Isolation and molecular typing of Mycobacterium avium subsp. paratuberculosis from faeces of dairy cows.

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis mainly in domestic and wild ruminants; paratuberculosis is also known as Johne's disease. This disease is endemic all over the world generating significant economic losses, especially in dairy herds, although, MAP is the cause of infection in many other species including primates. Currently, MAP mycobacteria are recognized as pathogens transmitted by food. They are a potential threat to animal and human health. Infected animals excreting mycobacteria with faeces are the main source of MAP. The development of control strategies and disease control are based on determination of the genetic diversity of the MAP strains causing Johne's disease. This study describes 43 strains isolated from a herd of dairy cows located in northern Poland. The types of MAP were determinted based on the polymorphism analysis of two insertion fragments: IS900 and IS1311. The polymorphism of IS900 was analyzed with the use of a PCR multiplex according to Collins' method and the IS1311 polymorphism with the use of the PCR-REA method. Based on the differences observed, the strains isolated were classified into two MAP types, cattle (C-type) and sheep (S-type), with the predominance of the cattle type.

Determination of polymorphism of IS-1311 sequence in Mycobacterium avium subspecies paratuberculosis strains isolated from milk samples.

The study was aimed at the genetic typing of Mycobacterium avium subspecies paratuberculosis (MAP) with PCR-REA method based on the polymorphism of IS-1311 sequence. MAP strains were isolated from milk samples collected from cows free of disease symptoms and anti-MAP antibodies. Samples of udder milk were collected from 310 cows originating from the herd with a low seroprevalence of paratuberculosis; every 5 samples were pooled. The polymorphism typing of the IS-1311 sequence of the isolated strains demonstrated the presence of two types of the mycobacterium: sheep and cattle ones, of which the bovine type constituted 98.5%.

Modeling the kinetics of survival of Staphylococcus aureus in regional yogurt from goat's milk.

The aim of this study was to determine the kinetics of the survival of the test strain of Staphylococcus aureus in the product investigated. Yogurt samples were contaminated with S. aure to an initial level of 10(3)-10(4) cfu/g. The samples were then stored at four temperatures: 4, 6, 20, 22°C. During storage, the number of S. aureus forming colonies in a gram of yogurt was determined every two hours. Based on the results of the analysis culture the curves of survival were plotted. Three primary models were selected to describe the kinetics of changes in the count of bacteria: Cole's model, a modified model of Gompertz and the model of Baranyi and Roberts. Analysis of the model fit carried out based on the average values of Pearson's correlation coefficient, between the modeled and measured values, showed that the Cole's model had the worst fit. The modified Gompertz model showed the count of S. aureus as a negative value. These drawbacks were not observed in the model of Baranyi and Roberts. For this reason, this model best reflects the kinetics of changes in the number of staphylococci in yogurt.

Evaluation of methods for Mycobacterium avium ssp. paratuberculosis detection in milk samples from the cattle herd showing low seroprevalence of Johne's disease.

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of chronic gastroenteritis in cattle called the Johne's disease (JD). The disease causes significant economic losses in cattle production. MAP is also supposed to be involved in the Crohn's disease and inflammatory bowel disease (IBD) in people. The detection of the cattle infection based on investigations of milk samples and evaluation of the capacity of the methods used to detect the disease was the objective of the present study. Following methods were applied for milk samples testing: detection of MAP in bacterial culture, detection of the specific IS-900 fragment of MAP in the genetic material isolated directly and detection of MAP antibodies. The results obtained were compared with the "golden standard" results, i.e. the isolation of MAP from the faeces. PQStat-the program for diagnostic reliability estimation, was used for evaluation of the sensitivity, specificity and predictive value. The method based on detection of the specific IS-900 fragment of MAP in the genetic material isolated directly from milk samples was found to possess the highest sensitivity. Detection of anti-MAP antibodies on the other hand showed the lowest sensitivity. The method of detecting anti-MAP antibodies in milk was the most specific while detection of the IS-900 fragment in the genetic material was the least specific method. These results obtained may serve as a guide to choose the most appropriate method for diagnosis of MAP infections by milk sample testing.

