Analysis of possible biofilm formation from different Salmonella serovars isolated from animal food in in vitro conditions.

Cilj ovog istraživanja je bio ispitivanje sposobnosti formiranja biofilma kod serovarijeteta Salmonella izolovanih iz hrane za životinje, poreklom iz 18 mešaona stočne hrane na području Južnobačkog okruga. Izolati korišćeni u ispitivanju (n=100) izolovani su poreklom iz uzoraka hrane za životinje uzorkovanih tokom dve godine ispitivanja (period 2012-2014). Identifikacija izolata izvršena je na osnovu kulturelnih, biohemijskih i seroloških karakteristika na osnovu preporuka standarda SRPS EN ISO 6579:2008. Potvrda i serološka tipizacija izolata Salmonella izvedena je u Nacionalnoj referentnoj laboratoriji za Salmonella, Shigela, Vibrio cholerae i Yersinia enterocolitica, Instituta za javno zdravlje Srbije „Dr Milan Jovanović Batut“ u Beogradu. Kao kontrolni sojevi, u ispitivanju su korišćene referentne kulture S. Typhimurium ATCC 14028 i S. Enteritidis ATCC 13076. Kao supstrat za formiranje biofilma korišćene su površine od polistirena i nerđajućeg čelika. Sposobnost formiranja biofilma ispitana je primenom sledećih metoda: Congo red agar test, pelikula test, test na mikrotitracionim pločama upotrebom kristal violet boje, i skenirajuća elektronska mikroskopija (SEM). Sva ispitivanja izvedena su upotrebom dve hranljive podloge (tripton soja bujona i Luria Bertani bujona) pri temperaturi inkubacije od 20C i 37C. Iz hrane za životinje izolovani su sledeći serovarijeteti Salmonella: Tennessee (18%), Agona (9%), Montevideo (15%), Enteritidis (12%), Stanleyville (6%), Infantis (12%), Typhimurium (3%), Typhimurium monofazni (1%), Mbandaka (6%), Senftenberg (7%), Jerusalem (2%), Thompson (1%), Amsterdam (1%), Colindale (1%), Dahra (1%), Livingstone (1%). Kod četiri izolata nije ustanovljen serovarijetet (Salmonella enterica subspecies enterica (1,3,19:i:-)). Prema rezultatima Congo red agar testa, na temperaturi od 20C, 98% izolata Salmonella ekspresioniralo je jednu ili obe komponente matriksa biofilma i formiralo kolonije RDAR, PDAR i BDAR morfotipova...The aim of this research was the investigation into the biofilm-forming ability of Salmonella serovars isolated from animal feed samples originating from 18 feedproducing facilities located in South Bačka District. The isolates which were tested (n=100) were isolated from feed samples collected in a two-year period (from 2012 to 2014). The identification of the isolates was made based on the cultural, biochemical and serological characteristics, following the recommendations given in the SRPS EN ISO 6579:2008 standard. The conformation and serological typing of Salmonella isolates were done in the National Reference Laboratory for Salmonella, Shigela, Vibrio cholerae and Yersinia enterocolitica, at the Institute of Public Health of Serbia 'Dr Milan Jovanović Batut' in Belgrade. In this study reference cultures of S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076 were used as the control strains. Surfaces made of polystyrene and of stainless steel were used as substrates for biofilm formation. The ability of Salmonella to form biofilm was tested with the following methods: Congo red agar test, pellicle test, microtiter plate assay with the use of crystal violet dye, and scanning electron microscopy (SEM). The whole investigation was carried out at two temperatures of incubation (20C and 37C). The following Salmonella serovars were isolated from animal feed: Tennessee (18%), Agona (9%), Montevideo (15%), Enteritidis (12%), Stanleyville (6%), Infantis (12%), Typhimurium (3%), Typhimurium monofazni (1%), Mbandaka (6%), Senftenberg (7%), Jerusalem (2%), Thompson (1%), Amsterdam (1%), Colindale (1%), Dahra (1%), Livingstone (1%). In four isolates serovars were not determined (Salmonella enterica subspecies enterica (1,3,19:i:-)). According to the results of Congo red agar test, when grown at 20C, 98% of the isolates expressed one or both components of the Salmonella biofilm matrix and formed colonies of various morphotypes (RDAR, PDAR and BDAR)..

