Comparison of PCR assay and bacteriological culture method for the detection of Brucella melitensis in stomach content samples of aborted sheep fetuses

The aim of this study was to evaluate the polymerase chain reaction (PCR) assay for detection of Brucella melitensis in stomach content samples of aborted sheep fetuses and to compare its performance with bacteriological culture method. It was also aimed to determine the agreement between PCR and Rose Bengal plate test (RBPT). Materials were collected from aborted sheep from 109 different sheep flocks in the region of Van during the lambing seasons of 2004-2005 and 2005-2006. Stomach contents from 135 aborted sheep fetuses were examined by bacteriological culture and PCR, and 135 sera from these aborting ewes were tested by RBPT. Identification and typing of Brucella strains were performed using standard classification test. B. melitensis biovar 3 was isolated from 26 (19.2%) of foetal stomach contents. B. melitensis was detected by PCR in 29 (21.4%) stomach content samples. Twenty five sera (18.5%) from aborting ewes tested positive by RBPT. The detection limit of B. melitensis 16 M strain by PCR was 1.7 x 10(3) cfu (colony forming units) /ml in spiked stomach contents. Diagnostic sensitivity and specificity of the PCR were detected as 100% and 97.2%, respectively. The agreement between PCR and RBPT was found to be 97%. In conclusion, PCR assay would have an advantage over conventional bacteriological culture method, but in particular for its ability to meet the specificity requirements for the detection of B. melitensis in stomach content samples of aborted sheep fetuses.     

Isolation and characterisation of local strains of Chlamydophila abortus (Chlamydia psittaci serotype 1) from Tunisia

Chlamydiosis is one of the major diseases that can lead to abortion in ewes. Since 1997, in 5 regions of Tunisia, Chlamydia-related abortions have been reported in 15 sheep and goat flocks. One hundred and sixty-six sera and 50 vaginal swab samples were collected from adult ewes. Chlamydial antigens were detected in 29 (58%) of the vaginal swabs using Enzyme Linked Immunosorbent Assay (ELISA) while 9 (18%) were positive by cell culture. Five strains were recovered from 4 different sheep flocks. Monoclonal antibody profiles and restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA spacer region showed that these isolates were C. abortus. Using amplified fragment length polymorphism (AFLP), these Tunisian strains were shown to exhibit the same pattern as strains isolated in France.     

Nutritional factors associated with vaginal prolapse in ewes

Serum calcium, magnesium and phosphate values of ewes recently affected by vaginal prolapse were compared with unaffected ewes in four flocks. Subclinical hypocalcaemia was demonstrated in some affected and unaffected ewes in three flocks. Magnesium and phosphate values were normal. In two flocks the body condition of ewes recently affected by vaginal prolapse was variable and reflected the variation in condition found in the flock. In a third flock affected ewes had significantly lower body condition scores than unaffected ewes (P less than 0.001). Analysis of the fourth flock was not possible. Oestrogenic mycotoxins were not detected in any of the feed samples taken from these flocks. The following year the management, nutrition and energy, and the protein and calcium status of ewes in 12 flocks of greyface/mule ewes with a history of a regular high (greater than 3 per cent) or low (less than 1 per cent) prevalence of vaginal prolapse were compared. A high prevalence was not associated with any particular feedstuff. A high or intermediate (1 to 3 per cent) prevalence of vaginal prolapse was found in three of the four flocks managed as a single group and these three flocks were fed on an unrestricted basis. Body condition scoring and beta-hydroxybutyrate estimation confirmed that ewes in these flocks were overfed. The prevalence of vaginal prolapse in the flocks was not related to the serum albumin, calcium or urea of the ewes. Therefore subclinical hypocalcaemia was probably a consequence of vaginal prolapse rather than a cause.     

