Chemical stability of active ingredients in diluted veterinary disinfectant solutions under simulated storage conditions.

Introduction: The product labels of veterinary disinfectants specify their expiration dates to prevent the use of outdated products, as these may result in disinfection and biosecurity failures during outbreak situations. However, a clear standard for the storage conditions of diluted disinfectant solutions has not yet been established, and the effects of storage conditions have scarcely been investigated. To fill this research gap, our study examined the stability of the active ingredients of diluted veterinary disinfectants based on their change in concentrations when stored at various temperatures for various time periods. Methods: Twenty veterinary disinfectants effective against either foot-and-mouth disease or avian influenza viruses were selected. The disinfectants were diluted to effective concentrations following the manufacturer's instructions. Using selective analytical techniques, the concentrations of the active ingredients of the samples that had been stored for varying intervals at different temperatures (4, 20, 30, and 45°C) were determined. These samples included soaps and detergents, acids, oxidizing agents, aldehydes, and copper compounds. The active ingredient concentrations of two of the samples were determined following freezing/thawing cycle, to establish their stability when exposed to simulated winter conditions. Results: Our results showed that most of the active ingredients had concentrations of 90% or greater of their initial concentrations, indicating ≥90% stability over a 21-day period under the experimental storage conditions. However, there were some exceptions. Glutaraldehyde, formaldehyde, and malic acid are over 90% stable at ≤ 30°C for 21 days, but their concentrations decreased to below 90% of their initial concentrations at 45°C, indicating a decline in stability when stored at 45°C for 21 days. The concentrations of potassium peroxymonosulfate and peracetic acid rapidly declined with increasing time and temperature to less than 90% of their initial concentrations. Discussion: Based on our findings, we propose that diluted disinfectant solutions should preferably be prepared daily. However, if the daily preparation of a diluted disinfectant solution is not feasible, then our results can be used as a reference, providing basic scientific data on the chemical stability of diluted disinfectant solutions commonly used in the veterinary field, thus indicating suitable storage conditions.

Complete genome sequence of Brucella canis strain HSK A52141, isolated from the blood of an infected dog.

Brucella canis infection can be clinically inapparent in dogs, and when infection goes unnoticed, there is a chance for dog-to-human transmission. A new strain of B. canis was isolated from the blood of an infected dog in order to analyze the pathogenic mechanism, compare genetic properties, and develop new genetic tools for early diagnosis of canine brucellosis. Herein, we report the complete genome sequence of the strain B. canis HSK A52141. This is the second complete genome sequence and biological annotation available for a member of B. canis.

Complete genome sequence of Brucella abortus A13334, a new strain isolated from the fetal gastric fluid of dairy cattle.

Brucella abortus is a major pathogen that infects livestock and humans. A new strain of B. abortus (A13334) was isolated from the fetal gastric fluid of a dairy cow, with the aim of using it to compare genetic properties, analyze virulence factor, and survey the epidemiological relationship to other Brucella species. Here, we report the complete and annotated genome sequence of B. abortus A13334.

Modified Vaccinia Virus Ankara as a Potential Biosafety Level 2 Surrogate for African Swine Fever Virus in Disinfectant Efficacy Tests.

In South Korea, despite the increase in emerging viral pathogens in the veterinary industry, only efficacy-tested, virus-specific disinfectants are allowed to be used. Moreover, domestic testing of disinfectants for their virucidal efficacies against foreign, malignant, infectious pathogens that are unreported within the country and/or contagious livestock diseases that require special attention regarding public hygiene are legally restricted. Therefore, the Animal and Plant Quarantine Agency (APQA) designed a study to select a potential biosafety level 2 surrogate of African swine fever virus (ASFV) for efficacy testing to improve the disinfectant approval procedures. For this, the modified vaccinia virus Ankara (MVA) was compared to ASFV in terms of its susceptibility to disinfectants. Effective concentrations of active substances of disinfectants (potassium peroxymonosulfate, sodium dichloroisocyanurate, malic acid, citric acid, glutaraldehyde, and benzalkonium chloride) against ASFV and MVA were compared; similarly, efficacies of APQA-listed commercial disinfectants were examined. Tests were performed according to APQA guidelines, and infectivities of ASFV and MVA were confirmed by hemadsorption and cytopathic effect, respectively. The results reveal that the disinfectants are effective against MVA at similar or higher concentrations than those against ASFV, validating the use of MVA as a potential biosafety level 2 surrogate for ASFV in efficacy testing of veterinary disinfectants.

