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.

Characterisation of Putative Outer Membrane Proteins from Leptospira borgpetersenii Serovar Hardjo-Bovis Identifies Novel Adhesins and Diversity in Adhesion across Genomospecies Orthologs.

Leptospirosis is a zoonotic bacterial disease affecting mammalian species worldwide. Cattle are a major susceptible host; infection with pathogenic Leptospira spp. represents a public health risk and results in reproductive failure and reduced milk yield, causing economic losses. The characterisation of outer membrane proteins (OMPs) from disease-causing bacteria dissects pathogenesis and underpins vaccine development. As most leptospire pathogenesis research has focused on Leptospira interrogans, this study aimed to characterise novel OMPs from another important genomospecies, Leptospira borgpetersenii, which has global distribution and is relevant to bovine and human diseases. Several putative L. borgpetersenii OMPs were recombinantly expressed, refolded and purified, and evaluated for function and immunogenicity. Two of these unique, putative OMPs (rLBL0972 and rLBL2618) bound to immobilised fibronectin, laminin and fibrinogen, which, together with structural and functional data, supports their classification as leptospiral adhesins. A third putative OMP (rLBL0375), did not exhibit saturable adhesion ability but, together with rLBL0972 and the included control, OmpL1, demonstrated significant cattle milk IgG antibody reactivity from infected cows. To dissect leptospire host-pathogen interactions further, we expressed alleles of OmpL1 and a novel multi-specific adhesin, rLBL2618, from a variety of genomospecies and surveyed their adhesion ability, with both proteins exhibiting divergences in extracellular matrix component binding specificity across synthesised orthologs. We also observed functional redundancy across different L. borgspetersenii OMPs which, together with diversity in function across genomospecies orthologs, delineates multiple levels of plasticity in adhesion that is potentially driven by immune selection and host adaptation. These data identify novel leptospiral proteins which should be further evaluated as vaccine and/or diagnostic candidates. Moreover, functional redundancy across leptospire surface proteins together with identified adhesion divergence across genomospecies further dissect the complex host-pathogen interactions of a genus responsible for substantial global disease burden.

Phenotypic and genotypic characterization of colistin-resistant Escherichia Coli with mcr-4, mcr-5, mcr-6, and mcr-9 genes from broiler chicken and farm environment.

BACKGROUND: Colistin is an antibiotic used as a last-resort to treat multidrug-resistant Gram-negative bacterial infections. Colistin had been used for a long time in veterinary medicine for disease control and as a growth promoter in food-producing animals. This excessive use of colistin in food animals causes an increase in colistin resistance. This study aimed to determine molecular characteristics of colistin-resistant Escherichia coli in broiler chicken and chicken farm environments. RESULTS: Four hundred fifty-three cloacal and farm environment samples were collected from six different commercial chicken farms in Kelantan, Malaysia. E. coli was isolated using standard bacteriological methods, and the isolates were tested for antimicrobial susceptibility using disc diffusion and colistin minimum inhibitory concentration (MIC) by broth microdilution. Multiplex PCR was used to detect mcr genes, and DNA sequencing was used to confirm the resistance genes. Virulence gene detection, phylogroup, and multilocus sequence typing (MLST) were done to further characterize the E. coli isolates. Out of the 425 (94%; 425/453) E. coli isolated from the chicken and farm environment samples, 10.8% (48/425) isolates were carrying one or more colistin-resistance encoding genes. Of the 48 colistin-resistant isolates, 54.2% (26/48) of the mcr positive isolates were genotypically and phenotypically resistant to colistin with MIC of colistin ≥ 4 µg/ml. The most prominent mcr gene detected was mcr-1 (47.9%; 23/48), followed by mcr-8 (18.8%; 9/48), mcr-7 (14.5%; 7/48), mcr-6 (12.5%; 6/48), mcr-4 (2.1%; 1/48), mcr-5 (2.1%; 1/48), and mcr-9 (2.1%; 1/48) genes. One E. coli isolate originating from the fecal sample was found to harbor both mcr-4 and mcr-6 genes and another isolate from the drinking water sample was carrying mcr-1 and mcr-8 genes. The majority of the mcr positive isolates were categorized under phylogroup A followed by phylogroup B1. The most prevalent sequence typing (ST) was ST1771 (n = 4) followed by ST206 (n = 3). 100% of the mcr positive E. coli isolates were multidrug resistant. The most frequently detected virulence genes among mcr positive E. coli isolates were ast (38%; 18/48) followed by iss (23%; 11/48). This is the first research to report the prevalence of mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8 genes in E. coli from broiler chickens and farm environments in Malaysia. CONCLUSION: Our findings suggest that broiler chickens and broiler farm environments could be reservoirs of colistin-resistant E. coli, posing a risk to public health and food safety.

