Ljiljana Suvajdžić

Outbreak of endemic form of protothecal mastitis on a dairy farm

In this paper an outbreak of mastitis in cows resistant to antibiotic treatment on a Holstein-Friesian cattle farm in Vojvodina (Serbia and Montenegro) was described. Microbiological examination of 73 milk samples from lactating cows revealed the presence of Prototheca zopfii in 22 samples (30.13%). Identification of the isolates was performed on the basis of growth features, microscopic appearance, assimilation patterns and antimicrobial susceptibility. Protothecal infection of the mammary gland resulted in clinical symptoms of chronic mastitis in 19 animals. Histopathological findings revealed distinct granulomatous inflammations of the mammary gland interstitium.

Mastitis associated with Prototheca zopfii - an emerging health and economic problem on dairy farms

Abstract Increased incidence of protothecal mastitis has been recorded in several countries in the past ten years. The main goal of this article is to draw the attention of scientific and professional community to the emerging issue of mammary protothecosis. The article collates currently known facts about infection reservoirs, predisposing factors for the development of mastitis, clinical manifestations of the disease, and potential transmission routes within the herd as well as the measures for control and eradication. We would like to point out that identification of protothecal mastitis on a dairy farm is associated with a range of problems. Early detection of infected animals can be difficult because of predominantly subclinical course of early-stage infection, which easily spreads between cows via the milking system. Spontaneous recovery has not been recorded and infected cows typically develop chronic mastitis with granulomatous infiltration and progressive loss of functional parenchyma of the mammary gland. Substantial economic losses and health damages associated with mammary protothecosis strongly emphasise the need for developing effective prevention strategies aimed at control of the infection.

Influence of the Sodium Salt of 3α,7α-Dihydroxy-12-Oxo-5β-Cholanate on Antimicrobial Activity of Ampicillin In Vitro

Background: Multiple resistances to antibiotics are an emergent problem worldwide. Scientists intensively search for new substances with the antimicrobial potential or the mode to restore the activity of old-generation antibiotics. Ampicillin is the antibiotic with the expanded range of antimicrobial activity, but its use has decreased due to the poor absorption and highly developed resistance. In vivo studies showed that ampicillin has better absorption and bioavailability if combined with bile acid salts. The aim of this study was to examine antimicrobial effects of ampicillin alone and its combination with semisynthetic monoketocholic acid salt (MKH) in vitro. Materials, Methods & Results: In this study, commercial preparation of ampicillin and sodium salt of 3α,7α-dihydroxy-12oxo-5β-cholanate were used. Their effects were evaluated on Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium), obtained from urine specimens of dogs with clinically manifested cystitis. The first two investigated strains were ampicillin-sensitive, while E. faecium was resistant to ampicillin. Modified macrodilution method according to Clinical and Laboratory Standards Institute Guidelines (M7-A8) was performed. Bacterial suspension equivalent to 0.5 McFarland was prepared in saline, compared to the standard (Biomerieux) ad oculi. The density was checked spectrophotometrically at a wavelength of 625 nm and adjusted if necessary to the desired absorbance from 0.08 to 0.1. The resultant suspension was diluted 1:100 and inoculated in test tubes. Number of bacteria was counted on Petri plates using dilutions from 10-3 to 10-7 in order to obtain valid and countable plates. One hundred microliters of appropriate dilutions were aseptically plated in triplicate onto nutrient agar. Plates were incubated on 37°C for 72 h, under aerobic conditions. The number of colony forming units (CFU) was determined by direct counting. As a valid for enumeration, we took plates with 30 to 300 CFU. Percentage of killed bacteria for ampicillin was from 69.33-95.19% for E. coli, 87.1296.92% for E. faecalis and 7.20-33.30% for E. faecium. Ampicillin applied in the combination with MKH killed 99.99% to 100% of E. coli, 94.59% to 99.91% of E. faecalis and 31.73% to 64.76% of E. faecium. Mean percentage of killed bacteria for ampicillin was 81.93% for E. coli, 91.64% for E. faecalis, and 18.13% for E. faecium, while in combination with MKH percentage was 99.96% for E. coli, 98.23% for E. faecalis and 47.54% for E. faecium. Discussion: Results are presented as pharmacological minimal inhibitory concentration (MIC) values. Ampicillin was applied at the concentration higher than the therapeutic one, which could explain high MIC values for E. coli and E. faecalis. The combination of ampicillin with MKH showed the best improvement of antimicrobial effect on E. faecium (Δ = 29.41%), isolate that was resistant to ampicillin when applied alone. In all the investigated isolates, the combinations with MKH were more effective than ampicillin administered alone. It seems that MKH demonstrates a synergistic antimicrobial activity with ampicillin in vitro, which considerably decreases MIC values for all investigated isolates. These results implicate that MKH could restore the previous activity of ampicillin against some bacteria, which could be a significant benefit for clinical practice.

