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Protection by Milk Immunoglobulin Concentrate against Oral Challenge with Enterotoxigenic Escherichia coli
Abstract
Enterotoxigenic Escherichia coli is a common cause of traveler's diarrhea. Prophylaxis against traveler's diarrhea has been associated with side effects from bismuth subsalicylate and the development of resistance to antimicrobial agents. We undertook a double-blind controlled trial in which a bovine milk immunoglobulin concentrate with high titers of antibodies against enterotoxigenic E. coli was used as prophylaxis against E. coli challenge in volunteers. Lyophilized milk immunoglobulins were prepared from the colostrum of cows immunized with several enterotoxigenic E. coli serotypes and fimbria types, E. coli heat-labile enterotoxin, and cholera toxin. As a control, an immunoglobulin concentrate with no anti-E. coli activity was prepared. Ten volunteers received buffered immunoglobulin concentrate against enterotoxigenic E. coli, and 10 received the control immunoglobulin concentrate, dissolved in water, three times a day. No side effects were observed. On the third day of immunoglobulin prophylaxis, the volunteers were given 10(9) colony-forming units of enterotoxigenic E. coli H10407 (O78:H11). This strain produces colonization factor antigen I and heat-labile and heat-stable enterotoxins. None of the 10 volunteers receiving the immunoglobulin concentrate against E. coli had diarrhea, but 9 of the 10 controls did (P less than 0.0001). All volunteers excreted E. coli H10407. We conclude from these preliminary results that milk immunoglobulin concentrate may be an effective prophylaxis against traveler's diarrhea.
... In ETEC CHIM studies that have used CFA/I-producing ETEC, such as H10407, anti-CFA/I IgA responses have been shown to peak at variable time points, and, in most of the studies, the fold increase in anti-CFA/I IgA was not very high except for the study by Coster et al. [60]. They found a 40-and a 26-fold increase in serum and ALS IgA levels, respectively, and with a high response ratio for both sample types (Table 1). ...
... It should also be noted that increased anti-colonization factor IgA levels in serum after infection appeared to be more frequent in volunteers who did not develop diarrhea [17,[60][61][62]. ...
... Several of the strains used in CHIM studies produce CS6, which is the most common colonization factor produced by human ETEC. Although IgA responses to CS6 have been poor in most of these studies [23,60,92,93], two studies have managed to show higher levels of immunogenicity in humans after both natural infection and administration of a vaccine candidate containing CS6 [94,95]. ...
Infection with enterotoxigenic Escherichia coli (ETEC) is a major contributor to diarrheal illness in children in low- and middle-income countries and travelers to these areas. There is an ongoing effort to develop vaccines against ETEC, and the most reliable immune correlate of protection against ETEC is considered to be the small intestinal secretory IgA response that targets ETEC-specific virulence factors. Since isolating IgA from small intestinal mucosa is technically and ethically challenging, requiring the use of invasive medical procedures, several other indirect methods are used as a proxy for gauging the small intestinal IgA responses. In this review, we summarize the literature reporting on anti-ETEC human IgA responses observed in blood, activated lymphocyte assayss, intestinal lavage/duodenal aspirates, and saliva from human volunteers being experimentally infected with ETEC. We describe the IgA response kinetics and responder ratios against classical and noncanonical ETEC antigens in the different sample types and discuss the implications that the results may have on vaccine development and testing.
... [4][5][6][7][8] This is further substantiated by studies demonstrating passive oral administration of hyperimmune bovine immunoglobulin (BIgG) generated against inactivated whole cell ETEC and against purified CFs protects against moderate to severe diarrhea (MSD) in CHIM using ETEC strains expressing homologous CFs. [9][10][11][12] CS6 is an atypical polymeric antigen that is highly prevalent among ETEC disease isolates globally. 4,13,14 Individuals naturally infected with CS6-expressing ETEC strains exhibit mucosal and serologic immune responses against CS6; [15][16][17] however, despite data supporting the role of CS6 as a key CF in ETEC-mediated disease, immunological correlates of protection are lacking. ...
... The products were safe and welltolerated, similar to other studies with orally administered bovine antibodies. [9][10][11][12]18 Additionally, antibodies targeting the B7A whole cell yielded a 50% reduction in MSD and a reduced disease severity score; however, anti-CS6 yielded no significant protection against MSD. There are several potential reasons for the lack of observed efficacy. ...
... products directed against the colonization factors, can reduce ETEC-mediated disease. [9][10][11][12] Interestingly, in this study, we did not see significant protection with a product designed against the major colonization factor of B7A, CS6, whereas the anti-whole cell product did provide protection. The lack of protection provided by the anti-CS6 product may be specific to the B7A challenge strain, or it may reflect a different role or function of this CF as compared to some of the others (e.g. ...
Background. Oral administration of bovine antibodies active against enterotoxigenic Escherichia coli (ETEC) have demonstrated safety and efficacy against diarrhea in human challenge trials. The efficacy of bovine serum immunoglobulins (BSIgG) against recombinant colonization factor CS6 or whole cell ETEC strain B7A was assessed against challenge with the CS6-expressing B7A.
Methods. This was a randomized, double-blind, placebo-controlled trial in which healthy adults received oral hyperimmune BSIgG anti-CS6, anti-B7A whole cell killed or non-hyperimmune BSIgG (placebo) in a 1:1:1 ratio then challenged with ETEC B7A. Two days pre-challenge, volunteers began a thrice daily, seven day course of immunoprophylaxis. On day 3, subjects received 1 × 10¹⁰ CFUs of B7A. Subjects were observed for safety and the primary endpoint of moderate-severe diarrhea (MSD).
Results. A total of 59 volunteers received product and underwent ETEC challenge. The BSIgG products were well-tolerated across all subjects. Upon challenge, 14/20 (70%) placebo recipients developed MSD, compared to 12/19 (63%; p = .74) receiving anti-CS6 BSIgG and 7/20 (35%; p = .06) receiving anti-B7A BSIgG. Immune responses to the ETEC infection were modest across all groups.
Conclusions. Bovine-derived serum antibodies appear safe and well tolerated. Antibodies derived from cattle immunized with whole cell B7A provided 50% protection against MSD following B7A challenge; however, no protection was observed in subjects receiving serum antibodies targeting CS6. The lack of observed efficacy in this group may be due to low CS6 surface expression on B7A, the high dose challenge inoculum and/or the use of serum derived antibodies versus colostrum-derived antibodies.
... Tacket et al performed at CVD a groundbreaking clinical trial in conjunction with Swiss investigators from Nestlé. 118 An ETEC immunoglobulin concentrate was prepared from the colostrum and milk of dairy cows hyper-immunized with an array ETEC strains of different serotypes expressing the major CFs, LT and ST. During the last eight weeks of gestation, milking cows were injected with 7 or 8 subcutaneous injections of 2 × 10 9 CFU of the mix of ETEC strains that were heat-or glutaraldehyde-treated, along with Freund's incomplete adjuvant, LT and cholera toxin (CT). ...
