Insect-based diets (house crickets and mulberry silkworm pupae): A comparison of their effects on canine gut microbiota.

Background and Aim: The gut microbiome plays an important role in the overall health and well-being of dogs, influencing various physiological processes such as metabolism, nutrient absorption, and immune function. Edible insects are a sustainable and nutritious alternative protein source attracting increasing attention as a potential component of animal feeds, including pet food. However, little is known about the effects of insect-based diets on the gut microbiota of dogs. This study aimed to examine the fecal microbiota of dogs fed a diet that substituted common protein sources (poultry meal) with the house cricket (Acheta domesticus [AD]) or mulberry silkworm pupae (Bombyx mori pupae [BMp]) at different levels. Materials and Methods: Fifteen healthy adult mixed-breed dogs were systemically randomized and assigned into each block under a completed randomized block design into the following five experimental dietary groups: control diet, 10% AD, 20% AD, 7% BMp, or 14% BMp for 29 days. The amounts fed to the dogs were based on the daily energy requirement. Fecal samples were collected on days 14 and 29 and analyzed for bacterial community structure using 16S ribosomal ribonucleic acid gene sequencing. Results: At the phylum and genus levels, microbiota and their diversity were generally relatively similar among all treatments. The diets containing insects did not significantly alter the major phyla in the gut microbiome of dogs (p > 0.05). A few significant changes were found in the relative abundance of bacterial genera, with the levels of Allobaculum and Turicibacter being reduced in dogs fed a higher level of BMp. In contrast, only a decrease in Turicibacter was found in dogs fed the lower level of AD than the control diet (p < 0.05). Corynebacterium and Lactobacillus levels in the dogs fed 14% BMp were significantly increased compared with those in the control group (p < 0.05). Conclusion: These findings suggest that insect-based diets may slightly alter the gut microbiota of dogs. Further research is needed to fully understand the mechanisms by which insect-based diets influence the gut microbiota of dogs and the long-term potential health implications.

Encapsidated-CpG ODN enhances immunogenicity of porcine circovirus type 2 virus-like particles.

A DNA fragment containing CpG motifs (CpG ODN) is one of the potent immunopotentiators used to improve vaccine efficacy. It can enhance a protective immunity by stimulating both innate and adaptive immune responses. In this study, we designed and constructed a recombinant plasmid carrying the combined CpG ODN to generate an immunopotentiator for boosting the immunogenicity of porcine circovirus type 2 (PCV2) virus-like particles (VLPs). The capsid protein of PCV2b was expressed in insect cells and purified by affinity chromatography. The purified capsid protein was incubated with the CpG ODN in the reaction that allowed VLPs formation and encapsidation of the CpG ODN to occur simultaneously. Morphology of the reassembled VLPs was similar to the PCV2 virions as observed using an electron microscope. When the CpG ODN-encapcidated VLPs was treated with DNase I, the VLPs could protect the packaged CpG ODN from the enzyme digestion. Moreover, we immunized mice subcutaneously with VLPs, CpG ODN-loaded VLPs, or phosphate buffer saline for three times at two-week intervals. The results showed that the CpG ODN-loaded VLPs could elicit significantly higher levels of PCV2-specific neutralizing antibodies and interferon gamma (IFN-γ) expression in the immunized mice compared to those conferred by the VLPs alone. Conclusively, we have proved that the CpG ODN incorporated in VLPs can serve as a potent immunopotentiator for PCV2 vaccine development.

In Vitro Anti-Inflammatory and Regenerative Effects of Autologous Conditioned Serum from Dogs with Osteoarthritis.

Osteoarthritis (OA) is mostly incurable and non-regenerative with long-term complications. Autologous conditioned serum (ACS), which is enriched in Interleukin 1 receptor antagonists (IL-1RA) and growth factors, could be an alternative treatment to accelerate the positive therapeutic effects. ACS is proposed to alleviate inflammation by blocking IL-1 receptors. However, to date, there is no report focusing on the cell-mediated anti-inflammation and regenerative effect caused by ACS, especially the ACS from patients. Therefore, this study aims to investigate the therapeutic potential of ACS generated from dogs with spontaneous OA, focusing on its promising anti-inflammatory and regenerative properties in vitro compared to the matched plasma. We found that ACS prepared from ten OA dogs contained significant concentrations of IL-1RA, vascular endothelial growth factor, and transforming growth factor beta, which are key cytokines in anti-inflammation and angiogenesis. Furthermore, we found that ACS suppressed T cell activity by reducing proliferation of effector T cells and simultaneously expanding numbers of immune suppressive FOXP3+ T cells. Lastly, we showed that ACS enhanced the proliferation of osteocytes and fibroblasts and promoted extracellular matrix gene expression in primary chondrocyte culture. Therefore, these studies indicate that ACS prepared from dogs with OA is active as an immunomodulatory and regenerative strategy for use in OA management.

