shelled semi-slug) were found as important hosts for A. malaysiensis. Host species, sampling site and macrohabitat type are risk factors associated with the prevalence of A. malaysiensis infection in rats. For gastropods, host species and sampling site are risk factors that correlate with the parasite detection. In total, 128 adult A. malaysiensis were recovered from the infected rats. The mean intensity of infection with adult A. malaysiensis was 4.65 for Rattus rattus complex and 4.90 for R. tiomanicus. Adult worms were found in the pulmonary artery or right ventricle, while eggs and first-stage larvae were found in capillaries of the caudal lung lobe. Infected lungs showed extravasated red blood cells in the alveolar spaces. The pulmonary arteries in the infected lung lobe were thickened. Kepong Metropolitan Park is the hotspot area for A. malaysiensis in Kuala Lumpur. These results provide essential information for public health officials to develop targeted interventions to reduce the transmission of A. malaysiensis in urban areas, particularly in recreational parks.

Clinicopathological and Radiological Features of Cats Presented with Infectious Respiratory Disease Signs: A Focus on Rhodococcus equi and Klebsiella pneumoniae.

The objective of this study was to evaluate the prevalence of involvement of common viral organisms R. equi and K. pneumoniae and their clinicopathological and radiological features in respiratory disease of Malaysian domestic cats. A total of 34 feline cases with acute/chronic infectious respiratory disease signs were followed prospectively to investigate respiratory disease due to R. equi and K. pneumoniae and their relationship with concurrent viral infections in disease manifestation. All sampled cats (n = 27) were positive for FCoV antibodies and negative for FeLV. A significantly high antibody titer for FCV in n = 26 cases was also noticed. A single sample of pyothorax from a 3-months-old, non-vaccinated kitten was positive for R. equi. Bronchopneumonia with severe infiltration of the polymorphs and mononuclear inflammatory cells were prominent features of lungs histopathology from the kitten positive for R. equi. K. pneumoniae subsp. pneumoniae was confirmed from tracheal swabs of two cats. Histologically, the tracheal tissues of the two cats positive for K. pneumoniae were normal. In diagnostic imaging, epicenter of the infectious URT disease was nasal conchae rostrally and nasal turbinates caudally, however for infectious LRT disease was bronchial tree. Conclusively, infectious respiratory disease is a complex illness in cats, predominantly for unvaccinated kittens and young adult cats, especially those kept in multi-cat household or shelter environments because of the involvement of multiple bacterial and viral organisms as primary or secondary invaders. Clinicians should not preclude feline rhodococcosis from differentials, especially in kittens with pyothorax and less than one year of age. Unlike R. equi, K. pneumoniae has the potential to colonize URT of cats which might be disseminating further to cause LRT disease.

Protective effects of Labisia pumila against neuropathy in a diabetic rat model.

Purpose: Diabetes accelerates peripheral, distal symmetric polyneuropathy, small fiber predominant neuropathy, radiculoplexopathy, and autonomic neuropathy. This study investigated the neuroprotective effects of gallic acid and myricetin-rich Labisia pumila extract in a diabetic neuropathy rat model and evaluated the neuropathy correlationship with serum inflammatory biomarkers. Methods: Thirty male rats were divided into 5 groups (n = 6), namely: healthy control; non-treated diabetic control; and diabetic-rats treated with 200 mg/kg metformin; Labisia pumila ethanol extract (LP) at 150 mg/kg or 300 mg/kg doses. Diabetes was induced by 60 mg streptozotocin /kg intraperitoneal injection. Rats were orally treated daily for ten weeks. Their fasting blood glucose (FBG), neurological functions (hot plate and tail immersion; thermal hyperalgesia; cold allodynia; motor walking function), biomarkers for inflammation and oxidative stress, the neuro-histopathological changes, and brain somatic index were measured. Results: The extract significantly prevented abnormal increases in FBG and decreases in body weight gain. It attenuated behavioral dysfunctions (hot plate and tail immersion; thermal hyperalgesia; cold allodynia; motor walking function), systemic inflammation (serum TNF-α, prostaglandin-E2) oxidative tension (malondialdehyde), histological brain and sciatic nerve injuries in the diabetic-rats, better than Metformin. Conclusion: LP mitigated neural dysfunction better than metformin partly by amending diabetic systemic inflammation, oxidative tension, and diabetic abnormalities. The nerve injuries were strongly correlated to serum prostaglandin-E2, TNF-α levels, and walking functions. The motor function was correlated to sensory neuronal functions, inflammation, and oxidation. The sensory neuronal functions were more affected by TNF-α than prostaglandin-E2 or oxidation. Diabetic brain and sciatic nerve deteriorations were influenced by serum TNF-α, PGE2, and MDA levels. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-021-00905-0.

