John K. House

Salmonella DNA Adenine Methylase Mutants Elicit Protective Immune Responses to Homologous and Heterologous Serovars in Chickens

ABSTRACT Salmonella DNA adenine methylase (Dam) mutants that lack or overproduce Dam are highly attenuated for virulence in mice and confer protection against murine typhoid fever. To determine whether vaccines based on Dam are efficacious in poultry, aSalmonella Dam− vaccine was evaluated in the protection of chicken broilers against oral challenge with homologous and heterologous Salmonella serovars. A Salmonella enterica serovar Typhimurium Dam− vaccine strain was attenuated for virulence in day-of-hatch chicks more than 100,000-fold. Vaccination of chicks elicited cross-protective immune responses, as evidenced by reduced colonization (10- to 10,000-fold) of the gastrointestinal tract (ileum, cecum, and feces) and visceral organs (bursa and spleen) after challenge with homologous (Typhimurium F98) and heterologous (Enteritidis 4973 and S. entericaO6,14,24: e,h-monophasic) Salmonella serovars that are implicated in Salmonella infection of poultry. The protection conferred was observed for the organ or the maximum CFU/tissue/bird as a unit of analysis, suggesting that Dam mutant strains may serve as the basis for the development of efficacious poultry vaccines for the containment of Salmonella.

Haemolytic anaemia in cattle in NSW associated with Theileria infections

Several outbreaks of anaemia, jaundice, abortion and mortality in cattle in New South Wales were attributed to the intracellular parasite, Theileria buffeli. Disease occurred predominantly in periparturient animals that had been moved from inland to coastal areas. Diagnosis was made via exclusion of other causes of haemolytic anaemia and observation of the parasite in blood smears. Treatments included both registered and non-registered products. There is a possibility of a new strain of Theileria sp. in Australia and the possible vectors encountered in NSW are discussed.

Human Salmonella clinical isolates distinct from those of animal origin.

ABSTRACT The global trend toward intensive livestock production has led to significant public health risks and industry-associated losses due to an increased incidence of disease and contamination of livestock-derived food products. A potential factor contributing to these health concerns is the prospect that selective pressure within a particular host may give rise to bacterial strain variants that exhibit enhanced fitness in the present host relative to that in the parental host from which the strain was derived. Here, we assessed 184 Salmonella enterica human and animal clinical isolates for their virulence capacities in mice and for the presence of the Salmonella virulence plasmid encoding the SpvB actin cytotoxin required for systemic survival and Pef fimbriae, implicated in adherence to the murine intestinal epithelium. All (21 of 21) serovar Typhimurium clinical isolates derived from animals were virulent in mice, whereas many (16 of 41) serovar Typhimurium isolates derived from human salmonellosis patients lacked this capacity. Additionally, many (10 of 29) serovar Typhimurium isolates derived from gastroenteritis patients did not possess the Salmonella virulence plasmid, in contrast to all animal and human bacteremia isolates tested. Lastly, among serovar Typhimurium isolates that harbored the Salmonella virulence plasmid, 6 of 31 derived from human salmonellosis patients were avirulent in mice, which is in contrast to the virulent phenotype exhibited by all the animal isolates examined. These studies suggest that Salmonella isolates derived from human salmonellosis patients are distinct from those of animal origin. The characterization of these bacterial strain variants may provide insight into their relative pathogenicities as well as into the development of treatment and prophylactic strategies for salmonellosis.

Intraspecies Variation in the Emergence of Hyperinfectious Bacterial Strains in Nature

Salmonella is a principal health concern because of its endemic prevalence in food and water supplies, the rise in incidence of multi-drug resistant strains, and the emergence of new strains associated with increased disease severity. Insights into pathogen emergence have come from animal-passage studies wherein virulence is often increased during infection. However, these studies did not address the prospect that a select subset of strains undergo a pronounced increase in virulence during the infective process- a prospect that has significant implications for human and animal health. Our findings indicate that the capacity to become hypervirulent (100-fold decreased LD50) was much more evident in certain S. enterica strains than others. Hyperinfectious salmonellae were among the most virulent of this species; restricted to certain serotypes; and more capable of killing vaccinated animals. Such strains exhibited rapid (and rapidly reversible) switching to a less-virulent state accompanied by more competitive growth ex vivo that may contribute to maintenance in nature. The hypervirulent phenotype was associated with increased microbial pathogenicity (colonization; cytotoxin production; cytocidal activity), coupled with an altered innate immune cytokine response within infected cells (IFN-β; IL-1β; IL-6; IL-10). Gene expression analysis revealed that hyperinfectious strains display altered transcription of genes within the PhoP/PhoQ, PhoR/PhoB and ArgR regulons, conferring changes in the expression of classical virulence functions (e.g., SPI-1; SPI-2 effectors) and those involved in cellular physiology/metabolism (nutrient/acid stress). As hyperinfectious strains pose a potential risk to human and animal health, efforts toward mitigation of these potential food-borne contaminants may avert negative public health impacts and industry-associated losses.