... In this instance the antibodies were provided passively. 118 The dilemma faced by vaccine developers is to devise an effective strategy to elicit the appropriate type and quantity of antibodies following active immunization. In the meantime, the study of Tacket et al launched product development for passive protection, one of which, Travelan, is an available commercial product to prevent ETEC travelers' diarrhea (vide infra) ...
... 121,122 The administration of NaHCO 3 (or a similar buffer) pretreatment to neutralize gastric acid, thereby allowing lower inocula to be administered to volunteers and increasing the consistency of clinical responses, has become routine even among the most recent research groups that perform ETEC challenge studies. 30,47,53,70,118,120,123 Attempts to increase further the consistency of experimental challenge by lengthening the period of fasting prior to ingestion of ETEC inocula and evaluating buffers other than NaHCO 3 have also been pursued. 37 ...
Enterotoxigenic Escherichia coli (ETEC) is a major cause of travelers’ diarrhea and of diarrhea among young children in developing countries. Experimental challenge studies in adult volunteers have played a pivotal role in establishing ETEC as an enteric pathogen, elucidating its pathogenesis by identifying specific virulence attributes, characterizing the human immune response to clinical and sub-clinical ETEC infection and assessing preliminarily the clinical acceptability, immunogenicity and efficacy of prototype ETEC vaccines. This review provides a historical perspective of experimental challenge studies with ETEC. It summarizes pioneering early studies carried out by investigators at the University of Maryland School of Medicine to show how those studies provided key information that influenced the directions taken by many research groups to develop vaccines to prevent ETEC. In addition, key experimental challenge studies undertaken at other institutions will also be cited.
... Passive oral administration of hyperimmune bovine milk immunoglobulin (bIgG) has been experimentally evaluated as a preventive or therapeutic modality for a variety of enteric infections including ETEC [14][15][16][17][18]. Notably, hyperimmune bIgG generated against a cocktail of inactivated whole-cell ETEC and against purified CFA/I fimbriae conferred 76%-100% protection against challenge with a prototype CFA/I-ETEC strain in volunteers [14,15,19], corroborating other lines of evidence that anti-CF immunity is associated with protection against ETEC diarrhea [20,21]. ...
... Passive oral administration of hyperimmune bovine milk immunoglobulin (bIgG) has been experimentally evaluated as a preventive or therapeutic modality for a variety of enteric infections including ETEC [14][15][16][17][18]. Notably, hyperimmune bIgG generated against a cocktail of inactivated whole-cell ETEC and against purified CFA/I fimbriae conferred 76%-100% protection against challenge with a prototype CFA/I-ETEC strain in volunteers [14,15,19], corroborating other lines of evidence that anti-CF immunity is associated with protection against ETEC diarrhea [20,21]. ...
... Effectively, this is the third in a progression of trials using the same volunteer challenge model that has incrementally shown the protective efficacy of bovine milk immunoglobulins first against a cocktail of ETEC antigens (including CFA/I) [14], then against CFA/I fimbriae [15], and here against dscCfaE, a stable, native-like variant of the CFA/I fimbrial tip adhesin. These findings indicate that CfaE represents an antigen of intrinsic importance to this protective effect. ...
Background
Tip-localized adhesive proteins of bacterial fimbriae from diverse pathogens confer protection in animal models, but efficacy in humans has not been reported. Enterotoxigenic Escherichia coli (ETEC) commonly elaborate colonization factors comprising a minor tip adhesin and major stalk-forming subunit. We assessed the efficacy of antiadhesin bovine colostral IgG (bIgG) antibodies against ETEC challenge in volunteers.
Methods
Adults were randomly assigned (1:1:1) to take oral hyperimmune bIgG raised against CFA/I minor pilin subunit (CfaE) tip adhesin or colonization factor I (CFA/I) fimbraie (positive control) or placebo. Two days before challenge, volunteers began a thrice-daily, 7-day course of investigational product administered in sodium bicarbonate 15 minutes after each meal. On day 3, subjects drank 1 × 10⁹ colony-forming units of colonization factor I (CFA/I)-ETEC strain H10407 with buffer. The primary efficacy endpoint was diarrhea within 120 hours of challenge.
Results
After enrollment and randomization, 31 volunteers received product, underwent ETEC challenge, and were included in the per protocol efficacy analysis. Nine of 11 placebos developed diarrhea, 7 experiencing moderate to severe disease. Protective efficacy of 63% (P = .03) and 88% (P = .002) was observed in the antiadhesin bIgG and positive control groups, respectively.
Conclusions
Oral administration of anti-CFA/I minor pilin subunit (CfaE) antibodies conferred significant protection against ETEC, providing the first clinical evidence that fimbrial tip adhesins function as protective antigens.
... Intestinal IgA responses toward pathogenic bacteria can block bacterial adherence to epithelial cells and play a beneficial role in reduction of bacterial colonization in mouse models of pathogenic O157 [60][61][62][63] . Similarly, cattle that were immunized against enterotoxigenic E. coli produced secretory immunoglobulins in milk; which did confer some benefits by protecting against and reducing severity of symptoms of enteropathogenic E. coli infections on human subjects which consumed the hyperimmunized milk 64,65 . Therefore, in some part, mucosal E. coli-specific secretory immunoglobulins may play an important role in reducing pathogenic E. coli colonization, adherence, or clinical pathogenicity 64,65 . ...
... Similarly, cattle that were immunized against enterotoxigenic E. coli produced secretory immunoglobulins in milk; which did confer some benefits by protecting against and reducing severity of symptoms of enteropathogenic E. coli infections on human subjects which consumed the hyperimmunized milk 64,65 . Therefore, in some part, mucosal E. coli-specific secretory immunoglobulins may play an important role in reducing pathogenic E. coli colonization, adherence, or clinical pathogenicity 64,65 . One reported vaccination study, which examined EspA of the T3SP complex, demonstrated that EspA-specific IgA did not impact cattle-GIT colonization of O157 66 but vaccination may reduce shedding through O157-specific IgG, TLR5-ligation or other unknown mechanism 60,67,68 . ...
Shiga toxin-producing Escherichia coli O157:H7 (O157) can cause mild to severe gastrointestinal disease in humans. Cattle are the primary reservoir for O157, which colonizes the intestinal tract without inducing any overt clinical symptoms. Parenteral vaccination can reduce O157 shedding in cattle after challenge and limit zoonotic transmission to humans, although the impact of vaccination and vaccine formulation on cellular and mucosal immune responses are undetermined. To better characterize the cattle immune response to O157 vaccination, cattle were vaccinated with either water-in-oil-adjuvanted, formalin-inactivated hha deletion mutant of Shiga toxin 2 negative (stx2−) O157 (Adj-Vac); non-adjuvanted (NoAdj-Vac); or non-vaccinated (NoAdj-NoVac) and peripheral T cell and mucosal antibody responses assessed. Cattle in Adj-Vac group had a higher percentage of O157-specific IFNγ producing CD4+ and γδ+ T cells in recall assays compared to the NoAdj-Vac group. Furthermore, O157-specific IgA levels detected in feces of the Adj-Vac group were significantly lower in NoAdj-Vac group. Extracts prepared only from Adj-Vac group feces blocked O157 adherence to epithelial cells. Taken together, these data suggest parenteral administration of adjuvanted, inactivated whole-cell vaccines for O157 can induce O157-specific cellular and mucosal immune responses that may be an important consideration for a successful vaccination scheme.
