Cryptosporidium in commercially produced turkeys on-farm and postslaughter.

AIMS: To determine the presence of Cryptosporidium species in commercially produced turkey flocks on farm and postslaughter. METHODS AND RESULTS: Three separate turkey flocks were sampled at a single farm and again postslaughter at a commercial processing facility. DNA was extracted and purified from faecal (farm) or caecal (postslaughter) samples and a fragment of 18S rDNA was amplified using a nested PCR approach. Amplified fragments were sequenced, aligned and a neighbour joining tree was constructed. Cryptosporidium meleagridis was not identified in any of the flocks tested. However, all flocks tested positive for Cryptosporidium parvum species. One of the flocks tested positive at the farm and postslaughter. CONCLUSIONS: While C. parvum was present in birds at the farm and postslaughter, turkeys at this facility are not likely to be a significant reservoir for this species. SIGNIFICANCE AND IMPACT OF THE STUDY: Cryptosporidium meleagridis infects avian and human hosts and is increasingly being recognized as a significant human pathogen. However, this study found no evidence of C. meleagridis in commercially produced turkeys at a single location.

Cloning and sequencing of cnf1 from Escherichia coli incriminated in mink and bovine colibacillosis.

Colibacillosis is responsible for significant losses to the mink and cattle industries. Previous work in our laboratory and by others has suggested that possession of cnf1, the gene encoding cytotoxic necrotizing factor (CNF1), may contribute to the virulence of isolates of E. coli from mink and cattle. The cnf1 gene from E. coli isolated from a mink with colisepticaemia and a bovid with scours was amplified and cloned as a 3.5 kb fragment, and the fragment was sequenced. The cnf1 sequences from the mink and bovine isolates of E. coli were compared to each other and to cnf1 sequences of E. coli from urinary tract and diarrhoea-associated infections of humans. The difference was only 7 nucleotides between the cnf1 sequences of the mink and bovine isolates of E. coli, which translated into 7 differences in amino acids. The cnf1 sequence of the mink isolate of E. coli had 15 nucleotide differences from the cnf1 sequences of the human isolate of E. coli (GenBank X70670), which translated into 11 differences in amino acids between these proteins. The cnf1 sequence of the bovine isolate of E. coli had 14 nucleotide differences from the cnf1 sequence of the human isolate of E. coli (GenBank X70670), which translated into 10 differences in amino acids between these proteins. The highly conserved sequences of the amino acids of CNF1 proteins make them a promising target for detection and control of the CNF1-producing E. coli involved in disease among various host species.

Characterization of Escherichia coli isolates incriminated in colisepticaemia in mink.

Colisepticaemia is a major health and economic concern for the mink industry, yet little information is available about the Escherichia coli that cause this disease. In this study, 40 E. coli, isolated from mink clinically diagnosed with colisepticaemia that had been submitted to the North Dakota State University Veterinary Diagnostic Laboratory, were randomly selected for characterization. These isolates were serotyped and screened for resistance to 18 antimicrobials, possession of transmissible R plasmids, and the presence of several virulence traits or genes using bioassays or the polymerase chain reaction. Several serotypes were identified that have previously been associated with septicaemia in other animal species. The majority of the isolates exhibited multiple antimicrobial resistance phenotypes. Common resistance phenotypes observed included those to tetracycline, sulfamethoxazole, streptomycin, ampicillin and kanamycin. Several of the isolates that could be studied by conjugation contained transmissible R plasmids coding for multiple antimicrobial resistance phenotypes. About half of the isolates produced colicin; all produced enterobactin: and all but one-quarter produced aerobactin. None of the isolates tested produced enterohaemolysin, and one-fifth were considered to be beta haemolytic. About half appeared to contain the gene encoding cytotoxic necrotizing factor-1; three contained the gene encoding EAE, but none appeared to contain the genes coding for LT, Sta/b, SLT-I/II or CNF-II toxins or K99 antigen. Approximately one-third of the isolates elaborated capsule. The results show that the E. coli isolates implicated in mink colisepticaemia possess similar virulence traits and antimicrobial resistance phenotypes to those associated with diarrhoeal diseases in food animals.