Comparison between hydroxyapatite and polycaprolactone in inducing osteogenic differentiation and augmenting maxillary bone regeneration in rats.

Background: The selection of appropriate scaffold plays an important role in ensuring the success of bone regeneration. The use of scaffolds with different materials and their effect on the osteogenic performance of cells is not well studied and this can affect the selection of suitable scaffolds for transplantation. Hence, this study aimed to investigate the comparative ability of two different synthetic scaffolds, mainly hydroxyapatite (HA) and polycaprolactone (PCL) scaffolds in promoting in vitro and in vivo bone regeneration. Method: In vitro cell viability, morphology, and alkaline phosphatase (ALP) activity of MC3T3-E1 cells on HA and PCL scaffolds were determined in comparison to the accepted model outlined for two-dimensional systems. An in vivo study involving the transplantation of MC3T3-E1 cells with scaffolds into an artificial bone defect of 4 mm length and 1.5 mm depth in the rat's left maxilla was conducted. Three-dimensional analysis using micro-computed tomography (micro-CT), hematoxylin and eosin (H&E), and immunohistochemistry analyses evaluation were performed after six weeks of transplantation. Results: MC3T3-E1 cells on the HA scaffold showed the highest cell viability. The cell viability on both scaffolds decreased after 14 days of culture, which reflects the dominant occurrence of osteoblast differentiation. An early sign of osteoblast differentiation can be detected on the PCL scaffold. However, cells on the HA scaffold showed more prominent results with intense mineralized nodules and significantly (p < 0.05) high levels of ALP activity with prolonged osteoblast induction. Micro-CT and H&E analyses confirmed the in vitro results with bone formation were significantly (p < 0.05) greater in HA scaffold and was supported by IHC analysis which confirmed stronger expression of osteogenic markers ALP and osteocalcin. Conclusion: Different scaffold materials of HA and PCL might have influenced the bone regeneration ability of MC3T3-E1. Regardless, in vitro and in vivo bone regeneration was better in the HA scaffold which indicates its great potential for application in bone regeneration.

Palatability assessment of prescribed diets on domestic shorthair cats.

Background and Aim: The value of the pet food industry, which is majorly the prescribed diet, exponentially increased over the years due to increased awareness among pet owners to provide a healthy lifestyle for their pets. However, several factors such as aroma, flavor, texture, and shape of prescribed diets greatly influenced the palatability in cats. Therefore, this study aimed to determine the palatability of the prescribed diet for domestic shorthair (DSH) cats. Materials and Methods: The two-bowl method was employed to determine the palatability of prescribed diets on five DSH cats for 6 days. Furthermore, the four types of prescribed diet assessed in this study were struvite, renal, hypersensitivity, and intestinal. Furthermore, the pet food palatability was analyzed using "First Approached," "First Consumed," "Total Consumption," and "Intake ratios." Results: Our findings revealed that "Total Consumption" and "Intake Ratios" were significantly different in struvite, renal, and intestinal diets compared to the hypersensitivity diet. In addition, this result indicates that the hypersensitivity diet is the most unpalatable compared with other diets. Conclusion: A detailed investigation of the diet ingredients showed that a hypersensitivity diet lacks herbs and spices than the other diets. Therefore, these ingredients lacking in the hypersensitivity diet influence the palatability of pet foods.

Synovial fluid proteome profile of surgical versus chemical induced osteoarthritis in rabbits.

