Development of a novel in-water vaccination protocol for DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine in adult sheep.

Intensive livestock production is associated with an increased incidence of salmonellosis. The risk of infection and the subsequent public health concern is attributed to increased pathogen exposure and disease susceptibility due to multiple stressors experienced by livestock from farm to feedlot. Traditional parenteral vaccine methods can further stress susceptible populations and cause carcass damage, adverse reactions, and resultant increased production costs. As a potential means to address these issues, in-water delivery of live attenuated vaccines affords a low cost, low-stress method for immunization of livestock populations that is not associated with the adverse handling stressors and injection reactions associated with parenteral administration. We have previously established that in-water administration of a Salmonella enterica serovar Typhimurium dam vaccine conferred significant protection in livestock. While these experimental trials hold significant promise, the ultimate measure of the vaccine will not be established until it has undergone clinical testing in the field wherein environmental and sanitary conditions are variable. Here we show that in-water administration of a S. Typhimurium dam attenuated vaccine was safe, stable, and well-tolerated in adult sheep. The dam vaccine did not alter water consumption or vaccine dosing; remained viable under a wide range of temperatures (21-37°C); did not proliferate within fecal-contaminated trough water; and was associated with minimal fecal shedding and clinical disease as a consequence of vaccination. The capacity of Salmonella dam attenuated vaccines to be delivered in drinking water to protect livestock from virulent Salmonella challenge offers an effective, economical, stressor-free Salmonella prophylaxis for intensive livestock production systems.

Protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine when delivered in-water to sheep challenged with Salmonella enterica serovar Typhimurium.

Stimulation of acquired immunity to Salmonella in livestock is not feasible in neonates (which can be infected within 24h of birth) and is challenging in feedlots, which typically source animals from diverse locations and vendors. Induction of innate immune mechanisms through mass vaccination of animals upon arrival to feedlots is an alternative approach. Transport, environmental conditions, changes in social grouping, and further handling during feedlot assembly are significant stressors. These factors, as well as concurrent exposure to a diversity of pathogens, contribute to the risk of disease. We have shown that oral immunization of calves with a modified live Salmonella enterica serovar Typhimurium vaccine strain, which lacks the DNA adenine methylase gene (S. Typhimurium dam), attenuates the severity of clinical disease, reduces fecal shedding, and promotes clearance of salmonellae following virulent homologous and heterologous challenge. This study examines the safety and efficacy of a S. Typhimurium dam vaccine in sheep via oral delivery in drinking water (ad libitum), as a means to effectively vaccinate large groups of animals. Adult merino sheep were vaccinated in drinking water -28 days, -7 days and 24h pre and 24h post-virulent Salmonella Typhimurium challenge which was administered via the oral route. Significant attenuation of clinical disease (temperature, appetite, and attitude) and reduction in mortality and virulent Salmonella Typhimurium fecal shedding and tissue colonization was observed in animals that received the vaccine 28 and 7 days pre-challenge. Further, vaccination did not pose a risk to stock previously infected with virulent salmonellae as mortalities and clinical disease in sheep vaccinated prior to or following virulent challenge did not differ significantly from the non-vaccinated controls. The capacity of S. Typhimurium dam vaccines delivered in drinking water to protect livestock from virulent Salmonella challenge offers an effective, economical, stressor free Salmonella prophylaxis for intensive livestock production systems.

Cross-protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine in calves challenged with Salmonella serovar Newport.

Intensive livestock production and management systems are associated with increased fecal-oral pathogen transmission and a resultant high prevalence of multiple Salmonella serovars in many large dairy farms and feedlots. Thus, it is imperative to develop livestock vaccines that are capable of eliciting potent states of cross-protective immunity against a diversity of serovars of a given species. Immunization with modified live Salmonella enterica serovar Typhimurium vaccine strains, that lack the DNA adenine methylase (Dam), confers cross-protective immunity in murine and avian models of typhoid fever as well as in a bovine model of salmonellosis. Here we examined whether a dam mutant Typhimurium vaccine (serogroup B) has the capacity to elicit cross-protection against a virulent challenge with an emerging, clinically relevant, and multi-drug resistant strain of serovar Newport (serogroup C2-C3) that has been associated with clinical disease in recent salmonellosis outbreaks in calves. Vaccinated animals challenged with Newport exhibited a significant attenuation of clinical disease (improved attitude scores, increased daily weight gains and reduced fever and diarrhea) and a concomitant reduction in Newport fecal shedding and colonization of mesenteric lymph nodes and lungs compared to non-vaccinated control animals. The capacity to elicit cross-protective immunity in calves suggests that dam mutant vaccines have potential application toward the prevention and control of Salmonella infection in commercial livestock production systems wherein livestock are exposed to a diversity of Salmonella serovars.