Quality of milk of cows in the first lactation vs. presence of anti-Ostertagia antibodies in their milk.

Invasions of gastrointestinal nematodes in dairy cows may affect animals productivity. The most frequently detected internal parasite of dairy cattle is Ostertagia ostertagi. The objective of this study was to determine O. ostertagi invasion extensiveness in selected herds of dairy cattle, with special consideration to cows being in the first lactation, and to analyze the milk yield and contents of basic constituents of milk originating from sero-positive cows. Five herds of dairy cattle (403), with different populations of cows, were selected for the study. Invasion extensiveness in particular herds was determined and ranged from 11.9% to 27.27%. Cows being in the first lactation, the udder milk of which was shown to contain anti-O. ostertagi antibodies, were producing on average 470 kg of milk annually less than cows being in the same lactation period. The analysis of results did not confirm the statistical significance of this difference, likewise it did not demonstrate any statistically significant differences in contents of fat, protein and dry matter. Despite a lack of the statistical significance a producer suffers great economic losses. The conducted study proves that the occurrence of O. ostertagi invasion in herds of dairy cattle is a global problem and that it affects cost-effectiveness of milk production.

The presence of anti-Hypoderma antibodies in udder milk samples, and correlation with selected parameters of dairy performance.

Hypodermosis is responsible for significant economic losses and remains a current problem in animal husbandry. This study determined the extent of Hypoderma spp. invasion in north-eastern Poland in dairy herds. The extent of the invasion, determined on the basis of positive results of an ELISA assay (Bovine Hypodermosis Milk Screening of Institut Pourquier), varied in herds. Statistical analysis performed on the results of milk productivity and content of basic components revealed a decrease in the milk yield and changes in milk protein in animals revealing positive, doubtful and negative results. Statistically significant differences were absent as regards the content of milk fat or milk total solids. The milk yield and the content of particular milk components were determined using the AT4 method, as part of a control program carried out by the Polish Federation of Cattle Breeders and Dairy Farmers (PFHBiPM).

Prevalence and antimicrobial resistance of Campylobacter in raw milk in the selected areas of Poland.

During the recent years, an immense increase in the number of food poisoning cases in people caused by Campylobacter (C.) species has occurred. Raw milk, next to poultry meat, is considered the most frequent cause of food poisoning in people caused by the subject bacteria, although it is not always possible to isolate Campylobacter cells from the incriminated milk. Most probably this difficulty is caused by low concentration of the pathogen in milk at the level of 2/3 cells/ml although even such low concentration represents risk to human health. The present study was aimed at determining the occurence of Campylobacter bacteria in milk originating from selected regions of Poland. The isolation method applied in this work was effective in recovering as few as 0.1 cell of Campylobacter per g of food. Among 150 bulk milk samples tested, Campylobacter spp. was isolated from 7 (4.6%) ones. The biochemical identification of the isolated strains conducted by means of conventional biochemical tests as well as by applying the API - Campy tests revealed that all the isolates belonged to the C. jejuni species. Determination of resistance to antibiotics was performed by means of the diffusion disks method for the following antibiotics: gentamicin, ciprofloxacin, ampicillin, chloramphenicol, erythromycin, doxycyclin and tetracycline. Among 7 isolates tested, all were susceptible to ampicillin, chloramphenicol, erythromycin and gentamicin, 28.5% to doxycyclin and 14.2% to tetracycline and ciprofloxacin.

Analysis of correlations between the occurrence of anti-MAP antibodies in blood serum and the presence of DNA-MAP in milk.

Paratuberculosis (Johne's disease) is a chronic, infectious enteritis of both domestic and wild ruminants. Unfortunately, the problem of MAP infections is not linked only with the health status of animals and potential direct and indirect economic losses in bovine herds (of dairy cattle in particular). MAP bacilli present in food of animal origin (milk in particular) are likely to lead to the development of the disease in humans. Fast and effective diagnosis of the disease in animals, especially of its subclinical form, may prevent the transmission of the germ to humans. The study was aimed at analyzing the correlations between the occurance of seropositive and serodoubtful reaction in the ELISA test and the presence of DNA-MAP in udder milk. The results suggest that half of the population of animals with positive and doubtful serological responces against John's disease are likely to be a potential source of germ transmission into humans. The fact of detecting DNA-MAP in 1/3 of all milk samples points to the likelihood of occurrence of MAP bacilli in milk of animals not displaying seropositive or serodoubtful responses.