BACKGROUND: Animal models are significant for understanding human osteoarthritis (OA). This study compared the synovial fluid proteomics changes in surgical and chemical induced OA models. METHODS: Thirty rabbits either had anterior cruciate ligament transection (ACLT) procedure or injected intra-articularly with monosodium iodoacetate (MIA, 8 mg) into the right knee. The joints were anatomically assessed, and the synovial fluid proteins analyzed using two-dimensional polyacrylamide gel electrophoresis (2DGE) and MALDI TOF/TOF mass spectrometry analysis at 4, 8 and 12 weeks. The proteins' upregulation and downregulation were compared with control healthy knees. RESULTS: Seven proteins (histidine-rich glycoprotein, beta-actin-like protein 2 isoform X1, retinol-binding protein-4, alpha-1-antiproteinase, gelsolin isoform, serotransferrin, immunoglobulin kappa-b4 chain-C-region) were significantly expressed by the surgical induction. They characterized cellular process (27%), organization of cellular components or biogenesis (27%), localization (27%) and biological regulation (18%), which related to synovitis, increased cellularity, and subsequently cartilage damage. Three proteins (apolipoprotein I-IV precursor, serpin peptidase inhibitor and haptoglobin precursor) were significantly modified by the chemical induction. They characterized stimulus responses (23%), immune responses (15%), biological regulations (15%), metabolism (15%), organization of cellular components or biogenesis (8%), cellular process (8%), biological adhesions (8%) and localization (8%), which related to chondrocytes glycolysis/death, neovascularization, subchondral bone necrosis/collapse and inflammation. CONCLUSIONS: The surgical induced OA model showed a wider range of protein changes, which were most upregulated at week 12. The biological process proteins expressions showed the chemical induced joints had slower OA progression compared to surgical induced joints. The chemical induced OA joints showed early inflammatory changes, which later decreased.

Risk and Predictive Factors of Leptospirosis in Dogs Diagnosed with Kidney and/or Liver Disease in Selangor, Malaysia.

Canine leptospirosis is commonly associated with kidney and/or liver disease. It has been widely reported and causes public health concerns due to its zoonotic potential and its re-emergence, resulting from close contact between humans and dogs. The current study identified potential risk and predictive factors for dogs diagnosed with kidney and/or liver disease due to leptospirosis. A total of 124 client-owned dogs were recruited, and information such as signalment, medical history, management, and clinical findings were documented. Samples collected from the recruited dogs were directly tested using polymerase chain reaction (PCR) and subsequently inoculated for bacterial isolation. Statistical analyses were descriptively analyzed, and risk analyses were performed using Pearson chi-square tests and logistic regression. A total of 53 dogs (42.7%) were positive for leptospiral infection based on PCR, and 10 leptospiral isolates were successfully recovered from eight dogs. The mortality rate of infected dogs was 34.0% (18/53). Medium and large dog breeds, with a history of exposure to rats, and managed outdoors had a greater risk for leptospirosis (p < 0.05). The significant predictors for the dogs' positivity were the presence of rats and acute clinical illness (p < 0.05). Administration of antibiotics and the detection of clinical illness at an early stage of the disease improved the survivability of the dogs (p < 0.05). Identifying the profile of dogs that are at risk to leptospirosis could be useful in the design of diagnostic and treatment strategies, as well as to increase awareness for prevention of the disease.

Comparison of bone and articular cartilage changes in osteoarthritis: a micro-computed tomography and histological study of surgically and chemically induced osteoarthritic rabbit models.

BACKGROUND: Osteoarthritis (OA) is a multifaceted condition that affects both the subchondral bones and the articular cartilage. Animal models are widely used as an effective supplement and simulation for human OA studies in investigating disease mechanisms and pathophysiology. This study is aimed to evaluate the temporal changes of bone and cartilage in surgically and chemically induced osteoarthritis using micro-computed tomography and histology. METHODS: Thirty rabbits underwent either anterior cruciate ligament transection (ACLT) procedure or injected intraarticularly with monosodium iodoacetate (MIA, 8 mg) at the right knee joint. The subchondral bones were scanned via micro-CT, and articular cartilage was assessed histologically at 4-, 8- and 12-week post-induction. RESULTS: Based on bone micro-architecture parameters, the surgically induced group revealed bone remodelling processes, indicated by increase bone volume, thickening of trabeculae, reduced trabecular separation and reduced porosity. On the other hand, the chemically induced group showed active bone resorption processes depicted by decrease bone volume, thinning of trabeculae, increased separation of trabecular and increased porosity consistently until week 12. Histologically, the chemically induced group showed more severe articular cartilage damage compared to the surgically induced group. CONCLUSIONS: It can be concluded that in the ACLT group, subchondral bone remodelling precedes articular cartilage damage and vice versa in the MIA group. The findings revealed distinct pathogenic pathways for both induction methods, providing insight into tailored therapeutic strategies, as well as disease progression and treatment outcomes monitoring.