Istraživanje sposobnosti tvorbe biofilma u sojeva Salmonella enterica serovar Tennessee izdvojenih iz hrane za životinje

Salmonella enterica subspecies enterica serovar Tennessee was the most frequent Salmonella serovar isolated from plant-based animal feed throughout a two-year period (2012-2013) of research conducted in the region of the Autonomous Province of Vojvodina. In this study, the ability of biofilm formation was investigated on 20 isolates of S. Tennessee. The starting point for this research was the established biofilm-forming ability of serovar Tennessee strains, which significantly contributes to their wide distribution in the environment (plant materials), and hence in animal feed. In this study, biofilm formation was investigated on polystyrene (microplate biofilm assay) and stainless steel (scanning electron microscopy) surfaces by culturing in Luria Bertani broth (LB) and Triptone Soya Broth (TSB). The expression of major matrix components of Salmonella biofilm (curli fimbriae and cellulose) was examined by cultivation on Congo Red agar. All isolates of S. Tennessee produced a biofilm on a polystyrene surface in a microtiter plate test, by cultivation in LB at an incubation temperature of 20 °C. Scanning electron microscopy revealed biofilm formation of S. Tennessee on a stainless steel surface by cultivation in LB, but not in TSB. The expression of curli fimbriae and cellulose, as well as the formation of characteristic “rdar” colonies on Congo Red agar has been observed in all isolates of S. Tennessee. This serovar is not specifically adapted to humans and animals, so the available epidemiological data do not indicate its major implication in food-borne infection outbreaks. However, this serovar manifests a pronounced ability for adherence to plant surfaces, biofilm formation and increased resistance to long-term desiccation, which is the most likely explanation for its frequent identification in low water activity feed.Salmonella enterica subsp. enterica serovar Tennessee bio je najčešće ustanovljen u hrani za životinje biljnog podrijetla u dvogodišnjem razdoblju istraživanja (2012. – 2013.) na području Autonomne pokrajine Vojvodine. Istražena je sposobnost proizvodnje biofilma kod 20 izolata serovara Tennessee, s polaznom pretpostavkom da sojevi toga serovara imaju dobru sposobnost tvorbe biofilma i da ta osobina značajno doprinosi njihovoj rasprostranjenosti u prirodnom okruženju (biljna materija), a posljedično i u hrani za životinje. Biofilm je bio proizveden na površinama od polistirena (test na mikrotitracijskim pliticama) i nehrđajućeg čelika (skenirajuća elektronska mikroskopija) uzgojem u Luria Bertani bujonu (LB) i tripton soja bujonu (TSB). Ekspresija glavnih komponenti matriksa biofilma salmonela (fimbrije i celuloza) istražena je uzgojem izolata na agaru s kongo crvenilom. Svi izolati serovara Tennessee tvorili su biofilm na površini polistirena u testu na mikrotitracijskim pliticama, uzgojem u LB na temperaturi inkubacije od 20 °C. Skenirajućom elektronskom mikroskopijom, na površini nehrđajućeg čelika ustanovljena je proizvodnja biofilma uzgojem u LB, ali ne i u TSB. Svi izolati tvorili su kovrčave fimbrije i celulozu te oblikovali kolonije karakterističnog rdar morfotipa na agaru s kongo crvenilom. Serovar Tennessee nije specifično adaptiran na ljude i životinje i prema epidemiološkim podatcima nema osobito značenje u pojavi hranom prenosivih infekcija. Međutim, on posjeduje izraženu sposobnost adherencije na površinu biljaka, sposobnost produkcije biofilma i otpornost na isušivanje, što vjerojatno doprinosi njegovom čestom nalazu u hrani za životinje s niskim sadržajem vlage

Clonal persistence of Salmonella enterica serovars Montevideo, Tennessee, and Infantis in feed factories

Introduction: Novel molecular techniques applied in biotechnology research have provided sound evidence on clonal persistence of distinct serovars of Salmonella in feed factory environments, over long periods of time (months, even years), which can be responsible for repeated in-house contamination of final products. In this study, we examined the possibility of clonal persistence of isolates of three Salmonella serovars that have been repeatedly identified in animal feed samples from three feed factories throughout a two-year period. Methodology: The isolates Salmonella enterica serovars Tennessee (n = 7), Montevideo (n = 8), and Infantis (n = 4) were tested for genetic diversity using pulsed-field gel electrophoresis (PFGE) and multicellular behavior patterns by applying the Congo red agar test. Results: SpeI and XbaI macro-restriction profiles indicated that isolates S. Montevideo and S. Infantis were identical, whereas isolates of S. Tennessee demonstrated greater genetic diversity, although the genetic differences did not exceed 10%. All Salmonella serovars demonstrated the ability to produce predominant matrix compounds essential for biofilm formation, curli fimbriae and cellulose. Conclusions: The identification of identical clones of S. Montevideo and S. Infantis, as well as the minor genetic diversity of S. Tennessee, which have been repeatedly isolated from animal feed in three production plants throughout a two-year period, indirectly suggests the possibility of their persistence in feed factory environments. Their ability to express the key biofilm matrix components further supports this hypothesis

