Veterinary Microbiology

Background: Melioidosis is a fatal emerging infectious disease of both man and animal caused by bacteria Burkholderia pseudomallei. Variations were suggested to have existed among the different B. pseudomallei clinical strains/genotypes which may implicate bacterial susceptibility and resistance toward antibiotics.

Aim: This study was designed to determine whether the phenotypic antibiotic resistance pattern of B. pseudomallei is associated with the source of isolates and the genotype.

Materials and Methods: A collection of 111 B. pseudomallei isolates from veterinary cases of melioidosis and the environments (soil and water) were obtained from stock cultures of previous studies and were phylogenetically characterized by multilocus sequence typing (ST). The susceptibility to five antibiotics, namely meropenem (MEM), imipenem, ceftazidime (CAZ), cotrimoxazole (SXT), and co-amoxiclav (AMC), recommended in both acute and eradication phases of melioidosis treatment were tested using minimum inhibitory concentration antibiotics susceptibility test.

Results: Majority of isolates were susceptible to all antibiotics tested while few resistant strains to MEM, SXT, CAZ, and AMC were observed. Statistically significant association was found between resistance to MEM and the veterinary clinical isolates (p<0.05). The likelihood of resistance to MEM was significantly higher among the novel ST 1130 isolates found in veterinary cases as compared to others.

Conclusion: The resistance to MEM and SXT appeared to be higher among veterinary isolates, and the novel ST 1130 was more likely to be resistant to MEM as compared to others.

Aim: Newcastle disease (ND) is an important viral disease for poultry caused by avian paramyxovirus which can be identified by its nature of agglutination activity with red blood cell (RBC) of different species. The study was aimed to characterize the hemagglutinating (HA) activity of ND virus (NDV) at three different temperatures using RBC of five avian species, six mammalian species, and eight different human blood groups. Materials and Methods: The study was conducted from January to December 2014 at Chittagong Veterinary and Animal Sciences University. Five avian and six different mammalian species were selected for the study. In each species, two blood samples were collected aseptically. Eight different blood groups (A+, A−, B+, B−, AB+, AB−, O+, and O−) were studied in human. HA test was performed using two virus strains ND lasota and field isolate of very virulent NDV (VVNDV) with mentioned species of RBC at chilling (4°C), incubating (37°C), and room temperature (24°C). Results: Avian RBC requires less time for agglutination than mammalian RBC. Incubation temperature (37°C) requires lowest time and chilling temperature requires highest time for agglutination of RBC. Duck RBC requires lowest time (17.81 min) while chicken RBC needs highest (57.5 min) time for HA at incubation temperature and at chilling temperature, respectively, against ND lasota virus and with field strain. Goat RBC requires significantly higher time for HA (184.68 min) at chilling temperature than other mammalian species. Human RBC requires almost similar time but O+ and O− blood group do not show any HA activity. Significant variation (p<0.05) found in quail RBC at incubation temperature. In mammalian species, a significant difference (p<0.05) has been observed in goat and horse RBC at chilling; horse and dog RBC at incubation; goat, horse, buffalo, and dog RBC at room temperature. In human, significant variation (p<0.05) has been found in A+, A− and B− blood group in chilling, in B+ blood group at incubation and A+, B+, B−, AB− blood group at room temperature against two virus strains. Conclusion: ND is considered as an economically significant disease which is highly contagious in nature infecting many avian species. The threat of ND outbreak to poultry industry necessitates effective control measures to reduce the burden in commercial and backyard farming in Bangladesh.

A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1). # 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 -1 1 3 5 ( 9 9 ) 0 0 0 0 4 -8

Background and Aim: Cattle are the main reservoir of Escherichia coli O157:H7 and other verotoxigenic E. coli (VTEC); therefore, there is an increased risk of infection to humans by either direct or indirect mode of transmissions. However, the prevalence of E. coli O157:H7 in the healthy cattle population of India is yet to be ascertained. This study aimed to screen the dairy cattle in and around Pune, Maharashtra, India, for verotoxin-producing E. coli O157:H7.

Materials and Methods: A total of 257 rectal swabs were collected from 15 different organized and unorganized dairy farms of Pune during the period, January-March 2015. The screening involved enrichment in EC broth followed by differential identification on MacConkey sorbitol agar. The presumptive positive isolates were further confirmed by multiplex polymerase chain reaction (PCR) using primers specific to rfbE (O157), fliC (H7), VT1 (MK1), and VT2 (MK2). Vero-toxicity and antibiotic sensitivity were examined in PCR confirmed isolates.

