Bacteriophages as antimicrobial agents against major pathogens in swine:a review

In recent years,the development of antibiotic resistant bacteria has become a global concern which has prompted research into the development of alternative disease control strategies for the swine industry.Bacteriophages(viruses that infect bacteria) offer the prospect of a sustainable alternative approach against bacterial pathogens with the flexibility of being applied therapeutically or for biological control purposes.This paper reviews the use of phages as an antimicrobial strategy for controlling critical pathogens including Salmonella and Escherichia coll with an emphasis on the application of phages for improving performance and nutrient digestibility in swine operations as well as in controlling zoonotic human diseases by reducing the bacterial load spread from pork products to humans through the meat.     

Adherence of Actinobacillus pleuropneumoniae serotype 1 to swine buccal epithelial cells involves fibronectin

The swine pathogen Actinobacillus pleuropneumoniae serotype 1 was investigated for its ability to adhere to swine, rat, and human buccal epithelial cells (BEC). The highest number of bacteria adhered was to swine BEC. This binding ability was affected by heating, extreme pH, treatment with sodium dodecyl sulfate, ethylenediamine tetraacetate, or periodate, and proteolysis, suggesting that cell-surface glycoproteins participate in adherence and that adherence is based mostly on ionic interactions. Mannose and swine fibronectin may play a direct role in this interaction. Convalescent-phase serum from naturally infected pigs inhibited the adhesion. There was a correlation between bacterial pathogenicity as well as host specificity and the capacity for adherence to swine BEC. Adhesion to swine BEC provides a convenient method to study in vitro the adherence of A. pleuropneumoniae and other pathogens of the pig respiratory tract.     

Comparing the minimum inhibitory and mutant prevention concentrations of selected antibiotics against animal isolates of Pasteurella multocida and Salmonella typhimurium

Historically, the use of antibiotics was not well regulated in veterinary medicine. The emergence of antibiotic resistance (ABR) in pathogenic bacteria in human and veterinary medicine has driven the need for greater antibiotic stewardship. The preservation of certain antibiotic classes for use exclusively in humans, especially in cases of multidrug resistance, has highlighted the need for veterinarians to reduce its use and redefine dosage regimens of antibiotics to ensure efficacy and guard against the development of ABR pathogens. The minimum inhibitory concentration (MIC), the lowest concentration of an antibiotic drug that will prevent the growth of a bacterium, is recognised as a method to assist in antibiotic dosage determination. Minimum inhibitory concentrations sometimes fail to deal with first-step mutants in bacterial populations; therefore dosing regimens based solely on MIC can lead to the development of ABR. The mutant prevention concentration (MPC) is the minimum inhibitory antibiotic concentration of the most resistant first-step mutant. Mutant prevention concentration determination as a complementary and sometimes preferable alternative to MIC determination for veterinarians when managing bacterial pathogens. The results of this study focused on livestock pathogens and antibiotics used to treat them, which had a MIC value of 0.25 µg/mL for enrofloxacin against all 27 isolates of Salmonella typhimurium. The MPC values were 0.50 µg/mL, with the exception of five isolates that had MPC values of 4.00 µg/mL. The MPC test yielded 65.52% (18 isolates) Salmonella isolates with florfenicol MICs in the sensitive range, while 11 isolates were in the resistant range. Seventeen isolates (58.62%) of Pasteurella multocida had MIC values in the susceptible range and 41.38% (12 isolates) had an intermediate MIC value. Mutant prevention concentration determinations as done in this study is effective for the antibiotic treatment of bacterial infections and minimising the development of resistance. The MPC method can be used to better control to prevent the development of antibiotic drug resistance used in animals.     