Fumonisins and co-occurring mycotoxins in north Serbian corn

The presence of fumonisin has not been regulated in the legislation of the Republic of Serbia. Therefore, the data on contamination of cereals, especially corn, which is highly susceptible to contamination by this toxin, are not sufficient. This paper presents the results of testing the corn samples collected in the autumn 2009 on the territory of Bačka. Samples were analyzed for the contents of fumonisins and it was determined whether there is a correlation between the moisture content, total number and class of fungi, as well as the content of aflatoxin, ochratoxin and zearalenone. Using enzymatic immunoaffinity method it was discovered that the highest percentage of samples were contaminated with fumonisins, which was probably due to the presence of Fusarium molds as the most abundant ones. The positive samples contained fumonisin in the concentrations from 0.030 to 1.52 mg kg−1. The influence of the climate and moisture content of grain on fungal contamination and mycotoxin production was analyzed in order to investigate the predictability of the presence of mycotoxins

Analysis of possible biofilm formation from different Salmonella serovars isolated from animal food in in vitro conditions.

Cilj ovog istraživanja je bio ispitivanje sposobnosti formiranja biofilma kod serovarijeteta Salmonella izolovanih iz hrane za životinje, poreklom iz 18 mešaona stočne hrane na području Južnobačkog okruga. Izolati korišćeni u ispitivanju (n=100) izolovani su poreklom iz uzoraka hrane za životinje uzorkovanih tokom dve godine ispitivanja (period 2012-2014). Identifikacija izolata izvršena je na osnovu kulturelnih, biohemijskih i seroloških karakteristika na osnovu preporuka standarda SRPS EN ISO 6579:2008. Potvrda i serološka tipizacija izolata Salmonella izvedena je u Nacionalnoj referentnoj laboratoriji za Salmonella, Shigela, Vibrio cholerae i Yersinia enterocolitica, Instituta za javno zdravlje Srbije „Dr Milan Jovanović Batut“ u Beogradu. Kao kontrolni sojevi, u ispitivanju su korišćene referentne kulture S. Typhimurium ATCC 14028 i S. Enteritidis ATCC 13076. Kao supstrat za formiranje biofilma korišćene su površine od polistirena i nerđajućeg čelika. Sposobnost formiranja biofilma ispitana je primenom sledećih metoda: Congo red agar test, pelikula test, test na mikrotitracionim pločama upotrebom kristal violet boje, i skenirajuća elektronska mikroskopija (SEM). Sva ispitivanja izvedena su upotrebom dve hranljive podloge (tripton soja bujona i Luria Bertani bujona) pri temperaturi inkubacije od 20C i 37C. Iz hrane za životinje izolovani su sledeći serovarijeteti Salmonella: Tennessee (18%), Agona (9%), Montevideo (15%), Enteritidis (12%), Stanleyville (6%), Infantis (12%), Typhimurium (3%), Typhimurium monofazni (1%), Mbandaka (6%), Senftenberg (7%), Jerusalem (2%), Thompson (1%), Amsterdam (1%), Colindale (1%), Dahra (1%), Livingstone (1%). Kod četiri izolata nije ustanovljen serovarijetet (Salmonella enterica subspecies enterica (1,3,19:i:-)). Prema rezultatima Congo red agar testa, na temperaturi od 20C, 98% izolata Salmonella ekspresioniralo je jednu ili obe komponente matriksa biofilma i formiralo kolonije RDAR, PDAR i BDAR morfotipova...The aim of this research was the investigation into the biofilm-forming ability of Salmonella serovars isolated from animal feed samples originating from 18 feedproducing facilities located in South Bačka District. The isolates which were tested (n=100) were isolated from feed samples collected in a two-year period (from 2012 to 2014). The identification of the isolates was made based on the cultural, biochemical and serological characteristics, following the recommendations given in the SRPS EN ISO 6579:2008 standard. The conformation and serological typing of Salmonella isolates were done in the National Reference Laboratory for Salmonella, Shigela, Vibrio cholerae and Yersinia enterocolitica, at the Institute of Public Health of Serbia 'Dr Milan Jovanović Batut' in Belgrade. In this study reference cultures of S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076 were used as the control strains. Surfaces made of polystyrene and of stainless steel were used as substrates for biofilm formation. The ability of Salmonella to form biofilm was tested with the following methods: Congo red agar test, pellicle test, microtiter plate assay with the use of crystal violet dye, and scanning electron microscopy (SEM). The whole investigation was carried out at two temperatures of incubation (20C and 37C). The following Salmonella serovars were isolated from animal feed: Tennessee (18%), Agona (9%), Montevideo (15%), Enteritidis (12%), Stanleyville (6%), Infantis (12%), Typhimurium (3%), Typhimurium monofazni (1%), Mbandaka (6%), Senftenberg (7%), Jerusalem (2%), Thompson (1%), Amsterdam (1%), Colindale (1%), Dahra (1%), Livingstone (1%). In four isolates serovars were not determined (Salmonella enterica subspecies enterica (1,3,19:i:-)). According to the results of Congo red agar test, when grown at 20C, 98% of the isolates expressed one or both components of the Salmonella biofilm matrix and formed colonies of various morphotypes (RDAR, PDAR and BDAR)..

