Bovine Fecal Samples Research Articles

Molecular detection and genetic characterization of kobuviruses in fecal samples collected from diarrheic cattle in Korea

Kobuviruses are small, non-enveloped viruses with single-stranded, positive-sense genomic RNA, belonging to the family Picornaviridae, a highly diverse family of important pathogens of humans and animals. In spite of discovery of bovine kobuvirus was first reported in 2003, which is several years ago, information about the virus is lacking. In this study, we first identified bovine kobuviruses from Korean diarrheic cattle by RT-PCR using the generic kobuvirus primers with subsequent cloning and sequencing, and reported genetic characterization of Korean bovine kobuviruses by phylogenetic analysis. Among 62 diarrheic fecal samples collected from 36 cattle farms in 2008–2010 in Korea, 16 (25.8%) samples and 13 (36.1%) farms were positive for kobuvirus, and this indicated that kobuvirus infections are endemic in diarrheic cattle in Korea. The rates of evolutionary substitution of kobuvirus and age of MRCA of Korean kobuvirus were estimated on the basis of temporal information of the sequences by divergence analysis. Phylogenetic analysis showed that six strains except CPF4293 belonged to bovine kobuviruses and formed the exclusive branch with other bovine kobuvirus reference strains; The CPF4293 strain, however, was especially close to porcine kobuviruses rather than to any other kobuviruses, and showed the close genetic relationship with porcine kobuvirus strains isolated in 2009 in Japan, even though it was originated from bovine fecal sample. These data raise questions as to whether interspecies transmission of porcine kobuvirus to cattle may occur in nature.

Shiga toxin-producing Escherichia coli O26 strains in bovine feces in East-Azarbaijan, Iran

Shiga toxin-producing Escherichia coli (STEC) strains are a diverse group of organisms associated with severe gastrointestinal and systemic disease in human. Among non-O157 STEC family, E. coli O26 strains are the common serogroup causing disease such as HUS in human. Cattle are thought to be the main source of this microorganism. We collected 211 samples of bovine feces from different area in East-Azarbaijan province and cultured them in Rhamnose–MacConkey agar supplemented with Cefixime and potassium-tellurite, (CT- RMAC agar). Colorless colonies in CT-RMAC agar medium confirmed by multiplex PCR assay using primer specific for genes encoding biosynthesis of the O antigens ( O-antigen flippase and O-antigen polymerase ) and shiga toxins (stx 1 and stx 2). There were colorless colonies in CT-RMAC agar medium of 18 samples (8.53 %) that 17 samples ( 8.05 %) of them were positive for the presence of the respective genes. Relatively high number of STEC O26 positive cattle found in our region can be a potential vehicle for transmission of this pathogen directly or indirectly (from food) to human. Key words: Shiga toxin-producing E coli 026, Bovine, HUS, CT-RMAC agar, multiplex PCR.

An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

Due to the lipid rich cell wall of Mycobacterium avium subspecies paratuberculosis (MAP), the complex nature of bovine feces, and intermittent organism shedding by infected cattle, it is difficult to recover a sufficient amount of high-quality MAP DNA from fecal samples, directly affecting the sensitivity of downstream polymerase chain reaction (PCR) tests. In the current study, a DNA extraction method, designated the Mississippi Veterinary Research and Diagnostic Laboratory (MVRDL) method, was developed for PCR-based detection of MAP in bovine fecal samples. The MVRDL method combined multiple procedures, including chemical pretreatment, 1-tube cell lysis and extraction, chelex matrix absorption, and mini-column purification. The DNA yield and purity, as measured by spectrophotometry, was 3.36 fg per colony forming unit (CFU) MAP and A260/280 absorbance ratio of 2, respectively. This method was further evaluated by real-time PCR. A linear correlation was found between cycle-threshold (Ct) and log input CFU (ranging from 7.2 to 7.2 × 10(7) CFU per ml or CFU per g). The detection limit of the real-time PCR assay was 3 CFU per ml of MAP culture or per g of MAP-spiked feces. In addition, the MVRDL method was validated by performing 7 Johne's direct fecal PCR proficiency tests administered by the National Veterinary Service Laboratories. Based on culture results as the "gold standard," the specificity of MVRDL PCR was 100%, and the sensitivity was 98.46% for samples containing more than 1.5 CFU per tube of fecal cultures. To the authors' knowledge, this is the most efficient MAP DNA extraction method in comparison with all previously published protocols.