Salmonella and on-farm risk factors in healthy slaughter-age cattle and sheep in eastern Australia.

OBJECTIVE: To examine healthy slaughter-age cattle and sheep on-farm for the excretion of Salmonella serovars in faeces and to identify possible risk factors using a questionnaire. PROCEDURE: The study involved 215 herds and flocks in the four eastern states of Australia, 56 with prior history of salmonellosis. Production systems examined included pasture beef cattle, feedlot beef cattle, dairy cattle, prime lambs and mutton sheep and animals were all at slaughter age. From each herd or flock, 25 animals were sampled and the samples pooled for Salmonella culture. All Salmonella isolated were serotyped and any Salmonella Typhimurium isolates were phage typed. Questionnaires on each production system, prepared in Epi Info 6.04, were designed to identify risk factors associated with Salmonella spp excretion, with separate questionnaires designed for each production system. RESULTS: Salmonellae were identified in all production systems and were more commonly isolated from dairies and beef feedlots than other systems. Statistical analysis revealed that dairy cattle were significantly more likely to shed Salmonella in faeces than pasture beef cattle, mutton sheep and prime lambs (P<0.05). A wide diversity of Salmonella serovars, all of which have been isolated from humans in Australia, was identified in both cattle and sheep. Analysis of the questionnaires showed access to new arrivals was a significant risk factor for Salmonella excretion on dairy properties. For beef feedlots, the presence of large numbers of flies in the feedlot pens or around stored manure were significant risk factors for Salmonella excretion. CONCLUSION: Dairy cattle pose the highest risk of all the slaughter-age animals tested. Some of the identified risk factors can be overcome by improved management practices, especially in relation to hygiene.

Salmonella Typhimurium persistence in a Hunter Valley dairy herd.

OBJECTIVE: An epidemiological study was undertaken at a Hunter Valley dairy with persistent Salmonella Typhimurium infection. The aim of the study was to identify cattle currently or previously infected with Salmonella, possible sources of the organism, patterns of spread, and husbandry practices that could be improved. METHODOLOGY: Faecal samples, feed, water and environmental samples were cultured for Salmonella and blood samples were tested for antibodies against Salmonella (Dublin and Typhimurium). A questionnaire was designed to identify possible risk factors associated with Salmonella excretion. RESULTS: S Typhimurium was apparently introduced from an old to a new dairy through manure spread as fertiliser. Salmonella apparently persisted in the effluent pond, and the following year clinical cases occurred after pasture, irrigated with water from the pond, was grazed by dry cows, and adult cattle became clinically ill with salmonellosis. The disease spread to other cows and calves. Poor design of calf pens assisted spread of Salmonella from sick to healthy calves. In addition, there was suspected transmission to the dairy farmer's 9-month-old daughter. Salmonellosis on a farm is a potential zoonotic risk to farm workers and their families. There is also the risk that cull cows may carry Salmonella to the abattoir and subsequently into the human food chain. Methods of waste management, and the design of calf pens, were identified as major risk factors that could be improved to minimise the spread of salmonellosis on this property.

Genetic diversity among Mycoplasma species bovine group 7: clonal isolates from an outbreak of polyarthritis, mastitis, and abortion in dairy cattle.

A comprehensive genetic analysis of 60 Mycoplasma sp. bovine group 7 isolates from different geographic origins and epidemiological settings is presented. Twenty-four isolates were recovered from the joints of calves during sporadic episodes of polyarthritis in geographically distinct regions of Queensland and New South Wales, Australia, including two clones of the type strain PG5O. A further three Australian isolates were also recovered from the tympanic bulla, retropharyngeal lymph node and the lung and another three isolates had unconfirmed histories. Six isolates originated from Germany, Portugal, Nigeria, and France. Twenty-four epidemiologically related isolates of Mycoplasma sp. bovine group 7 were recovered from multiple tissue sites and body fluids of infected calves with polyarthritis, mastitic milk, and from the stomach contents, lung and liver from aborted foetuses in three large, centrally managed dairy herds in New South Wales, Australia. Restriction endonuclease analysis (REA) of genomic DNA differentiated 29 Cfol profiles among these 60 isolates and grouped all 24 epidemiologically related isolates in a defined pattern showing a clonal origin. Three isolates of this clonal cluster were recovered from mastitic milk and the synovial exudate of clinically-affected calves and appeared sporadically for periods up to 18 months after the initial outbreak of polyarthritis indicating a persistent, close association of the organism with cattle in these herds. The Cfol profile representative of the clonal cluster was distinguishable from profiles of isolates recovered from multiple, unrelated cases of polyarthritis in Queensland and New South Wales and from other countries. All 24 isolates from the clonal cluster possessed a plasmid (pBG7AU) with a molecular size of 1022 bp. DNA sequence analysis of pBG7AU identified two open reading frames sharing 81 and 99% DNA sequence similarity with hypothetical replication control proteins A and B respectively, previously described in plasmid pADB201 isolated from M. mycoides subspecies mycoides. Other isolates of bovine group 7, epidemiologically unrelated to the clonal cluster, including two clones of the type strain PG5O, possessed a similar-sized plasmid. These data confirm that Mycoplasma sp. bovine group 7 is capable of migrating to, and multiplying within, different tissue sites within a single animal and among different animals within a herd.