Resistance to serum complement, iss, and virulence of avian Escherichia coli.

Control of avian colibacillosis is hampered by lack of easily identifiable markers for virulent Escherichia coli. Resistance to serum complement appears to be a widespread trait of virulent avian E. coil, suggesting that bacterial factors promoting survival in serum may be useful in discriminating between virulent and avirulent isolates. Such distinguishing factors may prove useful in diagnostic protocols or as targets in future colibacillosis control protocols. Interestingly, the factors responsible for resistance to complement differ in the E. coli isolated from mammalian and avian hosts, which may reflect differences in the nature of avian and mammalian colibacillosis. In some cases, genetic determinants for serum complement resistance in avian E. coli are found on aerobactin- or Colicin V-encoding plasmids. One such gene, iss, first described for its role in the serum resistance associated with a ColV plasmid from a human E. coli isolate, occurs much more frequently in isolates from birds with colibacillosis than in faecal isolates from healthy birds. Efforts to identify the genomic location of iss in a single, virulent avian E. coli isolate have revealed that it occurs in association with several purported virulence genes, all linked to a large conjugative R plasmid. At this time, it is not known whether iss merely marks the presence of a larger pathogenicity unit or is itself a contributor to virulence. Nevertheless, the presence of the complement-resistance determinant, iss, may be a marker of virulent avian E. coli exploitable in controlling avian colibacillosis.

Virulence factors of Escherichia cofi from cellulitis or colisepticemia lesions in chickens.

This study was designed to compare virulence factors of cellulitis-derived Escherichia coli to colisepticemic E. coli in order to clarify whether E. coli associated with cellulitis comprise a unique subset of pathogenic E. coli. Isolates were tested for serotype, capsule, aerobactin production, colicin production, the presence of the iss gene, and serum resistance. Untypable isolates made up the greatest percentage of each group. Serotypes O2 and O78 were the most commonly identified among both groups of isolates. No statistical differences in the distribution of aerobactin or colicin production, capsule, or iss gene were observed between groups. Cluster analysis showed that 90% of the E. coli isolates had greater than 42% livability in serum-resistance tests. No separation of colisepticemic vs. cellulitis E. coli isolates was observed on the basis of SR. Colicin production by E. coli was highly correlated with serum resistance (P = 0.0029). These data suggest that cellulitis E. coli have virulence traits similar to those of colisepticemic E. coli.

Complement resistance-related traits among Escherichia coli isolates from apparently healthy birds and birds with colibacillosis.

In this study, 294 Escherichia coli isolates from birds with colibacillosis were collected from disease outbreaks throughout the United States and were compared with 75 fecal E. coli isolates of apparently healthy chickens by their possession of several purported virulence genes, resistance to rough-lipopolysaccharide-specific bacteriophages (rLPSr), and elaboration of capsule. Traits were selected for study on the basis of their association with complement resistance. The genes targeted in this study included those encoding colicin V (cvaC) and the outer membrane proteins TraT (traT), OmpA (ompA), and Iss (iss). No significant differences were found between the two groups of isolates in the occurrence of cvaC-, traT-, or ompA-homologous sequences or in rLPSr. Only a few isolates were encapsulated, and the isolates of healthy birds were significantly more likely to be encapsulated than were the isolates of sick birds. However, iss, whether detected through hybridization or amplification, was found in more of the disease-associated isolates than in those of healthy birds. This difference was highly significant. Further, iss sequences were widely distributed among isolates of different serotypes from various avian host species and sites within these hosts. Such results suggest that possession of the iss sequence by an avian E. coli isolate may be a good indicator of that isolate's potential to cause disease. This association warrants further study because iss and the protein it encodes may be useful targets of future colibacillosis control efforts.