Prevalence Of Cryptosporidium Oocysts From Calves In Kurdistan Province, Of Iran

Prevalence of Cryptosporidium spp. oocysts in calves (n=412) in five selected farms in Saghez, Iran, was determined by using the modified Ziehl Nielsen staining technique. Of 412 bovine faecal samples, 8.5% were positive for Cryptosporidium spp. The prevalence of Cryptosporidium in less than 3-month age’s calves was higher than weaned calves and adults. Cryptosporidium spp. oocysts were detected in both diarrheic and non-diarrheic animals, but there was a significantly higher prevalence (p<.05) of oocyst shedding in diarrheic than in non – diarrheic animals. Confirmation of the diagnosis was performed using antiCryptosporidium monoclonal antibody specific for Cryptosporidium muris, Cryptosporidium parvum and Cryptosporidium baileyi.

Isolation and detection of Mycobacterium avium subsp. paratuberculosis (MAP) from cattle in Ireland using both traditional culture and molecular based methods

BackgroundMycobacterium avium subsp. paratuberculosis (MAP) causes a chronic gastroenteritis affecting many species. Johne's disease is one of the most widespread and economically important disease of ruminants. Since 1992 and the opening of the European market, the exposure and the transmission of MAP in cattle herds considerably increased. Improvements in diagnostic strategies for Ireland and elsewhere are urgently required. In total, 290 cattle from seven Irish herds with either a history or a strong likelihood of paratuberculosis infection were selected by a veterinary team over 2 years. Faecal samples (290) were collected and screened for MAP by a conventional culture method and two PCR assays. In order to further evaluate the usefulness of molecular testing, a nested PCR was also assessed.ResultsM. paratuberculosis was isolated and cultured from 23 faecal samples (7.9%) on solid medium. From a molecular perspective, 105 faecal samples (36%) were PCR positive for MAP specific DNA. A complete correlation (100%) was observed between the results of both molecular targets (IS900 and ISMAP02). Sensitivity was increased by ~10% with the inclusion of a nested PCR for ISMAP02 (29 further samples were positive). When culturing and PCR were retrospectively compared, every culture positive faecal sample also yielded a PCR positive result for both targets. Alternatively, however not every PCR positive sample (n = 105, 36%) produced a corresponding culture isolate. Interestingly though when analysed collectively at the herd level, the correlation between culture and PCR results was 100% (ie every herd which recorded at least 1 early PCR +ve result later yielded culture positive samples within that herd).ConclusionPCR on bovine faecal samples is a fast reliable test and should be applied routinely when screening for MAP within herds suspected of paratuberculosis. Nested PCR increases the threshold limit of detection for MAP DNA by approximately 10% but proved to be problematic in this study. Although slow and impractical, culturing is still regarded as one of the most reliable methods for detecting MAP among infected cattle.

Comparing plastic syringes and disposable columns to extract n-alkanes in forage and concentrate feeds

Saturated aliphatic hydrocarbons (n-alkanes) were extracted from feed, orts, and bovine fecal samples using disposable, plastic 5mL-syringes as an alternative material to disposable columns, which are normally used in the liquid-solid extraction phase of n-alkanes. For both methods, the n-alkane extracts (carbon chain length between 31 and 36 atoms) were identified using gas chromatography. The linear regression between methods were: 1) feces: column Alkane=2.63+0.92×syringeAlkane [r²=0.94, square root of the mean square error (RMSE)=13.7mg kg-1, n=30] from which the intercept and the slope did not simultaneously differ from zero and unity (P&gt;0.05), respectively; 2) feeds: column Alkane=0.36+1.12×syringeAlkane (r²=0.85, RMSE=1.9mg kg-1, n=21) from which the intercept and the slope did not simultaneously differ from zero and unity (P&gt;0.05), respectively; 3) orts: column Alkane=0.49+0.92×syringeAlkane (r²=0.98, RMSE=1.2mg kg-1, n=15) from which the intercept and the slope did not simultaneously differ from zero and unity (P&gt;0.05), respectively. Materials with low concentration of n-alkanes may affect the values obtained in both methods. These results suggested that disposable plastic syringes might be a viable alternative to columns thus, reducing analytical costs.