Effectiveness of Mycobacterium paratuberculosis isolation from raw milk by means of direct isolation of DNA and classic culture.

Detection of Mycobacterium paratuberculosis (MAP) in tissues of patients suffering from Crohn's disease has given rise to speculation that this mycobacterium may play some role in the development of this disease in humans. Food products, especially milk obtained from animals infected with paratuberculosis, may be a potential vector of MAP to humans, yet the detection of this pathogen poses a number of difficulties. This study was aimed at comparing the effectiveness of MAP isolation from milk samples. Mycobacteria were detected by means of two methods: direct isolation of DNA using a QIAamp DNA Mini Kit by Qiagen, and a culture method with the use of HEYM culture medium. Analyses were carried out on 87 samples of udder cow milk originating from a herd that exhibited seropositive and serodoubtful reactions against paratuberculosis. The presence of an insertion sequence IS-900 was detected in 18 samples of udder milk analyzed with the method of direct DNA isolation and in two samples analyzed by means of the culture method.

Flow cytometry as a method for the evaluation of raw material, product and process in the dairy industry.

Producing dairy products which are safe for consumers requires the constant monitoring of the microbiological quality of raw material, the production process itself and the end product. Traditional methods, still a "gold standard", require a specialized laboratory working on recognized and validated methods. Obtaining results is time- and labor-consuming and do not allow rapid evaluation. Hence, there is a need for a rapid, precise method enabling the real-time monitoring of microbiological quality, and flow cytometry serves this function well. It is based on labeling cells suspended in a solution with fluorescent dyes and pumping them into a measurement zone where they are exposed to a precisely focused laser beam. This paper is aimed at presenting the possibilities of applying flow cytometry in the dairy industry.

Optimisation of the time of milk sample decontamination with 0.75% cethylpirydinium chloride upon identification of Mycobacterium avium subsp. paratuberculosis.

Isolation of MAP from biological material, including milk, is very difficult. No matter how the fresh milk is obtained, it contains a certain number of rapidly growing microorganisms. Therefore, the decontamination process is applied in the methods of MAP isolation. Investigations were therefore undertaken to determine the optimal time of milk sample decontamination with 0.75% CPC. The experiment was divided into two parts: determination of the survival time of M.smegmatis and E. coli in 0.75% CPC and determination of the time of raw milk decontamination. The results suggest that the optimal time of milk sample decontamination is 15 min.

Detection methods of Mycobacterium paratuberculosis.

Mycobacterium paratuberculosis is receiving increasingly wider interest of scientific groups worldwide. This slow-growing mycobacterium does not only evoke paratuberculosis--an infectious cattle disease that brings huge economic losses--but it is also regarded as a potential cause of human Crohn;s disease. It is very difficult to diagnose precisely this kind of animal infection in its very early stages, as well as to detect occurrence of M. paratuberculosis cells in the environment, including food of animal origin. This paper reviews currently known and employed diagnostic techniques for M. paratuberculosis cell detection and identification.

Investigation on the inhibition of Campylobacter jejuni growth with the applications of some preservatives, medicines, herbs and herb preparations.

The aim of this study was to determine the influence of chosen preservatives, medicines, herbs and herb preparations on 39 Campylobacter jejuni strains, which were isolated from fresh poultry carcasses. De-Nol and ventrisol were used in the original concentration. Propolis, propolan, artecholin and aukalen were of pharmacies origin. The rest of substances examined were 10% water solutions. Out of 11 substances examined, potassium sorbate, sodium benzoate, De-Nol and ventrisol exerted the strongest inhibitory action on Campylobacter jejuni.

The occurrence of infectious and parasitic diseases in poultry slaughtered in the district of Olsztyn, Poland, 1986-91.