Surrogate Selection for Foot-and-Mouth Disease Virus in Disinfectant Efficacy Tests by Simultaneous Comparison of Bacteriophage MS2 and Bovine Enterovirus Type 1.

In South Korea, testing disinfectants against foot-and-mouth disease virus (FMDV) that are contagious in livestock or that require special attention with respect to public hygiene can be manipulated only in high-level containment laboratories, which are not easily available. This causes difficulties in the approval procedure for disinfectants, such as a prolonged testing period. Additionally, the required biosafety level (BSL) in the case of FMDV has hindered its extensive studies. However, this drawback can be circumvented by using a surrogate virus to improve the performance of the efficacy testing procedure for disinfectants. Therefore, we studied bacteriophage MS2 (MS2) and bovine enterovirus type 1 (ECBO) with respect to disinfectant susceptibility for selecting a surrogate for FMDV according to the Animal and Plant Quarantine Agency (APQA) guidelines for efficacy testing of veterinary disinfectants. Effective concentrations of the active substances in disinfectants (potassium peroxymonosulfate, sodium dichloroisocyanurate, malic acid, citric acid, glutaraldehyde, and benzalkonium chloride) against FMDV, MS2, and ECBO were compared and, efficacies of eight APQA-listed commercial disinfectants used against FMDV were examined. The infectivity of FMDV and ECBO were confirmed by examination of cytopathic effects, and MS2 by plaque assay. The results reveal that the disinfectants are effective against MS2 and ECBO at higher concentrations than in FMDV, confirming their applicability as potential surrogates for FMDV in efficacy testing of veterinary disinfectants.

Prevalence of Korean cats with natural feline coronavirus infections.

BACKGROUND: Feline coronavirus is comprised of two pathogenic biotypes consisting of feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV), which are both divided into two serotypes. To examine the prevalence of Korean cats infected with feline coronavirus (FCoV) type I and II, fecal samples were obtained from 212 cats (107 pet and 105 feral) in 2009. RESULTS: Fourteen cats were FCoV-positive, including infections with type I FCoV (n = 8), type II FCoV (n = 4), and types I and II co-infection (n = 2). Low seroprevalences (13.7%, 29/212) of FCoV were identified in chronically ill cats (19.3%, 16/83) and healthy cats (10.1%, 13/129). CONCLUSIONS: Although the prevalence of FCoV infection was not high in comparison to other countries, there was a higher prevalence of type I FCoV in Korean felines. The prevalence of FCoV antigen and antibody in Korean cats are expected to gradually increase due to the rising numbers of stray and companion cats.

Immunogenicity and safety of virus-like particle of the porcine encephalomyocarditis virus in pig.

BACKGROUND: In this study, porcine encephalomyocarditis virus (EMCV) virus-like particles (VLPs) were generated using a baculovirus expression system and were tested for immunogenicity and protective efficacy in vivo. RESULTS: VLPs were successfully generated from Sf9 cells infected with recombinant baculovirus and were confirmed to be approximately 30-40 nm by transmission electron microscopy (TEM). Immunization of mice with 0.5 µg crude protein containing the VLPs resulted in significant protection from EMCV infection (90%). In swine, increased neutralizing antibody titers were observed following twice immunization with 2.0 µg crude protein containing VLPs. In addition, high levels of neutralizing antibodies (from 64 to 512 fold) were maintained during a test period following the second immunization. No severe injection site reactions were observed after immunization and all swine were healthy during the immunization period. CONCLUSION: Recombinant EMCV VLPs could represent a new vaccine candidate to protect against EMCV infection in pig farms.

Three clusters of bovine kobuvirus isolated in Korea, 2008-2010.

Fecal samples (n = 107) were collected from cattle with ascertained or suspected diarrheal disease on Korean farms during 2008-2010. Of these, 37 samples tested positive for bovine kobuvirus. The 37 positive samples came from 32 cattle that exhibited diarrhea and five cattle that were non-diarrhetic. The majority of the virus-positive feces samples were from calves under 1 month of age (n = 25). Nine of the 37 cattle infected with bovine kobuvirus were confirmed to have a co-infection with other viruses including bovine rotavirus (n = 3), bovine coronavirus (n = 1), bovine viral diarrhea virus (n = 1), and both bovine coronavirus and bovine viral diarrhea virus (n = 4). A neighbor-joining tree grouped 36 of the Korean kobuvirus strains (with the exception of the KB8 strain) into three clusters (G1, G3, and G4), while strains derived from Thailand and Japan (except the U1 strain) were included in the G2 cluster. The results indicated that Korean bovine kobuvirus has diverse lineages regardless of disease status and species.