... This dynamic uncoiling and recoiling is a common property of pili from bacteria that colonize aqueous host environments M A J O R A R T I C L E (eg, reference [15]), and has the effect of dampening the incessant shear forces exerted on the adhered bacteria by their host. Thus, this spring-like mechanism inherent to pili in combination with adhesion mediated by the CfaE tip adhesin is essential to initiating and maintaining adhesion to the target tissue [16,17]. ...
... In Figure 1, we display the effect of several peptides on ETEC adherence. We found that histatin-5 and anti-CfaE antibodies (against the tip adhesin), which have previously been shown to prevent colonization in human subjects [16,17], decrease both the number of bacteria bound to each Caco-2 cell as well as the fraction of cells with adherent bacilli. Neither a peptide with the same amino acids as histatin-5 but in a scrambled order (scrambled peptide) nor an unrelated peptide of similar size, villin headpiece HP36 [28], recapitulated this inhibition of adherence ( Figure 1). ...
Background:
Diarrheal disease from enterotoxigenic Escherichia coli (ETEC) causes significant worldwide morbidity and mortality in young children residing in endemic countries and is the leading cause of traveler's diarrhea. As ETEC enters the body through the oral cavity and cotransits the digestive tract with salivary components, we hypothesized that the antimicrobial activity of salivary proteins might extend beyond the oropharynx into the proximal digestive tract.
Results:
Here, we show that the salivary peptide histatin-5 binds colonization factor antigen I pili, thereby blocking adhesion of ETEC to intestinal epithelial cells. Mechanistically, we demonstrate that histatin-5 stiffens the typically dynamic pili, abolishing their ability to function as spring-like shock absorbers, thereby inhibiting colonization within the turbulent vortices of chyme in the gastrointestinal tract.
Conclusions:
Our data represent the first report of a salivary component exerting specific antimicrobial activity against an enteric pathogen and suggest that histatin-5 and related peptides might be exploited for prophylactic and/or therapeutic uses. Numerous viruses, bacteria, and fungi traverse the oropharynx to cause disease, so there is considerable opportunity for various salivary components to neutralize these pathogens prior to arrival at their target organ. Identification of additional salivary components with unexpectedly broad antimicrobial spectra should be a priority.
... 109 E. coli is a signi cant enteric pathogen wherein prophylaxis through passive immunity has been demonstrated in several clinical studies. [110][111][112][113] Tacket and colleagues was able to passively protect human subjects against experimentally induced E. coli diarrhea with speci c bovine antibody. 110 Using heatinactivated or glutaraldehyde-inactivated E. coli for vaccination, pregnant cows were hyperimmunized with a large number of enterotoxigenic O serogroups. ...
... [110][111][112][113] Tacket and colleagues was able to passively protect human subjects against experimentally induced E. coli diarrhea with speci c bovine antibody. 110 Using heatinactivated or glutaraldehyde-inactivated E. coli for vaccination, pregnant cows were hyperimmunized with a large number of enterotoxigenic O serogroups. Milk collected during the rst 10 days of lactation was puri ed, concentrated, lyophilized and formulated for oral administration. ...
... Two products manufactured by Immuron Ltd. were the focus of our study: Travelan, a commercially available anti-ETEC HBC for prevention of TD, and IMM-124E, a more recent anti-ETEC HBC intended not only for prevention of TD but also as a therapeutic to improve systemic chronic inflammatory conditions such as metabolic syndrome and nonalcoholic steatohepatitis (NASH). Travelan has been shown to reduce both the incidence and severity of ETEC-induced diarrhea in up to 90% of volunteers (24,25), and Our analysis of innate immune components revealed an abridged profile of cytokines (TNF-␣) in HBC products compared to fresh unprocessed colostrum (IL-1, IL-4, and TNF-␣). The difference might reflect the intrinsic composition of these products (HBC was produced in New Zealand, whereas the fresh colostrum was harvested in the United States) and/or changes due to processing. ...
... The high levels of antibodies against the target vaccine antigens O6 and O78 polysaccharides, CFA/I, CFA/II, CS3, CS4, CS6, and LT detected in IMM-124E and Travelan reflect the purposeful hyperimmunization. These antibodies are believed to provide the basis for the clinical protection afforded by HBC in ETEC-challenged volunteers, which included reduced rate of illness and abdominal pain (24,25). HBC antibodies also recognized ETEC O serotypes not included in the vaccines, including O44, O55, and O127, as well as Shigella and Salmonella O polysaccharides, likely due to the presence of shared epitopes. ...
Diarrhea is a common illness among travelers to resource-limited countries, the most prevalent attributable agent being enterotoxigenic Escherichia coli (ETEC). There are presently no vaccines licensed specifically for the prevention of ETEC-induced traveler's diarrhea (TD), and this has propelled investigation of alternative preventive methods. Colostrum, the first milk expressed after birthing, is rich in immunoglobulins and innate immune components for protection of newborns against infectious agents. Hyperimmune bovine colostrum (HBC) produced by immunization of cows during gestation (and containing high levels of specific antibodies) is a practical and effective prophylactic tool against gastrointestinal illnesses. A commercial HBC product, Travelan®, is available for prevention of ETEC-induced diarrhea. Despite its demonstrated clinical efficacy, the underlying immune components and anti-microbial activity that contribute to protection remain undefined. We investigated innate and adaptive immune components of several HBC formulated to reduce the risk of ETEC diarrhea, including Travelan® and IMM-124E, a newer product that has broader gastrointestinal health benefits. The immune components measured included total and ETEC-specific IgG, total IgA, cytokines, growth factors, and lactoferrin. HBC products contained high levels of IgG specific for multiple ETEC antigens, including O-polysaccharide 78 and Colonization Factor Antigen-1 (CFA/I) present in the administered vaccines. Anti-microbial activity was measured in vitro using novel functional assays. HBC greatly reduced ETEC motility in soft agar and exhibited bactericidal activity in the presence of complement. We have identified immune components and antimicrobial activity potentially involved in the prevention of ETEC infection by HBC in vivo .