Effects of environmental condition, size, coat type, and body condition score on rectal temperature prediction in dogs using infrared auricular and surface temperature.

Background and Aim: Generally, rectal body temperature (BTrectum) is used to prefer as core body temperature in dogs. However, this procedure is time- and labor-consuming with stress induction. Therefore, infrared auricular temperature (BTear) and surface temperature (ST) could be applied to estimate BTrectum. This study aimed to estimate BTrectum from BTear or ST in various areas and determined the factors that influenced the accuracy of prediction equations. Materials and Methods: Under controlled temperature (n=197) and ambient temperature (n=183), the parameters BTrectum, BTear, and ST at internal pinna, auricular canal, lateral aspect of shoulder, hip, axillary area, inguinal area, footpad, and anal area (STrectum) were measured. In addition, temperature and humidity levels of the surrounding environment were recorded. The correlation between each measurement technique was calculated. The BTrectum prediction equation was created using all measured data and several influencing factors (environmental condition, size, coat type, and body condition score [BCS]). Results: The highest correlation with BTrectum was observed for BTear (r=0.61, p<0.01), which was similar to STrectum (r=0.61, p<0.01). Based on multiple linear regression model results using BTrectum as the dependent variable, BTear or STrectum were first selected as independent variables in all estimation equations. Ambient temperatures (R2=0.397), small breed (R2=0.582), long hair (R2=0.418), and/or a BCS of 2 (R2=0.557) provided the highest coefficients of determination of the prediction equation. Conclusion: The most appropriate predictors for estimating BTrectum were STrectum and BTear, which were impacted by the dog's signalments and the environment. To obtain satisfactory outcomes, the equation must be selected depending on the dog's signalments and the environmental conditions. However, based on the findings of this investigation, the accuracy remains low in several equations, and further studies are needed to improve the accuracy of the equation, mainly by increasing the sample size and developing a specific equation for each dog's signaling and environmental condition.

Possibility of Using House Cricket (Acheta domesticus) or Mulberry Silkworm (Bombyx mori) Pupae Meal to Replace Poultry Meal in Canine Diets Based on Health and Nutrient Digestibility.

There has been increasing interest in using insects as sustainable protein sources for humans and animals. Therefore, this study aimed to explore the possibility of substituting poultry meal with house cricket (Acheta domesticus: AD) or mulberry silkworm (Bombyx mori: BM) pupae. Fifty healthy adult mixed-breed dogs were selected and divided into five experimental groups, which were fed, in amounts based on daily energy requirement, with a control diet, a diet with 10% AD, with 20% AD, with 7% BM, or with 14% BM. Days 0-22 and 23-28 of the experiment served as the adaptation and collection phases, respectively. Haematology and blood chemistry were collected at days 0, 14, and 28, and body weight, body condition score, feed intake, faecal output, faecal score, faecal moisture, and apparent total tract digestibility of dry matter and nutrients were measured during the collection phase. The results from this study suggested that AD and BM can replace poultry meal without any adverse consequences on all measured parameters (p > 0.05). Therefore, AD at 20% or BM at 14% can be used in canine diet formulations. However, long-term feeding trials should be conducted and should focus on clinical signs relating to hypersensitivity disorders.

A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production.

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses.

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CLpro activities with IC50 of 3.57 ± 0.36 µM to 25.90 ± 1.40 µM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 µM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC50 (6.11 ± 1.90 to 7.75 ± 0.48 µM and 1.99 ± 0.30 to 4.03 ± 0.60 µM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CLpro-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs.

The effectiveness of marine based fatty acid compound (PCSO-524) and firocoxib in the treatment of canine osteoarthritis.