Porcine kobuvirus from pig stool in Korea.

To survey for porcine kobuvirus infection, a total of 119 pig fecal samples in Korea were collected from three pig farms with good breeding facilities in three provinces. Forty-three (36.1%) of the fecal samples tested were positive for porcine kobuvirus. In addition, porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) infections were not identified in any of the fecal samples, but porcine group A rotavirus (GAR) infection was identified in 11.8% (14/119) of the samples. Nucleotide sequence analysis showed sharp divergence between the five major Korean lineages by the Neighbor-joining method, and each of these lineages had high levels of bootstrap support (99 or 100%). Porcine kobuvirus is widespread in Korea regardless of the clinical condition (diarrhea or non-diarrhea). Piglets under the age of 3 weeks were the main age group infected with porcine kobuvirus, which is the general case for other species of kobuviruses.

Genetic characterization of the Korean LATC06 rinderpest vaccine strain.

We sequenced the genome of LATC06 generated by in vitro passage in Vero cells of the lapinized-avianized (LA) strain and compared its sequence to those of other rinderpest viruses. The LATC06 genome consists of 15882 nucleotides. Its transcriptional regulatory control sequences (TRSs) at gene boundaries are identical to those of the Kabete O strain. Cleavage sites for generating F1/F2 proteins were identified in the same amino acid position (aa 108) as F proteins in LATC06, L13, RBT1, Kabete O, and RBOK strains. There are three predicted N-glycosylation sites of H proteins in LA (Japan) and LATC06 strains. The six epitopes of H protein in the LA (Japan) strain that elicit immunodominant humoral responses are also found in the LATC06 strain.

Development and clinical evaluation of a rapid serodiagnostic test for toxoplasmosis of cats using recombinant SAG1 antigen.

Rapid serodiagnostic methods for Toxoplasma gondii infection in cats are urgently needed for effective control of transmission routes toward human infections. In this work, 4 recombinant T. gondii antigens (SAG1, SAG2, GRA3, and GRA6) were produced and tested for the development of rapid diagnostic test (RDT). The proteins were expressed in Escherichia coli, affinity-purified, and applied onto the nitrocellulose membrane of the test strip. The recombinant SAG1 (rSAG1) showed the strongest antigenic activity and highest specificity among them. We also performed clinical evaluation of the rSAG1-loaded RDT in 182 cat sera (55 household and 127 stray cats). The kit showed 0.88 of kappa value comparing with a commercialized ELISA kit, which indicated a significant correlation between rSAG1-loaded RDT and the ELISA kit. The overall sensitivity and specificity of the RDT were 100% (23/23) and 99.4% (158/159), respectively. The rSAG1-loaded RDT is rapid, easy to use, and highly accurate. Thus, it would be a suitable diagnostic tool for rapid detection of antibodies in T. gondii-infected cats under field conditions.

Virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus and avian influenza virus.

This study evaluated the virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus (ASFV) and avian influenza virus (AIV), according to the Animal and Plant Quarantine Agency (APQA) guidelines for efficacy testing of veterinary disinfectants. AEW (pH 5.0-6.5) was prepared using a commercially available "Electrolyzed Water Generator" with a free chlorine concentration (FCC) of 5-140 ppm, and its efficiency in reducing the titer of ASFV and AIV was tested in a suspension under low- and high-level organic soiling. Under low-level organic soiling conditions, AEW with FCC ≥40 ppm was effective against ASFV; under high-level organic soiling conditions, AEW with FCC ≥80 ppm was effective against ASFV. Under low-level organic soiling conditions, AEW with FCC ≥60 ppm was effective against AIV; under high-level organic soiling conditions, AEW with FCC ≥100 ppm was effective against AIV. The virucidal effect of AEW seemed dependent on the FCC and the presence of organic soiling. Based on these data, we recommend the following minimum FCCs in AEW treatment for routine disinfection in veterinary field under low- and high-level organic soiling conditions: for ASFV, 50 ppm and 100 ppm; and for AIV, 75 ppm and 125 ppm, respectively. In conclusion, the virucidal effects of AEW against ASFV and AIV emphasize its potential utility as a disinfectant, and we suggest considering organic soiling conditions while using AEW for implementing effective control measures for field applications.