Changing profile of the bovine rotavirus G6 population in the south of Ireland from 2002 to 2009

Bovine group A rotavirus is one of the main causes of neonatal diarrhoea in calves. This study examined the different G and P genotypes circulating in the bovine population, from 2002–2009, in the south of Ireland. Rotavirus positive bovine faecal samples ( n = 332) were collected from the Cork Regional Veterinary Laboratory, between 2002 and 2009 and subjected to RNA extraction, PAGE analysis, and G and P genotyping. Genotyping analysis identified G6, G10, P[5], and P[11] to be the predominant G and P genotypes in the present study, with G6 rotavirus responsible for 70–80% of rotavirus infections. The highest combination of G and P types found was G6 P[5], followed by G6 P[5+11] mixed infection. The prevalence of G6 and G10 has shifted over the years, with an increase in the amount of G10 P[11] being detected. Novel combinations (G6+G10P[11], G6+G10P[5+11] and G10P[5+11]) were also detected for the first time. In addition to this, sequence analysis of the VP7 RT-PCR amplicons has revealed that Irish G6 strains are falling within three different lineages, III–V. During this study, two samples, initially genotyped as G8P[11] were identified through sequence analysis as being true G6, lineage III with a high nucleotide identity to Hun4, a G6 human sample from Hungary. The increase in novel G and P type combinations, as well as changes seen in G6 samples could have an impact on rotavirus vaccination programmes, as the current vaccine available may not offer protection against all of these circulating types.

Escherichia coliO157 in Bovine Feces and Surface Water Streams in a Beef Cattle Farm of Argentina

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen, and ruminants are recognized as the main natural reservoir. The purposes of this study were to detect E. coli O157 in bovine feces and surface water in a beef cattle farm of Gualeguaychú, Argentina; to characterize the isolates; and to establish the clonal relatedness by pulsed-field gel electrophoresis. Between September 2005 and November 2006, 288 samples of bovine feces and 79 samples of water troughs were studied. E. coli O157 was detected by immunomagnetic separation and polymerase chain reaction as screening techniques. The rfb(O157) gene was detected in 3.8% of the 288 fecal samples and in 17.7% of the 79 water samples. The stx gene was detected in all rfb(O157)-positive fecal samples and in 5.1% of water samples. Eleven E. coli O157 strains isolated from bovine fecal samples and eight from water samples were characterized. The most frequent stx genotype identified was stx(1) and stx(2c(vh-a)). Twelve (63.2%) strains harbored fliC(H7), eae, and ehxA genes. Using pulsed-field gel electrophoresis with the enzyme XbaI, a total of eight patterns with at least 72.1% similarity were identified among the 19 strains. The patterns of 15 strains were grouped into four clusters: two of them included only bovine strains and the other two only aquatic strains. No genetic correlation was established between the bovine and water STEC strains detected. The prevalence of STEC O157:H7 established in the herd studied was higher than that previously reported for Argentine grazed cattle.

Performance Assessment PCR-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollution

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.

PXII-14 Detection and characterization of kobuviruses (in family picornaviridae) in new host species

Kobuviruses are small, non-enveloped viruses with single-stranded, positive-sense genomic RNA, belonging to the family Picornaviridae, a highly diverse family of important pathogens of humans and animals. In spite of discovery of bovine kobuvirus was first reported in 2003, which is several years ago, information about the virus is lacking. In this study, we first identified bovine kobuviruses from Korean diarrheic cattle by RT-PCR using the generic kobuvirus primers with subsequent cloning and sequencing, and reported genetic characterization of Korean bovine kobuviruses by phylogenetic analysis. Among 62 diarrheic fecal samples collected from 36 cattle farms in 2008–2010 in Korea, 16 (25.8%) samples and 13 (36.1%) farms were positive for kobuvirus, and this indicated that kobuvirus infections are endemic in diarrheic cattle in Korea. The rates of evolutionary substitution of kobuvirus and age of MRCA of Korean kobuvirus were estimated on the basis of temporal information of the sequences by divergence analysis. Phylogenetic analysis showed that six strains except CPF4293 belonged to bovine kobuviruses and formed the exclusive branch with other bovine kobuvirus reference strains; The CPF4293 strain, however, was especially close to porcine kobuviruses rather than to any other kobuviruses, and showed the close genetic relationship with porcine kobuvirus strains isolated in 2009 in Japan, even though it was originated from bovine fecal sample. These data raise questions as to whether interspecies transmission of porcine kobuvirus to cattle may occur in nature.