Chicken egg yolk antibodies (IgY) as non-antibiotic production enhancers for use in swine production: a review

In recent years, the use of in-feed antibiotics for growth and disease prevention in livestock production has been under severe scrutiny. The use and misuse of in-feed antibiotics has led to problems with drug residues in animal products and increased bacterial resistance. Chicken egg yolk antibodies (IgY) have attracted considerable attention as an alternative to antibiotics to maintain swine health and performance. Oral administration of IgY possesses many advantages over mammalian IgG such as cost-effectiveness, convenience and high yield. This review presents an overview of the potential to use IgY immunotherapy for the prevention and treatment of swine diarrhea diseases and speculates on the future of IgY technology. Included are a review of the potential applications of IgY in the control of enteric infections of either bacterial or viral origin such as enterotoxigenic Escherichia coli, Salmonella spp., rotavirus, porcine transmissible gastroenteritis virus, and porcine epidemic diarrhea virus. Some potential obstacles to the adoption of IgY technology are also discussed.     

Successful Eradication of Taylorella asinigenitalis,Pseudomonas aeruginosa,and Klebsiella pneumoniae Venereal Bacterial Pathogens Using Domestic Steam Disinfection: Implications for AI Practice

Steam disinfection has become established as a trusted method of microbial decontamination; however, there have been no reports on the use of this technology to disinfect equipment used in collection of semen in artificial insemination practice. Hence, it was the aim of this study to examine the survival of Taylorella asinigenitalis, Pseudomonas aeruginosa, and Klebsiella pneumoniae venereal bacterial pathogens using domestic steam disinfection. Sixteen bacterial pathogens from three genera Taylorella, Pseudomonas, and Klebsiella each at an inoculum density of approximately 1.5 × 10⁷ colony-forming units were subjected to a steam disinfection cycle. No bacteria were recovered after disinfection, including following recovery and nonselective cultural enrichment techniques. In the absence of full sterilization, domestic steam disinfection of equipment offers a cheap, simple, and widely available technology for the elimination of these pathogens, thereby enhancing infection control in equine breeding.     

猪细菌性传染病病原检测新方法研究进展

细菌性传染病是生猪养殖业的严重威胁之一，近年来其发病情况呈现出新变化，对动物产品质量和公共卫生安全造成严重危害。对病原进行快速、准确的检测，是预防和控制猪病的有效手段，对养猪业发展和人畜健康具有重要意义。伴随生命科学技术的发展和学科间的交叉融合，传统的病原菌分离培养、普通PCR方法和早期免疫学等检测技术均朝着精确高效的方向不断发展革新，并以传统方法为基础建立了多种新型检测技术。文章重点介绍了猪细菌性传染病病原检测的几种新技术：以核酸分子生物学为基础的实时荧光定量PCR法（qPCR）、环介导等温扩增法（LAMP）、夹心DNA杂交（DNAH）等；以免疫学为基础的免疫胶体金标记技术、时间分辨荧光免疫分析法（TRFIA）、免疫磁珠分离法（IMS）等；以蛋白质分子生物学为基础的细菌质谱鉴定技术；以及适用于现场快速诊断的即时检测（POCT）技术，并分析了这几种新技术的优缺点，为不同条件的检测机构和研究部门在开展猪细菌性传染病检测和研究时选择相应的方法提供一定的参考，以期在猪病预防控制中发挥更加积极的作用。     

Isolation and identification of bacteriophages infecting ayu Plecoglossus altivelis altivelis specific Flavobacterium psychrophilum

In order to investigate methods for controlling systemic bacterial coldwater disease (CWD), bacteriophages that infect Flavobacterium psychrophilum were isolated by the enrichment method from pond water collected from Japanese ayu farms. The five phages isolated were classified as members of Myoviridae (PFpW-3, PFpC-Y), Podoviridae (PFpW-6, PFpW-7), and Siphoviridae (PFpW-8) and had highly variable patterns of infectivity for different F. psychrophilum isolates (n = 128). The stability tests of the phages in different waters, pHs and temperatures were assessed, and the results indicated that none of the phages were affected by ayu farm conditions. Among the phages, PFpW-3 had high infectivity for F. psychrophilum isolated from ayu and other fish and demonstrated sufficient survivability in the stability tests. Thus, PFpW-3 and its indicator strain N2-3 were inoculated into cytophaga broth at different doses of multiplicity of infection (MOI) and proved to be efficient for the reduction of bacterial growth. This study may be the basis for a further evaluation of phage therapy in the treatment of CWD in Japanese ayu farms.     

