Isolation and characterization of bacteriophages with lytic activity against common bacterial pathogens

Research (Published online: 23-08-2017)

23. Isolation and characterization of bacteriophages with lytic activity against common bacterial pathogens

R. K. Shende, S. D. Hirpurkar, C. Sannat, Nidhi Rawat and Vinay Pandey

Veterinary World, 10(8): 973-978

ABSTRACT

Aim: Present investigation was conducted to isolate and characterize bacteriophages with lytic activity against common bacterial pathogens.

Materials and Methods: A total of 60 samples of animal waste disposal from cattle (42) and buffalo (18) farms were collected from three different strata, i.e., top, mid, and bottom of collection tank. Samples were primarily subjected to rapid detection methods, and then isolation of phage was done by double agar layer method using Bacillus subtilis (BsH) and Escherichia coli (EH) as host system. Phages were characterized on the basis of plaque morphology, temperature, pH susceptibility, and host range.

Results: Recovery of phages was higher from dairy cattle farm waste (78.57%) as compared to buffalo farm waste (72.22%) and bottom layer of tank showed maximum recovery. Bacillus subtilis (91%) supported the growth of more phages as compared to E. coli (9%). Three different phage morphotypes were observed each against Bacillus subtilis (BsHR1, BsHR2, and BsHR3) and E. coli (EHR1, EHR2, and EHR3). Mean phage titer of above six phage isolates ranged between 3x1010 and 5x1012 plaque forming units/ml. Viability of phages was by, and large unaffected at 70°C within 2-3 min, and phage isolates were completely inactivated below pH 3 and above 11. Coliphage EHR1 had widest host range followed by BsHR1 and BsHR2 while EHR2, EHR3, and BsHR3 had low lytic activity.

Conclusion: It could be concluded from the present study that the Bacillus and Coli phage has wide host range and thus exhibits the potential to be used as drug substitute tool against common bacterial pathogens.

Keywords: Bacillus subtilis, bacteriophage, Escherichia coli, host range, sewage.

Molecular etiopathology of naturally occurring reproductive diseases in female goats

Research (Published online: 22-08-2017)

22. Molecular etiopathology of naturally occurring reproductive diseases in female goats

V. Beena, R. V. S. Pawaiya, K. Gururaj, D. D. Singh, A. K. Mishra, N. K. Gangwar, V. K. Gupta, R. Singh, A. K. Sharma, M. Karikalan and Ashok Kumar

Veterinary World, 10(8): 964-972

ABSTRACT

Aim: The aim of the present study was to investigate the molecular etiopathology of occurrence of reproductive diseases in female goats. Reproductive diseases in goats account for major economic losses to goat farmers in terms of valuable loss of offspring and animal productivity.

Materials and Methods: A total of 660 female genitalia were examined for pathological conditions (macroscopic and microscopic lesions). The etiopathological study was carried out for the presence of pathogenic organisms such as Brucella, Chlamydia, and Campylobacter in the uterus and ovary. Based on the microscopic lesions, suspected samples were subjected to diagnostic polymerase chain reaction (PCR) for various etiological agents employing 16srRNA genus specific primers for Campylobacter and Chlamydophila and OMP31 gene-based PCR for Brucella melitensis and nested PCR using ITS-1 gene primers for Toxoplasma gondii. For Brucella suspected samples, immunohistochemistry (IHC) was also performed.

Results: In studied female genitalia, 108 (16.30%) showed gross abnormalities with overall 23.32% occurrence of pathological conditions (macroscopic and microscopic lesions). Pathological involvement of the uterus was the highest 68 (62.96%), followed by the ovaries 27 (25%) and other organs. Major uterine condition observed was endometritis (5.60%). In uterine infections, 35 (5.30%) samples were found positive for Campylobacter spp., 12 (1.81%) samples for B. melitensis, and 3 (0.45%) samples were positive for Chlamydophila spp. Among the samples positive for B. melitensis by PCR, 3 were found positive by IHC also. Corynebacterium ovis was detected by PCR using specific primers in a case of hydrosalpinx. It was concluded that many pathological lesions in female genitalia of functional significance play a major role in infertility in goats.