BACKGROUND: NSAIDs are accepted as the most predictably efficacious medical treatment of the clinical signs of osteoarthritis (OA). The marine-based fatty-acid compound PCSO-524 has been proposed as an adjunctive treatment for canine OA, however benefits of this agent is still controversial. The purpose of this study was to evaluate and compare the effectiveness of PCSO-524 combined with the NSAID firocoxib using force plate gait analysis, orthopedic assessment score (OAS) and canine brief pain inventory score (CBPI) in dogs with OA. A prospective, randomized, double-blinded study was conducted. Seventy-nine dogs that had hip and/or stifle OA were assigned randomly into three treatment groups: firocoxib, PCSO-524 and combination of firocoxib and PCSO-524, orally for 4 weeks. Peak vertical force (PVF, expressed as a percentage of bodyweight), OAS, CBPI, serum prostaglandin E2 concentration, hematology and blood chemistry values were evaluated before treatment (Day0), as well as at the second (Day14) and fourth week (Day28) during treatment. RESULTS: Within group analysis revealed significant increases in PVF over the 4-week treatment period for firocoxib, PCSO-524 and the combination (p < 0.05). Mean increases in PVF were 3.25 ± 4.13, 2.01 ± 3.86, 4.11 ± 4.69%BW (mean ± SD) respectively. The OAS showed non-significant change in all treatment groups. There were significant decreases in CBPI pain severity score (PSS) and CBPI pain interference scores (PIS) within some groups over time, however no significant differences were found between the groups. Significantly decreased serum PGE2 concentration (p < 0.05) was found in the combination group. Significant increases in BUN and creatinine (p < 0.05) compared to pre-treatment values were found in the firocoxib and combination groups but not in the PCSO-524 group at day28, but all values in all dogs remained within the normal ranges. CONCLUSIONS: The results of this study suggested combination of both PCSO-524 and firocoxib is more effective in alleviation of inflammation and improvement of weight bearing ability when compared to the uses of either PCSO-524 or firocoxib alone. Further clinical studies are needed to confirm this, and to determine if there is any benefit of PCSO-524 over placebo.

In vitro antibacterial activity of mangosteen (Garcinia mangostana Linn.) crude extract against Staphylococcus pseudintermedius isolates from canine pyoderma.

BACKGROUND: Staphylococcus pseudintermedius, commonly involved in canine pyoderma, can be classified as meticillin-susceptible S. pseudintermedius (MSSP) or meticillin-resistant S. pseudintermedius (MRSP). MRSP infections may be difficult to treat due to broad ß-lactam resistance of MRSP and typically additional multidrug-resistance. Topical antibacterial treatment is the preferred treatment modality for surface and superficial skin infections. HYPOTHESIS/OBJECTIVES: Mangosteen crude extract containing the antibacterial compound α-mangostin will have in vitro activity against MSSP and MRSP isolated from canine pyoderma. BACTERIAL ISOLATES: Twenty-three samples, MSSP (n = 12) and MRSP (n = 11), isolated from canine pyoderma. METHODS AND MATERIALS: Minimum inhibitory concentrations (MICs) were determined for mangosteen crude extract by broth microdilution. High-performance liquid chromatography (HPLC) analysis was used to determine the amount of α-mangostin in mangosteen crude extract. A time-kill assay was performed at 30 min and 2 h after exposure to a high concentration of crude extract (100× MIC). Antibacterial activity for α-mangostin was calculated according to HPLC results. RESULTS: The concentration of α-mangostin was 17.72 ± 1.42% w/w. The mean MIC of α-mangostin towards MSSP was 0.53 ± 0.35 µg/mL, whereas the mean value for MRSP was 0.47 ± 0.27 µg/mL. There was no difference between the mean MIC of MRSP and MSSP (P = 0.84). After a 30 min exposure to 100× MIC of the crude extract, a 95% reduction in colony forming units was found. CONCLUSIONS AND CLINICAL IMPORTANCE: The results showed that α-mangostin in mangosteen crude extract was effective in inhibiting S. pseudintermedius (both MRSP and MSSP). Clinical studies are needed to investigate this effectiveness further in vivo.

The immunogenicity of the secretory GΔTM protein of bovine ephemeral fever virus stably expressed by mammalian cells.