Gallic acid and myricetin-rich Labisia pumila extract mitigated multiple diabetic eye disorders in rats.

Diabetes affected about a quarter of a billion people globally, and one out of four diabetics has eye or vision problems. This study investigated whether gallic acid and myricetin-rich Labisia pumila extract (LP) consumption would help prevent diabetic eye disorders and some probable biochemistry involved relating to inflammation, vascular leakage, and oxidative tension. Male rats were divided into four groups (n = 6), namely healthy control, diabetic non-treated control, and hyperglycemic rats treated with 150 or 300 mg/kg LP. Intraperitoneal injection of 60 mg/kg streptozotocin was used to induce diabetes. Rats were fed in the morning and evening. Diabetic retinopathy was graded in rats using a dilated retinal digital ophthalmoscopy. Rats were sacrificed at 12 weeks and the retina, optic nerve, cornea, lens, sclera, ciliary bodies, iris, and conjunctiva were examined histologically. The diabetic rats consuming LP for 10 weeks showed dose-dependent, histopathologically-reduced eye abnormalities (keratopathy, cataract, sclera, conjunctiva, ciliary bodies, iris, limbus, corneal edema, epithelial barrier inefficiency, shallow punctate keratitis, lower basal layer cell density, retinopathy, glaucoma, and corneal changes). The LP significantly suppressed inflammation [increased serum tumor necrosis factor-α (TNF-α), prostaglandin-E2 (PGE2)], vascular leakage [claudin-1], abnormal vascularization [vascular endothelial growth factor (VEGF)], oxidative tension [malondialdehyde/reduced glutathione ratio], and hyperglycemia [fasting blood glucose] of the diabetic rats. The LP consumption was significantly protective against diabetic eye disorders and optic nerve dysfunction which were related to inflammation, vascular leakage, abnormal vascularization, and oxidative tension, which most likely influenced eye hemorrhage and collagen cross-linkage. PRACTICAL APPLICATIONS: The study shows that gallic acid and myricetin-rich Labisia pumila (LP) leaf consumption may be used as a complementary therapy for managing diabetes (fasting blood glucose) and preventing diabetic eye disorders (keratopathy, cataract, sclera, conjunctiva, ciliary bodies, iris, limbus, corneal edema, epithelial barrier inefficiency, shallow punctate keratitis, lower basal layer cell density, retinopathy, glaucoma, and corneal abnormalities). The LP consumptions reduced the serum biomarkers for inflammation (serum tumor necrosis factor-α TNF-α; prostaglandin-E2), vascular leakage/abnormalities (claudin-1 and vascular endothelial growth factor VEGF), and oxidative tension (malondialdehyde/reduced glutathione MDA/GSH ratio). The LP was eye-protective probably by normalizing fasting blood glucose, reducing inflammation, oxidative tension, vascular leakage, and irregular vascularization.

A Retrospective Study on Bacteriology, Clinicopathologic and Radiographic Features in 28 Cats Diagnosed with Pyothorax.

This retrospective study aimed to determine the etiological, clinicopathological, and radiographic features and outcome of feline pyothorax cases. Medical records from twenty-eight cats with pyothorax aged from 4 months to 10 years (median 10 months) diagnosed between 2013 and 2020 were reviewed. Dyspnoea (75.0%), abnormal lung sounds (75.0%) and open-mouth breathing (64.3%) were the predominant respiratory signs. Leucocytosis (61.5%), particularly monocytosis (68.0%), and hyperglobulinaemia (65.4%) were among the most prominent findings in blood analysis. Bilateral pleural effusion was found in 67.9% of the thoracic radiographs. A total of 47.4% of the cytological samples revealed the presence of bacteria, while all had positive bacterial growth. Pasteurella multocida, E. coli, Streptococcus spp., and Staphylococcus spp. were the predominant aerobic bacteria isolated from pleural effusion samples. A chest tube was placed in 64.3% of the cats and 66.7% of cats with chest tubes survived. In total, 46.4% of cats with pyothorax recovered. Amoxicillin-clavulanate was the antimicrobial of choice against aerobic bacteria found in this study and should be given in combination with antimicrobials that cover anaerobic bacteria. Chest tube placement is crucial for treatment success. Cytological results and bacterial culture may not be consistent; thus, bacterial culture should be performed for every case.