Quorum Signal Inhibitors and Their Potential Use against Fish Diseases

Quorum sensing (QS) is a process of bacterial communication used to control group behaviors, including bioluminescence, virulence factor production, biofilm formation, and biofilm antimicrobial tolerance. Many aquatic bacterial pathogens such as Aeromonas, Vibrio, and Edwardsiella spp. use QS to regulate virulence factor production. The disruption of QS has been shown to be an effective strategy in the competition between higher organisms and bacteria and more recently between bacterial species. For this reason, QS disruption has been proposed as a strategy to prevent bacterial pathogenicity. In this review, we summarize the current literature and illustrate the value of QS inhibitors in controlling virulence production in aquatic bacterial pathogens. This represents a new, nonantibiotic strategy to combat fish diseases.

Received August 11, 2015; accepted January 26, 2016     

Use of Negative Air Ionization for Reducing Bacterial Pathogens and Spores on Stainless Steel Surfaces

The use of chemicals in food plant sanitation for removing and killing microorganisms could be reduced by the use of alternative nonchemical interventions. Supercharged negative air ionizers have shown potential to effectively reduce airborne and surface microorganisms. In our earlier studies, a small chamber, controlled at 85% relative humidity and supercharged with a −25 kV electrostatic space charge system (ESCS), was used to transfer a strong negative charge to bacteria on stainless steel surfaces. The ESCS treatment caused the levels of biofilm bacteria from chicken carcass rinses to be significantly decreased with 99.8% efficiency at a distance of 18 cm with ion densities of 10⁶ negative ions/cm³ or more. In the present study, effects of the −25 kV charge of ESCS on specific pathogens important for food safety were studied. Treatment of Campylobacter jejuni, Escherichia coli, Salmonella enteritidis, Listeria monocytogenes, and Staphylococcus aureus achieved up to a 4 log reduction with 99.9% reduction efficiency in 3 h. Treatment of bacterial spores of Bacillus stearothermophilus achieved up to a 3 log reduction with a 99.8% reduction efficiency in 3 h. These significant reductions for 5 species of pathogenic bacteria and bacterial spores suggest that the ESCS is a promising alternative treatment for reduction of microbial load in a food-processing facility with the potential to reduce the amount of antimicrobials used.     

Levels of nicotinamide adenine dinucleotide in extracellular body fluids of pigs may be growth-limiting for Actinobacillus pleuropneumoniae and Haemophilus parasuis

During infection, nutrient deprivation can alter bacterial phenotype. This, in turn, may have implications for pathogenesis and prophylaxis. Actinobacillus pleuropneumoniae (biotype 1) and Haemophilus parasuis, respiratory tract pathogens of swine, are both V-factor-dependent. The concentrations of V factor in the extracellular fluids of pigs are unknown and may limit the growth of these bacteria in vivo. The aim of this study was to determine the concentrations of nicotinamide adenine dinucleotide (NAD) in select porcine body fluids and to compare the availability of NAD in vivo with the affinities of the organisms for this compound. Levels in plasma, tissue fluids (peritoneal, pleural, synovial, and cerebrospinal), and laryngeal, tracheal, and lung washings were determined with an enzymatic cycling assay. We concluded that, although the NAD supply in the respiratory tract is probably not growth-limiting, it may become limiting if the organisms are disseminated.     