Bovine ephemeral fever virus (BEFV) causes an acute febrile disease in cattle and water buffalo. The disease has an impact on dairy and beef production in tropical and subtropical countries. Vaccination is used for disease prevention and control. In this study, we developed a recombinant lentivirus to produce mammalian stable cells expressing histidine-tagged BEFV G protein with a deleted transmembrane domain (GΔTM) as a secretory protein. In addition, guinea pigs were immunised with the purified GΔTM protein and booster immunised at a 3-week interval. The mammalian stable cells were able to continuously produce GΔTM protein for a minimum of 25 passages. All of the mammalian stable cells expressing GΔTM protein could react specifically with a BEFV convalescent bovine serum. Serum samples from the immunised guinea pigs could react strongly and specifically with the purified GΔTM protein. Moreover, post-immunised guinea pig sera contained antibodies that could neutralise BEFV. These results indicate that the G protein without a transmembrane domain can be used as a subunit vaccine for the prevention and control of BEFV. The availability of the mammalian stable cells, which constitutively express GΔTM protein, could facilitate the potential use of the secretory protein for BEFV diagnosis and vaccine development.

Interferon gamma induces cellular protein alteration and increases replication of porcine circovirus type 2 in PK-15 cells.

Porcine circovirus type 2 (PCV2) infections may lead to the development of subclinical signs or chronic systemic syndromes, collectively known as "porcine circovirus-associated disease" (PCVAD) in swine. Interferon gamma (IFN-γ) is known to enhance PCV2 replication in vitro, and immune mediators may act as pivotal factors in triggering PCV2 infection progression toward PCVAD. We determined the effects of IFN-γ on PCV2 replication in PK-15 cells. PCV2 was cultured in the presence or absence of exogenous swine IFN-γ (swIFNγ). Growth curve analysis in PK-15 cells revealed that PCV2 could replicate to a significantly higher titer in swIFNγ medium. To investigate the host cell response upon PVC2 infection, differential expression of proteins in PCV2-infected PK-15 cells with or without swIFNγ stimulation was analyzed by proteomics (LC-MS/MS) analysis. A large proportion of the differentially expressed proteins in swIFNγ-treated PCV2-infected cells were found to be involved in apoptosis, cellular stress responses, cell survival/proliferation pathways, and inflammatory responses. We further confirmed the expression of these differentially expressed proteins at the mRNA levels by qRT-PCR. PCV2 infection in PK-15 cells in the presence of IFN-γ resulted in upregulation of cellular proteins in responses to stress, cell survival, and cell proliferation (Hsp90, MAP3K7, RAS-GTPase, c-myc, and 14-3-3 epsilon) as well as in an increase in the levels of proteins (CASP9 and TRAF5) related to the apoptosis pathways. Thus, PCV2 exploits several cellular biological processes through IFN activation for enhancing viral replication. This is the first evidence of IFN-γ promoting PCV2 replication in vitro via a mechanism similar to that used by several human viruses.

A field trial in Thailand of the efficacy of oral fluralaner for the treatment of dogs with generalized demodicosis.

BACKGROUND: Generalized demodicosis in dogs can be challenging to manage, especially in adult-onset cases. HYPOTHESIS/OBJECTIVES: This study evaluated the efficacy of oral fluralaner at the dose of 25-50 mg/kg for the treatment of canine generalized demodicosis. ANIMALS: Client-owned dogs from Bangkok, Thailand, diagnosed with generalized demodicosis according to published criteria. METHODS: Deep skin scrapings were performed at three to five affected areas to evaluate numbers of mites. Repeat examinations were performed monthly until parasitological cure; dogs were then followed up for two to 12 months. Parasitological cure was defined as two negative skin scrapings achieved one month apart. RESULTS: One hundred and fifteen dogs were included in the study, 73 with adult-onset demodicosis and 42 with juvenile-onset demodicosis. Twenty one dogs were lost to follow-up and 27 had one negative skin scraping but did not return. Sixty seven dogs (21 with juvenile-onset and 46 with adult-onset disease) reached parasitological cure, which occurred after two, three and four months in 63%, 85% and 100% (respectively) of dogs with adult-onset demodicosis, cumulatively, and after two and three months in 81% and 100% of dogs with juvenile-onset demodicosis. Underlying causes associated with adult-onset demodicosis included atopic dermatitis, neoplasia, metabolic diseases and idiopathy. No adverse effects of fluralaner were observed in any of the dogs. CONCLUSION: Fluralaner given at the label dose for flea and tick prevention is effective for the treatment of canine generalized demodicosis.

Screening for lead compounds and herbal extracts with potential anti-influenza viral activity.

