Antimicrobial resistance and virulence factors profile of Salmonella spp. and Escherichia coli isolated from different environments exposed to anthropogenic activity.

OBJECTIVE: The study aimed to identify the antimicrobial resistance (AMR) determinants and virulence factors in Salmonella spp. and Escherichia coli recovered from different anthropogenic areas in North Carolina. METHODS: Soil samples were collected from different anthropogenic areas, urban and natural. The minimum inhibitory concentration (MIC) was determined by using the broth microdilution method. Whole-genome sequencing (WGS) and analysis were done to identify the AMR determinants and virulence factors. RESULTS: A higher prevalence of Salmonella spp. and E. coli was detected in the urban environment. The Salmonella spp. isolates showed resistance to sulfisoxazole and streptomycin, whereas E. coli was resistant to sulfisoxazole, cefoxitin and ampicillin. Salmonella serotypes Schwarzengrund and Mississippi were identified based on WGS analysis. Aminoglycoside resistance genes and IncFIB and IncFIC(FII) plasmids were detected among Salmonella spp. In general, E. coli was predominated by isolates from phylogroups B1, B2 and D. The multidrug transporter mdfA gene was detected in most of the E. coli from both the urban (100%) and natural (84.5%) environments. The FosA7 gene was detected in an isolate from a residential yard. The pCoo and pB171 plasmids were detected in an urban environment; col(156) and pHN7A8 plasmids were detected in natural environments. CONCLUSIONS: The detection of AMR determinants and virulence factors in these bacteria is significant in understanding the occurrence and even the development of AMR. The presence of these determinants in different anthropogenic areas suggests the need to conduct longitudinal studies for comparing the profile of pathogens across different environments.

Characterization of drug resistance-associated TevAT1 gene of Trypanosoma evansi from Philippine water buffaloes (Bubalus bubalis)

This study detected and characterized the TevAT1 gene of Trypanosoma evansi isolates from Philippine water buffaloes (Bubalus bubalis). A total of 68 blood samples from Philippine water buffaloes were subjected to DNA extraction and PCR assay was performed using RoTat 1.2 gene to detect T. evansi. Those samples positive for T. evansi subsequently underwent another PCR assay to detect the presence of TevAT1 gene. Trypanosoma evansi was detected in 26.47% (18/68) blood samples in which distributed throughout the main islands of the country (4 from Luzon, 2 from Visayas and 12 from Mindanao). However, only 10 of these samples were positive for TevAT1 gene. Sequence alignment of the TevAT1 gene from local isolates showed no single nucleotide polymorphisms when compared to other strains in various countries. Those T. evansi without the gene of interest could be possibly resistant to some trypanocidal drugs but this needs to be further investigated in-vitro or in-vivo.

Characterisation of porcine epidemic diarrhea virus isolates during the 2014-2015 outbreak in the Philippines.

The viral agent of the porcine epidemic diarrhea (PED) was investigated during the reported 2014-2015 outbreaks in commercial farms in Central Luzon, Philippines. The study covered detection of PED virus (PEDV) in fecal and intestinal samples through reverse transcription PCR and sequence analysis of the nucleocapsid (N) gene. Results showed that 10 out of 34 fecal and intestinal samples examined were positive for PEDV. The partial nucleotide sequence of the N gene of the field samples showed 98-99% homologous to PEDV sequences registered in the GenBank. It was also noted that N gene sequences between field samples were 98% homologous. Interestingly, the partial sequences of the N genes of the field samples were genetically similar to the PEDV isolates from USA, China, Mexico, Canada and Japan. The phylogenetic tree analysis revealed that the Philippine samples clustered in group 2-1 of the PEDV, wherein the isolates of this group were responsible for the outbreaks in Asia and the USA. Analysis of the partial nucleotide and amino acid sequences revealed polymorphisms, deletions and insertions in the N-gene of the PEDV. Amino acid sequence alignment also showed deletions and insertion in the PEDV detected in the Philippines.

Molecular detection of ephemeral fever virus among large ruminants in the Philippines.

In the Philippines, bovine ephemeral fever (BEF) is currently undetected and considered as an exotic disease of both cattle and water buffaloes. The Philippines until now has no official data regarding the occurrence of BEF. There were no existing control programs or vaccine used for the prevention of the disease. However, there are claims of BEF existence in different water buffalo and cattle farms based on the clinical signs but never confirmed using laboratory test yet. Detection of BEF virus in cattle and water buffalo blood samples was conducted using reverse-transcription PCR targeting the glycoprotein (G) gene, a conserved region in the BEF virus genome. The samples were collected from 22 cattle and 50 water buffaloes with clinical signs suggesting of BEF infection. All water buffalo blood samples were negative while four cattle blood samples turned positive for BEF virus. The G gene partial sequence analysis from two BEF virus positive samples showed close relationship to Australian isolates.