Characterization of antimicrobial resistance of recent Salmonella enterica serovar Gallinarum isolates from chickens in South Korea.

Salmonella enterica serovar Gallinarum isolates (n=105) from chickens in South Korea between 2002 and 2007 were tested for antimicrobial susceptibility by determining minimum inhibitory concentrations of 16 antimicrobials, and their predominant resistance profiles were genetically characterized. Most isolates (99/105; 94.3%) were resistant to nalidixic acid and resistant/intermediately resistant to fluoroquinolones, and 63.8% (67/105) of the isolates were resistant to three or more antimicrobials. Forty-two quinolone-resistant isolates, of which the quinolone resistance-determining regions of the gyrA genes were sequenced, contained a substitution of a Ser to a Phe or Tyr at position 83 (71.4%), or a substitution of an Asp to an Asn, Gly, or Tyr at position 87 (28.6%). Fifty-seven sulphamethoxazole-resistant isolates were tested for the presence of class 1 integrons by polymerase chain reaction, and their resistance gene cassettes were analysed by sequencing. Three different class 1 integrons containing the resistance-gene insert aadA (52.6%; n=30), aadB (12.3%; n=7), or aadB-aadA (12.3%; n=7) were identified. Most isolates harbouring the integron containing aadB-aadA displayed resistance to all three aminoglycosides tested and also showed increased resistance to fluoroquinolones. These findings suggest that fluoroquinolone resistance may be epidemiologically linked to multiple aminoglycoside resistance.

A serological survey of canine respiratory coronavirus and canine influenza virus in Korean dogs.

The relationship between canine respiratory coronavirus (CRCoV) and canine influenza virus (CIV) seropositivity in dogs in Korea was examined. Sixty-two of the 483 samples (12.8%) were seropositive for CRCoV by indirect fluorescent antibody (IFA) analysis. Nineteen animals were seropositive for CIV by ELISA out of the 385 samples tested. Serum antibodies for both viruses were detected in 6 of the 483 dogs sampled, suggesting that these viruses are present in dogs in Korea. Although the role of CRCoV in canine infectious tracheobronchitis has not been fully elucidated, co-infection with CIV may synergistically worsen respiratory clinical signs and result in more severe canine tracheobronchitis.

Evaluation of the disinfectant concentration used on livestock facilities in Korea during dual outbreak of foot and mouth disease and high pathogenic avian influenza.

BACKGROUND: A nationwide outbreak of foot-and-mouth disease (FMD) in South Korea caused massive economic losses in 2010. Since then, the Animal and Plant Quarantine Agency (QIA) has enhanced disinfection systems regarding livestock to prevent horizontal transmission of FMD and Avian influenza (AI). Although the amount of disinfectant used continues to increase, cases of FMD and AI have been occurring annually in Korea, except 2012 and 2013. OBJECTIVES: This study measured the concentration of the disinfectant to determine why it failed to remove the horizontal transmission despite increased disinfectant use. METHODS: Surveys were conducted from February to May 2017, collecting 348 samples from disinfection systems. The samples were analyzed using the Standards of Animal Health Products analysis methods from QIA. RESULTS: Twenty-three facilities used inappropriate or non-approved disinfectants. Nearly all sampled livestock farms and facilities-93.9%-did not properly adjust the disinfectant concentration. The percentage using low concentrations, or where no effective substance was detected, was 46.9%. Furthermore, 13 samples from the official disinfection station did not use effective disinfectant, and-among 72 samples from the disinfection station-88.89% were considered inappropriate concentration, according to the foot-and-mouth disease virus guidelines; considering the AIV guideline, 73.61% were inappropriate concentrations. Inappropriate concentration samples on automatic (90.00%) and semi-automatic (90.90%) disinfection systems showed no significant difference from manual methods (88.24%). Despite this study being conducted during the crisis level, most disinfectants were used inappropriately. CONCLUSIONS: This may partially explain why horizontal transmission of FMD and AI cannot be effectively prevented despite extensive disinfectant use.

Molecular insights into the phylogeny of canine parvovirus 2 (CPV-2) with emphasis on Korean isolates: a Bayesian approach.

