First detection of bovine tuberculosis by Ziehl-Neelsen staining and polymerase chain reaction at dairy farms in the Lekok Sub-District, Pasuruan Regency, and Surabaya region, Indonesia.

Background and Aim: Bovine tuberculosis (TB) is a zoonotic disease of great public health importance, particularly in Indonesia, where control measures are limited or are not implemented. This study aimed to detect the presence of Mycobacterium pathogens in milk samples from dairy cattle in Pasuruan regency and Surabaya City, East Java, using Ziehl-Neelsen acid-fast staining and polymerase chain reaction (PCR). Materials and Methods: Milk samples were aseptically collected from 50 cattle in the Lekok Subdistrict, Pasuruan Regency, and 44 from dairy farms in the Lakarsantri Subdistrict, Wonocolo Subdistrict, Mulyorejo Subdistrict, and Kenjeran Subdistrict, Surabaya, East Java. To detect Mycobacteria at the species level, each sample was assessed by Ziehl-Neelsen staining and PCR using the RD1 and RD4 genes. Results: The results of PCR assay from 50 samples in Lekok Subdistrict, Pasuruan Regency showed that 30 samples (60%) were positive for Mycobacterium tuberculosis and two samples (4%) were positive for Mycobacterium bovis, although Ziehl-Neelsen staining did not show the presence of Mycobacterium spp. In the Surabaya region, 31 samples (70.45%) were positive for M. tuberculosis and three samples (6.8%) were positive for M. bovis. Six samples (13.63%) from all PCR-positive samples could be detected microscopically with Ziehl-Neelsen. Conclusion: The presence of bovine TB in this study supports the importance of using a molecular tool alongside routine surveillance for a better understanding of the epidemiology of bovine TB in East Java.

Antimicrobial resistance patterns and genes of Campylobacter jejuni isolated from chickens in Pasuruan, Indonesia.

Background: Poultry is one of the most prominent sources of Campylobacter jejuni, which is also a major means of transmission to people. Campylobacter jejuni contamination in chicken meat comes from chicken feces because it naturally exists in the intestines of chickens. Aim: The purpose of this study is to identify the antibiotic resistance patterns and genes of C. jejuni, which was found in chickens in Pasuruan, Indonesia. Methods: The samples used in this study were 200 contents of the small intestine of broiler chickens from 40 farms in Pasuruan Regency. The enriched sample was streaked on the selective media of modified charcoal cefoperazone deoxycholate agar containing the CCDA selective supplement. Antimicrobial susceptibility test utilizing the Kirby-Bauer diffusion test method in accordance with Clinical and Laboratory Standards Institute standards. The polymerase chain reaction (PCR) method was used to detect the (hipO), which encodes the C. jejuni strain, fluoroquinolone resistance (gyrA), beta-lactam resistance (blaOXA-61), and tetracycline resistance (tetO) genes. Results: The findings revealed a 14% (28/200) prevalence of C. jejuni in the small intestine of broiler chickens. These isolates showed high resistance to enrofloxacin (92.9%). All isolates (100%) were susceptible to amoxicillin-clavulanate. The PCR results showed all C. jejuni isolates (100%) detected the gyrA gene, 96.4% detected the blaOXA-61 gene, and 50% detected the tetO gene. Conclusion: The findings of antimicrobial resistance at a high level from the small intestine of broiler chickens illustrate the potential threat to human health. To lessen the effects now and in the future, coordinated and suitable action is needed, as well as steps to guarantee the poultry industry's economic survival and public health insurance.

First report of phenotypic and genotypic (blaOXA-61) beta-lactam resistance in Campylobacter jejuni from broilers in Indonesia.