Molecular detection of pathogenic Leptospira spp. in urban rodents from wet markets in northeast Malaysia.

Objective: This short study describes the occurrence of pathogenic Leptospira spp. in two major wet markets in Kota Bharu, Kelantan, Malaysia. Materials and Methods: 30 rodents (20 rats and 10 shrews) were caught in 2 wet markets, and a postmortem was performed to extract both kidneys. Molecular diagnosis via polymerase chain reaction (PCR) was conducted to detect leptospiral DNA using universal and pathogenic Leptospira primers, respectively. Results: The results showed that 20/28 (72%) rat samples were detected positive for Leptospira spp, and all shrews were negative. Further sequencing analysis identified L. interrogans and L. borgpetersenii as the most frequently Leptospirosis species from kidney samples. Conclusions: The presented study here sheds light on the presence of pathogenic leptospires harboring the rat population in both wet markets in Kelantan, which presents a great public health risk to wet market workers and visitors.

First case report on molecular detection of Trypanosoma lewisi in an urban rat in Kelantan, Malaysia: An accidental finding.

OBJECTIVE: This case report highlights the first detection of Trypanosoma lewisi, a blood protozoan parasite found in an urban rat in Kota Bharu, Kelantan. MATERIALS AND METHODS: Rat trapping was carried out within the Kota Bharu vicinity near a local wet market. A total of 38 rats were captured and subjected to peripheral blood smearing using Giemsa stain. Positive rats were sent for histopathological analysis for the evaluation of the organ samples. RESULTS: The presence of trypanosomes was found in one sample from a blood smear. This was connected to a histological lesion on kidney tissues, which revealed a high concentration of trypanosomes. Additionally, the positive sample was confirmed as T. lewisi based on molecular diagnosis via polymerase chain reaction and subsequent sequencing and phylogenetic analysis. CONCLUSIONS: This finding serves as a baseline for further surveillance on T. lewisi population among urban rats in Kelantan and possible zoonotic transmission to humans.

Complex chronic respiratory disease concurrent with coccidiosis in broiler chickens in Malaysia: A case report.

OBJECTIVE: The case study describes the cause of an increase in mortality rates among 35-day-old broilers that developed respiratory distress and bloody diarrhea on a farm in Malaysia. MATERIALS AND METHODS: The organ samples were subjected to laboratory testing and postmortem inspection. Escherichia (E.) coli and Mycoplasma (M.) gallisepticum were detected using bacterial isolation and molecular diagnostics using polymerase chain reaction. RESULTS: Chickens with the infection had widespread fibrin buildup in several organs and hemorrhages on the duodenal mucosa. Additional histology and laboratory analysis of organ samples revealed infection with M. gallisepticum, E. coli, and enteric Eimeria spp., all of which are consistent with complex chronic respiratory disease (CCRD) associated with coccidiosis. Tylosin tartrate 20% (w/w) (2.5 gm/l) was prescribed for 1 week along with a combination of the broad-spectrum bacteriostatic drug streptomycin (25 mg/kg) and coccidiostat (2 gm/5 l). CONCLUSION: CCRD and coccidiosis are both infectious diseases that can infect chicken flocks, resulting in production losses and carcass quality degradation. Early disease detection and proper treatment should be provided promptly, and tight farm biosecurity should be implemented to prevent chicken mortality on the farm, as was achieved successfully.

A case of aspergillosis outbreak in a broiler duck farm in Kelantan, Malaysia.

OBJECTIVE: This case report aims to discuss the veterinary approach taken to control a case of aspergillosis outbreak on a duck farm. MATERIALS AND METHODS: A broiler duck farm with a population of 900 Muscovy ducks was having a complaint of a 5% mortality rate in their 3-week-old ducklings. Upon presentation, 10% of the ducks appeared to be listless, dyspneic, ruffled feathers, and cyanotic. Postmortem examination of the dead birds was conducted. The collected samples were subjected to isolation and identification of the associated Aspergillus fumigatus under the microscope using the scotch tape method. RESULTS: Postmortem examination revealed whitish to creamy caseous nodules in the lungs, thoracic air sacs, gizzard, proventriculus, and intestines. Granuloma lesions and infiltration of inflammatory cells were observed in the lung and liver tissues. As for therapeutic management, all ducks were treated with copper sulfate, erythromycin, and multivitamins as the fungicide, antibiotic, and supplement, respectively, via drinking water. CONCLUSION: There is no effective treatment for Aspergillosis as the spores are difficult to destroy completely. Nonetheless, the disease can be controlled and prevented effectively with proper farm sanitation and providing a suitable feed storage environment to inhibit the growth of this opportunistic fungus.