Comparison of faecal culture and IS900 PCR assay for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faecal samples

Comparative efficacy of faecal culture and IS900 Polymerase chain reaction (PCR) assay of faecal samples was investigated in 40 clinically suspected cases of Johne's disease in dairy cattle. The sensitivity of faecal culture and PCR assay in this study was 52.5% (21/40) and 90% (36/40) respectively. All isolates appeared only on the mycobactin J supplemented Herrold's egg yolk medium (HEYM) at 8-16 weeks post-inoculation, were acid-fast and were positive for IS900 PCR yielding a single amplicon of 217 bp. A total of 28 faecal samples out of 40 were positive by IS900 primary PCR assay for Mycobacterium avium subsp. paratuberculosis (Map) yielding an expected product of size 217 bp. Twelve faecal samples, which gave negative results in the primary PCR, were subjected to secondary PCR assay. Of the 12 samples, 8 gave positive results in the IS900 nested PCR (nPCR), which yielded a PCR product of 167 bp, proving better sensitivity of nPCR assay than single amplification PCR. PCR could detect additionally 15 samples as positive which were negative by faecal culture. The chi-square analysis showed a highly significant difference between the tests (P< 0.01). This study suggests that IS900-PCR-based detection of Map could be used as a potential diagnostic tool for rapid and effective Johne's disease surveillance.

Bovine Kobuvirus in Europe

To the Editor: Picornaviruses (family Picornaviridae) are small, nonenveloped viruses with single-stranded, positive-sense genomic RNA. Picornaviruses are currently divided into 8 genera: Enterovirus, Aphthovirus, Cardiovirus, Hepatovirus, Parechovirus, Erbovirus, Teschovirus, and Kobuvirus (1). To date, the genus Kobuvirus consists of 2 officially recognized species, Aichi virus and Bovine kobuvirus, and 1 porcine kobuvirus as a candidate species (2–4). Aichi virus (strain A846/88) was first isolated in 1991 from feces of a person with acute gastroenteritis (2). Bovine kobuvirus (strain U-1) was detected in 2003 in bovine serum and fecal samples from clinically healthy cattle (3); in 2008, it was isolated from cattle with diarrhea (5). Aichi virus and bovine kobuvirus were first isolated in Japan. Porcine kobuvirus (strain S-1-HUN) was recently identified from domestic pigs in Hungary (4). Aichi viruses have been also detected in other countries in Asia (6), Europe (7,8), South America (7), and northern Africa (9). Bovine kobuvirus, however, has not been detected outside Asia (Japan and Thailand) (3,5).

Species identification, molecular sexing and genotyping using non‐invasive approaches in two wild bovids species: Bos gaurus and Bos javanicus

Since the second Indochina war, habitat destruction and overhunting has resulted in fragmentation of the remaining populations of Bos javanicus and B. gaurus. Nowadays, both species are in serious danger, especially the gaur. In Vietnam, where these species have become almost impossible to capture in the wild, non-invasive investigations are the only feasible approach to obtain data on populations. However, non-invasive derived DNA, especially in tropical areas, is usually characterized by low concentrations, poor quality and/or contamination from alien DNA. To assist in tropical conservation management, baseline information is provided here on assessing the reliability of species identification, molecular sexing and microsatellite genotyping using fecal DNA from B. gaurus and B. javanicus. For species identification using bovine fecal samples, cytochrome b fragment between positions 867 and 1140 was found to contain species diagnostic sites, which distinguishes the four species encountered in the region: B. gaurus, B. indicus, B. javanicus and B. taurus. For sex determination, primers were initially tested on DNA obtained from blood. Then, these primers were successfully used on DNA derived from fecal material. Finally, we also evaluate the feasibility of non-invasive microsatellite genotyping on fecal samples collected in Vietnamese nature reserves. The results presented here improve on current molecular methods based on fecal material obtained from tropical areas.

The Transmission of Thermotolerant Campylobacter spp. to People Living or Working on Dairy Farms in New Zealand

New Zealand has one of the highest rates of campylobacteriosis in the developed world with an incidence rate of 383.5 cases per 100,000 in 2006. Dairy farming has been suggested as a potential source of campylobacteriosis. To explore this connection, seven farm investigations were undertaken at dairy farms on which a campylobacteriosis case had been notified. Campylobacter spp. were isolated from a range of sources on the farm (including 66% of bovine faecal samples) and genotypes compared with that of the clinical isolate of the index case. In depth, epidemiological questionnaires were also administered to determine exposure risks from a wide range of possible sources. Contact with dairy cow faeces was the most likely source of infection in four of the seven cases investigated, and occurred exclusively in new farm workers and children. In one of the cases investigated, infection was likely to have been acquired from non-dairy related sources, and in two cases the source could not be determined. The relative risk of dairy farm worker being notified with campylobacteriosis was estimated to be 1.88 (95% confidence interval=1.6-2.2).