Background and Aim: Campylobacter is a zoonotic bacterium that is a major source of foodborne diseases. In humans, most cases of campylobacteriosis are caused by Campylobacter jejuni. Poultry is the main reservoir of Campylobacter for humans, because Campylobacter is part of the normal flora of the digestive tract of poultry. Antimicrobial resistance to several antibiotics in Campylobacter isolated from humans and food animals has increased rapidly. Beta-lactam is an antibiotic with a high prevalence of resistance in Campylobacter. This study aimed to investigate phenotypic and genotypic (blaOXA-61) beta-lactam resistance in C. jejuni from broilers in Indonesia. Materials and Methods: A total of 100 samples of broiler intestinal contents were obtained from 10 broiler farms in Pasuruan Regency, Indonesia. Campylobacter jejuni was identified using conventional and polymerase chain reaction (PCR)-based methods. Phenotypic detection of beta-lactam resistance was performed using an antimicrobial susceptibility test with antibiotic disks of aztreonam, ampicillin, and amoxicillin-clavulanic acid. Genotypic detection by PCR was performed using the blaOXA-61 gene, which encodes beta-lactamase. Results: Campylobacter jejuni was identified in 23% of the samples. Phenotypically, 100% (23/23) and 73.9% (17/23) C. jejuni isolates had high resistance to aztreonam and ampicillin, respectively, but all isolates were susceptible to amoxicillin-clavulanic acid. Genotypically, all isolates carried blaOXA-61, indicated by the presence of a 372-bp PCR product. Conclusion: Campylobacter jejuni is highly resistant to beta-lactams and is a serious threat to human health. Resistance to beta-lactams should be monitored because beta-lactamase genes can be transferred between bacteria. Public awareness must also be increased on the importance of using antibiotics rationally in humans and animals.

Molecular identification of blaTEM and blaCTX-M genes in multidrug-resistant Escherichia coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia.

Introduction: Escherichia coli is an opportunistic bacteria that can grow easily, produce toxins, and resist antibiotics. The phenomenon of E. coli developing multidrug resistance is currently the subject of extensive research. The objective of this study was to molecularly identify blaTEM and blaCTX-M genes in multidrug-resistant E. coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia. Material and Methods: One hundred and ten milk samples were collected from 45 dairy cattle farms in Tulungagung, Indonesia. Indole, methyl red, Voges-Proskauer and in citrate tests and triple iron sugar agar tests were used to identify E. coli. Multidrug resistance was determined in isolates through antibiotic sensitivity tests using tetracycline, streptomycin, trimethoprim, chloramphenicol and aztreonam. Extended-spectrum beta lactamase enzyme production was confirmed by double-disc synergy test (DDST). Molecular identification was performed to confirm the blaTEM and blaCTX-M genes. Results: One hundred and one (91.82%) E. coli strains were isolated from the samples. The antibiotic sensitivity test showed four (3.96%) multidrug-resistant (MDR) and one (0.99%) ESBL-positive E. coli by DDST confirmation. There were three (77.78%) blaTEM genes and one (0.99%) blaCTX-M gene discovered in the MDR E. coli isolates using PCR for molecular identification. Conclusion: The findings of the blaTEM and blaCTX-M genes encoding ESBL E. coli in dairy cattle milk in Tulungagung, Indonesia is concerning and argues for prompt action to stop the emergence of antibiotic resistance which has an impact on public health.

Prevalence and antibiotic resistance of Staphylococcus aureus and Escherichia coli isolated from raw milk in East Java, Indonesia.

Background and Aim: Raw milk can be a source of food-borne disease transmission and a medium for spreading antibiotic-resistant bacteria. Staphylococcus aureus and Escherichia coli are bacteria that have the pathogenic ability to attack host cells and are capable of harboring antibiotic-resistant genes. This study estimated the prevalence and antibiotic resistance of S. aureus and E. coli isolated from raw milk in East Java, Indonesia. Materials and Methods: Two hundred and fifty raw milk samples were collected from five dairy farms in East Java. S. aureus and E. coli were isolated using their respective selective media, whereas antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method. The methicillin-resistant S. aureus (MRSA) was confirmed using the oxacillin resistance screen agar test, and extended-spectrum beta-lactamase (ESBL)-producing E. coli was determined using the double-disk synergy test. The presence of mecA and blaTEM genes were screened by the polymerase chain reaction method. Results: Results indicated that the prevalence of S. aureus was 138 (55.2%) and that E. coli was 176 (70.4%). Of the 138 S. aureus isolated, 27 (19.6%) were MRSA, and among the 176 E. coli isolates identified, 3 (1.7%) were ESBL producers. The mecA gene was observed in 2 (7.4%) MRSA and all 3 (100%) ESBL-producing E. coli isolated harbored blaTEM genes. Conclusion: The presence of MRSA and ESBL-producing E. coli in raw milk is a serious public health threat, and public awareness should be raised about the dangers posed by these pathogenic organisms.

Seroprevalence of Toxoplasma gondii and associated alterations in hematology and serum biochemistry of one-humped camels (Camelus dromedarius) in Pakistan.