Shedding and Genetic Diversity of Leptospira spp. From Urban Stray Dogs in Klang Valley, Malaysia.

Leptospirosis is an endemic zoonoses of global proportions. Stray dogs have been postulated to play a role in disease transmission; however, supporting information are still limited. Roaming behavior may not only predispose the dogs to infection, but could also contribute to disease spread. In this study, the susceptibility of urban stray dogs in shedding Leptospira spp. was determined. Blood, urine, and tissue samples of kidney and liver were collected from 100 dogs from 2 animal control facilities. Serological testing using microscopic agglutination test (MAT) were performed on blood against 20 leptospiral serovars with a cut-off titre of ≥ 1:100. Samples were cultured onto semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media. Isolates were identified using molecular polymerase chain reaction (PCR) using 2 primers (16s rRNA and LipL32) and hyperimmune serum (HIS) MAT. The seroprevalence for the dogs positive for leptospirosis was 32% (n=32/100) with the following detected serovars: Javanica (n=13), Bataviae (n=10), Icterohaemorrhagiae (n=3), Autumnalis (n=2), Canicola (n=1), Pyrogenes (n=1), Copenhageni (n=1), and Australis (n=1). Six Leptospira spp. isolated were procured from urine (n=2), kidney (n=2) and liver (n=2). All 6 isolates belonged to L. interrogans, a pathogenic variant of Leptospira spp. Serotyping and phylogenetic analysis suggested serovar Bataviae (n=5) and serovar Canicola (n=1). Presence of vaccinal serovars (Icterohaemorrhagiae and Canicola) suggested potential post-vaccination antibodies but the predominance of non-vaccinal serovars (Javanica and Bataviae) indicate the possibility of current infection or post-exposure. Isolation of Leptospira spp. directly from urine sample not only suggested an active infection but highlighted the potential shedding capability among these stray dogs. These findings further strengthen speculations that urban stray dogs could play a role in transmission and dissemination of leptospirosis through their constant movement. The urine of infected dogs may contaminate the environment, posing a major public health threat.

Detection and characterization of Leptospira spp. in dogs diagnosed with kidney and/or liver disease in Selangor, Malaysia.

Leptospirosis is a serious bacterial disease that affects both humans and animals. A wide range of symptoms have been described in humans; the disease in dogs is commonly associated with kidney and/or liver disease. In Malaysia, information about the common serovars infecting dogs is limited. Therefore, we investigated the occurrences of leptospirosis in 124 pet dogs diagnosed with kidney and/or liver disease. Blood, urine, abdominal effusion, and/or kidney and liver were collected from the dogs. Based on microscopic agglutination testing, 53 of 124 (42.7%) dogs were seropositive for leptospiral exposure. Sera were frequently positive to serovars Bataviae (n = 12), Javanica (n = 10), and Icterohaemorrhagiae (n = 10). Direct detection using PCR showed that 42 of 124 (33.9%) of the whole blood and 36 of 113 (31.9%) urine samples were positive for pathogenic Leptospira spp. By PCR, 2 of 23 (9.1%) kidney and 2 of 23 (9.1%) liver were positive for pathogenic Leptospira spp. Abdominal effusion from 4 dogs were PCR-positive for pathogenic Leptospira spp. The species detected were L. interrogans, L. borgpetersenii, L. kirschneri, and L. kmetyi by partial 16S rRNA sequencing. We further identified and characterized 11 Leptospira spp. isolates from 8 dogs as serovars Bataviae, Javanica, and Australis. The mortality rate of the Leptospira-infected dogs was high (18 of 53; 34%).

Prevalence of Toxoplasma gondii Antibodies in Stray Dogs from Various Locations in West and East Malaysia.