Serological investigations on reticuloendotheliosis in turkey flocks

Four meat turkey and one turkey breeding flocks were surveyed for antibodies against reticuloendotheliosis virus (REV) at different intervals using commercial enzyme-linked immunosorbent assays. In addition, serum samples collected from 18 flocks at different ages were also tested for antibodies against REV. No antibodies were detected in any of the four meat turkey flocks that were surveyed. In the breeder flock, 20%) of tested samples from 1-day-old poults were positive. Between the fourth and 12th weeks all samples that were tested yielded negative results. At 16 weeks of age 15% of samples yielded a positive reaction, but antibodies could not be detected 4 weeks later. Examination of serum samples from 18 different flocks at various ages revealed that antibodies could be detected in five flocks. The percentage of positive sera per flock ranged between 10 and 40%.     

Ulcerative vulvitis and balanitis in sheep flocks

Outbreaks of ulcerative vulvitis and balanitis occurred in three commercial sheep flocks in England and Wales. Between 29 and 44 per cent of the ewes were affected; most of the lesions resolved in three weeks. Pathogens such as mycoplasmas, which have previously been associated with these conditions, were not detected despite using improved laboratory techniques. In one of the flocks, ovine herpesvirus type 2 was detected by pcr in the blood of two acutely affected ewes, from the vulval ulcers of one of them, and from the penis of an affected ram.     

Salmonella contamination of poultry flocks in The Netherlands

The contamination of poultry in the Netherlands with Salmonella enteritidis was tested. For this, different methods (detection of S. enteritidis in faecal samples of 25 g; detection of S. enteritidis in cloacal swabs; detection of S. enteritidis by serological testing of antibodies in serum) were compared for their efficiency to detect S. enteritidis in flocks of poultry. Testing of faecal samples clearly yielded the best results. This method was used in a transmission study, in which 14 flocks descending from a contaminated primary mother flock were screened for the presence of S. enteritidis. The method was also used for screening 49 flocks of laying hens and 52 flocks of broiler chickens throughout the Netherlands. From the transmission study it became clear that S. enteritidis, phage type 2 (Dutch phage set) was isolated both from the mother flock and from five of the descendent flocks. Screening of poultry flocks for the presence of salmonella revealed that salmonella was present in 47% of the layer flocks and in 94% of the broiler flocks. S. enteritidis was isolated from 15% of the flocks screened.     

Comparison of five real-time PCR assays for detecting virulence genes in isolates of Escherichia coli from septicaemic neonatal foals

Fifty-five isolates of Escherichia coli from septicaemic neonatal foals were used to validate five real-time pcr assays targeting different known virulence factor genes: curli fibre (csgD), ferric hydroxamate uptake (fhuA), type 1A pilin (fimA), aerobactin (lutA) and yersiniabactin (fyuA). A pcr assay targeting a universal sequence of the bacterial 16S rrna gene served as quality control. The pcr assays showed good analytical specificity and sensitivity on the basis of sequencing the pcr products, their lack of cross-reactivity with non-E coli organisms, high amplification efficiency and a limit of detection as low as 25 E coli colony-forming units. There were differences between the detection rates and amplification efficiencies for the five virulence genes. The pcr assays targeting genes csgD, fhuA and fyuA were able to detect all 55 E coli isolates, with gene csgD having the best amplification efficiency. The lowest detection rate and amplification efficiency of the E coli isolates was found for the lutA gene.     

Epidemiology of ovine brucellosis in Awassi sheep in Northern Jordan

We used a combined cross-sectional and longitudinal design to estimate seroprevalence of Brucella antibodies in Awassi sheep and the incidence of abortion due to brucellosis during one lambing season, and to test risk factors. The Brucella organisms isolated from aborted fetuses and vaginal swabs were characterized as Brucella melitensis biotype 3. Seventy Awassi sheep flocks were selected randomly from Northern Jordan. Sixty two of the 70 flocks were used in the cross-sectional study and 8 flocks were monitored for three consecutive months to estimate the incidence of abortion. Questionnaire data and 602 serum samples were collected and analyzed. Thirty five flocks (56%) were brucellosis-seropositive by the Rose Bengal plate-agglutination test (RBT) and 28 (45%) by enzyme-linked immunosorbent assay (ELISA). The crude seroprevalence of brucellosis at the individual-animal level was 14.3% by RBT, 7.2% by ELISA and 2.2% using both tests in series. The flock-specific, animal-level abortion risk ranged between 2.5 and 50% (median=22.6%). The flock brucellosis-status was used as the outcome variable in a multivariable logistic regression. Grazing at communal pasture increased odds, but usage of disinfectants, previous vaccination for brucellosis, and tap water were protective. The animal-level incidence of abortion was 20% and the specific incidence risk of abortion due to brucellosis was 13%.     

