80 research outputs found
Role of Staphylococcus aureus GapC and GapB in immunity and pathogenesis of bovine mastitis
Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of S. aureus is a major economically important disease affecting the dairy industry worldwide. S. aureus is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure S. aureus intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against S. aureus mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of S. aureus to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. S. aureus also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against S. aureus mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of S. uberis and S. dysgalactiae have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with
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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from S. aureus isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of S. aureus as model antigens for mastitis and to determine the regulation of expression of gapB and gapC genes and their roles in the pathogenesis of bovine S. aureus mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of S. aureus as antigens will protect against bovine S. aureus mastitis. To test this hypothesis we took the approach of using the gapB and gapC genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by S. aureus. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2,
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and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of S. aureus mastitis using the S. aureus wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in S. aureus mastitis. In aim 6 we examined the role of sar and agr loci on the expression of gapC and gapB genes by qRT- PCR using S. aureus RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both gapB and gapC expression were down regulated in the mutant strains, indicating that the expression of the gapB and gapC genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of gapB and gapC using q-RT-PCR. Our results showed that the expression of gapB and gapC genes in different strains of S. aureus was not consistent under the above-mentioned environmental conditions
Current Status of Antimicrobial Resistance and Prospect for New Vaccines against Major Bacterial Bovine Mastitis Pathogens
Economic losses due to bovine mastitis is estimated to be $2 billion in the United States alone. Antimicrobials are used extensively in dairy farms for prevention and treatment of mastitis and other diseases of dairy cattle. The use of antimicrobials for treatment and prevention of diseases of dairy cattle needs to be prudent to slow down the development, persistence, and spread of antimicrobial-resistant bacteria from dairy farms to humans, animals, and farm environments. Because of public health and food safety concerns regarding antimicrobial resistance and antimicrobial residues in meat and milk, alternative approaches for disease control are required. These include vaccines, improvements in housing, management practices that reduce the likelihood and effect of infectious diseases, management systems and feed formulation, studies to gain a better understanding of animal behavior, and the development of more probiotics and competitive exclusion products. Monitoring antimicrobial resistance patterns of bacterial isolates from cases of mastitis and dairy farm environments is important for treatment decisions and proper design of antimicrobial-resistance mitigation measures. It also helps to determine emergence, persistence, and potential risk of the spread of antimicrobial-resistant bacteria and resistome from these reservoirs in dairy farms to humans, animals, and farm environments
Bovine Mastitis: Part I
Bovine mastitis is one of the most important bacterial diseases of dairy cattle throughout the world. Mastitis is responsible for great economic losses to the dairy producer and to the milk processing industry resulting from reduced milk production, alterations in milk composition, discarded milk, increased replacement costs, extra labor, treatment costs, and veterinary services. Economic losses due to bovine mastitis are estimated to be 400 million in Canada (Canadian Bovine Mastitis and Milk Quality Research Network-CBMQRN) and $130 million in Australia per year. Many factors can influence the development of mastitis; however, inflammation of the mammary gland is usually a consequence of adhesion, invasion, and colonization of the mammary gland by one or more mastitis pathogens such as Staphylococcus aureus, Streptococcus uberis, and Escherichia coli
Control and Prevention of Mastitis: Part Two
Current mastitis control measures are based upon good milking time hygiene; use of properly functioning milking machines; maintaining clean, dry, comfortable housing areas; segregation and culling of persistently infected animals; dry cow antibiotic therapy; proper identification and treatment of cows with clinical mastitis during lactation; establishing udder health goals; good record-keeping; regular monitoring of udder health status and periodic review of mastitis control program. Despite significant effect of these control measures when fully adopted, especially on contagious mastitis pathogens, these measures are not equally adopted by all farmers, and mastitis continues to be the most common and costly disease of dairy cattle throughout the world
Molecular epidemiology and pathogenomics of extended-spectrum beta-lactamase producing- Escherichia coli and - Klebsiella pneumoniae isolates from bulk tank milk in Tennessee, USA
IntroductionThe rise in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in dairy cattle farms poses a risk to human health as they can spread to humans through the food chain, including raw milk. This study was designed to determine the status, antimicrobial resistance, and pathogenic potential of ESBL-producing -E. coli and -Klebsiella spp. isolates from bulk tank milk (BTM).MethodsThirty-three BTM samples were collected from 17 dairy farms and screened for ESBL-E. coli and -Klebsiella spp. on CHROMagar ESBL plates. All isolates were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS).ResultsTen presumptive ESBL-producing bacteria, eight E. coli, and two K. pneumoniae were isolated. The prevalence of ESBL-E. coli and -K. pneumoniae in BTM was 21.2% and 6.1%, respectively. ESBL-E. coli were detected in 41.2% of the study farms. Seven of the ESBL-E. coli isolates were multidrug resistant (MDR). The two ESBL-producing K. pneumoniae isolates were resistant to ceftriaxone. Seven ESBL-E. coli strains carry the