Conclusion: The present study concluded that many pathological lesions in female genitalia of functional significance play a major role in infertility in goats.

Keywords: Brucella melitensis, Campylobacter spp., Chlamydophila spp., Corynebacterium ovis, etiopathology, female genitalia, goat, ovary, reproductive diseases, uterus.

Subchronic toxicity of Nile tilapia with different exposure routes to Microcystis aeruginosa: Histopathology, liver functions, and oxidative stress biomarkers

Research (Published online: 21-08-2017)

21. Subchronic toxicity of Nile tilapia with different exposure routes to Microcystis aeruginosa: Histopathology, liver functions, and oxidative stress biomarkers

H. M. R. Abdel-Latif and A. M. Abou Khashaba

Veterinary World, 10(8): 955-963

ABSTRACT

Background: Toxic cyanobacterial blooms (Microcystis aeruginosa contains microcystins [MCs]) have been reported to induce clinicopathological alterations as well as different oxidative stress in aquatic biota.

Aim: Three-week subchronic exposure experiment was carried out on Nile tilapia, to determine their effects on fish behavior, tissues, liver functions, antioxidant enzymes, and lipid peroxidation.

Materials and Methods: Fish were exposed to four main treatments; orally fed diet plus toxic cells of M. aeruginosa (containing 3500 μg/g MC-LR), immersion in 500 μg MC-LR/L, intraperitoneal injection of M. aeruginosa MC-LR with a dose of 0.1 ml of extracted toxin at a dose of 200 μg/kg bwt, and the fourth one served as a control group, then the fish were sacrificed at the end of 3rd week of exposure.

Results: The results revealed no recorded mortality with obvious behavioral changes and an enlarged liver with the congested gall bladder. Histopathology demonstrated fragmentation, hyalinization, and necrosis of the subcutaneous musculature marked fatty degeneration, and vacuolation of hepatopancreatic cells with adhesion of the secondary gill lamellae associated with severe leukocytic infiltration. Furthermore, liver functions enzymes (aspartate aminotransferase and alanine aminotransferase, and the activities of glutathione peroxidase, glutathione reductase, lipid peroxidase, and catalase enzymes) were significantly increased in all treatments starting from the 2nd week as compared to the control levels.

Conclusion: In this context, the study addresses the possible toxicological impacts of toxic M. aeruginosa contain MC-LR to Nile tilapia, and the results investigated that MC-LR is toxic to Nile tilapia in different routes of exposure as well as different doses.

Keywords: catalase, lipid peroxidation, Microcystis aeruginosa, microcystins, Nile tilapia.

Beta-lactamase antimicrobial resistance in Klebsiella and Enterobacter species isolated from healthy and diarrheic dogs in Andhra Pradesh, India

Research (Published online: 20-08-2017)

20. Beta-lactamase antimicrobial resistance in Klebsiella and Enterobacter species isolated from healthy and diarrheic dogs in Andhra Pradesh, India

N. Mohammad Sharif, B. Sreedevi, R. K. Chaitanya and D. Sreenivasulu

Veterinary World, 10(8): 950-954

ABSTRACT

Aim: The aim of this study was to characterize beta-lactamase antimicrobial resistance in Klebsiella and Enterobacter species isolated from healthy and diarrheic dogs in Andhra Pradesh.

Materials and Methods: A total of 136 rectal swabs were collected from healthy (92) and diarrheic (44) dogs, bacteriological cultured for Klebsiella and Enterobacter growth and screened for beta-lactamase antimicrobial resistance phenotypically by disc diffusion method and genotypically by polymerase chain reaction targeting blaTEM, blaSHV, blaOXA, blaCTX-M Group 1, 2, blaAmpC, blaACC, and blaMOX genes.

