PCR based early detection and antibiotic resistance pattern of Salmonella Gallinarum isolates from Pakistan poultry.

The poultry industry in developing countries is still combating mortality and economic loss due to Salmonella contamination. Salmonella Gallinarum is a common pathogen of poultry birds, being the etiologic agent of fowl typhoid, which specifically infects adult birds via the oral-fecal route. Timely detection of S. Gallinarum in poultry flocks can allow early treatment intervention leading to a decrease in economic losses. Detection of S. Gallinarum is challenging, while its PCR-based detection is a promising strategy, however, due to its high genomic similarity with other commonly existing Salmonella spp., identification of S. Gallinarum from poultry samples with high specificity is still a challenge. The current study was conducted to isolate S. Gallinarum from different districts of Pakistan, assess their antibiotic susceptibility profile, and develop a method for its early detection. A total of 20 strains were isolated using buffer peptone water, selenite cysteine broth, and Xylose Lysine Tergitol-4 (XLT-4) agar supplemented with tergitol and characterized by biochemical procedures. The antibiotic sensitivity profile highlighted the highest resistance of isolates towards novobiocin and nalidixic acid, commonly used antibiotics in Pakistan Poultry production. The primers designed to amplify a unique genomic region of S. Gallinarum, showed successful detection of twenty S. Gallinarum strains, while no amplification with genomic DNA from other common Salmonella spp. The reported method can be utilized to detect S. Gallinarum from tissue samples of infected birds in a short time leading to early diagnosis and timely treatment intervention.

Isolation, toxinotyping and antimicrobial susceptibility testing of Clostridium perfringens isolated from Pakistan poultry.

BACKGROUND AND OBJECTIVES: Clostridium perfringens is a causative agent of enteric infections in animals including poultry by producing twenty different types of toxins. A single strain produces only a subset of these toxins, which form the basis of its classification into seven toxinotypes (A-G). C. perfringens toxinotype A is a widespread cause of necrotic enteritis (NE) in poultry. The current study was conducted to determine the prevalence of different toxins and antimicrobial susceptibility of C. perfringens isolated from Pakistan NE affected poultry. METHODS: A total of 134 intestinal samples of the diseased birds were collected postmortem and processed for isolation of C. perfringens using tryptose sulphite cycloserine (TSC) agar supplemented with d-cycloserine. Isolates were confirmed by Gram's staining, biochemical and molecular analyses. Toxinotyping was performed by multiplex PCR. Antimicrobial susceptibility profile of isolates was performed by Kirby Bauer disc diffusion method. RESULTS: A total of 34 strains of C. perfringens were isolated from 134 samples with prevalence rate of 25.37%. All the isolated strains were toxinotype A, as they were positive for alpha toxin (CPA) and negative for other tested toxins such as beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), toxin perfringens large (TpeL) and necrotic B-like toxin (NetB). Interestingly, all the isolated strains of C. perfringens were multidrug resistant. The highest resistance was observed against Neomycin, Trimethoprim, Tetracycline and Lincomycin which are routinely used at Pakistan poultry production. CONCLUSION: C. perfringens toxinotype A is prevalent in Pakistan poultry. Incidence of C. perfringens with prevalence rate of 25.37% can pose serious threat to Pakistan's poultry industry given that all the isolated strains were multidrug resistant. Our findings highlight the need for new antibiotics and antibiotic alternatives to overcome multidrug resistance.

SGP-C: A Broad Host Range Temperate Bacteriophage; Against Salmonella gallinarum.

Salmonella gallinarum is a poultry restricted-pathogen causing fowl-typhoid disease in adult birds with mortality rates up-to 80% and exhibit resistance against commonly used antibiotics. In this current study, a temperate broad host range bacteriophage SGP-C was isolated against S. gallinarum from poultry digesta. It showed infection ability in all the 15 tested field strains of S. gallinarum. The SGP-C phage produced circular, turbid plaques with alternate rings. Its optimum activity was observed at pH 7.0 and 37-42°C, with a latent period of 45 min and burst size of 187 virions/bacterial cell. The SGP-C lysogens, SGPC-L5 and SGPC-L6 exhibited super-infection immunity against the same phage, an already reported feature of lysogens. A virulence index of 0.5 and 0.001 as MV50 of SGP-C suggests its moderate virulence. The genome of SGP-C found circular double stranded DNA of 42 Kbp with 50.04% GC content, which encodes 63 ORFs. The presence of repressor gene at ORF49, and absence of tRNA sequence in SGP-C genome indicates its lysogenic nature. Furthermore, from NGS analysis of lysogens we propose that SGP-C genome might exist either as an episome, or both as integrated and temporary episome in the host cell and warrants further studies. Phylogenetic analysis revealed its similarity with Salmonella temperate phages belonging to family Siphoviridae. The encoded proteins by SGP-C genome have not showed homology with any known toxin and virulence factor. Although plenty of lytic bacteriophages against this pathogen are already reported, to our knowledge SGP-C is the first lysogenic phage against S. gallinarum reported so far.

Hepatitis C Virus-Associated Extrahepatic Manifestations in Lung and Heart and Antiviral Therapy-Related Cardiopulmonary Toxicity.

Besides liver cirrhosis and hepatocellular carcinoma, chronic hepatitis C virus (HCV) infection is associated with many extrahepatic manifestations (EHMs). HCV exhibits lymphotropism that is responsible for various EHM. An important characteristic of HCV is escape from the immune system, which enables it to produce chronic infections and autoimmune disorders along with accumulation of circulating immune complexes. These EHMs have large spectrum, because they affect many organs such as heart, lungs, kidney, brain, thyroid, and skin. HCV-related cardiac and pulmonary manifestations include myocarditis, cardiomyopathies, cardiovascular diseases (i.e., Stroke, ischemic heart disease), chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma, and interstitial lung diseases. This review discusses etiology and pathogenesis of HCV-associated cardiac and pulmonary manifestations and how different genes, immune system, indirectly linked factors (mixed cryoglobulinemia), liver cirrhosis, and antiviral treatment are involved in HCV-related heart and lung diseases, however, their exact mechanism is not clear.