blaCTX-M gene, and five of them co-harbored blaTEM-1. ESBL-E. coli co-harbored blaCTX-M with other resistance genes, including qnrB19, tet(A), aadA1, aph(3’’)-Ib, aph(6)-Id), floR, sul2, and chromosomal mutations (gyrA, gyrB, parC, parE, and pmrB). Most E. coli resistance genes were associated with mobile genetic elements, mainly plasmids. Six sequence types (STs) of E. coli were detected. All ESBL-E. coli were predicted to be pathogenic to humans. Four STs (three ST10 and ST69) were high-risk clones of E. coli. Up to 40 virulence markers were detected in all E. coli isolates. One of the K. pneumoniae was ST867; the other was novel strain. K. pneumoniae isolates carried three types of beta-lactamase genes (blaCTX-M, blaTEM-1 and blaSHV). The novel K. pneumoniae ST also carried a novel IncFII(K) plasmid ST.ConclusionDetection of high-risk clones of MDR ESBL-E. coli and ESBL-K. pneumoniae in BTM indicates that raw milk could be a reservoir of potentially zoonotic ESBL-E. coli and -K. pneumoniae
Protective effect of anti-SUAM antibodies on \u3cem\u3eStreptococcus uberis\u3c/em\u3e mastitis
In the present study, the effect of anti-recombinant Streptococcus uberis adhesion molecule (SUAM) antibodies against S. uberis intramammary infections (IMI) was evaluated using a passive protection model. Mammary quarters of healthy cows were infused with S. uberis UT888 opsonized with affinity purified anti-rSUAM antibodies or hyperimmune sera. Non-opsonized S. uberis UT888 were used as a control. Mammary quarters infused with opsonized S. uberis showed mild-to undetectable clinical symptoms of mastitis, lower milk bacterial counts, and less infected mammary quarters as compared to mammary quarters infused with non-opsonized S. uberis. These findings suggest that anti-rSUAM antibodies interfered with infection of mammary gland by S. uberis which might be through preventing adherence to and internalization into mammary gland cells, thus facilitating clearance of S. uberis, reducing colonization, and causing less IMI
Occurrence and antimicrobial resistance of Escherichia coli O157: H7 and Salmonella in the milk and feces of lactating dairy cows and camels in Borana pastoral community, Ethiopia
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Prevalence and antimicrobial resistance of Escherichia coli O157:H7 and Salmonella, and the prevalence of Staphylococcus aureus in dairy cattle and camels under pastoral production system
Escherichia coli O157:H7, Salmonella and Staphylococcus aureus are common foodborne pathogens. We determined the prevalence of E. coli O157:H7 and Salmonella in feces and milk and the prevalence of S. aureus in milk from dairy cattle and camels in the Borana pastoral community in the Southern Oromia Region of Ethiopia. Paired individual cow composite (pooled from all quarters in equal proportions) milk and fecal samples were collected from cows (n = 154) and camels (n = 158). Samples were cultured on bacterial isolation and identification media. E. coli O157:H7 and Salmonella isolates were further tested for susceptibility against nine antimicrobial drugs. Different risk factors associated with hygienic milking practices were recorded and analyzed for their influence on the prevalence of these bacteria in milk and feces. The prevalence of E. coli O157:H7 and Salmonella in feces were 3.9% and 8.4%, respectively, in cows, and 0.6% and 2.5%, respectively, in camels. E. coli O157:H7 and Salmonella were detected in the composite milk samples of 2.6% and 3.9% of the cows, respectively, and 0% and 1.3% of the camels, respectively. S. aureus was detected in composite milk samples of 33.4% of the cows and 41.7% of the camels. All E. coli O157:H7 (n = 11) and Salmonella (n = 25) isolates from both animal species and sample types were resistant to at least one antimicrobial drug. Multidrug resistance was observed in 70% (7/10) of the E. coli O157:H7 fecal and milk isolates from cows and 33.3% (2/6) of the Salmonella fecal and milk isolates from camels. The prevalence of these bacteria in feces and milk was not affected by risk factors associated with milking practices. Given the very close contact between herders and their animals and the limited availability of water for hand washing and udder cleaning, these bacteria are most likely present in all niches in the community. Improving community awareness of the need to boil milk before consumption is a realistic public health approach to reducing the risk of these bacteria
Molecular correlates of host specialization in Staphylococcus aureus
The majority of Staphylococcus aureus isolates that are recovered from either serious infections in humans or from mastitis in cattle represent genetically distinct sets of clonal groups. Moreover, population genetic analyses have provided strong evidence of host specialization among S. aureus clonal groups associated with human and ruminant infection. However, the molecular basis of host specialization in S. aureus is not understood.We sequenced the genome of strain ET3-1, a representative isolate of a common bovine mastitis-causing S. aureus clone. Strain ET3-1 encodes several genomic elements that have not been previously identified in S. aureus, including homologs of virulence factors from other gram-positive pathogens. Relative to the other sequenced S. aureus associated with human infection, allelic variation in ET3-1 was high among virulence and surface-associated genes involved in host colonization, toxin production, iron metabolism, antibiotic resistance, and gene regulation. Interestingly, a number of well-characterized S. aureus virulence factors, including protein A and clumping factor A, exist as pseudogenes in ET3-1. Whole-genome DNA microarray hybridization revealed considerable similarity in the gene content of highly successful S. aureus clones associated with bovine mastitis, but not among those clones that are only infrequently recovered from bovine hosts.Whole genome sequencing and comparative genomic analyses revealed a set of molecular genetic features that distinguish clones of highly successful bovine-associated S. aureus optimized for mastitis pathogenesis in cattle from those that infect human hosts or are only infrequently recovered from bovine sources. Further, the results suggest that modern bovine specialist clones diverged from a common ancestor resembling human-associated S. aureus clones through a combination of foreign DNA acquisition and gene decay
A meta-analysis of the proportion of animal Salmonella isolates resistant to drugs used against human salmonellosis in Ethiopia
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