Results: A total of 33 Klebsiella and 29 Enterobacter isolates were recovered. Phenotypic beta-lactamase resistance was detected in 66.6% and 25% of Klebsiella and Enterobacter isolates, respectively, from healthy dogs and 66.6% and 60% of Klebsiella and Enterobacter isolates, respectively, from diarrheic dogs. Overall, incidence of extended-spectrum beta-lactamase (ESBL) phenotype was found to be 21.2% (7/33) in Klebsiella isolates, whereas none of the Enterobacter isolates exhibited ESBL phenotype. Predominant beta-lactamase genes detected in Klebsiella species include blaSHV (84.8%), followed by blaTEM (33.3%), blaCTX-M Group 1 (15.1%), and blaOXA (6.1%) gene. Predominant beta-lactamase genes detected in Enterobacter species include blaSHV (48.2%), followed by blaTEM (24.1%), blaAmpC (13.7%), and blaOXA (10.3%) gene.

Conclusion: The present study highlighted alarming beta-lactamase resistance in Klebsiella and Enterobacter species of canine origin in India with due emphasis as indicators of antimicrobial resistance.

Keywords: beta-lactamase resistance, dogs, Enterobacter, extended-spectrum beta-lactamase, Klebsiella.

Vaccination with Salmonella Typhi recombinant outer membrane protein 28 induces humoral but non-protective immune response in rabbit

Research (Published online: 19-08-2017)

19. Vaccination with Salmonella Typhi recombinant outer membrane protein 28 induces humoral but non-protective immune response in rabbit

Anjani Saxena, Rajesh Kumar and Mumtesh Kumar Saxena

Veterinary World, 10(8): 946-949

ABSTRACT

Aim: Typhoid is one of the most important food and water borne disease causing millions of deaths over the world. Presently, there is no cost effective vaccine available in India. The outer-membrane proteins (Omps) of Salmonella have been exhibited as a potential candidate for development of subunit vaccine against typhoid. The objective of the present study was to evaluate the use of recombinant Omp 28 protein for immunization of rabbit to elucidate its protection against virulent Salmonella Typhi.

Materials and Methods: Immune potential of recombinant Omp28 was tested in New Zealand Rabbits. Rabbits were divided into two groups, i.e., control and test group. Control group was injected with phosphate buffer saline with adjuvant while test group were injected with recombinant Omp28 along with adjuvant. Rabbits were bleed and serum was collected from each rabbit. Serum was tested by Enzyme-linked immunosorbent assay (ELISA) for humoral response. Rabbits were challenged with virulent culture to test the protective immunity.

Results: Humoral response was provoked at 15th day and maintained till 30th day. The mean ELISA titer at 15th day was 1 : 28000 (mean titer log 10 : 4.4472) and on the 30th day was 1 : 25866 (mean titer log 10 : 4.4127). Protective immune potential of Omp 28 was assessed by challenge studies in rabbits for which vaccinated and control rabbits were challenged with 109 cells of virulent culture of S. Typhi. In control group, out of six, no rabbit could survive after 48 days while in vaccinated group, three out of six rabbit were survived.

Conclusion: Immunization of rabbit with recombinant Omp 28 induced a strong humoral response which was exhibited by high antibody titer in ELISA. Subsequently, intraperitoneal homologous challenge of the immunized New Zealand rabbit resulted in lack of significant protection. These findings indicate that Omp 28 though provoked the humoral immunity but could not provide the protective immunity in rabbit model.

Keywords: adjuvant, outer-membrane proteins, recombinant protein, Salmonella, vaccine.