A Bayesian approach was utilized to further investigate the molecular phylogeny of canine parvovirus 2 (CPV-2) with emphasis on Korean isolates, and the complete VP2 gene sequences for 23 Korean isolates in 2007 and two vaccine strains were determined. Of the 44 Korean CPVs, 40 were identified as CPV-2a, three as CPV-2b, and the remaining one as a CPV-2a variant having a Gly300Asp mutation. Compared with CPV-2a, our findings also showed the presence of six additional amino acid substitutions (Thr440Ala, Ile418Thr, Pro435Ser, Asp413Asn, Thr322Ser, and Tyr324Ile) within the VP2 gene of the Korean isolates. In the phylogenetic tree, the overall Korean CPVs did not emerge as an independent clade within the global CPVs, nor did they show a close relationship to the CPVs from any other country. Our VP2 sequence data also revealed no geographic influence on the spread of CPVs worldwide. dN/dS analysis showed that purifying selection is acting on the VP2 gene of Korean CPVs.

Encephalomyocarditis in Korea: serological survey in pigs and phylogenetic analysis of two historical isolates.

Serum samples from 3315 pigs from 363 farms located throughout all nine Korean provinces were tested for the presence of encephalomyocarditis virus (EMCV) antibodies using the virus neutralization test. The seroprevalence of EMCV in the total pig population was 9.1%, whereas in the herd the prevalence was 43.5%. The first two EMCVs isolated were K3 and K11; these strains were isolated in 1990 from a mummy and a stillborn fetus, respectively, suspected of having EMCV. Phylogenetic analyses of the capsid coding region and the VP3/VP1 genes using the Bayesian approach, and a neighbor-joining analysis, revealed that the EMCV strains fell into two clusters: groups 1 and 2, with two sub-clusters within group 1, group 1a and 1b. The Korean isolates belonged to the group 1a cluster, along with strains BJC3 (China), B424/90 (Greece) and BEL-2887A/91 (Belgium), whereas five strains isolated from Sus scrofa in Belgium (B279/95, B440/95), Italy (I001/96, I136/86), and Cyprus (C108/95) belonged to the group 2 cluster.

Adjuvant effect of bovine heat shock protein 70 on piroplasm surface protein, p33, of Theileria sergenti.

In this study, we investigated the immunological effects of bovine heat shock protein 70 (HSP70) on the major Theileria sergenti surface protein (p33). The gene encoding p33 was expressed as a fusion protein with bovine HSP70 from a plasmid vector. The adjuvant function of HSP70 on p33 was evaluated with regard to antibody response, cytokine production, and a challenge experiment in mice or cattle. HSP-p33 fusion protein provoked higher humoral and cellular immunity than either Escherichia coli-expressed p33 or piroplasm soluble protein. The HSP adjuvant activity toward p33 was also possible to detect in the inoculated cattle. The overall growth of parasites in cattle was significantly restrained in the HSP-p33-inoculated group, up to 50-52 days longer than in the controls. The present results indicate that HSP-p33 fusion protein is a promising candidate vaccine for clinical theileriosis in the field.

Highly pathogenic avian influenza virus (H5N1) in domestic poultry and relationship with migratory birds, South Korea.

During the 2006-2007 winter season in South Korea, several outbreaks of highly pathogenic avian influenza virus (H5N1) were confirmed among domestic poultry and in migratory bird habitats. Phylogenetic analysis showed that all isolates were closely related and that all belong to the A/bar-headed goose/Qinghai/5/2005-like lineage rather than the A/chicken/Korea/ES/2003-like lineage.

The Far East taiga forest: unrecognized inhospitable terrain for migrating Arctic-nesting waterbirds?

The degree of inhospitable terrain encountered by migrating birds can dramatically affect migration strategies and their evolution as well as influence the way we develop our contemporary flyway conservation responses to protect them. We used telemetry data from 44 tagged individuals of four large-bodied, Arctic breeding waterbird species (two geese, a swan and one crane species) to show for the first time that these birds fly non-stop over the Far East taiga forest, despite their differing ecologies and migration routes. This implies a lack of suitable taiga refuelling habitats for these long-distance migrants. These results underline the extreme importance of northeast China spring staging habitats and of Arctic areas prior to departure in autumn to enable birds to clear this inhospitable biome, confirming the need for adequate site safeguard to protect these populations throughout their annual cycle.