Virginia L. Mohler

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 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.

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.

Antimicrobial susceptibility of Salmonella isolates recovered from calves with diarrhoea in Australia.

OBJECTIVE To identify the common serotypes and antimicrobial resistance patterns of Salmonella spp. associated with diarrhoea in Australian dairy calves under the age of 6 weeks. DESIGN Cross-sectional study. METHODS Faecal samples were collected from outbreaks of diarrhoea in dairy and dairy beef calves less than 6 weeks old. Samples were screened for Salmonella using standard enrichment culture techniques. The antimicrobial susceptibility to 12 commonly used veterinary and human antimicrobials was assessed using the Kirby-Bauer disk diffusion method and the susceptibility profiles of dairy and dairy beef properties were compared using Fishers exact test. RESULTS Salmonella ser. Dublin, S. ser. Typhimurium and S. ser. Bovismorbificans were the three most common salmonella serotypes isolated. The majority of properties had one serotype. Most of the Salmonella isolates were not resistant to any of the antimicrobials tested. No resistance was seen to amikacin and nalidixic acid, and only one isolate was resistant to ceftiofur or amoxicillin-clavulanic acid. The most common antimicrobial resistance was to streptomycin, ampicillin or combination sulfonamides. Multi-drug resistance was detected in S. ser. Anatum, S. ser. Bovismorbificans, S. ser. Muenster, S. ser. Newport and S. ser. Typhimurium. Isolates from dairy beef properties were more likely to be resistant to ampicillin, kanamycin, neomycin, sulfamethoxazole/trimethoprim and tetracycline (P < 0.05) and were more likely to exhibit multi-drug resistance. CONCLUSION The prevalence of antimicrobial resistance in Salmonella isolates from dairy calves in Australia is low compared with that reported overseas. From a human health perspective, resistance to antimicrobials used in the treatment of human salmonellosis was infrequent.

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.

Milk acidification to control the growth of Mycoplasma bovis and Salmonella Dublin in contaminated milk

Bacterial contamination of milk fed to calves compromises calf health. Several bacterial pathogens that infect cows, including Mycoplasma bovis and Salmonella enterica ssp. enterica serovar Dublin, are shed in milk, providing a possible route of transmission to calves. Milk acidification lowers the milk pH so that it is unsuitable for bacterial growth and survival. The objectives of this study were to (1) determine the growth of M. bovis and Salmonella Dublin in milk, and (2) evaluate the efficacy of milk acidification using a commercially available acidification agent (Salstop, Impextraco, Heist-op-den-Berg, Belgium) to control M. bovis and Salmonella Dublin survival in milk. For the first objective, 3 treatments and a positive control were prepared in 10 mL of milk and broth, respectively, and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 104 cfu/mL. Each treatment was retained at 5, 23, or 37°C with the positive control at 37°C. Aliquots were taken at 4, 8, 24, 28, 32, 48, 52, and 56 h after inoculation and transferred onto agar medium in triplicate following a 10-fold dilution series in sterile phosphate-buffered saline. All plates were incubated and colonies counted. For the second objective, 4 treatments and a positive control were prepared with 100 mL of milk and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 106 cfu/mL. With the use of Salstop, treatments were adjusted to an approximate pH of 6, 5, 4, or 3.5. The positive control was left untreated. At 1, 2, 4, 6, 8, and 24 h after treatment, triplicate aliquots were taken, the pH measured, and then the aliquots were transferred onto agar medium and into broth for enrichment. Following incubation, agar colonies were counted, while broths were plated and incubated prior to colonies being counted. All trials were repeated. Mycoplasma bovis did not grow in milk, but Salmonella Dublin proliferated. The pH of all acidification treatments remained stable for 24 h. No viable M. bovis organisms were detected at 1 h of exposure to pH 3.5 and 4 or at 8 h of exposure to pH 5. Following 24 h of exposure to pH 6 M. bovis remained viable. No viable Salmonella Dublin organisms were detected at 2 and 6 h of exposure to pH 3.5 and 4, respectively. Salmonella Dublin remained viable following 24 h of exposure to pH 5 and 6. These results demonstrate that milk acidification using Salstop is effective at eliminating viable M. bovis and Salmonella Dublin organisms in milk if the appropriate pH and exposure time are maintained.