Bioactivity of orange-spotted grouper (Epinephelus coioides) cathepsin L: Proteolysis of bacteria and regulation of the innate immune response.

Cathepsin L (CTSL) is a cysteine endopeptidase involved in protein degradation mainly in lysosomes. Following activation in an acidic environment, it plays a key role in a variety of physiological, immunological, and pathological processes. The biological function of CTSL in teleost remains unclear. Immunohistochemical analysis revealed that CTSL was expressed mainly in lymphoid organs, head kidney, trunk kidney, and liver, which particularly was expressed in leukocyte-like cells. We performed two forms of recombinant CTSL (rCTSL and rTCTSL) derived from orange-spotted grouper (Epinephelus coioides) to elucidate the role of CTSL in teleost innate immunity, based on differences in immune-related gene expression. We determined that rCTSL has a proteolytic function whereas rTCTSL does not. Under CTSL activation, we observed increases in IL-1ß, IL-6, IL-12, IFNγ, CCL-1, CCL-3, epinecidin-1, lysozyme, and IgM. The bacteriolytic activity of rCTSL was more pronounced against Gram-positive bacteria than Gram-negative bacteria. Our findings indicate CTSL plays multiple roles in the reactions of innate immunity.

BACTERIAL IDENTIFICATION AND ANTIBIOTIC SENSITIVITY FROM THE ABSCESSES OF CAPTIVE TORTOISES-CLINICAL ANTIBIOTIC RECOMMENDATIONS.

Bacterial abscesses are commonly seen in tortoises. The morbidity and the resultant mortality are high. Multifactorial problems, antibiotics misapplication. and antibiotic-resistant bacteria make abscess treatment complicated and ineffective. This study identifies the etiological bacterial species and determines the best antibiotics for abscess treatment in captive tortoises. Sterile swab specimens from 40 tortoises with abscesses were analyzed using the Analytical Profile Index (API) system. Sixty-five bacteria species were identified covering facultative anaerobic gram-negative (n = 30, 46.2%), facultative anaerobic gram-positive (n = 19, 29.2%), and aerobic gram-negative bacteria (n = 16, 24.6%). The antibiotic sensitivity of these bacteria to 30 antibiotics was assessed using the Kirby-Bauer disk-diffusion method. Greater than 80% anaerobic gram-negative bacterial species showed sensitivity to amikacin and ceftazidime. Greater than 80% anaerobic gram-positive bacterial species were sensitive to amoxicillin, ampicillin, carbenicillin, and penicillin. In addition, more than 80% aerobic gram-negative bacterial species were sensitive to ceftazidime, colistin sulphate, amikacin, gentamicin, kanamycin, polymyxin B, and tobramycin. This study provides clinicians significant information for initial antibiotic options, which could elevate the abscess therapy success rate and improve the life quality of tortoises.

ANIMAL TRAINING AND ACUPUNCTURE IN A BENGAL TIGER ( PANTHERA TIGRIS TIGRIS) WITH HIND LIMB PARAPARESIS.

An 18-yr-old, captive-born male Bengal tiger ( Panthera tigris tigris) presented a 1-yr history of chronic and progressive paraparesis in both hind limbs. Lateral and hind limb radiographs were revealed normal except for severe spondylosis deformans, forming a bony bridge between the last lumbar and the first sacral vertebra and disc mineralization between the second and third lumbar vertebra. Medical therapies were instituted, including corticosteroids, hydroacupuncture and electroacupuncture. Animal training allowed veterinarians to perform acupuncture safely without having to anesthetize the animal. Animal training made intensive treatment possible. Neither corticosteroids nor hydroacupuncture alone provided much clinical improvement. The tiger reacted positively after electroacupuncture was performed. The tiger began showing clinical improvement after three electroacupuncture treatments and could eventually walk on all four limbs at the end of the treatment.

Influenza A(H6N1) Virus in Dogs, Taiwan.

We determined the prevalence of influenza A virus in dogs in Taiwan and isolated A/canine/Taiwan/E01/2014. Molecular analysis indicated that this isolate was closely related to influenza A(H6N1) viruses circulating in Taiwan and harbored the E627K substitution in the polymerase basic 2 protein, which indicated its ability to replicate in mammalian species.

Immunochromatographic strip assay development for avian influenza antibody detection.