Gold nanoparticle-based probes for the colorimetric detection of Mycobacterium avium subspecies paratuberculosis DNA.

Gold nanoparticle (AuNP) is considered to be the most stable metal nanoparticle having the ability to be functionalized with biomolecules. Recently, AuNP-based DNA detection methods captured the interest of researchers worldwide. Paratuberculosis or Johne's disease, a chronic gastroenteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), was found to have negative effect in the livestock industry. In this study, AuNP-based probes were evaluated for the specific and sensitive detection of MAP DNA. AuNP-based probe was produced by functionalization of AuNPs with thiol-modified oligonucleotide and was confirmed by Fourier-Transform Infrared (FTIR) spectroscopy. UV-Vis spectroscopy and Scanning Electron Microscopy (SEM) were used to characterize AuNPs. DNA detection was done by hybridization of 10 µL of DNA with 5 µL of probe at 63 °C for 10 min and addition of 3 µL salt solution. The method was specific to MAP with detection limit of 103 ng. UV-Vis and SEM showed dispersion and aggregation of the AuNPs for the positive and negative results, respectively, with no observed particle growth. This study therefore reports an AuNP-based probes which can be used for the specific and sensitive detection of MAP DNA.

Molecular detection and phylogenetic analysis of Ehrlichia canis in a Philippine dog.

Canine monocytic ehrlichiosis (CME), caused by a rickettsial bacterium, Ehrlichia canis, is distributed worldwide, particularly in tropical and subtropical regions. Transmission of E. canis is primarily mediated by the vector tick, Rhipicephalus sanguineus sensu lato and the bacteria then infect and replicate in monocytes and macrophages. Many cases are seen in veterinary hospitals and treated routinely; however, the genetic variation of E. canis strains found in the Philippines has been poorly investigated to date. In this study, the 16S rRNA gene and the gp200 gene of E. canis were detected by polymerase chain reaction from an infected dog in the Philippines, and the deduced amino acid sequence of the gp200 gene was subjected to a phylogenetic analysis. The Philippine genotype formed a cluster with the Taiwan genotype, and was somewhat divergent from the USA and Brazil strains. This suggested that E. canis underwent evolution in East and Southeast Asia, confirming the utility of the gp200 gene for the assessment of genetic relationships among strains.

Evaluation of LAMP for detection and/or screening of Leptospira spp. infection among domestic animals in the Philippines.

OBJECTIVE: This study assessed the applicability of loop-mediated isothermal amplification (LAMP) for the detection of leptospirosis among domesticated animals and sewage rats. Specifically, it evaluated the ability of LAMP to amplify Leptospira spp. targeting the 16s rRNA gene in boiled urine samples. MATERIALS AND METHODS: A total of 140 samples from different domestic animals were tested for the presence of the antigen. A nested-polymerase chain reaction (nPCR) protocol was used to compare and determine the sensitivity of LAMP in detecting Leptospira spp. The LAMP was also evaluated by comparing its amplification result using agarose gel electrophoresis and color change using dye. RESULTS: Positivity rate of Leptospira spp. antigen was 29.0% (40/140) for LAMP and 9.3% (13/140) for nPCR. Also, LAMP results for gel electrophoresis and dye color change varied in some samples that may be due to the interpretation of the result in dye color change. CONCLUSION: Overall, LAMP is a rapid, sensitive, and cost-effective diagnostic method compared with nPCR. Also, LAMP has a potential application as pen-side screening, surveillance, and clinical diagnostic kits of infectious diseases without requiring advance equipment and skilled personnel.

Green synthesis of gold nanoparticles reduced and stabilized by sodium glutamate and sodium dodecyl sulfate.

The Turkevich method has been used for many years in the synthesis of gold nanoparticles. Lately, the use of plant extracts and amino acids has been reported, which is valuable in the field of biotechnology and biomedicine. The AuNPs was synthesized from the reduction of HAuCl4 3H2O by sodium glutamate and stabilized with sodium dodecyl sulfate. The optimum concentrations for sodium glutamate and sodium dodecyl sulfate in the synthesis process were determined. The characteristics of the synthesized AuNPs was analysed through UV-Vis Spectroscopy and SEM. The AuNPs have spherical shape with a mean diameter of approximately 21.62 ± 4.39 nm and is well dispersed. FTIR analysis of the AuNPs reflected that the sulfate head group of sodium dodecyl sulfate is adsorbed at the surface of the AuNPs. Thus, we report herein the synthesis of AuNPs using sodium glutamate and sodium dodecyl sulfate.

