WITHDRAWN: Correlation of SPINK1 gene polymorphisms in canine pancreatitis.

Ahead of Print article withdrawn by publisher.

Sound exposure-induced cytokine gene transcript profile changes in captive bottlenose dolphin (Tursiops truncatus) blood identified by a probe-based qRT-PCR.

Cetacean health may be potentially affected by anthropogenic sound. We have initiated investigations on the effect of low-frequency underwater sound on immunological gene transcript profiles of captive bottlenose dolphins (Tursiops truncatus) using a probe-based quantitative gene expression assay. Six immunologic genes (IL-2Rα, -4, -10, -12, TNFα and IFNγ) were selected for analysis using two validated housekeeping genes (PGK1 and HPRT1) as reference genes. Twenty-four blood samples from six clinically healthy individuals and six blood samples from individuals after sound exposures were available. The gene transcript profile of sound-exposed dolphins was consistent with a stress-induced TH2 shift profile as compared to controls. This study may lead to better understanding of the effects of anthropogenic sound on immune responses of cetaceans.

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas).

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

Development and validation of a new loop-mediated isothermal amplification for detection of pathogenic Leptospira species in clinical materials.

Leptospirosis, a global zoonotic disease, is often neglected but has a significant impact on human health. Here we reported a new loop mediated isothermal amplification (LAMP) assay targeting lipL32 gene of pathogenic Leptospira spp. Polymerase chain reaction (PCR), nested-PCR and real-time PCR assays were included in this study for the comparison of analytic and diagnostic sensitivity and specificity. LipL32 LAMP we designed enables detection of 10 copies of L. interrogans and has a higher diagnostic sensitivity (91.67%) and specificity (100%) than other PCR-based methods. The high sensitivity, specificity and flexible reaction conditions of the lipL32 LAMP assay makes it feasible for resource-limited countries and on-site application.

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.

Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus).

Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans.

Serological and PCR detection of feline leptospira in southern Taiwan.

Taiwan is in the subtropical zone and has typhoons every year. Leptospirosis is an endemic disease in Taiwan, and feline leptospirosis in Taiwan remains unknown so far. From January, 2010, to September, 2011, 233 cats in south Taiwan (159 stray cats and 74 household cats) were sampled in this research. Leptospira antibody titer was detected by the serology gold standard, the microscopic agglutination test (MAT). Both serum and urine were examined for Leptospira DNA by polymerase chain reaction (PCR) with two sets of primers. In this study, the serological survey showed 21 (9.3%) examined sera contained antibodies specific for pathogenic Leptospira serogroups. The results of PCR revealed that 25 (19.1%) serum and 80 (67.8%) urine samples were found positive for leptospiral DNA sequences. All products amplified from PCR reactions were sequenced by an automated method for further confirmation. This is the first study concerning the epidemiology of pathogenic Leptospira in stray and household cats' urine, and the results demonstrate that some of the cats are susceptible to pathogenic Leptospira and have the potential to shed pathogenic Leptospira into the environment. This could be an issue of public health.

Hematologic and biochemistry values for black-faced spoonbills (Platalea minor) with and recovering from botulism.

Type C1 botulism outbreaks in Black-faced Spoonbills (Platalea minor) occurred in Taiwan from 2002 to 2003, and hematologic and biochemistry parameters from botulism-paralyzed birds and recovered birds were compared. Values for creatinine and uric acid were higher (P<0.0025) in birds with botulism than in recovered birds. Lower white blood cell counts (P<0.005) and values for alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and triglycerides (P<0.025) were observed in recovered birds. Based on these observations, we suggest that hematologic and biochemistry analyses should be performed to assess the health condition of birds recovering from botulism.

Genomic diversity and molecular differentiation of Riemerella anatipestifer associated with eight outbreaks in five farms.

Riemerella anatipestifer causes infectious serositis of ducks and geese. The genomic diversity of R. anatipestifer associated with outbreaks in waterfowls was studied using 24 multidrug-resistant R. anatipestifer isolates collected from the visceral organs of ducks and geese from seven outbreaks in four goose farms and one outbreak in one duck farm. Seven methods were used to differentiate these isolates. Plasmid patterns differed in plasmid number and size, ranging from 2.9 kb to 20 kb, and provided seven profiles. Divergent nucleotide sequences (predominant in 670 to 830 base pairs) of the ompA gene categorized the 24 isolates into three groups based on cluster analysis and polymerase chain reaction-restriction fragment length polymorphism. Repetitive-sequence polymerase chain reaction and pulsed-field gel electrophoresis analysis revealed the highest genotypic variations among the isolates. Genotypes and serotypes differed among farms and within the same farm and even within a single goose. In conclusion, a difference in R. anatipestifer genotypes and serotypes was observed for multiple outbreaks in waterfowls.

Prevalence and characterization of multidrug-resistant (type ACSSuT) Salmonella enterica serovar Typhimurium strains in isolates from four gosling farms and a hatchery farm.

Salmonella enterica serovar Typhimurium strains of phage types DT104 and U302 are often resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (the ACSSuT resistance type) and are major zoonotic pathogens. Increased consumption of goose meat may enhance the risk of transferring S. enterica serovar Typhimurium and other enteric pathogens from geese to human due to the consumption of meats from infected geese or improper preparation of meats. Therefore, we characterized S. enterica serovar Typhimurium strains isolated from four goose farms (farms A, B, C, and D) and one hatchery farm (farm E) to determine the epidemic and genetic differences among them. Antibiotic susceptibility tests and multiplex PCR confirmed that 77.6% (52/67) of strains were ACSSuT strains isolated from farms A, C, and E. Antibiotic-susceptible strains were isolated mostly from farm B, and no strain was observed in farm D. All ACSSuT strains harbored a 94.7-kb virulence plasmid and contained one 1.1-kb conserved segment identical to that of Salmonella genomic island 1. Four genotypes were determined among these S. enterica serovar Typhimurium isolates by pulsed-field gel electrophoresis analysis of XbaI-digested DNA fragments. Most isolates (85.29%; 29/34) of major genotype Ib were ACSSuT strains isolated mainly from goslings of farm C and egg membranes of farm E, a hatchery farm, suggesting that S. enterica serovar Typhimurium strains in isolates from goslings might originate from its hatchery, from the egg membranes to the gosling fluff after hatching. Multiple phage types, types 8, 12, U283, DT104, and U302, were identified. In conclusion, geese were a reservoir of diverse multidrug-resistant (type ACSSuT) S. enterica serovar Typhimurium strains, and each farm was colonized with genetically closely related S. enterica serovar Typhimurium strains.