New Triplex Real-Time PCR Assay for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Feces

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan(mgb) and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10(2) CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.

Comparison of flow cytometry and immunofluorescence microscopy for the detection of Giardia duodenalis in bovine fecal samples

The performance of flow cytometry (FC) was compared with immunofluorescence microscopy (IM) for detection of Giardia duodenalis in bovine feces. Samples from 36 adult dairy cows and 208 dairy calves were collected. Flow cytometry test characteristics were calculated using continuous, ordinal, and dichotomized results. Spearman correlation coefficients comparing the results of the 2 tests were 0.47 and 0.68 for cows and calves, respectively. Using IM as indicative of presence or absence of G. duodenalis cysts in each sample, likelihood ratios of FC results with 0, 1, and > or = 2 gated events indicated that samples with 1 gated event were likely to be positive in the cows but not in the calves. Immunofluorescence microscopy detected G. duodenalis in 69.7% and 48.1% of cows and calves, respectively. When dichotomizing the FC results at a cut-off point of 1 or 2 gated events, 46.3% and 19.9% of the cow and 51.9% and 35.1% of the calf samples, respectively, were classified as G. duodenalis-positive. Relative to IM, the sensitivity in the cows was 0.59 and 0.28, respectively, and 0.76 and 0.64, respectively, in the calves. At a cut-off point of 1, 65.7% and 73.1% of the cow and calf samples, respectively, were correctly classified in FC, and at a cut-off point of 2, 49.3% and 78.4% were correctly classified in the cows and calves, respectively. Flow cytometry was less sensitive than IM. Possible reasons and research needed to improve FC for G. duodenalis detection are discussed.

Influence of Type of Culture Medium on Characterization ofMycobacterium aviumsubsp.paratuberculosisSubtypes

We evaluated the effects of culture and/or enrichment methods on the selection of Mycobacterium avium subsp. paratuberculosis subtypes. M. avium subsp. paratuberculosis isolates from bovine fecal samples processed using a centrifugation protocol were investigated in both liquid (MGIT ParaTb tubes) and solid (Herrold's egg yolk medium) culture media. For this evaluation, M. avium subsp. paratuberculosis subtyping was based on the sequence variation in two of the most discriminatory short sequence repeat loci, i.e., mononucleotide G and trinucleotide GGT, in isolates from liquid and solid cultures. This study identified the existence of one major predominating fingerprint (>13G-5GGT) in bovine fecal samples, regardless of the type of medium used for isolation. Matched-pair analysis of subtypes showed that 69% of samples presented unique subtypes in the two culture media used, while 31% shared the same G-GGT allele. Furthermore, the liquid culture method appeared to select for a more genotypically diverse subtype population than the solid culture method. The variety of subtypes observed between liquid and solid cultures obtained from the same fecal samples suggests that the culture method could provide a "microbiological" bias and lead to a discrepancy in the growth of M. avium subsp. paratuberculosis strains. In conclusion, this study identified that these two types of culture media determined differential growth of M. avium subsp. paratuberculosis strains and that this should be considered in evaluating detection capabilities of diagnostic tests or interpreting data from molecular epidemiological studies performed using conventional solid fecal culture or automated liquid medium culture methods.

Detection and Identification of Salmonella Typhimurium in Bovine Diarrhoeic Fecal Samples by Immunomagnetic Separation and Multiplex PCR Assay