The heart microbiome of insectivorous bats from Central and South Eastern Europe

Host associated microbiome not only may affect the individual health-status or provide insights into the species- or group specific bacterial communities but may act as early warning signs in the assessment of zoonotic reservoirs, offering clues to predict, prevent and control possible episodes of emerging zoonoses. Bats may be carriers and reservoirs of multiple pathogens such as viruses, bacteria and parasites, showing in the same time robust immunity against many of them. The microbiota plays a fundamental role on the induction, training and function of the host immune system and the immune system has largely evolved in order to maintain the symbiotic relationship of the host with these diverse microbes. Thus, expanding our knowledge on bat-associated microbiome it can be usefully in understanding bats’ outstanding immune capacities. The aim of this study was to investigate the presence of different bacterial communities in heart tissue of insectivorous bats, Nyctalus noctula, Pipistrellus pipistrellus and Rhinoplophus hipposideros, from Central and Eastern Europe using high-throughput sequencing of variable regions of the 16S rRNA. In addition, species-specific PCRs were used to validate the presence of the vector-borne pathogens Bartonella spp. and Rickettsia spp. In this study we identified a wide variety of bacterial groups, with the most abundant phyla being Proteobacteria and Firmicutes. The results showed that at individual level, the year or location had no effect on the diversity and composition of the microbiome, however host species determined both structure and abundance of the bacterial community. We report the presence of vector-borne bacteria Bartonella spp. in samples of N. noctula and indications of Rickettsia spp. in R. hipposideros. Our results provide a first insight into the bacterial community found in heart tissue of bats from Central and South Eastern Europe.     

Pathogen re-colonization of in-house composted and noncomposted broiler litter

In-house litter composting has been reintroduced to the industry and shown to reduce bacteria by as much as 2 orders of magnitude. Other industries have demonstrated that pathogens can recolonize a waste-residual when microbial competition has been reduced or inhibited following composting. Poultry growers, in the process of shifting to in-house composting for pathogen control, should be aware of this potential problem. A laboratory microcosm study investigated pathogenic bacteria recolonization into composted and noncomposted broiler litter over a simulated broiler grow-out cycle. Objectives were to: 1) determine colonization potential for zoonotic and poultry bacterial pathogens, 2) identify beneficial bacteria which reduce pathogen recolonization, and 3) identify the effects of ammonia on pathogen recolonization. Composted broiler litter allowedListeria andCampylobacter to colonize within the first 2 wk of the grow-out period while noncomposted litter resisted colonization. Colonization was nearly identical by the end of the grow-out period, and showed that bacterial pathogens had essentially been overtaken by commensal or normal bacteria. 16S rRNA libraries demonstrated reductions in Proteobacteria associated with composted litter (48 vs. 16%), which may indicate that this phylum occupies a niche which zoonotic pathogens prefer to occupy. Ammonia generation neither inhibited nor promoted bacterial colonization, as levels were high for both litter treatments. This study neither suggests nor condemns the continued use of this cost-effective, litter-treatment process; findings suggest that while the beneficial microbial population was initially reduced, it quickly recovered and pathogen colonization was neither enhanced nor inhibited because of this. This study demonstrates that the recently adopted in-house composting process may continue to be used, provided poultry health gains continue to be seen.     

Bacterial zoonoses of fishes: A review and appraisal of evidence for linkages between fish and human infections

Human contact with and consumption of fishes presents hazards from a range of bacterial zoonotic infections. Whereas many bacterial pathogens have been presented as fish-borne zoonoses on the basis of epidemiological and phenotypic evidence, genetic identity between fish and human isolates is not frequently examined or does not provide support for transmission between these hosts. In order to accurately assess the zoonotic risk from exposure to fishes in the context of aquaculture, wild fisheries and ornamental aquaria, it is important to critically examine evidence of linkages between bacteria infecting fishes and humans. This article reviews bacteria typically presented as fish-borne zoonoses, and examines the current strength of evidence for this classification. Of bacteria generally described as fish-borne zoonoses, only Mycobacterium spp., Streptococcus iniae, Clostridium botulinum, and Vibrio vulnificus appear to be well-supported as zoonoses in the strict sense. Erysipelothrix rhusiopathiae, while transmissible from fishes to humans, does not cause disease in fishes and is therefore excluded from the list. Some epidemiological and/or molecular linkages have been made between other bacteria infecting both fishes and humans, but more work is needed to elucidate routes of transmission and the identity of these pathogens in their respective hosts at the genomic level.     