Toxoplasmosis is caused by an obligate intracellular protozoan parasite; Toxoplasma gondii, which is one of the most important zoonotic parasite worldwide. In dogs, the sexual reproductive cycle of T. gondii is lacking, and the animals are not widely consumed as food, but they are vital in the mechanical transmission of the parasite. However, there is no present data on the exposure of stray dogs to T. gondii in Malaysia. The objective of this serological survey was to determine the prevalence of T. gondii antibodies (IgG) and associated factors in stray dogs in East and West Malaysia. Antibodies to T. gondii were determined in serum samples from 222 stray dogs from 6 different states in East and West Malaysia (Peninsular Malaysia) using an Indirect ELISA. The seroprevalence for T. gondii was 23.4% (Confidence interval: CI 17.8-29.2%). Stray dogs from Selangor and Kuala Lumpur had the highest seroprevalence (32.4%; CI 13.2-45.5%) and lowest in those from Penang and Kedah (12.5%; CI 1.3-23.5%). Gender and breed were not associated with T. gondii seropositivity. However, adult dogs were more likely to be seropositive for T. gondii (OR=2.89; CI 1.1-7.7) compared with younger dogs. These results revealed that T. gondii is prevalent in stray dogs in the studied areas in Malaysia, and indicative of the level of environmental contamination of this parasite especially in urban areas.

Citrus hystrix leaf extract attenuated diabetic-cataract in STZ-rats.

Diabetic cataract causes severe vision loss. This study evaluated the effects of hesperidin-standardized Citrus hystrix leaf flavonoids-rich extract (CLE) on diabetic-cataract development. Streptozotocin-induced diabetic rats were orally given 150 and 300 mg CLE/kg body-weight. These were compared with non-treated diabetic or healthy rats as controls, over 8 weeks. The CLE gradually attenuated fasting blood glucose (FBG), biomarkers for inflammation (Tumor necrosis factor alpha TNF-α; prostaglandin E2 PGE2); vascular permeability, (Vascular endothelial growth factor VEGF); and oxidative stress, (malondialdehyde MDA). The diabetic cataract was significantly mitigated by the 150 mg CLE/kg dose. Good correlations were found between cataract incidence with FBG (r2  = 0.90), serum PGE2 (r2  = 0.91), MDA (r2  = 0.99), VEGF (r2  = 0.71), but not with TNF-α levels (r2  = 0.49) suggesting the serum FBG, PGE2, MDA, and possibly the VEGF levels may help to predict the cataract risks. The CLE mitigated cataract probably by attenuating hyperglycaemia, inflammation, lens fluid influx, vascular leakage, lens osmotic-imbalance, and fibers over-hydration. PRACTICAL APPLICATIONS: The study shows the flavonoids-rich Citrus hystrix leaf consumption, effectively attenuated diabetes (fasting blood glucose) and mitigated diabetic cataract. It help reduce diabetes-related hyperglycaemia, oxidative stress, inflammation, and vascular leakage. The evidences were the CLE consumptions reduced the serum biomarkers tumor necrosis factor-alpha TNF-α; prostaglandin E2 PGE2, vascular endothelial growth factor (VEGF), and malondialdehyde (MDA). The C. hystrix leaf contains hesperidin, apiin, diosmin, saponarin, apigetrin, rutin and xanthotoxol, and other flavonoid glucosides. The study also showed good correlations between cataract incidence with fasting blood glucose FBG (r2  = 0.90), serum PGE2 (r2  = 0.91), and MDA (r2  = 0.99), and less closely with VEGF (r2  = 0.71) suggesting these serum biomarkers may help predict cataract risks. The CLE indicated cataract mitigation properties probably by attenuating FBG, inflammation, lens fluid influx, lens osmotic-imbalance, and fibers over-hydration.

Clinicopathological and radiographic features in 40 cats diagnosed with pulmonary and cutaneous Rhodococcus equi infection (2012-2018).