Cloning and sequencing of the iss gene from a virulent avian Escherichia coli.

Control of colibacillosis is important to the poultry industry. We have found that the presence of a gene for increased serum survival, iss, is strongly correlated with Escherichia coli isolated from birds with colibacillosis. Therefore, the iss gene and its protein product, Iss, are potential targets for detection and control of avian colibacillosis. The iss gene was amplified from a virulent avian E. coli isolate and sequenced. The sequences of the gene and the predicted protein product were compared with those of iss from a human E. coli isolate and lambda bor. The iss gene from the avian E. coli isolate has 96.8% identity with the iss gene from the human E. coli isolate and 89.4% identity with lambda bor. The Iss protein from the avian isolate has 87% identity with Iss from the human isolate and 90% identity with Bor. The low identity between the two Iss proteins is because of a frame-shift in their respective coding sequences. In sum, iss from this avian E. coli isolate is very similar to iss from a human E. coli isolate, but because of a frameshift mutation in the coding sequence of iss from the human E. coli isolate, Iss proteins from avian and human E. coli isolates have only 87% identity. The strong association of iss with E. coli isolated from birds with colibacillosis, suggests that this sequence be studied for its value as a marker or target to be used in colibacillosis control.

Iss from a virulent avian Escherichia coli.

No single characteristic of virulent avian Escherichia coli has been identified that can be exploited in colibacillosis detection protocols. Research in our lab suggests a strong association between the presence of an iss DNA sequence with an isolate's disease-causing ability. The study presented here focuses on the techniques used in the expression, purification, and characterization of avian E. coli Iss protein. In brief, iss was cloned into an expression vector, the construct was transformed into a protease-deficient E. coli, and expression was induced. The protein was expressed as a glutathione-S-transferase (GST) fusion and purified by affinity chromatography. The GST portion was cleaved from Iss, Iss was harvested by affinity chromatography, and the identity of Iss was confirmed by N-terminal sequencing. Currently, purified Iss is being used to prepare hybridomas for production of monoclonal antibodies with the goal of evaluating anti-Iss as a reagent for the detection of virulent avian E. coli.

Further characterization of a complement-sensitive mutant of a virulent avian Escherichia coli isolate.

An attempt was made to characterize the mechanism of complement resistance operating in a virulent avian Escherichia coli isolate. Using flow cytometry to detect antibody to C3, we found that there was significantly more antibody bound to a complement-sensitive mutant of this wild type than to the parent organism, suggesting that more C3 subunits were bound to the wild type. Neither the wild type nor the mutant degraded C3. Further, the mutant was phagocytosed to a significantly greater degree than the wild type by cultured phagocytes in the presence of C5-deficient serum. These data suggest that the wild type is resistant to complement, at least in part, because of its ability to restrict C3 deposition on its surface. Therefore, the decrease in virulence seen in the mutant may be related to its increased sensitivity to complement-mediated bacteriolysis or its enhanced susceptibility to complement-opsonized phagocytosis or both.

Large plasmids of avian Escherichia coli isolates.

The plasmid DNA of 30 Escherichia coli isolates from chickens was extracted and examined using techniques designed to isolate large plasmids. This plasmid DNA was examined for the presence of certain known virulence-related genes including cvaC, traT, and some aerobactin-related sequences. Seventeen of the 30 isolates contained from one to four plasmids greater than 50 kb in size. Eleven of these 17 strains possessed plasmids greater than 100 kb in size. Therefore, E. coli isolates of chickens frequently contain large plasmids, and many of these plasmids are likely to contain virulence-related sequences.

The distribution of invA, pagC and spvC genes among Salmonella isolates from animals.