To detect antibody on pen-side is a rapid way to know the avian influenza (AI) infectious status in a chicken flock. The purpose of this study was to develop an immunochromatographic strip (ICS) assay to detect the antibody against the AI virus (AIV) for field applications. The ICS was constructed by fixing an AIV strain A/chicken/Taiwan/2838V/2000 (H6N1) onto a nitrocellulose membrane as the antigen at the test line and goat anti-rabbit IgG antibody at the control line. The colloidal gold conjugated with rabbit anti-chicken IgG was used as the tracer. The present ICS was used to detect antibodies against avian influenza virus in 326 chicken serum samples from the field. Compared with HI, this ICS could detect antibodies against H5 and H6 AIVs. The hemagglutination inhibition (HI) test was used as the standard to evaluate the ICS accuracy. The results showed that the sensitivity and specificity of this ICS reached 95.2% (159/167) and 94.3% (150/159), respectively. The Kappa value of the HI and ICS was 0.896 (P < 0.001). In conclusion, this ICS could be used as a rapid test to detect antibodies against AIVs in the field.

Natural A(H1N1)pdm09 influenza virus infection case in a pet ferret in Taiwan.

Ferrets have demonstrated high susceptibility to the influenza virus. This study discusses a natural 2009 pandemic influenza A (H1N1) (A(H1N1)pdm09) virus infection in a pet ferret (Mustela putorius furo) identified in Taiwan in 2013. The ferret was in close contact with family members who had recently experienced an influenza-like illness (ILI). The ferret nasal swab showed positive results for influenza A virus using one-step RT-PCR. The virus was isolated and the phylogenetic analysis indicated that all of the eight segmented genes were closely related to the human A(H1N1)pdm09 virus linage isolated in Taiwan. This study may provide a perspective view on natural influenza A virus transmission from the local human population into pet ferrets.

A 20-year disease survey of captive formosan serows (Capricornis swinhoej) at the Taipei Zoo (1991-2011).

The Formosan serow (Capricornis swinhoei) is endemic to Taiwan. The wild population has declined dramatically over the past few decades and the species is listed as a "precious and rare species" protected under law in Taiwan. Disease investigations have been rare except for sporadic observations of wild individuals, and no long-term disease survey has been performed on this species. The objective of this study was to identify and report on the most common diseases in captive Formosan serows and determine the potential causes. Medical records of Formosan serows (n = 62) housed at the Taipei Zoo over a 20-yr period (1991-2011) were collected and analyzed for this study. The most common diseases affected the gastrointestinal system and the skin. Parasitic etiologies accounted for greater than 85% of these diseases, and coinfection was common. Coccidia and lice were the most common endo- and ectoparasites, respectively. High mortality was noted in serows less than 1 yr old associated with parasitism. The results from this study could provide vital information on disease prevention and species management, which may greatly help in rehabilitation of captive and wild populations.

Comparison of xylazine-ketamine and dexmedetomidine-ketamine anesthesia in captive Formosan serows (Capricornis swinhoei).

The Formosan serow (Capricornis swinhoei) is a rare endemic species found in Taiwan. Few studies, including studies on anesthetic protocols, have been conducted on this species. This study evaluates the anesthetic effectiveness of intramuscular xylazine-ketamine and dexmedetomidine-ketamine on captive Formosan serows. Fifty-seven anesthetic events were performed on 22 adults using a combination of xylazine (2.6 ± 0.5 mg/kg) and ketamine (3.7 ± 1.0 mg/kg). Forty-eight anesthetic events were performed on 29 adults using a combination of dexmedetomidine (33.9 ± 4.3 µg/kg) and ketamine (3.4 ± 0.4 mg/kg). Xylazine-ketamine anesthesia was antagonized with tolazoline (3.3 ± 0.8 mg/kg). Dexmedetomidine-ketamine anesthesia was antagonized with atipamezole (272.8 ± 78.2 µg/kg). Both drug combinations showed smooth anesthetic and recovery processes without statistical differences in respiratory rate, heart rate, rectal temperature and hemoglobin oxygen saturation. Dexmedetomidine-ketamine reversed by atipamezole showed a significantly shorter recovery time (1.8 ± 2.3 min) than xylazine-ketamine reversed by tolazoline (4.5 ± 1.7 min) (P < 0.05). Both anesthetic protocols indicated safe and reliable immobilization whereas atipamezole provided better reversal.