Activity of Serbian Aronia prunifolia against Prototheca wickerhamii and Prototheca zopfii

Background: Beneficial effects of berries have been known from 16th century, but their antimicrobial effects have been explained scientifically only recently. The two most common aronia species are black chokeberry, Aronia melanocarpa [Michx.] Elliot and red chokeberry, Aronia arbutifolia [L.] Elliot. Purple chokeberry (Aronia prunifolia) is a hybrid of these two species. Protothecosis is a disease caused by achlorophyllous algae Prototheca species. Infections with Prototheca species are more common in veterinary medicine. The purpose of this study was to investigate the activity of Aronia prunifolia berries against Prototheca zopfii (P. zopfii) and Prototheca wickerhamii (P. wickerhamii). Materials, Methods & Results: Purple chokeberry juice was made by squeezing the fruits and evaporated to dryness. Extracts of purple aronia were obtained by maceration with ethanol 80 % (v/v) for 24 h. Prototheca zopfii was obtained from udder of cow with mastitis and Prototheca wickerhamii was isolated from human oral cavity. Total phenolic and flavonoid contents were analized using spectrophotometric methods. The chemical composition of the tested substances was determined by a high performance liquid chromatography (HPLC) method. The examination was conducted by a micro-dilution method according to the guidelines of Clinical and Laboratory Standards Institute (CLSI). Microsoft Excel program 2007 was used for statistical analysis. The antialgal activity was expressed as minimal inhibitory concentrations MIC99, MIC90 and MIC80, the lowest concentration which kills 99%, 90% and 80% of organisms, respectively. Furthermore, minimal algaecide concentration (MAC), the lowest concentration which kills 99.9% of organisms is determined, as well as break point, the lowest concentration at which there is no algal growth. Discussion: The content of ascorbic acid was twice as high in the ethanolic extract as in the juice. Content of polyphenolic compounds was high in both juice and ethanolic extract. The quantity of phenocarbonic acids in juice and ethanolic extract was relatively low. Some of them were found only in juice (ellagic, coumaric and gentisic acids) as opposed to others found only in ethanolic extract (chlorogenic acid). Flavonoids were also detected in juice and ethanolic extract. Extract was much richer in flavonoid content when compared to aronia juice. Catechin was present in concentration of 186.3 mg/ 100 g of dry matter in the aronia juice, and 680.65 mg/ 100 g of dry matter in the ethanolic extract, which was more than 3.6 times higher. Quercetin was found only in the extract. The rutin content was 12 times and the chrysin content was 2.5 times higher in the aronia extract. The biggest difference could be noted in the quantitative contents of anthocyanins, 26 times higher concentration in extract than in juice. In general, higher content of bioactive compounds could be observed in the extract than in the juice. The results showed that the ethanolic extract of aronia fruits exhibited antialgal activity against both Prototheca species, while the juice showed no antialgal activity. This difference in antialgal effect is presumably related to the high content of several groups of biocompounds, especially catechin and anthocyanins, present in the ethanolic extract, and probably their synergistic action. There is no comparable data of antialgal effects of aronia in literature.

Characterization Of Tetracycline Resistance Of Salmonella Enterica Subspecies Enterica Serovar Infantis Isolated From Poultry In The Northern Part Of Serbia

Abstract Resistance to tetracycline was studied in Salmonella Infantis isolated from 28 poultry farms in the Northern part of Serbia (The Autonomous Province of Vojvodina). A total of 18 isolates were resistant to nalidixic acid (NAL) and tetracycline (TET). The minimal inhibitory concentration (MIC) to TET, ranged from 1-256 mg/L. Namely, 13 isolates exhibited MIC to TET at 256 mg/L, in four of the isolates, the MIC was 128 mg/L and one isolate had MIC 64 mg/L. Ten isolates were exhibiting a MIC of 1mg/L. It was evident that Salmonella Infantis had also spread to breeders and layers. In this work, we detected the tetA gene and the corresponding tetR gene (encoding the repressor protein) as well as the truncated transposon Tn1721, which are responsible for the resistance to TET. The presence of the non conjugative transposons from the conjugative plasmid has facilitated the spread of resistance to TET in Salmonella. It was concluded that higher biosecurity practice in poultry farming presents the best option to eliminate infections caused by Salmonella spp. from poultry flocks in Serbia. A rational use of antimicrobials is necessary to prevent any further spread of Salmonella Infantis resistant clones.