New molecular diagnostic techniques often rely on hybridization or amplification of specific DNA regions to detect pathogenic bacteria. The choice of genes to be used as probes or as the targets of amplification techniques is critical to the success of these procedures. The genes so used might best be those associated with virulent isolates and having a wide distribution among such isolates. In this study three genes, invA, pagC and spvC, thought to be associated with the virulence of salmonellae, were labelled and used to probe the total DNA from 103 Salmonella isolates from animals in an attempt to determine whether these genes might be useful in diagnostic procedures. pagC was detected in 99% of the Salmonella tested, and invA was detected in 94.2% of the isolates. Both pagC and invA were detected with a significantly higher frequency than spvC in isolates from chickens and swine, but no significant difference in detection of these three genes occurred when bovine isolates were examined. Failure to detect any of these genes occurred in only one isolate. Isolates from apparently healthy or from clinically ill chickens and swine could not be distinguished by detecting these three genes. The genes were not detected in the non-Salmonella strains tested. These results suggest that, of these three genes, pagC may be the best choice for use as a probe or polymerase chain reaction target in future detection protocols.

Detection and characterization of Salmonella typhimurium from a dairy herd in North Dakota.

Nasal secretions, faecal samples and buffy coats were obtained from 102 cattle from a North Dakota dairy herd with a history of calf scours. Treated buffy coats, faecal samples and nasal secretions were inoculated into tetrathionate broth (TB), incubated at 37 degrees C overnight, and plated onto brilliant green agar medium with novobiocin (BGAN). The TB was left at room temperature for 5 days and then used to inoculate fresh TB. The fresh TB was incubated at 37 degrees C over night and plated onto BGAN medium. All the plates were incubated at 37 degrees C over night and observed for Salmonella-like growth. Suspect colonies were further tested and Salmonella isolates were serotyped by the National Veterinary Services laboratory. Twenty-two of the 36 calves sampled harboured S. typhimurium in their faeces, but no samples from cows were positive. No Salmonella were isolated from the buffy coats, but 4 calves were shown to have Salmonella in their nasal secretions. Extended enrichment of the faecal cultures in TB resulted in a significant increase in Salmonella isolations, although 2 samples were positive following the initial enrichment period and not after secondary enrichment. The typical Salmonella isolate detected from this herd contained a transmissible R-plasmid encoding resistance to tetracycline, kanamycin, sulphisoxazole and ampicillin. This study confirmed that delayed secondary enrichment in TB is superior to primary enrichment for detection of Salmonella from cattle.

Characterization of an avirulent mutant of a virulent avian Escherichia coli isolate.

A virulent, complement-resistant avian Escherichia coli isolate and its avirulent, complement-sensitive, transposon-insertion mutant were compared for the purpose of revealing structures associated with complement resistance. Both had a smooth lipopolysaccharide layer, contained traT, and lacked a capsule, but the mutant possessed a 16.2-kilodalton outer-membrane protein (OMP) not present in the wild-type. This protein may be the product of a coding region interrupted by transposon insertion. Such results suggest that an OMP greater than 16.2 kilodaltons in size may be responsible for the complement resistance and virulence of this wild-type E. coli.

Association of K-1 capsule, smooth lipopolysaccharides, traT gene, and Colicin V production with complement resistance and virulence of avian Escherichia coli.

A group of complement-resistant, virulent avian Escherichia coli isolates were compared with a group of complement-sensitive, avirulent avian isolates for the presence of K-1 capsule, smooth lipopolysaccharides (LPS), the traT gene, and Colicin V (ColV) production. These parameters were selected because of their reported association with complement resistance and virulence in E. coli. Lethality in chicken embryos has also been shown to be correlated with virulence of avian E. coli for chickens. The complement-resistant, virulent E. coli isolates did not possess a K-1 capsule. Production of ColV and the presence of smooth LPS were significantly correlated with embryo lethality. There was no correlation between the presence of traT and embryo lethality. These results suggest that complement resistance and virulence in avian E. coli are associated with ColV production and smooth LPS but not with K-1 antigen or traT.