... Milk-fat-globule membrane (MFGM) ingestion increased the resistance to diarrheagenic E. coli in healthy adults [11], and fortifying infant formula with bovine milk lactoferrin was shown to reduce the incidence of diarrhea and respiratory tract infections in weaned infants [9]. Moreover, consumption of bovine immunoglobulins from Nutrients 2024, 16, 592 2 of 14 milk or colostrum of immunized cows decreased enterotoxigenic E. coli (ETEC)-induced diarrhea [12,13] as well as rotavirus infection [14,15]. In addition, infants consuming raw cow's milk have a reduced incidence of common respiratory infections [16], whereas TGF-β and other components present in raw bovine milk contribute to establishing a regulatory environment that decreases T helper 2 (Th2) responses associated with allergic reactions [17,18]. ...
Bovine milk contains bioactive proteins, carbohydrates, and phospholipids with immunomodulatory properties impacting human immunity, potentially contributing to resistance to infections and allergies through diverse mechanisms. One such mechanism is the enhancing of the innate immune response to secondary pathogen-related stimuli, termed innate immune training. Although in vitro studies demonstrate that milk immunoglobulin G (IgG) can train human monocytes, evidence for in vivo immune training is limited. To explore the potential of bovine IgG for inducing innate immune training in vivo, this human study utilized an IgG-rich whey protein concentrate (WPC). Healthy male volunteers were assigned to a high dose WPC, low dose WPC, or placebo group. Blood was collected pre- and post-two weeks of WPC consumption. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with TLR ligands, evaluating IL-6 and TNF-α production by monocytes, myeloid DCs, and plasmacytoid DCs. Additionally, RNA was isolated for differential gene expression (DGE) analysis. Results indicated that the two-week WPC intervention did not influence the ex vivo response of studied cells to TLR agonists. Furthermore, PBMC gene expression patterns showed no significant differences between the placebo and high dose WPC groups. The data suggests that oral WPC ingestion did not enhance immune responses in young, healthy male participants.
... 176 An early clinical study conducted by Tacket et al. showed that daily consumption of an ETEC hyperimmune bovine milk concentrate shortly after each meal protected volunteers from an experimental oral challenge with 1.2 × 10 9 CFU of ETEC H10407. 177 In a subsequent study by Otto and colleagues, an ETEC HBC delivered prior to each meal reduced the incidence of diarrhea in volunteers orally challenged with ETEC H10407. 178 In a controlled human infection model (CHIM) study, volunteers received oral bovine colostrum IgG antibodies against CS17 significantly prevent diarrhea caused by ETEC strain LSN03-016011/A expressing CS17. ...
Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers in developing countries. ETEC is characterized by the ability to produce major virulence factors including colonization factors (CFs) and enterotoxins, that bind to specific receptors on epithelial cells and induce diarrhea. The gut microbiota is a stable and sophisticated ecosystem that performs a range of beneficial functions for the host, including protection against pathogen colonization. Understanding the pathogenic mechanisms of ETEC and the interaction between the gut microbiota and ETEC represents not only a research need but also an opportunity and challenge to develop precautions for ETEC infection. Herein, this review focuses on recent discoveries about ETEC etiology, pathogenesis and clinical manifestation, and discusses the colonization resistances mediated by gut microbiota, as well as preventative strategies against ETEC with an aim to provide novel insights that can reduce the adverse effect on human health.
... Studies do support the fact that humans as well as animals (Steinsland et al., 2003), and even the test animals from the laboratories (Roy et al., 2008) may develop immunity against recurrent infection if they have been exposed to ETEC pathotypes. Some of the studies even support the idea during investigational human trial models of ETEC of the same pathotypes, that both active, as well as passive immunization, was strong enough to provide protection against further infection (Tacket et al., 1988). When the whole- Fig. 1. ...
Escherichia coli (E. coli) is a widespread bacterial species that comprise a broad variety of strains and can be highly pathogenic often. Diarrhea caused by E. coli pathotypes is a leading public health problem in underdeveloped countries such as Pakistan. Five different diarrheagenic E. coli pathotypes (Enterohaemorrhagic, Enteropathogenic, Enteroaggregative, Enteroinvasive and Enterotoxigenic E. coli) were isolated from 200 samples of diarrheal children in Quetta, Pakistan. Total-cell protein profiles of all the strains isolated were resolved via SDS-Polyacrylamide Gel Electrophoresis (PAGE). More than 15 protein bands were found ranging from 15 kDa to 153 kDa in size. A dendrogram plot explained the relatedness of the pathotypes amongst them. The protein bands of 70 and 15 kDa, analyzed through GelAnalyzer 2010a© were found common among all the isolated pathotypes. Purified bands of these two proteins (70 and 15 kDa) from the gel were injected in experimental mice groups for immunization purposes. During immunogenicity tests, the mice that were immunized with 70 and 15 kDa proteins stayed alive for seven days despite subsequently being challenged with the E. coli pathotypes, while higher mortality 80% were observed in the non-immunized control group. Upon the postmortem it was revealed that all the vital organs were covered by the E. coli strains injected for challenge studies. The immunized mice and control mice sera subjected to the Agar Gel Immunodiffusion assay (AGID) test for antibody/antigen reaction were found positive for antibodies presence. The exploration of immunogenic proteins among all the prevalent pathotypes can be a breakthrough in the development of a combined, effective, and safe vaccine. Further investigation of these immunogenic proteins would be of great importance for future immunization endeavors.
... To fill this important gap in public health and travel medicine, the US Naval Medical Research Center (NMRC) has been advancing a subunit candidate vaccine based on subunits of common colonization factors (CFs), surfaceexposed polymeric protein appendages that mediate initial adherence and colonization of the small intestine. Antibodies directed to CFs have been shown to be protective in natural and experimental infections [18][19][20][21][22][23][24]. One of the most prevalent and epidemiologically important CFs is an atypical polymeric antigen termed CS6 [22,25,26]. ...
Introduction
Enterotoxigenic Escherichia coli (ETEC) is a common cause of infectious diarrhoea and a leading cause of morbidity and mortality in children living in resource-limited settings. It is also the leading cause of travellers’ diarrhoea among civilian and military travellers. Its dual importance in global public health and travel medicine highlights the need for an effective vaccine. ETEC express colonization factors (CFs) that mediate adherence to the small intestine. An epidemiologically prevalent CF is coli surface antigen 6 (CS6). We assessed the safety and immunogenicity of a CS6-targeted candidate vaccine, CssBA, co-administered intramuscularly with the double-mutant heat-labile enterotoxin, dmLT [LT(R192G/L211A)].
Methods
This was an open-label trial. Fifty subjects received three intramuscular injections (Days 1, 22 and 43) of CssBA alone (5 µg), dmLT alone (0.1 µg) or CssBA (5, 15, 45 µg) + dmLT (0.1 and 0.5 µg). Subjects were actively monitored for adverse events for 28 days following the third vaccination. Antibody responses (IgG and IgA) were characterized in the serum and from lymphocyte supernatants (ALS) to CS6 and the native ETEC heat labile enterotoxin, LT.