The aim of this study was to use the immunomagnetic separation (IMS) test plus a multiplex polymerase chain reaction (m-PCR) assay to detect Salmonella at genus level and also for the identification of Salmonella enterica serovar Typhimurium in bovine diarrhoeic fecal samples. In all, 400 bovine diarrhoeic fecal specimens were examined by conventional bacterial culture, IMS, and m-PCR. For m-PCR assay, four set primers were selected: 139-141, specific for inv-A gene of Salmonella spp and the RfbJ, FliC and FljB, specific for the rfbJ, FliC and fljB genes of Salmonella Typhimurium or other Salmonella serovars with similar antigenic properties. Thirty-three (8.25%) out of the 400 fecal samples were culture positive for Salmonella serovars. Of these, 66.7% (22 of 33) were Salmonella enterica serovar Typhimurium, and 9.1% (three of 33) were serovar Dublin. In the IMS + m-PCR, four amplified product (663, 526, 284 and 183 bp) were found in all specimens that had serovar Typhimurium (4,5,12:i:1,2), they corresponded, respectively, to the rfbJ, fljB, inv-A and Flic genes of this serovar. In serovar Dublin (1,9,12:g,p:-), Georgia (6,7:b:e,n,z(15)) and, Enteritidis (1,9,12;g,m:-) only one PCR product (284 bp) was amplified from the inv-A gene. In serovars Augustenborg (6,7:i:1,2) and Lindenburg (6,8:i:1,2) three positive bands (526, 284 and 183 bp) were amplified corresponding to the fljB, inv-A and Flic genes, respectively. In serovar Virchow (6,7:r:1,2) two amplified products (284 and 526 bp) from the inv-A and FliC genes were observed. In serovar Gloucster (1,4,12(27):i:1,w) three fragments (183, 284 and 663) from the FliC, inv-A and, rfbJ genes respectively, were observed. In the positive control as expected, four PCR products were amplified corresponding to the FliC, inv-A, fljB and rfbJ genes, respectively. In conclusion, the results of this study showed that detection of Salmonella at genus level with universal ST139-141 primers and identification of Salmonella Typhimurium by using specific primers of O4, H(2):1, 2 and H(1) antigens can potentially permit to more readily evaluate fecal and other types of samples for the presence of these organisms. Compared to bacteriological culture the combination of IMS and m-PCR resulted a faster method for the detection and identification of Salmonella at genus and serovar level by using of universal and specific primers.

Prevalence of coccidia in beef cattle in western Turkey

A total of 504 bovine faecal samples collected from intensively managed beef farms in Afyon province of Turkey were examined to determine the types and prevalence of coccidian parasites present. Coccidian oocysts were found in 20.04% of all the samples examined by sucrose-flotation. The species detected and their prevalence were Eimeria bovis (34.55%), E. auburnensis (23.03%), E. canadensis (14.55%), E. brasiliensis (10.91%), E. zuernii (6.67%), E. bukidnonensis (3.03%), E. cylindrica (2.42%), E. ellipsoidalis (1.21%), E. illinoisensis (1.21%), E. alabamensis (1.21%) and Isospora sp. (1.21%). Mixed infections of two to four species were found in 43.6% of the animals. The overall prevalence of coccidial oocysts in faecal samples was 27.23% for calves, 15.65% for cows. Linear regression analysis showed that there is a significant reduction in the OPG (the number of oocysts per gram of feces) levels (P<0.05) in cows infected with Eimeria. No cases of clinical coccidiosis were observed in this survey.

Sensitivity of Direct Plating for Detection of High Levels of E. coli O157:H7 in Bovine Fecal Samples

To assess the sensitivity of direct plating of bovine fecal samples for detection of Escherichia coli O157:H7, calves (n = 28) were orally inoculated with 10(9) colony-forming units (cfu) per calf of a mixture of three strains of nalidixic acid-resistant E. coli O157:H7, and fecal samples were collected for analysis. One-gram samples from inoculated calves were mixed with 9 mL of Gram-negative broth with vancomycin, cefixime, and cefsoludin. From this suspension, serial dilutions were made (10(-1) to 10(-4)) and spread plated in triplicate on Sorbitol MacConkey agar with nalidixic acid for enumeration of E. coli O157:H7 in fecal samples. Direct plating samples were streaked for isolation on Sorbitol MacConkey agar with cefixime, and tellurite (SMACct). After incubation overnight at 37 degrees C, morphologically typical colonies from direct streak plates were plated onto blood agar and incubated overnight at 37 degrees C; then an indole test was performed on each colony. Indole-positive colonies were confirmed by O157 agglutination and were then plated on SMAC agar with 20 microg/mL nalidixic acid (SMACnal) to confirm nalidixic acid resistance. Overall sensitivity of detection was 32.5% (110/338 samples). Sensitivity to detect fecal samples shedding at above 5 x 10(4) cfu/g was 83% (71/86 samples). Based on these data, direct plating of fecal samples might be an effective way to identify cattle that are likely to be shedding E. coli O157 at high levels.