Microflora of Internal Organs and Muscles of Lambs and Pigs in Spontaneous Infection with Cysticercus Tenuicollis

PoPova, T. P. and K. Kanchev, 2013. Microflora of internal organs and muscles of lambs and pigs in spontaneous infection with Cysticercus tenuicollis. Bulg. J. Agric. Sci., 19: 325-330 Microbiological investigations for a carrier of pathogenic microorganisms were performed in internal organs of lambs and pigs spontaneously infected with Cysticercus tenuicollis, the larval form of Taenia hydatigena (Pallas, 1766). Studies were conducted in 2010 and first half of 2011. From the livers of the studied lambs were isolated Enterobacter agglomerans, Citrobacter diversus, Klebsiella pneumoniae and Dichelobacter nodosus, and of the pigs Proteus mirabilis, Pseudomonas fluorescens, Bacillus sp. and Clostridium sp. From the livers of both species were isolated Pseudomonas cepacia, Campylobacter coli, Enterococcus faecalis, Staphylococcus haemolyticus, Listeria innocua and Candida albicans. Lowest number of species was isolated from the lungs of the tested animals E. faecalis in the lambs and Streptococcus pneumoniae in the pigs. Most bacterial species were found in muscles of lamb: Escherichia coli, Citrobacter freundii, Campylobacter coli, Staphylococcus sciuri, E. faecalis and Bacillus sp. The data show that the parasites make prerequisites for penetration and growth of pathogenic microorganisms in internal organs of affected animals. Results from the studies of extracts of infested and healthy livers of lambs for antibacterial effect strongly suggest that Cysticercus tenuicollis has inhibitory effects on microorganisms isolated from infested organs. Even more pronounced was that effect on bacteria that were not in contact with this parasite. Microorganisms growing in organs with invasion of Cysticercus tenuicollis, to some extent develop resistance and less influenced by inhibiting factors emanating from it. Кey words: Cysticercus tenuicollis, lambs, pigs, microflora, antibacterial effect Bulgarian Journal of Agricultural Science, 19 (No 2) 2013, 325-330 Agricultural Academy E-mail: [email protected] Introduction Bacterial and parasitic liver diseases in farm animals cause losses associated with decreased productivity of affected animals, and forfeiture of damaged organs. Entrance to the microorganisms that cause liver abscesses, necroses and generalized infections is often created from migrating parasites through the liver parenchyma such as Cysticercus tenuicollis and Fasciola hepatica (Каmburov et al., 1994; Soulsby, 1982). Bacterial diseases of the liver often can lead to disturbances in bile secretion and hepatic functions. In addition, the internal organs and meat from infected animals may be source of food borne infections and intoxications of the consumers (El-Dakhly et al., 2007; Roberts et al., 2005). Liver damages caused by parasites, including C. tenuicollis, create favorable conditions for penetration and growth of pathogenic microorganisms. For instance, the development of necrotic hepatitis caused by Clostridium novyi type B, to which are sensitive the small ruminants, often is seen in animals with liver parasites (Abu-Samra et al., 1984). These authors and others (Hamid et al., 1992) have isolated Clostridium novyi type B in Sudan from liver samples of goats that died with signs of necrotic hepatitis at once infected 326 T. P. Popova and K. Kanchev with C. tenuicollis and Fasciola gigantica. The authors recommend the introduction of routine vaccination for prevention of necrotic hepatitis, whose development is obviously easier at liver infestations. Literature data show that traumatically and functional damages of the liver by the migration of young C. tenuicollis, can create favorable conditions for local growth of some pathogenic microorganisms. On the other hand, competitive interactions between parasites and microorganisms are possible which would impede the free development of the microbial population. Therefore, this work is focused on conducting research to establish the nature of the microflora, growing jointly with C. tenuicollis in the livers of spontaneously infected lambs and pigs, as well as on research for a possible inhibitory effect of products of this larval form to some bacteria. Material and Methods Organs. Microbiological tests on 12 samples of internal organs of clinically healthy lambs and pigs with spontaneous infection with Cysticercus tenuicollis were performed. Nine of them were from livers, 2 from lungs and one of the muscles. All the animals originated from the same farm near Pernik city. Of all the samples after burning on their surface and incision with sterile instruments slopes on elective and selective nutrient media for different groups of microorganisms were made. Nutrient media. 1. Solid: Blood agar, agar in Tsaysler, Folic-azide medium for enterococci, Bordet-Gengou (BUL BIO NCIPD Ltd. – Sofia); Cetrimide, Chapman Stone, Sabouraud (Antisel, Sharlau Chemie S. A., Spain); Chromocult® Coliform agar for Gram-negative bacteria, Campylobacter agar (Merck); Listeria Selective agar (Biolab Zrt., Budapest). 2. Liquid: Soy-casein medium for blood cultures; broth in Mosel, medium of Tarotsi (BUL BIO NCIPD Ltd. – Sofia); Listeria Enrichment Broth (Merck). Cultures were grown at 37°C for 24-72 h in aerobic, microaerophilic and anaerobic conditions (by аnaerob packs with palladium catalyst – H2 + co2 – BUL BIO NCIPD Ltd. – Sofia). Identification of the isolated microorganisms was made by microscopic examination of native (for motility) and stained by Gram and Klet preparations, as well as by reading of cultural characteristics on solid and in liquid media and of biochemical properties using Polymicrotest (BUL BIO NCIPD Ltd. – Sofia) and tests for oxidase and catalase. Isolation and identification of bacteria was conducted in accordance with the international identifier of Bergey (Holt et al., 1994) and of fungi according Dictionary of the Fungi (Hawksworth et al., 1983). Extracts. Microbiological tests were performed to determine the antimicrobial activity of extracts from livers of lambs with and without parasites. The organs were homogenized 1:2 with sterile saline solution in a tissue mortar, then filtered successively through a sieve with pores 0,2 and 0,1 mm and sterilized mechanically through bacterial filter with a pore size 0,2 μm (VWR N.A. PN: 28145-477, Europe PN: 514-0061). Determination of the sensitivity of isolated bacteria to organ extracts, as well as to antimicrobial means was done through the classic agar-gel diffusion method of Bauer et al. (1966). The bacterial suspensions in the exponential growth phase were inoculated at a dose of 2.106 cells/ml on Mueller – Hinton agar (Scharlau Antisel, Bulgaria) with pH 7,2 7,4 and thickness of 4 mm in Petri dishes 9 cm in diameter. The extracts were applied through instillation of 0,1 ml in 9-mm wells in agar, and antibiotics in the form of standard antibiotic discs (BUL BIO NCIPD Ltd. – Sofia) and such prepared by us. Incubation was performed at 37° C for 24 hours. The results were interpreted by the three-degree system of Bauer et al. (1966) after measuring the diameters of inhibitory zones in mm. In order to study the antimicrobial action of tissue extracts the following nine bacterial strains isolated from the organs tested were used: by one strain of Pseudomonas fluorescens, P. cepacia, Enterobacter agglomerans, Citrobacter diversus, Staphylococcus haemolyticus, Listeria innocua and 3 strains of Campylobacter coli. The same strains were tested for susceptibility to antimicrobial agents. For comparison of the results about the effects of the infected organs on the bacteria, in the research were included 5 more strains isolated from other animals (dogs) with pyodermatitis: P. aeruginosa 514, P. cepacia, S. aureus 319, Streptococcus pyogenes 321, S. pyogenes 321 and Enterococcus sp. Microflora of Internal Organs of Lambs and Pigs in Spontaneous Infection with Cysticercus Tenuicollis 327 Statistical analysis of the results was made using the classical method of Student – Fisher.