Detection of anti-reticuloendotheliosis virus antibody by blocking enzyme-linked immunosorbent assay with expression envelope protein.

The current reticuloendotheliosis virus (REV) antibody detection kit that uses enzyme-linked immunosorbent assay (ELISA) needs concentrated virus, which is difficult to obtain due to its poor propagation in cells. In addition, this kit detects only chicken antibody but not other species. To overcome these disadvantages, we cloned and expressed REV env gene to develop monoclonal antibodies (mAbs), which we used for antibody detection in ELISA. Three mAbs were prepared from mice. These three mAbs could recognize REVs from ducks and geese by immunodot assay. In addition, the epitopes that the three mAbs recognized were determined by using three different env protein fragments by western blotting. One mAb was used to develop a blocking ELISA (bELISA) coated with expressed env protein to detect anti-REV antibody in chicken serum. This assay had a 98.8% (79/80) agreement with a commercial ELISA kit. Another 146 chicken sera with known neutralization antibodies were used as positive controls to evaluate this bELISA. The sensitivity and specificity this bELISA were 88.9% (40/45) and 94.8% (91/96), respectively. Thus, this bELISA could be used for anti-REV antibody detection in birds.

Impact of prior infection and repeated vaccination on post-vaccination antibody titers of the influenza A(H1N1)pdm09 strain in Taiwan schoolchildren: Implications for public health.

BACKGROUND: The objective of this study was to evaluate the effects of prior-infection and repeated vaccination on post-vaccination antibody titers. METHODS: A(H1N1)pdm09 strain was included in 2009 pandemic monovalent, 2010-2011, and 2011-2012 trivalent influenza vaccines (MIVpdm09, TIV10/11, TIV11/12) in Taiwan. During the 2011-2012 influenza season, we conducted a prospective sero-epidemiological cohort study among schoolchildren from grades 1 - 6 in the two elementary schools in Taipei with documented A(H1N1)pdm09 vaccination records since 2009. Serum samples were collected at pre-vaccination, 1-month, and 4-months post-vaccination (T1, T2, T3). Anti-A(H1N1)pdm09 hemagglutination inhibition titers (HI-Ab-titers) were examined. We also investigated the impact of four vaccination histories [(1) no previous vaccination (None), (2) vaccinated in 2009-2010 season (09v), (3) vaccinated in 2010-2011 season (10v), and (4) vaccinated consecutively in 2009-2010 and 2010-2011 seasons (09v + 10v)] and pre-vaccination HI-Ab levels on post-vaccination HI-Ab responses as well as adjusted vaccine effectiveness (aVE) against serologically-defined infection from T2 to T3. RESULTS: TIV11/12 had zero serious adverse events reported. A(H1N1)pdm09 strain in TIV11/12 elicited seroprotective Ab-titers in 98% of children and showed promising protection (aVE: 70.3% [95% confidence interval (CI): 51.0-82.1%]). Previously unvaccinated but infected children had a 3.96 times higher T2 geometric mean titer (T2-GMT) of HI-Ab than those naïve to A(H1N1)pdm09 (GMT [95% CI]: 1039.7[585.3-1845.9] vs. 262.5[65.9-1045], p = 0.046). Previously vaccinated children with seroprotective T1-Ab-titers had a higher T2-GMT and a greater aVE than those with non-seroprotective T1-Ab-titers. Repeatedly vaccinated children had lower T2-GMT than those receiving primary doses of TIV11/12. However, after controlling prior infection and T1-Ab-titers, differences in T2-GMT among the four vaccination histories became insignificant (p = 0.16). CONCLUSION: This study supports the implementation of annual mass-vaccination with A(H1N1)pdm09 in schoolchildren for three consecutive influenza seasons when vaccine and circulating strains were well matched, and found that prior infection and pre-vaccination HI-Ab levels positively impacted post-vaccination HI-Ab responses.

Comparison of Chicken Immune Responses after Inoculation with H5 Avian Influenza Virus-like Particles Produced by Insect Cells or Pupae.