Nonstructural protein 1 (NS1) of the highly pathogenic avian influenza virus (H5N1) contains a conserved RNA binding domain (RBD) that inhibits antiviral functions of host-innate immune response. Dimerization of NS1 forms a central groove and binds to double stranded (ds) RNA. This region might serve as a potential drug target. In this study, three dimensional structure model of NS1 RBD protein was constructed and virtual screening was performed to identify lead compounds that bound within and around the central groove. The virtual screening showed that 5 compounds bound within the central groove with binding energy ranging between -16.05 and -17.36 Kcal/mol. Two commercially available compounds, estradiol and veratridine, were selected for using in an in vitro screening assay. The results showed that neither of the compounds could inhibit the association between dsRNA and NS1 RBD protein. In addition, 34 herbal extracts were examined for their inhibitory effects. Five of them were able to inhibit association between NS1 RBD and dsRNA in electrophoresis mobility shift assay. Four herbs, Terminalia belirica, Salacia chinensis, Zingiber montanum and Peltophorum pterocarpum, could reduce > 50% of infectivity of H5N1 in a cell-based assay, and it is worth further studying their potential use as source of antiviral drugs.

Hemostatic Markers in Congestive Heart Failure Dogs with Mitral Valve Disease.

Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, D-dimer, antithrombin III (AT III), protein C (PC), factor VII (F.VII), and factor VIII (F.VIII), as well as hematocrit (HCT), platelets number (PLT), total plasma protein (TP), and albumin (ALB), were studied on fifty-eight congestive heart failure (CHF) dogs with mitral valve disease (MVD) and fifty control dogs. All of variables of MVD group, except APTT, were significantly different (P < 0.5) from control group. The variables were also compared among functional classes of CHF dogs and control dogs. It was determined that the higher the functional class of CHF dogs was, the greater the levels of fibrinogen and D-dimer were, whereas the lesser the activities of AT III and PC were presented. Additionally, TP had linear correlation with fibrinogen, D-dimer, HCT, and PLT (r = 0.31, 0.30, 0.43, and 0.38, resp., P < 0.5). These findings suggested that fibrinogen and D-dimer were the factors predisposing hypercoagulability through an increase in blood viscosity. The hemorheological abnormalities would shift an overall hemostatic balance toward a more thrombotic state in CHF dogs with MVD.

Development of an inactivated 3C(pro)-3ABC (mu3ABC) ELISA to differentiate cattle infected with foot and mouth disease virus from vaccinated cattle.

Foot and mouth disease, a highly contagious disease of cloven-hoofed animals, is still endemic in Asia, Africa, and a few countries in South America. Subclinical and persistent infections usually occur in vaccinated cattle exposed to FMDV. Successful control and eradication measures need a diagnostic assay that can distinguish between immune responses to infection and vaccination. The non-structural 3ABC ELISA is the most reliable differential diagnostic assay. However, expression of the native 3ABC gene in insect cells yielded truncated versions of the proteins; thus, a monoclonal antibody to capture digested proteins is needed to develop the assay. The purpose of this study was to develop a simple indirect 3ABC ELISA using complete 3ABC protein. The full-length mutated 3ABC protein with inactive 3C(pro) (mu3ABC) gene was constructed. The histidine-tagged mu3ABC protein was produced in insect cells for easy purification and measuring. This permits simple assay design and reproducible assay development. mu3ABC ELISA had diagnostic specificity and sensitivity of 96.6% and 84%, respectively, compared to Ceditest(®) FMDV-NS. Agreement of both assays was excellent with κ value of 0.823 (p<0.05). The mu3ABC ELISA could distinguish infected from vaccinated animals. These factors are necessary for the successful development of an in-house NSP-based ELISA. Availability of a reliable assay with acceptable costs would facilitate successful disease control and the establishment of disease-free zones. Expansion of such zones may ultimately decrease the risk of introducing FMDV into disease-free countries, thus accelerating global FMD control.

Risk factors for urate uroliths in cats.