Molecular Investigation of <i>Eimeria</i> Species in Broiler Farms in the Province of Vojvodina, Serbia

Coccidiosis is a significant poultry disease caused by the Eimeria species. This study aims to determine the prevalence of Eimeria spp. on broiler farms in Vojvodina, along with the identification of parasite species, and assess the implemented biosecurity measures. The study was conducted on 100 broiler chicken farms (28 small-sized; 34 medium-sized; 38 large-sized farms) from June 2018 to December 2021. One pooled sample of faeces was collected from three to six-week-old chickens from each farm, and assessment of biosecurity measures was carried out using a questionnaire. Using the PCR method, DNA of Eimeria was found in 59 samples (59%), while 41 samples (41%) were negative. Four species of Eimeria were identified, and their prevalence was the following: E. acervulina (37%), E. maxima (17%), E. mitis (25%) and E. tenella (48%). A significant difference (p < 0.05) was established in the number of oocysts in flocks from small-sized farms compared to medium-sized farms. It was found that regular implementation of disinfection, disinsection and deratisation measures, as well as all the biosecurity measures, can significantly reduce the occurrence of coccidiosis. These results will help to develop better strategies for the control and prevention of coccidiosis on farms

Investigation of dissemination of aspergillosis in poultry and possible control measures

Fungi belonging to genus Aspegillus are ubiquitous saprophytic microorganisms which are, in certain circumstances, responsible for clinical infections of respiratory tract in all poultry, particularly in young birds. In case of a lung form, Aspergillus fumigatus, A. niger and A. glaucus are the most frequently isolated fungi. In general, poultry is constantly exposed to these fungi in its environment. Predisposing factors, such as long exposition and highly contaminated environment and litter, high humidity in poultry houses, poor ventilation, malnutrition and stress, all contribute to clinical aspergillosis. Some geographic and seasonal regularities are observed in relation to the distribution of disease outbreaks. In this sense, cases of aspergillosis in our country were more frequently noted in wild areas located northern from the rivers Sava and Danube. Influence of some factors on the outbreak and spreading, as well as predominant clinical features of aspergillosis in poultry were investigated in this paper. Possible prophylactic and intervention measures were discussed. The occurrence of Aspergillus sp. in poultry was analyzed according to the clinical and laboratory investigations performed during the two selected years, 2000 and 2010. Widespread aspergillosis was noted in poultry flocks of different age, both in young and adult birds. During the years 2000 and 2010, acute aspergillosis was found in 12 and 16 commercial flocks of chickens and turkeys, respectively. Ocular infection with Aspergillus was determined in 10 day old broilers from two flocks. Aspergillus sp. was isolated from unhatched eggs (6.86%), litter (23.07%), environmental (36.17%) and hatchery swabs (3.85%). Besides the appropriate antifungal therapy, enforcement of proper sanitary-hygiene measures on poultry farms and hatcheries, as well as microbiological control of feed are considered essential for an efficient control of infection and its spreading