Results
Across all dose cohorts, the vaccine was safe and well-tolerated with no vaccine-related severe or serious adverse events. Among vaccine-related adverse events, a majority (98%) were mild with 79% being short-lived vaccine site reactions. Robust antibody responses were induced in a dose-dependent manner with a clear dmLT adjuvant effect. Response rates in subjects receiving 45 µg CssBA and 0.5 µg dmLT ranged from 50 to 100% across assays.
Conclusion
This is the first study to demonstrate the safety and immunogenicity of CssBA and/or dmLT administered intramuscularly. Co-administration of the two components induced robust immune responses to CS6 and LT, paving the way for future studies to evaluate the efficacy of this vaccine target and development of a multivalent, subunit ETEC vaccine.
... Besides animal studies, clinical trials have shown benefit of immunoglobulin concentrate (IC) administration to patients with irritable bowel syndrome, reducing symptoms (improved stool consistency and frequency, pain, and bloating) and cytokine production [20]. Immunoglobulin concentrates have been also shown to be effective in the management of enteropathy associated with diarrhea-predominant in irritable bowel syndrome and human immunodeficiency virus infection [21,22], or diarrhea produced by E. coli or Shigella [23,24]. Supplementation of infant formula with an IC protects against diarrhea in infants [25]. ...
Serum protein concentrates have been shown to exert in vivo anti-inflammatory effects. Specific effects on different cell types and their mechanism of action remain unraveled. We aimed to characterize the immunomodulatory effect of two porcine plasma protein concentrates, spray dried serum (SDS) and an immunoglobulin concentrate (IC), currently used as animal nutritional supplements with established in vivo immunomodulatory properties. Cytokine production by the intestinal epithelial cell line IEC18 and by primary cultures of rat splenocytes was studied. The molecular pathways involved were explored with specific inhibitors and gene knockdown. Our results indicate that both products induced GROα and MCP-1 production in IEC18 cells by a MyD88/NF-κB-dependent mechanism. Inhibition of TNF production was observed in rat primary splenocyte cultures. The immunoglobulin concentrate induced IL-10 expression in primary splenocytes and lymphocytes. The effect on TNF was independent of IL-10 production or the stimulation of NF-kB, MAPKs, AKT, or RAGE. In conclusion, SDS and IC directly regulate intestinal and systemic immune response in murine intestinal epithelial cells and in T lymphocytes and monocytes.
... The efficacy of oral antibodies for the treatment of gastrointestinal infection was first demonstrated more than 40 years ago for the treatment of E. coli infection in human infants [60][61][62] . Additional successful human clinical trials against other GI pathogens, such as rotavirus and C. difficile, have been reported [63][64][65][66][67] . ...
Arthrospira platensis (commonly known as spirulina) is a photosynthetic cyanobacterium. It is a highly nutritious food that has been consumed for decades in the US, and even longer by indigenous cultures. Its widespread use as a safe food source and proven scalability have driven frequent attempts to convert it into a biomanufacturing platform. But these were repeatedly frustrated by spirulina's genetic intractability. We report here efficient and versatile genetic engineering methodology for spirulina that allows stable expression of bioactive protein therapeutics at high levels. We further describe large-scale, indoor cultivation and downstream processing methods appropriate for the manufacturing of biopharmaceuticals in spirulina. The potential of the platform is illustrated by pre-clinical development and human testing of an orally delivered antibody therapeutic against campylobacter, a major cause of infant mortality in the developing world and a growing antibiotic resistance threat. This integrated development and manufacturing platform blends the safety of food-based biotechnology with the ease of genetic manipulation, rapid growth rates and high productivity characteristic of microbial platforms. These features combine for exceptionally low-cost production of biopharmaceuticals to address medical needs that are unfeasible with current biotechnology platforms.
... In addition to normal IgG constituents, specific vaccination of cows against human or bovine pathogens (hyper-immunisation) results in the production of neutralizing antibodies that show benefits for preventing and treating infections, resulting in increased weight gain in clinical and veterinary situations. Examples include preventing and treating enteropathic infections by Escherichia coli [47] or rotavirus [48]. Similarly, using a tooth surface model, a concentrate of hyper-immune milk was shown to prevent adherence of Candida albicans [49], and a hyperimmune BC preparation reduced dental plaque [50]. ...
Colostrum is the milk produced during the first few days after birth and contains high levels of immunoglobulins, antimicrobial peptides, and growth factors. Colostrum is important for supporting the growth, development, and immunologic defence of neonates. Colostrum is naturally packaged in a combination that helps prevent its destruction and maintain bioactivity until it reaches more distal gut regions and enables synergistic responses between protective and reparative agents present within it. Bovine colostrum been used for hundreds of years as a traditional or complementary therapy for a wide variety of ailments and in veterinary practice. Partly due to concerns about the side effects of standard Western medicines, there is interest in the use of natural-based products of which colostrum is a prime example. Numerous preclinical and clinical studies have demonstrated therapeutic benefits of bovine colostrum for a wide range of indications, including maintenance of wellbeing, treatment of medical conditions and for animal husbandry. Articles within this Special Issue of Nutrients cover the effects and use bovine colostrum and in this introductory article, we describe the main constituents, quality control and an overview of the use of bovine colostrum in health and disease.
... In clinical trials, ingestion of bovine milk-or colostrum-derived immunoglobulins consisting mainly of IgG from immunized dairy cows is sufficient to significantly reduce ETEC infection in adult volunteers [7,35], indicating a role for IgG in passive oral immunizations. In fact, in a recent report, orally delivered anti-colonization factor antigen CFA/I IgG and SIgA human mAbs were equally effective at blocking ETEC infection in a mouse model [9]. ...
Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer’s patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection. In this report, we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to prevent infection of invasive Salmonella enterica serovar Typhimurium (STm) in mice. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer’s patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer’s patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer’s patch tissues. Our results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonetheless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.
... It is known that the immunoglobulins found in BC bind to many human pathogens and allergens and can neutralize infection of human cells, as well as treat gastrointestinal inflammation (Ulfman et al., 2018). Also, passive oral administration of BC has been experimentally evaluated as a preventive or therapeutic modality for a variety of enteric infections (Tacket et al., 1988;Davidson et al., 1989;Tacket et al., 1992;Greenberg and Cello, 1996;Freedman et al., 1998;Savarino et al., 2017). Food formulations with BC can compensate or have a beneficial effect on health due to the addition of specific components if those components are lacking in the daily diet (Santini and Novellino, 2018). ...