The common ovine Shiga toxin 2-containing Escherichia coli serotypes and human isolates of the same serotypes possess a Stx2d toxin type.

Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the stx(2) subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans. The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep. The major stx(2) subtype in the ovine isolates was shown to be stx(2d-Ount) (119 of 146 [81.5%]) and was predominantly associated with serotypes O75:H(-)/H8/H40, O91:H(-), O123:H(-), O128:H2, and OR:H2. However, 17 of 18 (94.4%) ovine isolates of serotype O5:H(-) possessed a stx(2d-O111/OX3a) subtype. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both clinical and nonclinical human infections also contained a stx(2d) (stx(2d-Ount/O111/OX3a)) subtype. These studies suggest that a specific stx(2) subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of E. coli during outbreaks of STEC-associated diseases in humans.

Virulence properties and serotypes of Shiga toxin-producing Escherichia coli from healthy Australian slaughter-age sheep.

A group of 1,623 ovine fecal samples recovered from 65 geographically distinct mutton sheep and prime lamb properties across New South Wales, Australia, were screened for the presence of Shiga toxin-producing Escherichia coli (STEC) virulence factors (stx(1), stx(2), eaeA, and ehxA). A subset was cultured for STEC isolates containing associated virulence factors (eaeA and/or ehxA), which were isolated from 17 of 20 (85%) and 19 of 20 (95%) tested prime lamb and mutton sheep properties, respectively. STEC isolates containing stx(1), stx(2), and ehxA were most commonly isolated (19 of 40 flocks; 47.5%), and this profile was observed for 10 different serotypes. Among 90 STEC isolates studied, the most common serotypes were O91:H(-) (22 isolates [24.4%]), O5:H(-) (16 isolates [17.8%]), O128:H2 (11 isolates [12.2%]), O123:H(-) (8 isolates [8.9%]), and O85:H49 (5 isolates [5.6%]). Two isolates (2.2%) were typed as O157:H(-). A total of 78 of 90 STEC isolates (86.7%) expressed Shiga toxin in Vero cell culture and 75 of 84 ehxA-positive isolates (89.3%) expressed enterohemolysin on washed sheep blood agar. eaeA was observed in 11 of 90 (12.2%) ovine STEC isolates, including serotypes O5:H(-), O84:H(-), O85:H49, O123:H(-) O136:H40, and O157:H(-). Although only 2 of 90 isolates were typed as O157:H(-), the predominant serotypes recovered during this study have been recovered from human patients with clinical disease, albeit rarely.

The detection of Shiga toxin-producing Escherichia coli in diagnostic bovine faecal samples using vancomycin-cefixime-cefsulodin blood agar and PCR.

The prevalence of complex Shiga toxin-producing Escherichia coli (STEC), i.e. STEC containing accessory virulence factors intimin (eaeA) and/or enterohaemorrhagic E. coli haemolysin (ehxA) and their serotypes were determined in diagnostic bovine faecal samples processed during a 3 months period. The presence of complex STEC was determined using PCR and vancomycin-cefixime-cefsulodin blood agar (BVCCA) using a dual approach which involved (i) direct culture of faecal samples on BVCCA followed by mutiplex PCR of BVCCA positive colonies and (ii) culture of faecal samples enriched in modified EC (mEC) broth (with a complex STEC profile determined by PCR) on BVCCA followed by multiplex PCR of BVCCA positive colonies. Using both techniques complex STEC were isolated from 23 (18.7%) of the 123 faecal samples. Complex STEC were isolated from 14 faecal samples by direct culture on BVCCA and 13 faecal samples yielded complex STEC by culture of mEC broths with a complex STEC profile on BVCCA. Only four samples were positive using both techniques. The serotypes isolated included O5:H-, O26:H-, O26:H11, O91:H21, O111:H-, O111:H8, O104:H11, O113:H21 and O157:H8. This study confirms that non-O157 STEC can be isolated from bovine faeces and that they carry types associated with human disease. This work also demonstrates that the use of a dual approach is advisable to increase the likelihood of isolating complex STEC.