Teat skin normal flora and colonization with mastitis pathogen inhibitors

Isolates of bacteria from normal teats were used to attempt colonization of teats of dry cows or neonatal calves. Isolates for inoculation were chosen on the basis of ability to inhibit mastitis pathogens in vitro, with the ultimate goal of in vivo inhibition of mastitis pathogens at the teat surface. Three bacterial normal flora isolates (Corynebacterium xerosis, Bacillus sp. and Aerococcus viridans) persisted less than 10 days on the teats of dry cows. The fourth isolate, Staphylococcus hominis 1, was studied in greatest detail because studies characterizing the normal teat flora showed staphylococci to be the predominant flora. The S. hominis 1 isolated used for inoculation was an inhibitor of Gram-positive mastitis pathogens. It was a biotype not found on these teats prior to inoculation, thus facilitating identification of the inoculated isolate on sequential sampling. Colonization of newborn calves, before other bacterial floral became established, resulted in recovery of inoculated S. hominis 1 for an average of 51 days or longer. On dry cow teats it was detected for up to 28 days. On several occasions the inoculated S. hominis 1 was found in pure culture. Since many new infections occur during the dry period, the colonization of dry cow tests with S. hominis 1 organisms inhibitory for Gram-positive pathogens should be tested as an adjunct to other methods of mastitis prevention.     

Exposure of feral swine (Sus scrofa) in the United States to selected pathogens

Feral swine (Sus scrofa) are widely distributed in the United States. In 2011 and 2012, serum samples and tonsils were recovered from 162 and 37 feral swine, respectively, in the US to evaluate exposure to important swine endemic pathogens. Antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) were found in 2.5% and 25.3% of tested sera, respectively. Positive serological reactions against Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae have been detected in 19.7% and 69.7% of animals. More than 15% of animals presented antibodies against these 2 pathogens simultaneously. Most animals were also seropositive for Lawsonia intracellularis. Feral swine can also be involved in transmission of zoonotic agents. Almost 50% of animals possessed antibodies against Salmonella. In addition, 94.4% of animals were carriers of Streptococcus suis in their tonsils. In conclusion, feral swine may be considered as a potential reservoir for different endemic diseases in domestic pigs, as well as for important zoonotic agents.     

Development of a multiplex PCR assay to detect Edwardsiella tarda,Streptococcus parauberis,and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.     

Application of mouse model for evaluation of recombinant LpxC and GmhA as novel antigenic vaccine candidates of Glaesserella parasuis serotype 13

Glaesserella parasuis (G. parasuis) has been one of the bacteria affecting the large-scale swine industry. Lack of an effective vaccine has limited control of the disease, which has an effect on prevalence. In order to improve the cross-protection of vaccines, development on subunit vaccines has become a hot spot. In this study, we firstly cloned the lpxC and gmhA genes from G. parasuis serotype 13 isolates, and expressed and purified their proteins. The results showed that LpxC and GmhA can stimulate mice to produce IgG antibodies. Through testing the cytokine levels of interleukin 4 (IL-4), IL-10 and interferon-γ (IFN-γ), it is found that recombinant GmhA, the mixed LpxC and GmhA can stimulate the body to produce Th1 and Th2 immune responses, while recombinant LpxC and inactivated bacteria can only produce Th2 immune responses. On the protection rate for mice, recombinant LpxC, GmhA and the mixture of LpxC and GmhA can provide 50%, 50% and 60% protection for lethal dose of G. parasuis infection, respectively. The partial protection achieved by the recombinant LpxC and GmhA supports their potential as novel vaccine candidate antigens against G. parasuis.     