This study evaluated the influence of fermentation with Lactobacillus plantarum LUHS135 and Lactobacillus paracasei LUHS244, ultrasonication, and different methods of dehydration on the content of immunoglobulins IgG, IgA, and IgM in bovine colostrum (BC), as well as the antimicrobial activity of the treated and fresh BC samples (fresh - BC; freeze-dried - BClyoph; vacuum-dried (+45°C) - BCvacdried; BC fermented with LUHS135 - BCLUHS135; BC fermented with LUHS244 - BCLUHS244; BC fermented with LUHS135 and freeze-dried - BCLUHS135lyoph; BC fermented with LUHS244 and freeze-dried - BCLUHS244 lyoph; BC fermented with LUHS135 and vacuum-dried - BCLUHS135 vacdried; BC fermented with LUHS244 and vacuum-dried - BCLUHS244 vacdried; BC ultrasonicated and freeze-dried - BCultr lyoph; BC ultrasonicated and vacuum-dried - BCultr vacdried). The antimicrobial activity was assessed against Klebsiella pneumoniae, Salmonella enterica, Pseudomonas aeruginosa, Acinetobacter baumanni, Proteus mirabilis, methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Enterococcus faecium, Bacillus cereus, Streptococcus mutans, Enterobacter cloacae, Citrobacter freundii, Staphylococcus epidermis, Staphylococcus haemolyticus and Pasteurella multocida using agar well diffusion method, as well as in liquid medium. In liquid medium performed analysis showed that the fermented BC samples had the broadest antimicrobial spectrum (of 15 tested pathogenic strains, BCLUHS135 vacdried and BCLUHS135lyoph inhibited 13; BCLUHS244 vacdried inhibited 12; and BCLUHS135, BCLUHS244 and BCLUHS244 lyoph inhibited 11). Based on the inhibition zones, BCLUHS135lyoph samples exhibited the broadest inhibition spectrum, inhibiting the growth of 12 of the 15 tested pathogenic strains). According to the lactic acid bacteria (LAB) strain selected for BC fermentation, different properties of the BC will be obtained. To ensure a broad antimicrobial spectrum and high IgG content, fermentation with LUHS135 can be recommended (IgG concentration in BCLUHS135 was retained), whereas fermentation will LUHS244 will provide a high IgM concentration (IgM concentration increased by 48.8% and 21.6% in BCLUHS244 and BC LUHS244lyoph samples, respectively). However, IgA is very sensitive for fermentation, and further studies are needed to increase IgA stability in BC. Finally, fermented BC can be recommended as a food/beverage ingredient, providing safety, as well as improved functionality through displaying a broad spectrum of antimicrobial activities.
... Oral feeding with specific colostrum antibodies to healthy volunteers, followed by challenge with a well-defined inoculum of a virulent challenge strain, has been used in different ETEC challenge experiments [35]. Usually, the volunteers ingest gram quantities of antibodies together with an antacid, starting 2À7 days before and during challenge, and continue this regimen during the evaluation phase. ...
Although enterotoxigenic Escherichia coli (ETEC) is the most common causes of bacterial diarrhea in children in developing countries and in travelers to these areas, no licensed vaccine against ETEC disease is yet available. In this chapter, we identify the most promising vaccine antigens and immunization routes as a basis for development of an effective ETEC vaccine. A number of different candidate vaccines, both purified antigens and whole cells, have been tested for safety and immunogenicity in preclinical trials, several with promising results. A few candidate vaccines have also reached testing in clinical trials, the most advanced being an oral inactivated whole cell vaccine, ETVAX, consisting of recombinant E. coli bacteria overexpressing the most prevalent colonization factors (CFs; fimbrial and nonfimbrial antigens) in combination with a heat-labile enterotoxoid (LCTBA) and dmLT double mutant heat-labile enterotoxin serving as an adjuvant. This vaccine has been tested successfully in different phase1 and 2 trials both in Swedish adults and in descending age groups in Bangladesh, and it is currently being evaluated for protective efficacy in a large phase 2b trial in European travelers to Africa.
... 4,5 Moreover, the cholera vaccine Dukoral ® (Valneva) provides short-term protection against LT-expressing ETEC by eliciting cholera toxin (CT)-specific antibodies that are cross-reactive to LT. 6 Finally, oral passive immunization studies with antibodies directed against the colonization factor CFA/I or the tip adhesin of CFA/I, CfaE, were protective against the CFA/I+ ETEC strain H10407 in a controlled human challenge trial. 5,7,8 These human challenge studies are especially important for ETEC vaccine development as the pathogenicity and specificity of ETEC strains differ between hosts (i.e., human and porcine ETEC strains express different CFs). ...
Although offering great potential for generating intestinal immunity, vaccination by the oral route suffers from several barriers such as the breakdown of protein vaccines in the stomach and/or the induction of oral tolerance. To investigate whether these barriers can be circumvented, Mark T. Orr and colleagues at the Infectious Disease Research Institute use a parenteral (intramuscular) vaccination protocol in mice. Intramuscular immunization with an enterotoxigenic E. coli (ETEC) vaccine plus a Toll-like receptor 4 adjuvant in stable emulsion (SLA-SE) elicits a functional antibody response in both the gut and serum. Importantly, this intramuscular vaccination triggers robust production of IgA in the gut. These findings suggest that with the right adjuvant combination it might possible to generate potent protective mucosal immunity following parenteral immunization.
... An effective ETEC vaccine could prevent deaths from these pathogens and reduce the substantial morbidity associated with diarrheal illnesses as well as their pernicious sequelae [43]. All ETEC vaccine candidates that have advanced to clinical trials to date have exclusively targeted the plasmid-encoded CFs and/ or LT [44], and seminal studies in humans clearly support the importance of CFs [45] or their associated subunits [13] as protective antigens. Nevertheless, development of a broadly protective ETEC vaccine has been confounded by the remarkable underlying genetic plasticity of E coli [8] and a still-developing understanding of mechanistic correlates of protection [46]. ...
Background:
Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal illness in the developing world. Enterotoxigenic E coli vaccinology has been challenged by genetic diversity and heterogeneity of canonical antigens. Examination of the antigenic breadth of immune responses associated with protective immunity could afford new avenues for vaccine development.
Methods:
Antibody lymphocyte supernatants (ALS) and sera from 20 naive human volunteers challenged with ETEC strain H10407 and from 10 volunteers rechallenged 4-6 weeks later with the same strain (9 of whom were completely protected on rechallenge) were tested against ETEC proteome microarrays containing 957 antigens.
Results:
Enterotoxigenic E coli challenge stimulated robust serum and mucosal (ALS) responses to canonical vaccine antigens (CFA/I, and the B subunit of LT) as well as a small number of antigens not presently targeted in ETEC vaccines. These included pathovar-specific secreted proteins (EtpA, EatA) as well as highly conserved E coli antigens including YghJ, flagellin, and pertactin-like autotransporter proteins, all of which have previously afforded protection against ETEC infection in preclinical studies.
Conclusions:
Taken together, studies reported here suggest that immune responses after ETEC infection involve traditional vaccine targets as well as a select number of more recently identified protein antigens that could offer additional avenues for vaccine development for these pathogens.