Genetic and biochemical diversity among isolates of Paenibacillus alvei cultured from Australian honeybee (Apis mellifera) colonies.

Twenty-five unique CfoI-generated whole-cell DNA profiles were identified in a study of 30 Paenibacillus alvei isolates cultured from honey and diseased larvae collected from honeybee (Apis mellifera) colonies in geographically diverse areas in Australia. The fingerprint patterns were highly variable and readily discernible from one another, which highlighted the potential of this method for tracing the movement of isolates in epidemiological studies. 16S rRNA gene fragments (length, 1,416 bp) for all 30 isolates were enzymatically amplified by PCR and subjected to restriction analysis with DraI, HinfI, CfoI, AluI, FokI, and RsaI. With each enzyme the restriction profiles of the 16S rRNA genes from all 30 isolates were identical (one restriction fragment length polymorphism [RFLP] was observed in the HinfI profile of the 16S rRNA gene from isolate 17), which confirmed that the isolates belonged to the same species. The restriction profiles generated by using DraI, FokI, and HinfI differentiated P. alvei from the phylogenetically closely related species Paenibacillus macerans and Paenibacillus macquariensis. Alveolysin gene fragments (length, 1, 555 bp) were enzymatically amplified from some of the P. alvei isolates (19 of 30 isolates), and RFLP were detected by using the enzymes CfoI, Sau3AI, and RsaI. Extrachromosomal DNA ranging in size from 1 to 10 kb was detected in 17 of 30 (57%) P. alvei whole-cell DNA profiles. Extensive biochemical heterogeneity was observed among the 28 P. alvei isolates examined with the API 50CHB system. All of these isolates were catalase, oxidase, and Voges-Proskauer positive and nitrate negative, and all produced acid when glycerol, esculin, and maltose were added. The isolates produced variable results for 16 of the 49 biochemical tests; negative reactions were recorded in the remaining 30 assays. The genetic and biochemical heterogeneity in P. alvei isolates may be a reflection of adaptation to the special habitats in which they originated.

Geographically diverse Australian isolates of Melissococcus pluton exhibit minimal genotypic diversity by restriction endonuclease analysis.

Melissococcus pluton, the causative agent of European foulbrood is an economically significant disease of honey bees (Apis mellifera) across most regions of the world and is prevalent throughout most states of Australia. 49 Isolates of M. pluton recovered from diseased colonies or honey samples in New South Wales, Queensland, South Australia, Tasmania and Victoria were compared using SDS-PAGE, Western immunoblotting and restriction endonuclease analyses. DNA profiles of all 49 geographically diverse isolates showed remarkably similar AluI profiles although four isolates (one each from Queensland, South Australia, New South Wales and Victoria) displayed minor profile variations compared to AluI patterns of all other isolates. DNA from a subset of the 49 Australian and three isolates from the United Kingdom were digested separately with the restriction endonucleases CfoI, RsaI and DraI. Restriction endonuclease fragment patterns generated using these enzymes were also similar although minor variations were noted. SDS-PAGE of whole cell proteins from 13 of the 49 isolates from different states of Australia, including the four isolates which displayed minor profile variations (AluI) produced indistinguishable patterns. Major immunoreactive proteins of approximate molecular masses of 21, 24, 28, 30, 36, 40, 44, 56, 60, 71, 79 and 95 kDa were observed in immunoblots of whole cell lysates of 22 of the 49 isolates and reacted with rabbit hyperimmune antibodies raised against M. pluton whole cells. Neither SDS-PAGE or immunoblotting was capable of distinguishing differences between geographically diverse isolates of M. pluton. Collectively these data confirm that Australian isolates of M. pluton are genetically homogeneous and that this species may be clonal. Plasmid DNA was not detected in whole cell DNA profiles of any isolate resolved using agarose gel electrophoresis.

Detection of shiga-like toxin (stx1 and stx2), intimin (eaeA), and enterohemorrhagic Escherichia coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR.

A multiplex PCR was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 (stx1 and stx2), intimin (eaeA), and enterohemolysin A (hlyA) in 444 fecal samples derived from healthy and clinically affected cattle, sheep, pigs, and goats. The method involved non-solvent-based extraction of nucleic acid from an aliquot of an overnight culture of feces in EC (modified) broth. The detection limit of the assay for both fecal samples and pure cultures was between 18 and 37 genome equivalents. stx1 and hlyA were the most commonly encountered virulence factors.