Bacteriophages are increasingly being used as biological control agents against pathogenic bacteria. In the present study, we isolate and characterize bacteriophage Akh-2 from Geoje Island, South Korea, to evaluate its utility in controlling motile Aeromonas septicemia. Akh-2 lysed four of the seven Aeromonas hydrophila strains tested. Transmission electron microscopy analysis showed that Akh-2 belongs to the Siphoviridae family, with head and tail sizes of 50 ± 5 and 170 ± 5 nm, respectively. One-step growth curve analysis revealed that the phage has a latent period of 50 ± 5 min and a burst size of 139 ± 5 plaque-forming units per infected cell. The phage appeared stable in a pH range of 6–8 and a temperature range of −80 to 46 °C. Based on next-generation sequencing analysis, its genome is 114,901 bp in size, with a 44.22% G + C content and 254 open reading frames. During an artificial induction of the disease, loach (Misgurnus anguillicaudatus) treated with Akh-2 showed an increased survival rate and time compared with the non-treated control. Our results suggest that Akh-2 is a potential biological agent for the treatment of Aeromonas infections in fish.

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The present study documented the first record of Euphlyctis kalasgramensis from Punjab, Pakistan. The specimens were collected during field visits from June through August 2018. Various morphometric measurements of E. kalasgramensis were taken and compared with Euphlyctis cyanophlyctis. Snout-vent length (SVL) was 38.11 ± 0.87 mm (n ¼ 5), snout length was 3% of SVL, foot length was 55% of SVL, head length was 32% of SVL and weight was 8.01 ± 0.12 g (n ¼ 5). A few specimens (n ¼ 2) were euthanized and preserved for molecular analysis through mitochondrial 16S rRNA gene sequences. The newly obtained DNA sequences of E. kalasgramensis were submitted to GenBank and accession numbers were obtained (MK881165.1 and MK920114.1). The Maximum likelihood and Neighbor-joining trees based on Kimura 2-parameter distance resulted in similar phylogenetic trees. Euphlyctis kalasgramensis was out group in both phylogenetic trees. The interspecific divergence of E. kalasgramensis and E. cyanophlyctis was high ranging from 4% to 6% as compared to low intraspecific divergence 0% and 1%. The diversity and distribution ranges of many amphibians species are not well known in Pakistan due to lack of taxonomic information. In our recommendation, a large scale DNA barcoding is required to report more cryptic or new species from Pakistan.

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Motile Aeromonas septicemia (MAS) is a common freshwater fish disease and major threat to the aquaculture in Pakistan. The present study was carried out on suspected fish samples to isolate and characterize local strains of Aeromonas hydrophila, a key pathogen responsible for the said disease in aquacultured fishes. A total of ninety suspected fish specimens were collected from fish farms in Kasur, Okara and Gujranwala districts of Punjab, Pakistan from June 2018 to April 2019. The specimens were processed and A. hydrophila strains were isolated. The primary identification of sixty seven isolates were verified by colony morphology, microscopy and phenotypic characterization with ten biochemical reactions. The A. hydrophila strains of test samples were molecularly characterized by polymerase chain reaction (PCR) using 16S rRNA at desired size of 356bp. The PCR amplified product was subjected to DNA sequencing and phylogenetic analysis showed homology with related strains of Aeromonas spp. By antibiotic sensitivity test, the isolates were checked for nine antibiotics in which the pathogen was sensitive to four and resistant to five drugs. Results of genetic analysis confirmed strains as A. hydrophila which are useful to take preventive measures against the said disease.

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