Prevalence of major enteric pathogens in Australian dairy calves with diarrhoea

Objective  Determine the prevalence of the major enteric pathogens in dairy and dairy beef calves with diarrhoea in Australia. Design  Cross‐sectional study. Methods  Faecal samples from 84 Australian dairy and dairy beef properties (597 samples) were screened for rotavirus and coronavirus using real‐time reverse transcription polymerase chain reaction, for Salmonella spp. using selective enrichment faecal culture, and for enterotoxigenic Escherichia coli (K99) and Cryptosporidium parvum using a commercial enzyme‐linked immunosorbent assay. A logistic regression with random effects model was used to compare prevalence of pathogens in dairy and dairy beef operations. Results  Enteric pathogens were isolated from 97.6% of outbreaks and 95.0% of samples. Rotavirus was the most common pathogen identified (477/597, 79.9%) followed by C. parvum (349/597, 58.5%), Salmonella spp. (142/597, 23.8%), coronavirus (129/597, 21.6%) and E. coli K99 (104/597, 17.4%). Multiple pathogens were identified on 96.4% of farms and from 71.0% of samples. Samples from dairy beef properties were more likely to have multiple pathogens than dairy properties (P < 0.05), whereas rotavirus and Salmonella spp. were more likely to be identified in samples collected from dairy beef than dairy properties (P < 0.05). Conclusion  Most outbreaks of calf diarrhoea in dairy and dairy beef operations involve multiple pathogens. Rotavirus and C. parvum were the most frequently identified pathogens across production systems. Salmonella spp. and rotavirus were more frequently identified in dairy beef operations.

Salmonella DNA adenine methylase mutants prevent colonization of newly hatched chickens by homologous and heterologous serovars.

Salmonella mutants lacking DNA adenine methylase (Dam) are highly attenuated for virulence and confer protection against oral challenge with homologous and heterologous Salmonella serovars in mice and chicken broilers. To determine whether vaccines based on Dam are efficacious in preventing early colonization of newly hatched chickens, a Salmonella typhimurium Dam(-) vaccine was evaluated for the protection of chicks against oral challenge with homologous and heterologous Salmonella serovars. Vaccination of chicks elicited protection 2 and 6 days post-challenge as evidenced by a significant reduction in colonization of the gastrointestinal tract (ileum, cecum and feces) and visceral organs (spleen and bursa) when challenged with homologous S. typhimurium. Moderate protection was observed following challenge with heterologous S. enteritidis and Salmonella O6, 14, 24:e, h-monophasic) serovars. These data suggest that Salmonella Dam mutant strains conferred cross-protection, presumably via competitive exclusion mechanisms that prevent superinfection of chicks by other Salmonella strains. Such protection may reduce pre-harvest Salmonella contamination in poultry, decreasing the potential for food-borne transmission of this pathogen to humans.

Salmonella DNA adenine methylase mutants elicit early and late onset protective immune responses in calves.

Salmonellosis is an important disease of livestock and Salmonella contamination of livestock-derived food products and effluents pose a significant risk to human health. Salmonella vaccines currently available to prevent salmonellosis in cattle have limited efficacy. Here we evaluated a Salmonella enterica serovar Typhimurium vaccine strain lacking the DNA adenine methylase (Dam) for safety and efficacy in calves. Vaccination was safe in calves, and following challenge with virulent Typhimurium 4 weeks post-immunization, vaccinated animals exhibited significantly lower mortality, diarrhea, and rectal temperatures, as well as reduced colonization of gastrointestinal tract and visceral organs compared to non-vaccinated control animals. Additionally, early onset protection (competitive exclusion) in vaccinated neonatal calves was demonstrated by attenuated clinical disease (as measured by rectal temperatures and attitude scores) and reduced mortality when challenged with virulent Typhimurium 24h after immunization. Taken together, these data suggest that vaccination with Salmonella Dam mutant strains confer significant protection against Salmonella infections in cattle via both adaptive immunity and competitive exclusion mechanisms.

Artificial insemination field data on the use of sexed and conventional semen in nulliparous Holstein heifers.

This study investigated conception rates and other reproductive outcomes achieved with artificial insemination (AI) of nulliparous Holstein heifers using sexed and conventional semen in a commercial Australian dairy herd in central western New South Wales from January 2004 to April 2009. Retrospective data from on-farm records of 9,870 inseminations of 4,456 heifers were analyzed using several mixed models to assess the effect of temperature and humidity surrounding breeding, insemination sire, artificial insemination technician, service number, and heifer weight and age at breeding on reproductive traits (conception rates, sex ratios, gestation length, and abortion and stillbirth rates). Semen was used from 15 sexed sires and 41 unsexed sires. Sexed semen was primarily used at first and second service. Empirical conception rates of 31.6 and 39.6% were achieved for sexed and unsexed semen respectively, whereas model-based predictions were lower, at 21.3 and 32.1%. Conception rates were significantly affected by insemination sire, sex-sorting, heifer age at breeding, temperature and humidity surrounding breeding, service number, and AI technician. Sexed semen yielded 86% heifers, compared with 48% for conventional semen. Significant predictors of calf sex included semen sexing, gestation length, and insemination sire. Twinning rate was high, at 3.6% for both semen types, and gestation length and heifer weight at breeding were significant predictors of twinning. Abortion rates for sexed and unsexed conceptions were similar at 6.1 and 6.5%, respectively, and were affected by heifer age at breeding. Stillbirth rate was affected by calf sex, twinning, gestation length, and AI technician; semen sorting, age at breeding, and temperature and humidity were marginally significant predictors. No abnormalities were observed in the development of offspring, except for a marginally higher stillbirth rate for sexed calves, a finding that needs further investigation. Many variables influence the breeding outcomes associated with the application of sex-sorted sperm on commercial dairy farms. Recognition and management of these variables will increase the economic return from the investment in sex-sorted sperm.

Salmonella in Calves

Salmonellae are endemic on most large intensive farms and salmonellosis is a common cause of neonatal morbidity and mortality. Disease and mortality usually reflect a variety of management events and environmental stressors that contribute to compromised host immunity and increased pathogen exposure. The diversity of salmonella serovars present on farms, and the potential for different serovars to possess different virulence factors, require the implementation of broad prophylactic strategies that are efficacious for all salmonellae. This article discusses strategies to promote host immunity and minimize pathogen exposure at the farm level. The benefits of control include a reduction in disease incidence and mortality, reduced drug and labor costs, and improved growth rates.

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.