Presence and characterization of Escherichia coli virulence genes isolated from diseased pigs in the central region of Argentina

Research (Published online: 18-08-2017)

18. Presence and characterization of Escherichia coli virulence genes isolated from diseased pigs in the central region of Argentina

Fernando A. Bessone, Gabriela Bessone, Sebastian Marini, Maria B. Conde, Fabrisio E. Alustiza and Gustavo Zielinski

Veterinary World, 10(8): 939-945

ABSTRACT

Background: The main pathogen of neonatal and post weaning diarrhea and edema disease (ED) is Escherichia coli and pathotypes involved are enterotoxigenic, enteropathogenic, and shiga toxigenic (ETEC, EPEC, and STEC, respectively). Those diseases cause economic loss in pig production.

Aim: The aim of this work was to evaluate the presence of strains expressing virulence markers genes and the antibiotic susceptibility profiles of E. coli from clinical cases of post weaning diarrhea and ED in farms in the central area of Argentina.

Materials and Methods: Intensive pig farms from the central region of Argentina were sampled. Intestinal mucosa swabs from pigs with diarrhea were taken, seeded on MacConkey agar plates, biochemically typified and tested by polymerase chain reaction (PCR). Antibiograms were made by disk-diffusion method.

Results: A total of 54 strains from clinical cases studied showed PCR findings: 88.88% (48/54) expressed at least one gene coding for a virulence factor. Colonization factors found were: 39.58% of strains had F18, 33.33% were F4 and 31.25% adhesin involved in diffuse adherence-I; 29.17%, 25%, and 2.1% expressed LT, STb, and STa, respectively. 25% were STx and 16.67% were eae positive. Only 2.1% were STx2. The most active antibiotics against most strains were gentamicin and ceftiofur, but resistance profiles against many antibiotics were found.

Conclusion: High circulation of pathogens strains of E. coli among pigs with diarrhea with an extended antibiotic resistance profile.

Keywords: antibiotic resistance profile, Escherichia coli, pig, virulence genes.

High-performance liquid chromatography ultraviolet-photodiode array detection method for aflatoxin B1 in cattle feed supplements

Research (Published online: 17-08-2017)

17. High-performance liquid chromatography ultraviolet-photodiode array detection method for aflatoxin B1 in cattle feed supplements

Lazuardi Mochamad and Bambang Hermanto

Veterinary World, 10(8): 932-938

ABSTRACT

Aim: The objective of the current study is to determine the concentration of aflatoxin B1 using high-performance liquid chromatography (HPLC) with a photodiode array (PDA) detector.

Materials and Methods: Aflatoxin B1 certified reference grade from Trilogy Analytical Laboratory dissolved acetonitrile (ACN) at 10 μg/mL was used for standard assessment. HPLC instruments such as ultraviolet-PDA detector used a Shimadzu LC-6AD pump with DGU-20A5 degasser, communication module-20A, and PDA detector SPD-M20A with FRC-10A fraction collector. The HPLC was set isocratic method at 354 nm with a reverse-phase ODS C18 column (LiChrospher® 100 RP-18; diameter, 5 μm) under a 20°C controlled column chamber. Rheodyne® sample loops were performed in 20 μL capacities. The mobile phase was performed at fraction 63:26:11 H2O: methanol:ACN at pH 6.8. A total of 1 kg of feed contained 10% bread crumbs and 30% concentrated, 40% forage, and 20% soybean dregs were using commercials samples. Samples were extracted by ACN and separated with solid phase extraction ODS 1 mL than elution with mobile phase to collect at drying samples performed. The samples were ready to use after added 1 mL mobile phase than injected into the system of HPLC.

Results: We found that the retention time of aflatoxin B1 was approximately 10.858 min. Linearity of 0.01-0.08 μg/mL aflatoxin B1 dissolved in mobile phase was obtained at R2=0.9. These results demonstrate that these methods can be used to analyze aflatoxin B1 and gain 89-99% recovery. The limit of detection of this assay was obtained at 3.5 x 10-6 μg/mL.

Conclusion: This method was easy to apply and suitable to analyzing at small concentrations of aflatoxin B1 in formulated product of feed cattle.

Keywords: feeds supplements, high-performance liquid chromatography photodiode array detector, isocratic methods, mycotoxin.