High seroprevalence of Histomonas meleagridis in Dutch layer chickens

Histomona meleagridis is a protozoan parasite that may cause outbreaks of histomonosis with high mortality, especially in turkey flocks. Chickens are less susceptible to the disease than are turkeys, but are considered to act as an important reservoir. To determine the seroprevalence of H. meleagridis in Dutch layer chicken flocks, a large scale seroepidemiologic study (3376 samples) was performed by sampling 12 organic flocks, 24 free-ranging flocks, 40 flocks with floor housing, and 40 flocks with cage housing. At the end of the laying period, approximately 30 blood samples per flock were collected for serology. The seroprevalence found was high. In every flock, at least one of the samples tested positive while in 87% of the flocks, at least one of the samples was strongly positive. There were no significant statistical differences in seropositivity between the housing types. To confirm the enzyme-linked immunosorbent assay (ELISA) results, a small-scale seroepidemiologic study (576 samples) was performed in 29 additional layer chicken flocks kept in different housing systems. Subsequently, a subset of five seropositive flocks was selected. Five birds were obtained from each of these flocks in order to detect the parasite using culture and PCR. In all five flocks, H. meleagridis was either isolated from (culture), detected in (PCR), or both, the birds sampled. Together with the previously performed validation studies, the latter results confirm that the positive ELISA serology found is genuine. We conclude that the seroprevalence of H. meleagridis in layers is, as anticipated, high.     

New data on the transmission of pigeon circovirus

Nineteen racing pigeons aged from one to five years were examined postmortem. pcr tests showed that the spleens of 16 of them were positive for pigeon circovirus, the livers of six were positive, and blood from one of them was positive for the virus. Five of 44 embryos in embryonated eggs collected from three lofts were positive by pcr, but swabs taken from the crops of 64 adult birds which were feeding one- to 10-day-old squabs in these three lofts were negative for the viral dna.     

Dynamics of Campylobacter spp. spread investigated in 14 broiler flocks in Switzerland

Ten conventional and four extensive outdoor broiler flocks, distributed over nine farms, were investigated twice per week during a 35-58-day rearing period to observe the dynamics of Campylobacter spp. spread within these flocks. Strains isolated during this period were genotyped by restriction fragment length polymorphism analysis of the flaA gene and macrorestriction profiling with pulsed field gel electrophoresis. A total of 4112 samples were collected; 157 (3.8%) of these samples were Campylobacter positive, with all C. jejuni. The positive samples were distributed over three conventional and two extensive outdoor flocks on five farms. These five positive flocks were colonized from the fifth to the seventh week of age and remained colonized until slaughter. Each of the flocks showed a flock-specific genotype of Campylobacter that predominated until slaughter. Presuming different ways of entry, a combination of this fact and the observed dynamics of C. jejuni spread within the flocks indicates that a single source from the environment may have been responsible for the colonization of each flock. These conclusions may serve to further develop combat strategies at farm level.     

Sensitive test for screening for Staphylococcus aureus in bovine mastitis by broth cultivation and PCR

An assay was developed and evaluated for screening for Staphylococcus aureus in milk samples from cases of bovine mastitis by overnight cultivation in a broth containing 7.5 per cent sodium chloride, followed by pcr to amplify the nuc gene. The assay could detect concentrations of S aureus as low as 1 colony-forming unit/ml milk. Among 106 milk samples collected from individual quarters of lactating cows in one dairy herd and from a bulk tank, S aureus was detected in nine samples by the pcr assay but in only three samples by conventional microbiological culture.     

Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples

PCR assay has been shown to be a promising option for the diagnosis of brucellosis. However, few studies have been performed with field samples in order to evaluate the assay as a diagnostic tool. In this study, routine use of a species-specific PCR assay previously developed for the identification of Brucella cultures was assessed for the detection of Brucella DNA directly from the stomach contents of aborted sheep fetuses. The assay is based on the insertion sequence IS711 in the Brucella chromosome. In the study, during 3 successive lambing seasons (1998-1999, 1999-2000 and 2000-2001) 126 aborted fetus samples each from different flocks and locations were examined. Brucella strains were isolated from 39 (31%) of the samples and all of the strains were identified as Brucella melitensis by biochemical characteristics, agglutination with monospecific A and M sera and PCR. Thirty-seven of 39 B. melitensis isolates were biotyped as biotype 3, and 2 isolates as biotype 1. From 38 of 39 culture positive fetal stomach contents B. melitensis-specific DNA was detected by PCR. PCR was found negative in all of the culture negative samples. Compared with culture, sensitivity and specificity of PCR were determined as 97.4 and 100%, respectively. The results indicate that this PCR procedure has a potential for use in routine diagnosis of sheep brucellosis.     

Prevalence of 'Candidatus Helicobacter suis' in pigs of different ages

Samples from the antrum and fundus of the stomachs of 457 pigs from 22 different herds were screened for the presence of 'Candidatus Helicobacter suis' by pcr, and samples from the antrum and/or fundus of 222 of the stomachs were tested for urease activity. The prevalence of the infection was very low before weaning, increased rapidly after weaning and reached 90 per cent in the adult boars and sows. The agreement between the results obtained with the pcr test and the urease test was very good for some age groups and sampling sites, but poor for other age groups and sampling sites.     

Detection ofCampylobacter in 100 commercial flocks–Evaluation of plating media and filtration method

Campylobacter is a natural member of the gut microflora in many commercial broilers and as such can become a contaminant on edible surfaces during processing. Culturing gut contents or feces can be a means to determine flock status prior to live-haul. The wide variety of non-Campylobacter background bacteria in these complex samples contaminates growth media and can make it very difficult to isolateCampylobacter. Over the course of 17 months, we cultured cecal contents from 100 different broiler flocks. For the last 50 flocks, we tested 3 selective plating media with and without the additional selection of a 0.45-μm filter for detection ofCampylobacter from cecal contents. Furthermore, from the last 50 flocks we also collected and cultured carcass rinse samples. Growth media tested included: Campy–Cefex Agar, Campy–Line Agar, and RF-Campylobacter jejuni/coli agar. About half (52%) of the 100 tested flocks were positive forCampylobacter; positive flocks were detected during each month of the year. Overall, theCampylobacter status of cecal contents from one carcass was predictive of the status of a carcass rinse from the same flock. Placing a complex sample such as cecal contents onto a 0.45-μm filter laid on top of the plating medium improved the detection ofCampylobacter by eliminating non-Campylobacter background colonies. All media allowed for detection ofCampylobacter from less complex carcass rinse samples without filtering. However, Campy–Cefex agar had higher numbers of competing bacterial colonies than did Campy–Line agar or RF-Campylobacter jejuni/coli agar.     

A census of the prevalence of vaginal prolapse in sheep flocks in the Borders region of Scotland

A postal census of vaginal prolapse in sheep flocks in the Borders region of Scotland yielded 540 replies from 963 owners (56 per cent). There were 262,250 ewes in 976 flocks and 2573 vaginal prolapses were reported. Analysis of the data revealed that 390 (40 per cent) of the flocks had no vaginal prolapses and in 237 (24.3 per cent) the reported prevalence was between 0.1 per cent and 1.0 per cent. Only 63 (6.5 per cent) of flocks had a greater than 5 per cent prevalence of vaginal prolapses. The greatest number of prolapses occurred in an upland flock of greyface ewes mated with Suffolk tups with 50 cases among 700 ewes (7.1 per cent) and the highest prevalence was in an upland Scottish blackface flock of ewes bred with Suffolk tups with 15.2 per cent (35 cases among 230 ewes). There were marked breed differences; very few hill breeds were affected and most cases occurred in greyface ewes mated with Suffolk tups.     