INTRODUCTION: Novel clade 2.3.4.4 H5 highly pathogenic avian influenza virus (HPAIV) outbreaks have occurred since early 2015 in Taiwan and impacted the island economically, like they have many countries. This research investigates the immunogenicity of two HPAIV-like particles to assess their promise as vaccine candidates. MATERIAL AND METHODS: The haemagglutinin (HA) gene derived from clade 2.3.4.4 H5 HPAIV and matrix protein 1 (M1) gene were cloned into the pFastBac Dual baculovirus vector. The resulting recombinant viruses were expressed in Spodoptera frugiperda moth (Sf)21 cells and silkworm pupae to generate Sf21 virus-like particles (VLP) and silkworm pupa VLP. Two-week-old specific pathogen-free chickens were immunised and their humoral and cellular immune responses were analysed. RESULTS: The silkworm pupa VLP had higher haemagglutination competence. Both VLP types elicited haemagglutination inhibition antibodies, anti-HA antibodies, splenic interferon gamma (IFN-γ) and interleukin 4 (IL-4) mRNA expression, and CD4+/CD8+ ratio elevation. However, chickens receiving silkworm pupa VLP exhibited a significantly higher anti-HA antibody titre in ELISA after vaccination. Although Sf21 VLP recipients expressed more IFN-γ and IL-4, the increase in IFN-γ did not significantly raise the CD4+/CD8+ ratio and the increase in IL-4 did not promote anti-HA antibodies. CONCLUSION: Both VLP systems possess desirable immunogenicity in vivo. However, in respect of immunogenic efficacy and the production cost, pupa VLP may be the superior vaccine candidate against clade 2.3.4.4 H5 HPAIV infection.

A novel strategy for avian species and gender identification using the CHD gene.

We report on a novel and rapid strategy for the simultaneous identification of both avian species and gender by analyzing a section of the CHD gene. The CHD gene is carried by the avian sex determining chromosomes where a female bird carries both a W and Z chromosome but a cock bird carries two copies of the Z chromosome. Two primer pairs, CHD1F/CHD1R and P2/P8, were used to amplify a part of the CHD gene from 144 samples corresponding to 58 avian species. For all species tested, two fragments were observed at least in one amplification for female samples. All tested species produced species specific size fragments allowing both sex determination and species identification using these primer pairs. However, special care is still warranted as so few samples have been characterised. This novel strategy for avian species and gender identification using the CHD gene was developed for a number of applications from ecology to forensic science.

Combination of multiplex reverse transcription recombinase polymerase amplification assay and capillary electrophoresis provides high sensitive and high-throughput simultaneous detection of avian influenza virus subtypes.

The pandemic of avian influenza viruses (AIVs) in Asia has caused enormous economic loss in poultry industry and human health threat, especially clade 2.3.4.4 H5 and H7 subtypes in recent years. The endemic chicken H6 virus in Taiwan has also brought about human and dog infections. Since wild waterfowls is the major AIV reservoir, it is important to monitor the diversified subtypes in wildfowl flocks in early stage to prevent viral reassortment and transmission. To develop a more efficient and sensitive approach is a key issue in epidemic control. In this study, we integrate multiplex reverse transcription recombinase polymerase amplification (RT-RPA) and capillary electrophoresis (CE) for high-throughput detection and differentiation of AIVs in wild waterfowls in Taiwan. Four viral genes were detected simultaneously, including nucleoprotein (NP) gene of all AIVs, hemagglutinin (HA) gene of clade 2.3.4.4 H5, H6 and H7 subtypes. The detection limit of the developed detection system could achieve as low as one copy number for each of the four viral gene targets. Sixty wild waterfowl field samples were tested and all of the four gene signals were unambiguously identified within 6 h, including the initial sample processing and the final CE data analysis. The results indicated that multiplex RT-RPA combined with CE was an excellent alternative for instant simultaneous AIV detection and subtype differentiation. The high efficiency and sensitivity of the proposed method could greatly assist in wild bird monitoring and epidemic control of poultry.

Novel Low-Voltage Electro-Ejaculation Approach for Sperm Collection from Zoo Captive Lanyu Miniature Pigs (Sus barbatus sumatranus).