... In the first study, Tacket et al. (81) showed a clear prophylactic effect of hyperimmune colostrum from cows immunized with a range of E.coli serotypes. In this study volunteers received an oral challenge with 10 9 enterotoxicogenic E.coli. ...
This review aims to provide an in depth overview of the current knowledge of the effects of bovine immunoglobulins on the human immune system. The stability and functional effects of orally ingested bovine immunoglobulins in milk products are described and potential mechanisms of action are discussed. Orally ingested bovine IgG (bovine IgG) can be recovered from feces, ranging from very low levels up to 50% of the ingested IgG that has passed through the gastrointestinal tract. In infants the recovered levels are higher than in adults most likely due to differences in stomach and intestinal conditions such as pH. This indicates that bovine IgG can be functionally active throughout the gastrointestinal tract. Indeed, a large number of studies in infants and adults have shown that bovine IgG (or colostrum as a rich source thereof) can prevent gastrointestinal tract infections, upper respiratory tract infections, and LPS-induced inflammation. These studies vary considerably in target group, design, source of bovine IgG, dosage, and endpoints measured making it hard to draw general conclusions on effectiveness of bovine immunoglobulin rich preparations. Typical sources of bovine IgG used in human studies are serum-derived IgG, colostrum, colostrum-derived IgG, or milk-derived immunoglobulins. In addition, many studies have used IgG from vaccinated cows, but studies using IgG from nonimmunized animals have also been reported to be effective. Mechanistically, bovine IgG binds to many human pathogens and allergens, can neutralize experimental infection of human cells, and limits gastrointestinal inflammation. Furthermore, bovine IgG binds to human Fc receptors which, enhances phagocytosis, killing of bacteria and antigen presentation and bovine IgG supports gastrointestinal barrier function in in vitro models. These mechanisms are becoming more and more established and explain why bovine IgG can have immunological effects in vivo. The inclusion of oral bovine immunoglobulins in specialized dairy products and infant nutrition may therefore be a promising approach to support immune function in vulnerable groups such as infants, children, elderly and immunocompromised patients.
... One approach that has been used to explore ETEC pathogen biology is the controlled human infection model, in which healthy immunologically naïve volunteers are challenged by ingestion of enterotoxigenic E. coli. Notably, this model was used initially to establish these toxin-producing E. coli as a cause of diarrheal illness in humans (34), and subsequently to examine virulence features of ETEC (35), immune responses to experimental infection (36) and to conduct vaccine studies (37)(38)(39)(40). The majority of ETEC human challenge studies conducted to date have involved H10407, a prototype strain originally isolated from an adult with severe, cholera like illness in Bangladesh (41). ...
Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, that may involve strain-specific virulence features as well as host factors, have not been elucidated. We demonstrate that when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete a novel adhesin molecule, EtpA. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and non-canonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A specific lectin/hemagglutinin. Importantly, we also show that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals, and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and the effective delivery both heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.
... Also topical delivery of antibodies to protect against sexually transmitted diseases or fertilization has been proven as a useful strategy [197]. Similarly, antibodies directed against intestinal pathogens have shown a protective function of the underlying epithelium [198]. In human midcycle cervical mucus samples, the diffusion coefficients of most of the tested fluorescent labelled proteins (exogenous), including IgG were similar to diffusion coefficients in buffer, indicating a lack of barrier function given by the mucus [15,197]. ...
The mucosa of the gastrointestinal tract, eyes, nose, lungs, cervix and vagina is lined by epithelium interspersed with mucus-secreting goblet cells, all of which contribute to their unique functions. This mucus provides an integral defence to the epithelium against noxious agents and pathogens. However, it can equally act as a barrier to drugs and delivery systems targeting epithelial passive and active transport mechanisms. This review highlights the various mucins expressed at different mucosal surfaces on the human body, and their role in creating a mucoid architecture to protect epithelia with specialized functions. Various factors compromising the barrier properties of mucus have been discussed, with an emphasis on how disease states and microbiota can alter the physical properties of mucus. For instance, Akkermansia muciniphila, a bacterium found in higher levels in the gut of lean individuals induces the production of a thickened gut mucus layer. The aims of this article are to elucidate the different physiological, biochemical and physical properties of bodily mucus, a keen appreciation of which will help circumvent the slippery slope of challenges faced in achieving effective mucosal drug and gene delivery.
... Subsequently, watery diarrhea is induced through the action of heat-stable (ST) and/or heat-labile (LT) enterotoxins. Experimental human challenge studies to investigate the pathogenesis of ETEC diarrhea in humans, study immunity, and evaluate vaccines have been performed at the Center for Vaccine Development (CVD) of the University of Maryland School of Medicine since the mid-1970s, as well as at other institutions [4][5][6][7][8][9][10][11][12][13][14][15][16]. ...
Enterotoxigenic Escherichia coli (ETEC) is a non-invasive enteric pathogen of considerable public health importance, being one of the most common attributable causes of diarrheal illness in infants and young children in developing countries and the most common cause of traveler’s diarrhea. To enhance study-to-study consistency of our experimental challenge model of ETEC in volunteers, and to allow concomitant multi-site trials to evaluate anti-ETEC immunoprophylactic products, hundreds of vials, each containing a standardized inoculum of virulent wild-type (wt) ETEC strain H10407 (serotype O78:H11 expressing colonization factor antigen I and heat-labile and heat-stable enterotoxins), were prepared under current Good Manufacturing Practices (cGMP) and frozen. Following thawing, the contents of each vial can be used (diluted as necessary) to prepare consistent challenge inoculum, even at different study sites. A preliminary human experimental challenge study using this cGMP inoculum was conducted on a research isolation ward and the clinical and cell-mediated immune responses evaluated. Of the 6 healthy adult volunteers challenged 83% (5/6) developed diarrhea and 50% developed moderate-to-severe diarrhea (MSD). Moderate and severe diarrhea were defined as passage of ≥ 1 liter or ≥ 3 liters of diarrheal stool respectively. We compared the CD4+ T cell responses of volunteers who developed MSD against those who did not and identified significant differences in ETEC-specific cytokine production and gut homing potential. We furthermore demonstrated that increased expression of the gut-homing molecule integrin α4β7 by peripheral T follicular helper cells (pTfh) correlated with decreased stool volume and increased ETEC-specific IgA B memory cell (BM) development. Collectively, despite small numbers of volunteers, our results indicate a potential role for CD4+ T cells, in particular pTfh, in modulating disease outcome following exposure to wt ETEC in a volunteer experimental challenge model.
... 1992). Moreover, bovine colostrum rich in antibodies to Shiga toxin and enterohemorrhagic E. coli-hemolysin reportedly diminishes the frequency of loose stools in children with E. coliassociated diarrhoea (Huppertz et al., 1999) and in individuals with traveller's diarrhoea caused by enterotoxigenic E. coli (Tacket et al., 1988). ...