Results: Out of the 257 samples analyzed, 1.9% (2/105) were positive for O157:H7 and 39% (41/105) were positive for VTEC. Two PCR confirmed positive O157:H7 strains and two randomly selected PCR-positive VT strains exhibited in vitro cytopathic effect on Vero cells on day-7 post-inoculation. Antibiotic sensitivity profiling of O157:H7 strains exhibited resistance against penicillin G, kanamycin, ampicillin, tetracycline, gentamycin, cefotaxime, streptomycin, and piperacillin.

Conclusion: These findings reveal the presence of pathogenic E. coli O157:H7 in the healthy cattle of Pune; in a situation, wherein regular surveillance for O157:H7 is not a norm. Therefore, the findings presented herein warrant routine surveillance and public awareness to prevent the transfer of such pathogens and manage health risks to the public.

Background and Aim: Newcastle disease (ND) is a viral infection that causes high mortality and economic loss in the poultry industry. The Office International des Epizooties (OIE) recommends several diagnostic methods for the detection of ND, including isolation and molecular tests. However, these detection methods are time-consuming and highly expensive. Therefore, this study was conducted to develop a coagglutination kit as a novel diagnostic tool for ND in the poultry industry.

Materials and Methods: Two adult male New Zealand White rabbits weighing 2.5 kg were vaccinated using ND life vaccine intraperitoneally. The vaccination was conducted once a week for 4 weeks with multilevel doses. Rabbits' serum was collected at week 6 and inactivated at 56°C for 30 min. The serum was precipitated using ammonium sulfate and reacted with protein A of Staphylococcus aureus to produce the agglutination kit for detecting ND virus. A total of 25 chickens suspected with ND infection from a local poultry farm in Yogyakarta were used as the test samples. The chickens were necropsied, and the brain, spleen, lung, intestine, and feces were collected. Half of these organs were subjected to tests using the coagglutination kit and reverse transcription-polymerase chain reaction (RT-PCR). The other half was processed for histopathology. Data were analyzed qualitatively.

Results: Of the 25 samples, 13 (52%) were positive for ND infection when tested using both the ND coagglutination kit and RT-PCR. The positive samples also exhibited several histopathological changes, including perivascular cuffing surrounding the cerebral blood-brain barrier, hemorrhagic pneumonia, splenitis, and necrotic hemorrhage enteritis.

Conclusion: This study confirmed that the ND coagglutination kit could be used as a novel diagnostic tool for the detection of ND virus infection in the poultry industry.

Aim: Subclinical mastitis in bovines is mainly responsible for the huge economic loss of the dairy farmers, of which Pseudomonas aeruginosa is one of the causative agents. The study was aimed at a screening of suspected milk samples from different cattle farms of West Bengal for detection and confirmation of P. aeruginosa strains followed by their characterization. Materials and Methods: Around 422 milk samples were screened from different dairy farms primarily by on-spot bromothymol blue (BTB) test and then in the lab by somatic cell counts (SCC) to finally consider 352 samples for detection of P. aeruginosa. Selective isolation and confirmation of the isolates were done using selective media, viz., cetrimide and Pseudomonas agar followed by confirmation by fluorescent technique. Molecular characterization of the strains was done by polymerase chain reaction for the presence of toxA (enterotoxin A, 352 bp) and exoS (exoenzyme S, 504 bp) genes. Results: Approximately, 371 (87.9%) samples were positive in on-spot BTB test among which 352 (94.8%) samples revealed high SCC values (more than 3 lakh cells/ml) showing infection when screened. Among these, 23 (6.5%) samples yielded typical Pseudomonas sp. isolates out of which only 19 (5.4%) isolates were confirmed to be P. aeruginosa which showed characteristic blue-green fluorescence due to the presence of pigment pyoverdin under ultraviolet light. Out of these 19 isolates, 11 isolates were positive for toxA, 6 isolates for exoS, and 2 for both these pathogenic genes. Conclusion: Approximately, 5.4% cases of bovine subclinical mastitis infections in South Bengal were associated with P. aeruginosa which possess pathogenic genes such as toxA (63.2%) and exoS (36.8%).

Dermatophytes are responsible for a majority of superficial fungal infections. They are distinctive from other pathogenic fungi in their ability to use keratin as living. They can sustain in different ecosystems that added in their morphological and ecological range with high flexibility to changing environmental conditions. Lately, Microsporum canis has evolved as a resistant pathogen against several antifungals and has been in the focus of interest for causing outbreaks and epidemics in humans from domestic animals. The increasing evolution of resistance, therapeutic failures, and reduced number of available drugs stimulated to investigate therapeutic alternatives by using some commonly available plants empirically used for their antifungal properties. Likewise, the present study reports the in vitro antifungal efficacy of crude as well as fractionalized extracts with different solvents of Ficus racemosa leaves (L.), Cassia fistula L. and Nerium oleander L. Antifungal activity was evaluated according to Clinical Laboratory Standards Institute (CLSI) protocol by determination of Minimum Inhibitory Concentration (MIC's) values on a clinical isolate of M. canis that showed resistance to some commonly used antifungals. All crude phyto-extracts except Nerium oleander L. effectively inhibited the growth of M. canis. The methanolic extract of Ficus racemosa L. and Cassia fistula L. completely inhibited the fungal growth at a concentration of 2 µg/mL and 8 µg/mL respectively. These two phyto-extracts proved to have significant antifungal activity against common antifungal resistant M. canis isolate. Further studies with the identified plant parts through in vivo studies and pinpoint isolation of the active compound may prove beneficial as an effective alternative therapy for the pathogen.