OBJECTIVES: This retrospective study aimed to describe clinical manifestations, diagnostic options, radiological features, therapeutic plans and outcomes for cats infected with Rhodococcus equi. METHODS: Forty cats aged between 2 months and 11 years old (median 6 months) that were definitively diagnosed with rhodococcosis between 2012 and 2018 were recruited in this study. Medical records were reviewed for information on signalment, history, clinical presentation, diagnostic testing, treatment plans and clinical outcomes. RESULTS: Of the 40 cats, 36 showed the pulmonary form of the disease, with 35 (87.5%) presenting with dyspnoea, while four cats presented with only cutaneous lesions. Mean body temperature was 38.7 ± 0.2°C. Dyspnoea was noted in 87.5% of the cats. Leukocytosis (58.3%) with band neutrophilia (83.3%), monocytosis (58.3%) and thrombocytopenia (55.5%) were prominent findings in the haematology reports. Hyperproteinaemia (61.1%) with hypoalbuminaemia (22.2%) and hyperglobulinaemia (63.8%) with a low albumin:globulin ratio (38.9%) were prominent features of blood biochemistry reports. An alveolar-interstitial pattern was noted in 75% of pre-thoracocentesis radiographs. Pleural effusion, hepatomegaly, thoracic lymphadenopathy and atelectasis of any lung lobe were seen in 88.9%, 75%, 41.7% and 36.1% of cats, respectively. Overall, the mortality rate was 67.5% in both forms. CONCLUSIONS AND RELEVANCE: Clinicians should be aware that feline rhodococcosis manifests as a pulmonary disease at a much higher rate than previously reported. Further studies are required to address the epidemiology, pathophysiology, disease management and prognosis of feline rhodococcosis. The role of immunosuppression as a predisposing factor in feline rhodococcosis requires further investigation.

Comparison of diclofenac with apigenin-glycosides rich Clinacanthus nutans extract for amending inflammation and catabolic protease regulations in osteoporotic-osteoarthritis rat model.

BACKGROUND: Osteoporotic-osteoarthritis is an incapacitating musculoskeletal illness of the aged. OBJECTIVES: The anti-inflammatory and anti-catabolic actions of Diclofenac were compared with apigenin-C-glycosides rich Clinacanthus nutans (CN) leaf extract in osteoporotic-osteoarthritis rats. METHODS: Female Sprague Dawley rats were randomized into five groups (n = 6). Four groups were bilateral ovariectomised for osteoporosis development, and osteoarthritis were induced by intra-articular injection of monosodium iodoacetate (MIA) into the right knee joints. The Sham group was sham-operated, received saline injection and deionized drinking water. The treatment groups were orally given 200 or 400 mg extract/kg body weight or 5 mg diclofenac /kg body weight daily for 28 days. Articular cartilage and bone changes were monitored by gross and histological structures, micro-CT analysis, serum protein biomarkers, and mRNA expressions for inflammation and catabolic protease genes. RESULTS: HPLC analysis confirmed that apigenin-C-glycosides (shaftoside, vitexin, and isovitexin) were the major compounds in the extract. The extract significantly and dose-dependently reduced cartilage erosion, bone loss, cartilage catabolic changes, serum osteoporotic-osteoarthritis biomarkers (procollagen-type-II-N-terminal-propeptide PIINP; procollagen-type-I-N-terminal-propeptide PINP; osteocalcin), inflammation (IL-1ß) and mRNA expressions for nuclear-factor-kappa-beta NF-κß, interleukin-1-beta IL-1ß, cyclooxygenase-2; and matrix-metalloproteinase-13 MMP13 activities, in osteoporotic-osteoarthritis rats comparable to Diclofenac. CONCLUSION: This study demonstrates that apigenin-C-glycosides at 400 mg CN extract/kg (about 0.2 mg apigenin-equivalent/kg) is comparable to diclofenac in suppressing inflammation and catabolic proteases for osteoporotic-osteoarthritis prevention. Graphical abstract.

First report of pathogenic Leptospira spp. isolated from urine and kidneys of naturally infected cats.