Semen collection can be achieved via hand penile massage or rectal stimulation using electro-ejaculation methods. Traditional electro-ejaculation procedure applied relatively high voltage of 3-15 volts with a maximum current of 900 mA. However, these manipulations often result in great stress and discomforts in animals. In this study, we showed low-voltage electro-ejaculation procedure using 2-3 volts with a maximum current of 500 mA can efficiently stimulated ejaculations in zoo captive lanyu miniature pigs with a high success rate of 81.3% (13/16). Besides normal semen properties (semen volume, pH, sperm concentration), we demonstrated that low-voltage electro-ejaculation caused less stress in the animals, and sperm cells obtained via low-voltage electro-ejaculation exhibit low abnormality (10.3%), high viability (84.3%), motility (75.7%), progressive motility (63.7%), and acrosome integrity (88%). However, cryopreservation protocol used in the current study requires further optimization, as sperm mitochondrial function was partially compromised during freezing procedures. Taken together, we demonstrated in this study that a low-voltage electro-ejaculation approach can be used to obtain quality sperm cells from zoo captive lanyu miniature pig with less physical stress during electro-ejaculation procedure.

Replication of a Dog-Origin H6N1 Influenza Virus in Cell Culture and Mice.

The world's first natural avian-origin H6N1 influenza A virus infection case in dogs was confirmed in Taiwan in 2014. The H6N1 virus in chickens has been endemic in Taiwan since 1972. Whether the dog H6N1 virus has interspecies transmission potential is the key issue we aim to understand. Following one virus passage in embryonated eggs and two further passages in MDCK cells, we obtained two virus derivatives, E01EE (PB1 739E and PB2 627E) and E01GK (PB1 739G and PB2 627K), respectively. The pathogenicity of E01EE and E01GK was investigated using plaque assay, growth dynamic analysis and cell viability quantification in cells from different animal species. The impact of amino acid mutation on PB1 739 and PB2 627 on viral ribonucleoprotein (RNP) activity was also analyzed. Further mouse infection experiments were performed. The results showed that both E01EE and E01GK decreased cell relative viability of canine MDCK cells, human A549 cells and chicken DF1 cells. E01Gk caused greater cellular harm in MDCK and A549 cells and had significantly higher virus titers in all of the cells compared to E01EE. The PB2 627K but not PB1 739G was the critical mutation that influenced the viral RNP activity. Both E01EE and E01GK caused mice pneumonia and considerable virus shedding, especially E01GK. This report verifies PB2 E627K mutation in virulence and spotlights the potential for the dog H6N1 virus to extend interspecies transmission.

Development of an oligonucleotide microarray for simultaneous detection of two canine MDR1 genotypes and association between genotypes and chemotherapy side effects.

Canine MDR1 gene mutations produce translated P-glycoprotein, an active drug efflux transporter, resulting in dysfunction or over-expression. The 4-base deletion at exon 4 of MDR1 at nucleotide position 230 (nt230[del4]) in exon 4 makes P-glycoprotein lose function, leading to drug accumulation and toxicity. The G allele of the c.-6-180T＞G variation in intron 1 of MDR1 (single nucleotide polymorphism [SNP] 180) causes P-glycoprotein over-expression, making epileptic dogs resistant to phenobarbital treatment. Both of these mutations are reported to be common in collies. This study develops a more efficient method to detect these two mutations simultaneously, and clarifies the genotype association with the side effects of chemotherapy. Genotype distribution in Taiwan was also investigated. An oligonucleotide microarray was successfully developed for the detection of both genotypes and was applied to clinical samples. No 4-base deletion mutant allele was detected in dogs in Taiwan. However, the G allele variation of SNP 180 was spread across all dog breeds, not only in collies. The chemotherapy adverse effect percentages of the SNP 180 T/T, T/G, and G/G genotypes were 16.7%, 6.3%, and 0%, respectively. This study describes an efficient way for MDR1 gene mutation detection, clarifying genotype distribution, and the association with chemotherapy.

Simultaneous detection and differentiation of Newcastle disease and avian influenza viruses using oligonucleotide microarrays.