Molecular characterization of infectious bronchitis virus strains isolated from the enteric contents of Brazilian laying hens and broilers

Infectious bronchitis virus (IBV) is the causative agent of avian infectious bronchitis, which is characterized by respiratory, reproductive, and renal signs. However, the role of IBV as an enteric pathogen in still controversial. In Brazil, antigenic groups of IBV divergent from the Massachusetts serotype used for vaccination schedules in that country have already been demonstrated. The present study aimed to assess the different genotypes of IBV in Brazilian commercial poultry flocks by partial sequencing of the S1 amino-terminus coding region using enteric contents as samples and examine their relationship with the vaccine serotype currently in use. Samples of enteric contents were taken as pools of five birds from each of 18 poultry farms (17 broiler and one laying farm) from five Brazilian states between 2002 and 2006. Birds were presenting watery diarrhea and poor general condition but were without respiratory, renal, or reproductive signs. Conventional antibacterial and anticoccidial therapies were unsuccessful and, furthermore, all samples proved negative for rotavirus, reovirus, and astrovirus. Eleven IBV samples were isolated in embryonated eggs and resulted in S1 sequences. Phylogenetic analysis showed that these segregated into an exclusive cluster, close to serotype D274, but distant from Massachusetts. Mean amino acid identity amongst these Brazilian strains was 94.07%; amongst these and serotypes D274, 4/91, and Massachusetts, mean amino acid identity was 77.17%, 69.94%, and 68.93%, respectively. In conclusion, the presence of genotype variant strains of IBV in Brazilian poultry flocks has been demonstrated and might be the reason for the unsuccessful control of IBV in Brazil. Furthermore, these results also strengthen the implications of IBV in enteric diseases of poultry.     

Incidence of Clostridium perfringens in broiler chickens and their environment during production and processing.

During a calendar year, a study was conducted involving 16 broiler flocks on four different farms, two farms belonging to each of two major U.S. poultry integrators. As determined by the detection of Clostridium perfringens in fecal or cecal samples, 15 (94%) of the flocks became positive for this bacterial enteropathogen, and only one remained negative throughout the 6-to-8-wk rearing period. Paper pads beneath chicks that were transported from the hatchery to the rearing house were contaminated with C. perfringens in 15 (94%) of the flocks. When sampled biweekly through grow out, 13 of the flocks were C. perfringens positive at 2 wk of age. These results suggest that colonization of the intestinal tract of broilers by C. perfringens is an early event. Of the environmental samples, all but feed in the hopper were contaminated before placement for at least one of the rearing periods. All sample types were contaminated at some point during the 6-to-8-wk grow-out period. Of the on-farm environmental samples, the highest incidences (percentage positive) of C. perfringens were detected in wall swabs (53%), fan swabs (46%), fly strips (43%), dirt outside the house entrance (43%), and swabs of workers' boots (29%). Birds were usually transported to the processing plant in coops that were already contaminated with C. perfringens. In the plant, C. perfringens was isolated more frequently from samples of scald water than from those of chill water. Clostridium perfringens was recovered from broiler carcasses after chilling in 13 (81%) of the 16 flocks. The proportion of C. perfringens-positive carcasses for the contaminated flocks ranged from 8% to 68% with a mean of 30%.     

Canine adenovirus type 2 infection in four puppies with neurological signs

Four nine- to 11-week-old puppies developed respiratory and neurological signs due to an infection with canine adenovirus type 2 (cav-2); three of these were euthanased. They had moderate, diffuse pneumonia but there were no histological abnormalities in the central nervous system. Adenovirus-specific nucleic acid was detected by pcr in samples of lung and brain and the amplified product was 99.8 per cent homologous with the cav-2 reference strain Toronto a26/61. The positive pcr result was confirmed by in situ hybridisation in samples of lung, liver and spleen, but not in brain, and cav was isolated in cell culture from lung material; pcrs for canine distemper virus and canine herpesvirus-specific nucleic acids were negative, but large amounts of Bordetella bronchiseptica were isolated from lung material.