Leptospirosis is one of the most widespread zoonotic diseases and can infect both humans and animals worldwide. Healthy cat, as a potential source of exposure to humans, are likely underestimated owing to the lack of overt clinical signs associated with Leptospira spp. infection in this species. The aim of the study was to determine the exposure, shedding, and carrier status of leptospires in shelter cats in Malaysia by using serological, molecular, and bacteriological methods. For this study, 82 healthy cats from two shelters were sampled. The blood, urine, and kidneys were tested using the microscopic agglutination test (MAT), polymerase chain reaction (PCR), and bacterial culture. On the basis of serological, molecular, and/or culture techniques, the total detection of leptospiral infection was 29.3% (n = 24/82). Through culture techniques, 16.7% (n = 4/24) of the cats that tested positive were carriers with positive kidney cultures, and one cat was culture positive for both urine and kidney. The Leptospira spp. isolates were identified as pathogenic L. interrogans serovar Bataviae through serological and molecular methods. Through serological techniques, 87.5% (n = 21/24) had positive antibody titers (100-1600) and most of the Bataviae serogroup (n = 19/21). Using PCR, 16.7% (n = 4/24) of cats were shown to have pathogenic Leptospira spp. DNA in their urine. Furthermore, three out of four culture positive cats were serology negative. The present study reports the first retrieval of pathogenic leptospires from urine and kidneys obtained from naturally infected cats. The results provide evidence of the potential role of naturally infected cats in the transmission of leptospires. Additionally, leptospiral infection occurs sub-clinically in cats. The culture isolation provides evidence that healthy cats could be reservoirs of leptospiral infection, and this information may promote the development of disease prevention strategies for the cat population.

Using field training in indigenous communities as a method of creating awareness of the one health concept among Malaysian university students: a non-experimental pre and post-test intervention study.

BACKGROUND: This paper describes the result of workshops conducted to increase the knowledge and awareness of university students using a multidisciplinary, collaborative, multisectoral and trans-disciplinary approach concerning One Health and the indigenous people of peninsular Malaysia called the Orang Asli. METHODS: A non-experimental pre and post-test intervention study was carried out among medical, veterinary and allied health students from six public and private universities who attended workshops on One Heath in two Orang Asli communities living by the Temenggor lake in Malaysia as part of the Malaysia One Health University Network (MYOHUN) efforts in training future and present One Health workforce. RESULTS: There was a significant increase in various aspects of knowledge and interest concerning One Health and the Orang Asli. The mean knowledge scores of One Health (p < 0.001) and Orang Asli (p < 0.001) increased significantly post workshop. A repeated measures ANOVA with a Greenhouse-Geisser correction showed the mean scores of knowledge of One Health F (1, 166) = 127.198, p < 0.001) and Orang Asli F (1, 166) = 214.757, p < 0.001) differed statistically significantly between the two time points. The test revealed that the score differences for knowledge on One Health (mean difference = 1.796, p < 0.001) and Orang Asli (mean difference = 4.940, p < 0.001) were statistically significant. Repeated measures ANOVA showed a significant difference in the knowledge on Orang Asli between the students of different courses F (4,166) = 3.734, p-0.006. The difference in the One Health knowledge scores between the students of different courses was not statistically significant F (4,166) = 0.998, p = 0.410. CONCLUSIONS: Emphasis on field training in relation to One health can provide university students greater levels of preparedness to combat zoonotic diseases.

Prevalence of Toxoplasma gondii Antibodies in Stray Dogs from Various Locations in West and East Malaysia

Toxoplasmosis is caused by an obligate intracellular protozoan parasite; Toxoplasma gondii, which is one of the most important zoonotic parasite worldwide. In dogs, the sexual reproductive cycle of T. gondii is lacking, and the animals are not widely consumed as food, but they are vital in the mechanical transmission of the parasite. However, there is no present data on the exposure of stray dogs to T. gondii in Malaysia. The objective of this serological survey was to determine the prevalence of T. gondii antibodies (IgG) and associated factors in stray dogs in East and West Malaysia. Antibodies to T. gondii were determined in serum samples from 222 stray dogs from 6 different states in East and West Malaysia (Peninsular Malaysia) using an Indirect ELISA. The seroprevalence for T. gondii was 23.4% (Confidence interval: CI 17.8-29.2%). Stray dogs from Selangor and Kuala Lumpur had the highest seroprevalence (32.4%; CI 13.2-45.5%) and lowest in those from Penang and Kedah (12.5%; CI 1.3-23.5%). Gender and breed were not associated with T. gondii seropositivity. However, adult dogs were more likely to be seropositive for T. gondii (OR=2.89; CI 1.1-7.7) compared with younger dogs. These results revealed that T. gondii is prevalent in stray dogs in the studied areas in Malaysia, and indicative of the level of environmental contamination of this parasite especially in urban areas.