Background and Aim: Antibiotic resistance has been a progressively documented problem, resulting in treatment failure in humans and animals. This study aimed to investigate the antimicrobial susceptibility and virulence of extensively drug-resistant (XDR) Aeromonas spp. in wild Mugil cephalus and its surrounding seawater along the coastal road of Port Said, Egypt.

Materials and Methods: Specimens were examined bacteriologically, confirmed biochemically, and tested for their sensitivity against 11 antimicrobial agents. Molecular confirmation of the obtained isolates by 16S rRNA was performed, followed by the detection of antimicrobial resistance and virulence genes.

Results: Aeromonas spp. was recovered from fish (44%) and water samples (36%). A. hydrophila was the most prevalent identified strain, followed by Aeromonas sobria, Aeromonas caviae, and Aeromonas schubertii. Moreover, 90% of the tested isolates were multidrug-resistant (MDR), while 26.67% were XDR. Tested isolates were resistant to β-lactams and sulfonamides (100%), oxytetracycline (90%), and streptomycin (62.22%) but completely susceptible to cefotaxime. XDR isolates successfully amplified resistance genes (blaTEM, sul1, and tetA(A)) but not the (aadA1) gene, although there was phenotypic resistance to streptomycin on plates. All XDR isolates carry the cytotoxic enterotoxin gene (act), but alt gene was detected in only one isolate (12.5%).

Conclusion: Data in this study provide a recent update and highlight the role of wild mullet and seawater as reservoirs for MDR and XDR Aeromonas spp. that may pose a risk to humans as food-borne infection or following direct contact.

Background and Aim: Enrofloxacin and tylosin can be combined into an antibiotic formulation which is expected to have a broader range of antibacterial activity against various infections in broilers. Validation method analysis of the levels of these two active compounds needs to be done for future use in pharmacokinetic or residual studies. The present study aims to determine a suitable validation method of isocratic high-performance liquid chromatography (HPLC) to measure the concentration of antibiotic combinations in the broiler liver, kidney, and muscles. Materials and Methods: The combination of enrofloxacin and tylosin in the liver, kidney, and muscle was validated by HPLC method to find the procedures, processes, equipment, and systems used, consistently provides the appropriate results. The chromatography system consisted of an Octadecyl-silica column of 5 μm in diameter and 150 mm in length with a mobile phase of a mixture of 0.05 M monobasic sodium phosphate (pH 2.5) and a...

Background and Aim: Q fever is a zoonotic disease caused by Coxiella burnetii. Cattle, sheep, and goat are the main reservoir of C. burnetii. In Egypt, the epidemiological data about C. burnetii in camels are limited. Therefore, the current study was conducted to identify C. burnetii infection in camels by different molecular tools and to estimate its seropositivity through the detection of anti-C. burnetii antibodies in camel sera.

Materials and Methods: Blood samples were collected 112 from camels in Giza and Cairo Provinces, Egypt. All blood samples were screened by trans-quantitative polymerase chain reaction (trans-qPCR) for C. burnetii and positive samples subjected to standard PCR using the superoxide dismutase enzyme coding gene of C. burnetii. Sera of studied camels were examined for the presence of antibodies against C. burnetii using enzyme-linked immunosorbent assay.

Results: Out of 112 camels, 19 were positive for C. burnetii by qPCR with an overall prevalence of 16.9% (18.6% in Giza and 15.1% in Cairo Provinces, respectively). The seroprevalence of anti-C. burnetii IgG antibodies in the examined camels was 4.5% (5/112).

Conclusion: Trans-qPCR assay is a rapid and sensitive tool for the detection of C. burnetii in acute stage. Camels should be considered one of the major reservoirs for C. burnetii in Egypt.

Successful detection of Mycoplasma synoviae (MS) by culture and PCR from samples collected in the environment of experimentally infected chickens and turkeys, or under ®eld conditions, is described. Results showed that in the experimental infection, 10/96 and 46/96 samples of food, drinking water, feathers, droppings or dust were positive by culture and Mycoplasma-PCR. In ®eld conditions, the number of positive results for environmental samples were respectively 7/28 and 17/ 28. These observations highlight the high disseminating capacities of this mycoplasma and show the usefulness of the PCR method for epidemiological studies. #