Background and Aim: Toxoplasma gondii is an intracellular protozoan that infects humans and animals. This study aimed to estimate the seroprevalence of T. gondii and the associated alterations in hematology and serum biochemistry of one-humped camels (Camelus dromedarius) in Mianwali district, Pakistan. Materials and Methods: A total of 350 blood samples were obtained from male and female camels of different ages (≤3 years old, 4-6 years old, and ≥7 years old). To validate T. gondii antibodies, the collected samples were subjected to indirect enzyme-linked immunosorbent assay using purified recombinant micronemal protein 3 as an antibody catching antigen. Results: The prevalence of T. gondii was 50.2% higher in male camels than in female camels (16.5%) (p<0.001). Furthermore, the prevalence of T. gondii in camels was directly proportional to age (p<0.001). It was 63.33% (57/90) in camels of ≥7 years of age, 32.54% in 4-6 years old age group, and 23.08% in ≤3 years old age group. The hematological analysis of infected camels revealed a significant increase in the values of glucocorticoid-remediable aldosteronism, lymphocyte percentage, monocyte percentage (MONO%), corpuscular hemoglobin (MCH), and procalcitonin. Furthermore, substantially higher levels of liver enzymes alanine aminotransferase, aspartate aminotransferase, and the macro-mineral potassium were found in the serum of T. gondii-infected camels. Conclusion: The seropositivity of T. gondii is directly associated with the age and sex of camels, which may be considered as potential risk factors. Furthermore, T. gondii infection directly impacts the hemato-biochemistry of infected camels.

Probiotic Pediococcus pentosaceus ABY 118 to Modulation of ChIFN-γ and ChIL-10 in Broilers Infected by Eimeria tenella Oocyst.

Eimeria causes coccidiosis, which has long been recognized as a disease in chickens that significantly affects the economy. The global chicken population continues to grow, and its contribution to food security increases, making it increasingly important to produce chicken meat that is safe for human and health. This study aims to prove Pediococcus pentosaceus ABY 118 to modulation of ChIFN-γ and ChIL-10 in chickens infected with E. tenella oocysts. This study used 100 of day-old chickens (DOC), randomly divided into 5 treatments; each treatment consists of 20 chickens. The treatments was as follows: P0 (-): negative control; P0 (+): positive control; P1: monensin; P2: probiotic 1.5 × 108 CFU/ml; and P3: probiotic 3.0 × 108 CFU/ml. At the age of 20 days, Eimeria tenella (E. tenella) oocysts were inoculated orally at a dose of 1 × 104. The probiotic P. pentosaceus ABY 118 was given orally through drinking water from DOC to 35 days. Monensin was given orally through feed from the age of 14-26 days. The results of statistical analysis showed that there was a significant difference (P < 0.05) between treatments on ChIFN-γ and ChIL-10 at 6 and 8 days postinfected with E. tenella oocysts. Based on the results of this study, it can be concluded that the use of P. pentosaceus ABY 118 isolates at a dose of 1.5 × 108 CFU/ml and 3.0 × 108 CFU/ml per liter of drinking water can increase health by stimulation of ChIFN-γ and ChIL-10 in broiler infected with E. tenella oocyst.

Isolation and Identification of Escherichia coli O157:H7 Lytic Bacteriophage from Environment Sewage.

Escherichia coli O157:H7 is one of the pathogenic bacteria causing foodborne disease. The use of lytic bacteriophages can be a good solution to overcome the disease. This study is aimed at isolating lytic bacteriophages from environmental sewage with E. coli O157:H7 bacterial cells. The sample used in this study was eight bacteriophages, and the technique used in identifying E. coli O157:H7 carriers of the stx1 and stx2 genes was PCR. The double layer plaque technique was used to classify bacteriophages. Plaque morphology, host specificity, and electron micrograph were used to identify the bacteriophages. The result obtained plaque morphology as a clear zone with the largest diameter size of 3.5 mm. Lytic bacteriophage could infect E. coli O157:H7 at the highest titer of 10 × 108 PFU/mL. Bacteriophages have been identified as Siphoviridae and Myoviridae. Phage 3, phage 4, and phage 8 could infect Atypical Diarrheagenic E. coli 1 (aDEC1) due to their host specificity. The Friedman statistical tests indicate that lytic bacteriophage can significantly lyse E. coli O157:H7 (p = 0.012). The lysis of E. coli O157:H7 by phage 1, phage 2, phage 3, and phage 5 bacteriophages was statistically significant, according to Conover's posthoc test (p < 0.05). The conclusion obtained from this study is that lytic bacteriophages from environmental sewage could lyse E. coli O157:H7. Therefore, it could be an alternative biocontrol agent against E. coli O157:H7 that contaminates food causing foodborne disease.