Prevalence of babesiosis (Babesia bovis and Babesia bigemina) in cattle and water buffalo in Nueva Ecija, Philippines using Nested Polymerase Chain Reaction

The study was conducted to determine the prevalence of Babesia bovis and Babesia bigemina infection in blood samples of cattle and water buffaloes using nested polymerase chain reaction (nested-PCR). It also aimed to generate a spot map showing areas in Nueva Ecija, the Philippines where B. bovis and B. bigemina were detected. Whole blood samples of cattle (148) and water buffalo (65) were collected for DNA extraction and subsequent nested-PCR to detect B. bovis and B. bigemina. To further confirm and validate the nested-PCR results, three selected positive samples for each B. bovis and B. bigemina were sequenced and examined for homology analysis. The results showed that the prevalence of B. bovis, B. bigemina and mixed infection in cattle were 11.49% (17/148), 10.81% (16/148) and 5.41% (8/148), respectively. Homology analysis of nucleotide sequence of three selected DNA samples for each B. bovis showed two 99% and one 96% (partial sequence analysis) identities with B. bovis Thailand strain, while B. bigemina positive samples showed all 100% identities with B. bigemina Philippine strain. The result did not demonstrate in all water buffalo samples. These findings provide information about the prevalence of B. bovis and B. bigemina in cattle and water buffaloes in Nueva Ecija, which can be beneficial for strategic planning, disease management, and control and prevention.

Molecular epidemiology of pathogenic Leptospira spp. among large ruminants in the Philippines.

The extent of Leptospira infection in large ruminants resulting to economic problems in livestock industry in a leptospirosis-endemic country like the Philippines has not been extensively explored. Therefore, we determined the prevalence and carrier status of leptospirosis in large ruminants using molecular techniques and assessed the risk factors of acquiring leptospirosis in these animals. Water buffalo and cattle urine samples (n=831) collected from 21 farms during 2013-2015 were subjected to flaB-nested PCR to detect pathogenic Leptospira spp. Leptospiral flaB was detected in both species with a detection rate of 16.1%. Leptospiral DNA was detected only in samples from animals managed in communal farms. Sequence analysis of Leptospira flaB in large ruminants revealed the formation of three major clusters with L. borgpetersenii or L. kirschneri. One farm contained Leptospira flaB sequences from all clusters identified in this study, suggesting this farm was the main source of leptospires for other farms. This study suggested that these large ruminants are infected with various pathogenic Leptospira species causing possible major economic loss in the livestock industry as well as potential Leptospira reservoirs that can transmit infection to humans and other animals in the Philippines.

Molecular characterization of the gag gene of caprine arthritis encephalitis virus from goats in the Philippines.

Caprine arthritis encephalitis virus (CAEV) causes caprine arthritis encephalitis syndrome, which is an emerging disease of goats in the Philippines. DNA sequence analysis showed homology of 86-93 % between Philippine CAEV and available CAEV sequences in GenBank. CAEV was detected using nested polymerase chain reaction (PCR), and new sets of primers were designed in order to amplify the gag gene, which is a highly conserved region of the viral genome. In addition, the Philippine CAEV isolate clustered in group B with the prototype caprine lentivirus. Based on amino acid sequence alignments, it is possible that the Philippine CAEV isolate is a new strain of CAEV, but it is also possible that it was already present in the country even before the start of goat importation. Molecular characterization of the CAEV gag gene is important for the development of a detection kit specific for the local strain of CAEV and the establishment of small ruminant lentivirus eradication programs in the Philippines. This study is the first report to describe the molecular characteristics of CAEV circulating in the Philippines.

Caprine arthritis encephalitis virus detection in blood by loop-mediated isothermal amplification (LAMP) assay targeting the proviral gag region.

Caprine arthritis encephalitis virus (CAEV), of the genus Lentivirus of the Retroviridae family, causes persistent disease, which is characterized by polyarthritis and mastitis in adult goats and progressive paresis (leukoencephalomyelitis) in kids. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CAEV in blood samples. Species-specific primers amplifying the gag gene region in the provirus were used for the detection of CAEV. The LAMP assay result was obtained 30 min after incubation on a constant temperature at 63 °C in a heat block. Resulting amplicons were visualized by addition of SYBR green dye after the reaction and checked by agarose gel electrophoresis. The sensitivity of LAMP assay was evaluated by comparing the result with the nested polymerase chain reaction. Based on the experiments, the result of the assay indicated a rapid and sensitive test for the detection of CAEV.