Newcastle disease (ND) and avian influenza (AI) are two of the most important zoonotic viral diseases of birds throughout the world. These two viruses often have a great impact upon the poultry industry. Both viruses are associated with transmission from wild to domestic birds, and often display similar signs that need to be differentiated. A rapid surveillance among wild and domestic birds is important for early disease detection and intervention, and is the basis for what measures should be taken. The surveillance, thus, should be able to differentiate the diseases and provide a detailed analysis of the virus strains. Here, we described a fast, simultaneous and inexpensive approach to the detection of Newcastle disease virus (NDV) and avian influenza virus (AIV) using oligonucleotide microarrays. The NDV pathotypes and the AIV haemagglutinin subtypes H5 and H7 were determined at the same time. Different probes on a microarray targeting the same gene were implemented in order to encompass the diversified virus strains or provide multiple confirmations of the genotype. This ensures good sensitivity and specificity among divergent viruses. Twenty-four virus isolates and twenty-four various combinations of the viruses were tested in this study. All viruses were successfully detected and typed. The hybridization results on microarrays were clearly identified with the naked eyes, with no further imaging equipment needed. The results demonstrate that the detection and typing of multiple viruses can be performed simultaneously and easily using oligonucleotide microarrays. The proposed method may provide potential for rapid surveillance and differential diagnosis of these two important zoonoses in both wild and domestic birds.

Sex identification of owls (family Strigidae) using oligonucleotide microarrays.

Molecular sexing of the diversified avian family Strigidae is difficult. Sex identification using the intron length difference between W and Z chromosomal CHD1 genes, as visualized by agarose gel electrophoreses, often produces ambiguous results. Here we describe a simple method for sexing a variety of Strigidae species using oligonucleotide microarrays, on which several sex-specific probes operated complementarily or in concert. The sex of 8 owl species was identified clearly on the microarrays through sequence recognition. This sequence-directed method can be easily applied to a wider range of Strigidae species.

Association between indoor air pollution and respiratory disease in companion dogs and cats.

BACKGROUND: Indoor air pollution (IAP) leads to important respiratory morbidity and mortality in humans. Companion dogs and cats share the same household environment with their owners and are exposed to IAP. HYPOTHESIS: Pets with respiratory disease are more commonly exposed to indoor air pollutants in their homes and to worse air quality than pets without respiratory disease. ANIMALS: Three hundred and forty-eight animals (230 dogs and 118 cats) were recruited. METHODS: Dogs and cats attending the National Taiwan University Veterinary Hospital were prospectively enrolled over a 12-month period. Questionnaires were collected from pet owners regarding the status of signs of respiratory problem of animals and air pollutants in their homes. Clinical assessment was performed by veterinarians on all animals included in the case-control study and the presence/absence of respiratory disease and diagnoses were recorded. Individual exposure to particulate matter of 2.5 µm or less (PM2.5) was estimated in the domestic microenvironment of the animals. RESULTS: Dogs with respiratory disease were more commonly exposed to incense burning than control dogs (30 versus 13%, P = .045), but household PM2.5 level was not different between dogs with and without respiratory disease [median 30.8 µg/m3 , range 10.8-214.2 versus median 38.2 µg/m3 , range 5.4-69.4, P = .57]. Signalment factors (age, body weight, and body condition score) instead of IAP factors were associated with respiratory disease in dogs using multivariable logistic regression. In contrast, household PM2.5 level was significantly higher in cats with respiratory disease than in control cats [median 38.6 µg/m3 , range 17.8-131.2 versus median 27.4 µg/m3 , range 15.4-70.0, P = .017]. Cats living in households with PM2.5 > 35 µg/m3 were more likely to have respiratory disease than those living in households with acceptable levels of PM2.5 (OR = 4.13, 95% CI 1.12-15.27, P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: The link between IAP and respiratory disease in dogs is complicated. An unacceptable level of household PM2.5 (>35 µg/m3 ) is significantly associated with respiratory disease in cats. The effect of IAP on the respiratory health of companion animals warrants further attention.

Sexing a wider range of avian species based on two CHD1 introns with a unified reaction condition.

Identifying the sex of a bird is important to ensure successful breeding strategies and effective conservation programs. Sex may be identified from the intron size of the CHD1 gene located on the avian sex chromosomes Z and W. However, because of the great nucleotide diversity across different avian species, no given intron is in widespread use without ambiguous results. Complicated modifications of the reaction condition are required to suit different species. Two CHD1 introns were used with a unified reaction condition in this study to simplify the procedure. Consequently, genders of 73 avian species covering 19 families were successfully identified based on this two-intron approach. This means the ability to sex a wider range of avian species using a simplified procedure, greatly assisting in population management at zoos. Zoo Biol 26:425-431, 2007. (c) 2007 Wiley-Liss, Inc.