The Analysis of OmpA and Rz/Rz1 of Lytic Bacteriophage from Surabaya, Indonesia.

A good strategy to conquer the Escherichia coli-cause food-borne disease could be bacteriophages. Porins are a type of ß-barrel proteins with diffuse channels and OmpA, which has a role in hydrophilic transport, is the most frequent porin in E. coli; it was also chosen as the potential receptor of the phage. And the Rz/Rz1 was engaged in the breakup of the host bacterial external membrane. This study aimed to analyze the amino acid of OmpA and Rz/Rz1 of lytic bacteriophage from Surabaya, Indonesia. This study employed a sample of 8 bacteriophages from the previous study. The OmpA analysis method was mass spectrometry. Rz/Rz1 was analyzed using PCR, DNA sequencing, Expasy Translation, and Expasy ProtParam. The result obtained 10% to 29% sequence coverage of OmpA, carrying the ligand-binding site. The Rz/Rz1 gene shares a high percentage of 97.04% to 98.89% identities with the Siphoviridae isolate ctTwQ4, partial genome, and Myoviridae isolate cthRA4, partial genome. The Mann-Whitney statistical tests indicate the significant differences between Alanine, Aspartate, Glycine, Proline, Serine (p=0.011), Asparagine, Cysteine (p=0.009), Isoleucine (p=0.043), Lysine (p=0.034), Methionine (p=0.001), Threonine (p=0.018), and Tryptophan (p=0.007) of OmpA and Rz/Rz1. The conclusion obtained from this study is the fact that OmpA acts as Phage 1, Phage 2, Phage 3, Phage 5, and Phage 6 receptors for its peptide composition comprising the ligand binding site, and Rz/Rz1 participates in host bacteria lysis.

Detection of invA virulence gene of multidrug-resistant Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java, Indonesia.

BACKGROUND AND AIM: The increasing number of multidrug-resistant (MDR) Salmonella species on poultry farms in Indonesia has caused concern regarding human health. This study was conducted to determine the presence of the virulence gene invA in MDR Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province, Indonesia. MATERIALS AND METHODS: Cloacal swab samples were collected by purposive sampling from 15 farms in four districts. Isolation and identification of bacteria were performed using standard microbiological techniques. Confirmation of MDR isolates was done using five different classes of antibiotics, including the beta-lactam, aminoglycoside, fluoroquinolone, phenicol, and monobactam groups. An antibiotic susceptibility test was conducted using the Kirby-Bauer disk diffusion method, and a polymerase chain reaction method was used to screen for the presence of invA. RESULTS: It was observed that 32.26% (50/155) of the samples were positive for Salmonella species. Of these 50 Salmonella isolates, 7 (14%) were identified as MDR strains. An important finding was the detection of invA in all the seven MDR Salmonella strains (100%) isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province. CONCLUSION: Veterinarians have an extremely important role in monitoring the use of antibiotics in farm animals to mitigate the rapid spread of MDR organisms in our environment, which can otherwise cause serious economic losses and also public health issues.

Detection of coagulase gene in Staphylococcus aureus from several dairy farms in East Java, Indonesia, by polymerase chain reaction.

AIM: This study was conducted to study the coagulase (coa) gene-based genetic diversity of Staphylococcus aureus, isolated from different samples of cattle from three different regions in East Java Province, Indonesia. MATERIALS AND METHODS: A total of 160 raw milk samples collected in East Java Province, Indonesia, were screened for the presence of S. aureus. The presumptive isolates were confirmed by coa test. The confirmed S. aureus isolates were subjected to coa gene polymerase chain reaction. RESULTS: Of 160 different samples, 20 (12.5%) isolates of S. aureus were confirmed by positive coa test. Of 20 S. aureus isolates, 19 (95%) isolates carried coa gene. Six different genotypes of coa gene, i.e., 440 bp, 510 bp, 547 bp, 680 bp, 740 bp, and 820 bp were obtained. One coa genotypes, 510 bp (10 isolates) were observed in polymorphism to be more prevalent than the others, and the genotype was present in at least one isolates from every region. CONCLUSION: It can be concluded that coa gene is easily epidemiological tool for detection of variation strain from S. aureus.