During 6 years, 1986-91, in poultry processing plants in the district of Olsztyn, Poland, 37,779,959 carcasses and internal organs of slaughtered fattened poultry were (1,691,188 hens, 23,681,855 chickens, and 12,226,016 turkeys) were examined. As a result of the antemortem and postmortem inspections, 744,499 birds were condemned for human consumption, which was 1.66% of the total number of birds examined. The highest percentage of condemned birds was registered in hens (2.4%) and the lowest in chickens (1.27%). By means of a medical examination, the following diseases were diagnosed most frequently in chickens: Marek's disease in 2,265 birds (0.095%), salmonellosis in 13,463 birds (0.056%), coccidiosis in 9,548 birds (0.04%), and chronic illness of the respiratory system in 377 birds (0.0016%). In hens, salmonellosis was found in 15,951 birds (0.94%), tuberculosis in 301 birds (0.018%), leukemia in 122 birds (0.007%), and aspergillosis in 71 birds (0.0042%). In turkeys, chronic illness of the respiratory system was found in 23,938 birds (0.196%), aspergillosis in 13,243 birds (0.11%), salmonellosis in 3,918 birds (0.032%), and leukemia in 29 birds (0.00024%).

[Effect of preservatives on survival of Campylobacter jejuni in ground pork meat]. / Wplyw srodków konserwujacych na przezywalnosc Campylobacter jejuni w mielonym miesie wieprzowym.

Campylobacter jejuni strains: Pen 2, 3, 6, 10 and 20 isolated from the food-borne infections in humans were tested. Fresh ground pork samples supplemented with chemical preservatives: sodium chloride--24,000 mg/kg, sodium nitrite--125 mg/kg, potassium nitrate--500 mg/kg, sodium ascorbate--300 mg/kg and polyphosphate (Hamine S)--3000 mg/kg were contaminated by C. jejuni strains. Survival of C. jejuni in ground pork was determined immediate after the contamination and over a 2-d period at 4 degrees C on Brucella agar (Difco) containing 10% horse blood, which were incubated 48 hrs at 42 degrees C under microaerobic conditions (5% O2, 5% CO2 and 90% N2). Campylobacter jejuni was isolated from all tested samples at the initial inoculum 2.5 x 10(5) to 1.7 x 10(8) cfu/1 g of meat. It was proved that chemical preservatives, added to meat samples in concentration usually used in meat processing, were affected in differential way on the survival of different strains of C. jejuni. Campylobacter jejuni Pen 2 was resistant to all preservatives used in this studies. Campylobacter jejuni Pen 3 and Pen 10 were sensitive to sodium nitrite, and Pen 10 was sensitive also to sodium chloride, potassium nitrate and composition of all tested chemicals. Also, Campylobacter jejuni Pen 20 was sensitive to sodium chloride, but potassium nitrate, sodium ascorbate and Hamine S stimulated growth of this strain.

[Effect of components of curing mixtures on the antagonistic properties of enterococci and other bacteria isolated from cured meat]. / Wplyw skladników mieszanek peklujacych na wlasciwosci antagonistyczne enterokoków oraz bakterii wyizolowanych z miesa peklowanego.

Effect of sodium chloride, glucose, sodium glutamate, potassium nitrate, sodium nitrate, sodium nitraite, ascorbic acid, sodium citrate, polyphosphates (Hamine) on antagonistic activity of enterococci and various bacteria isolated from cured meat against Bacillus cereus, Bacillus subtilis, Salmonella typhi-murium, Salmonella cholerae-suis, Staphylococcus aureus, Clostridium botulinum type A, Clostridium botulinum type B, Clostridium botulinum type E, Clostridium bifermentans, Clostridium perfringens type A, Clostridium sporogenes. It was found that some of the chemical compounds examined increased the antagonistic effect and other compounds partly or totally inhibited it. Sodium nitrate, sodium nitrite, potassium nitrate, glucose, and ascorbic acid in majority of cases increased antagonism and sodium citrate and polyphosphates restrained this phenomenon.