A Retrospective Study on Antimicrobial Resistance in Mannheimia (Pasteurella) haemolytica,Escherichia coli,Salmonella Species,and Bordetella avium from Chickens in Minnesota

A 5-yr retrospective study (1998 to 2002) was conducted to determine the rate of isolation of 4 different bacterial pathogens and their antimicrobial resistance from chickens submitted to the Minnesota Veterinary Diagnostic Laboratory, St. Paul, Minnesota. The in vitro antimicrobial resistance was evaluated using the disc diffusion method. A total of 218 bacteria were isolated with the frequency of isolation being Mannheimia (Pasteurella) haemolytica 92 (42.2%) > Escherichia coli 80 (36.7%) > Salmonella spp., 42 (19.3%) > Bordetella avium 4 (1.8%). All isolates were resistant to macrolides and tetracycline antimicrobials but showed varied resistance to aminoglycoside and beta-lactam antibiotics. A majority of the isolates showed high resistance to spectinomycin, sarafloxacin, tetracycline, sulfadimethoxine, and sulfachloropyridiazine. These results emphasize the need for continued surveillance of chicken pathogens to monitor their resistance patterns with a view to control the infections caused by these pathogens.     

Organic barn dust extract exposure impairs porcine macrophage function in vitro: Implications for respiratory health

Respiratory diseases are responsible for a significant amount of animal morbidity and mortality in the swine industry, including the majority of nursery and grower/finisher deaths. Innate immunity, including the maintenance of lung macrophage health and function, is an important defense mechanism against respiratory pathogens and their associated losses. Chronic exposure of swine industry workers to airborne barn dust results in significant predisposition to airway diseases and impairment of alveolar macrophage (AM?) function. Because of their importance in maintaining normal respiratory function, this study was designed to evaluate the impact of barn dust on swine macrophages. As measures of macrophage function, we evaluated the activation of NF-κB, cytokine production, cell surface marker expression and the phagocytic and antibacterial capabilities of porcine macrophages after in vitro exposure to an organic swine barn dust extract (ODE). ODE treatment induced AM? secretion of both pro- and anti-inflammatory cytokines, suggesting a complex activation profile. Additionally, ODE induced expression of genes (TLR2, NOD2) involved in sensing Gram-positive bacteria, a major component of barn dust. ODE exposure also enhanced the expression of several cell surface markers of activation, including a receptor for the porcine reproductive and respiratory syndrome virus. Moreover, two key functions of AM?, phagocytosis and bacterial killing, were impaired after exposure to ODE. Treatment with ODE for the first 72 h of differentiation also inhibited the ability of monocyte-derived macrophages to translocate NF-κB to the nucleus following endotoxin stimulation. Taken together, these results demonstrate, for the first time, that organic dust extract exposure negatively affects pig macrophage activation and function, potentially enhancing host susceptibility to a variety of respiratory infections.     

Clinical and histological features of brucellin skin test responses in Brucella suis biovar 2 infected pigs

Current serological tests for swine brucellosis detect antibodies to the Brucella O-polysaccharide (O/PS). However, when infections by bacteria carrying cross-reacting O/PS occur, these tests suffer from false positive serological reactions (FPSR), and the skin test with Brucella soluble protein extracts is the best diagnostic alternative to differentiate true Brucella suis infections from FPSR in pigs. Since this test has been seldom used in B. suis infected swine, the clinical and histological features involved have not been described properly. Here, we describe the clinical and histological events in B. suis biovar 2 infected pigs skin tested with a cytosoluble O/PS free protein extract from rough Brucella abortus Tn5::per mutant. A similar extract from rough Ochrobactrum intermedium was also used for comparative purposes. No relevant differences were evidenced between the homologous and heterologous allergens, and the main clinical feature was an elevated area of the skin showing different induration degrees. Moreover, an important vascular reaction with hyperemia and haemorrhage was produced in most infected sows 24–48 h after inoculation, thus facilitating the clinical interpretation of positive reactions. Histologically, combined immediate (type III) and delayed (type IV) hypersensitivity reactions were identified as the most relevant feature of the inflammatory responses produced.