OBJECTIVE: To identify demographic factors associated with urate urolithiasis in cats and determine whether the rate of urolith submission to a laboratory had changed over time. DESIGN: Case series and case-control study. ANIMALS: Cases consisted of 5,072 cats with urate uroliths submitted to the Minnesota Urolith Center between January 1, 1981, and December 31, 2008. Controls consisted of 437,228 cats without urinary tract diseases identified in records of the Veterinary Medical Database during the same period. PROCEDURES: Information on cat breed, age, sex, reproductive status, and location of uroliths was used to identify risk factors. Changes in annual urolith submission rates were evaluated. RESULTS: Purebred cats had significantly higher odds of developing urate uroliths than did cats of mixed breeding (reference group). On the other hand, cats of the Abyssinian, American Shorthair, Himalayan, Manx, and Persian breeds had significantly lower odds of developing urate uroliths than did mixed breeds. Neutered cats were 12 times as likely to develop urate uroliths as were sexually intact cats. Cats in all age groups had significantly increased odds of developing urate uroliths, compared with cats < 1 year of age (reference group). Cats ≥ 4 but < 7 years of age had the highest odds of all groups and were 51 times as likely to develop urate uroliths as were cats < 1 year of age. Urolith submission rates did not change significantly with time. CONCLUSIONS AND CLINICAL RELEVANCE: Findings of this study suggested that the typical cat with urate uroliths was a purebred neutered cat, 4 to 7 years old, with uroliths in the bladder or urethra. This information may be helpful in predicting mineral composition of uroliths in vivo. However, no conclusions can be made regarding cause-and-effect relationships.

Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses.

Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems.

Genetic characterization of porcine circovirus type 2 in piglets from PMWS-affected and -negative farms in Thailand.

Porcine circovirus type 2 (PCV2) is the major swine pathogen associated with Porcine circovirus associated disease (PCVAD) including post-weaning multisystemic wasting syndrome (PMWS). Currently, there are 4 subtypes of PCV2 (PCV2a, b, c and d) and some epidemiological evidences demonstrated that virulence of PCV2 may relate to its subtypes. Recently, PMWS was observed more frequently in swine farms in Thailand; however, the information regarding to PCV2 subtype involved was limited. Therefore, this study was aimed to determine the association between occurrence of PMWS and PCV2 subtypes as well as genetically characterize PCV2 in Thailand. PCV2 DNA was isolated from faecal swabs and whole blood of piglets from PMWS-affected and -negative farms. The full length ORF2 sequences were compared using multiple alignment. The results showed that PCV2 DNA was detected more frequently in PMWS-affected farms. The nucleotide identities of the ORF2 from 9 PCV2 isolates representing each PMWS-affected farm and one from the negative farm ranged from 92.4 to 99.5% suggesting that there is some genetic variation of PCV2 in Thai swine. The 10 PCV2 isolates were classified into 2 clusters, in which the 7 isolates from PMWS-positive farms were in PCV2b cluster 1 A/B. The remaining isolates were separated in the new subtype called PCV2e. The results suggest the presence of new PCV2 subtypes in addition to PCV2a and PCV2b in Asian swine population. However, correlation between subtypes and virulence of PCV2 infection is not conclusive due to limited number of the PCV2 sequences from PMWS negative farms.

Rate and frequency of recurrence of uroliths after an initial ammonium urate, calcium oxalate, or struvite urolith in cats.

OBJECTIVE: To determine frequency of and interval until recurrence after initial ammonium urate, calcium oxalate, and struvite uroliths in cats and whether breed, age, or sex was associated with increased risk for urolith recurrence. DESIGN: Case-control study. ANIMALS: 4,435 cats with recurrent uroliths. PROCEDURES: To identify recurrence of uroliths in cats for which uroliths were submitted for analysis at the Minnesota Urolith Center in 1998, the facility's database was searched for urolith resubmissions from the same cats between 1998 and 2003. Risk factors and differences in mean interval until recurrence were assessed. RESULTS: Of 221 cats with ammonium urate uroliths in 1998, 29 (13.1%) had a first and 9 (4.1%) had a second recurrence. Mean interval until recurrence was 22 and 43 months for the first and second recurrence, respectively. Of 2,393 cats with calcium oxalate uroliths in 1998, 169 (7.1%) had a first, 15 (0.6%) had a second, and 2 (0.1%) had a third recurrence. Mean interval until recurrence was 25, 38, and 48 months for the first, second, and third recurrence, respectively. Of 1,821 cats with struvite uroliths in 1998, 49 (2.7%) had a first and 3 (0.2%) had a second recurrence. Mean interval until recurrence was 29 months for first and 40 months for second recurrences. CONCLUSIONS AND CLINICAL RELEVANCE: These results provided insights into the frequency of urolith recurrence in cats. Because some uroliths associated with recurrent episodes probably were not submitted to our facility, our data likely represented an underestimation of the actual recurrence rate.