Clinically, there is a growing need to identify new means to limit the response of the gut mucosa to inflammatory stimuli. Bovine colostrum is increasingly recognised to promote human gastrointestinal health but the mechanism(s) involved are not entirely elucidated.
This study investigated specific anti-inflammatory and anti-bacterial effects of colostrum in an in vitro model of intestinal cell inflammation. Colostrum reduced interleukin (IL)-8 levels when Caco-2 and HT29 cells were stimulated with TNF-α. Increasing concentrations of colostrum also reduced IL-8 expression following co-culture with adherent-invasive Escherichia coli (AIEC), a finding that correlated with reduced bacterial adherence to the cells. Colostrum was also shown to have direct antibacterial activity.
The findings of this study add to the growing body of evidence that bovine colostrum may indeed be beneficial to gastrointestinal health due to modulation of intestinal epithelial inflammatory responses consequent to interruption of host:pathogen interactions.
... Antibodies which block the H antigen on fimbriae of enteropathogenic E. coli strains have proven useful as a prophylactic measure (45). Some studies have provided evidence for the protective and therapeutic effect of Igs enriched from regular bovine cheese or colostrum from non-immunized cows against non-specific diarrheal diseases of newborn farm animals (46,47) and humans (48). ...
The nutritional importance of colostrum in different farm animals and it's quality has two basic facts that the colostrum quality begins to degrade immediately after calving and colostrum rapidly loses quality after about six hours post calving. Six hour colostrum has 5.3% albumin by weigh but 12 hour colostrum has 2.96% albumin. Colostrum serves mainly three functions are laxative, nutritive and protective. Laxative to aid in the certainty of the muconium lining of the digestive tract. Nutritive has provided an excellent energy source for the newborn. Energy reserves in the newborn are limited, and the high fat content of colostrum serves that purpose well. Protective contains antibodies (Igs) to protect the newborn kid until its own immune system begins functioning about 3 weeks of age. The newborn kid can absorb the essential antibodies in the colostrum only during its first 24 hours or so of life, as there is a rapid reduction in the permeability of the intestinal wall of the newborn to the antibodies. These antibodies protect the kid for the first 8-10 weeks against many diseases. It may be difficult to rear healthy, well-growing kids if they do not receive colostrum. Kids that do not receive colostrum will be less resistant to scours (diarrhea) and other aliments later in life.
Background
Hyperimmunized bovine colostrum containing antibodies has been shown to be effective in the treatment of rotavirus diarrhea. Antibodies derived from eggs of immunized hens may be a less expensive and more practical alternative. In this study, children with proven rotavirus diarrhea were treated with immunoglobulin extracted from eggs of chicken immunized with human rotavirus strains.
Methods
In a randomized, double‐blind study, 79 children with known rotavirus diarrhea were assigned to receive either 10 g hyperimmune egg yolk (HEY) daily in four equally divided doses for 4 days (HEY group) or a similar preparation obtained from nonimmunized chicken (placebo group). The daily stool frequency and amount, oral rehydration solution (ORS) intake, and presence of rotavirus in the stool were monitored for 4 days.
Results
In the HEY‐treated group. there was significant reduction in stool output (in grams per kilogram per day; HEY vs. placebo; 87 ± 59 vs. 120 ± 75, P = 0.03), and significant reduction of ORS intake (in milliliters per kilogram per day) (HEY vs. placebo; 84 ± 46 vs. 122 ± 72, P = 0.008) on day 1 and clearance of virus on day 4 (HEY vs. placebo; 73% vs. 46%, P = 0.02). There was, however, no difference in diarrheal duration between the groups.
Conclusions
Treatment with HEY against four human rotavirus strains resulted in modest improvement of diarrhea associated with earlier clearance of rotavirus from stools. These results indicate an encouraging role of HEY in the treatment of rotavirus‐induced diarrhea in children. Further studies are needed to optimize the dose and neutralization titer and thus improve the efficacy of egg yolk immunoglobulin IgY derived from immunized hens.
Infectious diarrhea is a World Health Organization public health priority area due to the lack of effective vaccines and an accelerating global antimicrobial resistance crisis. New strategies are urgently needed such as immunoprophylactic for prevention of diarrheal diseases. Hyperimmune bovine colostrum (HBC) is an established and effective prophylactic for infectious diarrhea. The commercial HBC product, Travelan ® (Immuron Ltd, Australia) targets multiple strains of enterotoxigenic Escherichia coli (ETEC) is highly effective in preventing diarrhea in human clinical studies. Although Travelan ® targets ETEC, preliminary studies suggested cross-reactivity with other Gram-negative enteric pathogens including Shigella and Salmonella species. For this study we selected an invasive diarrheal/dysentery-causing enteric pathogen, Shigella , to evaluate the effectiveness of Travelan ® , both in vitro and in vivo . Here we demonstrate broad cross-reactivity of Travelan ® with all four Shigella spp . ( S . flexneri , S . sonnei , S . dysenteriae and S . boydii) and important virulence factor Shigella antigens. Naïve juvenile rhesus macaques (NJRM) were randomized, 8 dosed with Travelan ® and 4 with a placebo intragastrically twice daily over 6 days. All NJRM were challenged with S . flexneri 2a strain 2457T on the 4 th day of treatment and monitored for diarrheal symptoms. All placebo-treated NJRM displayed acute dysentery symptoms within 24–36 hours of challenge. Two Travelan ® -treated NJRM displayed dysentery symptoms and six animals remained healthy and symptom-free post challenge; resulting in 75% efficacy of prevention of shigellosis (p = 0.014). These results strongly indicate that Travelan ® is functionally cross-reactive and an effective prophylactic for shigellosis. This has positive implications for the prophylactic use of Travelan ® for protection against both ETEC and Shigella spp . diarrheal infections. Future refinement and expansion of pathogens recognized by HBC including Travelan ® could revolutionize current management of gastrointestinal infections and outbreaks in travelers’ including military, peacekeepers, humanitarian workers and in populations living in endemic regions of the world.
The timelines for developing vaccines against infectious diseases are lengthy, and often vaccines that reach the stage of large phase 3 field trials fail to provide the desired level of protective efficacy. The application of controlled human challenge models of infection and disease at the appropriate stages of development could accelerate development of candidate vaccines and, in fact, has done so successfully in some limited cases. Human challenge models could potentially be used to gather critical information on pathogenesis, inform strain selection for vaccines, explore cross-protective immunity, identify immune correlates of protection and mechanisms of protection induced by infection or evoked by candidate vaccines, guide decisions on appropriate trial endpoints, and evaluate vaccine efficacy. We prepared this report to motivate fellow scientists to exploit the potential capacity of controlled human challenge experiments to advance vaccine development. In this review, we considered available challenge models for 17 infectious diseases in the context of the public health importance of each disease, the diversity and pathogenesis of the causative organisms, the vaccine candidates under development, and each model's capacity to evaluate them and identify correlates of protective immunity. Our broad assessment indicated that human challenge models have not yet reached their full potential to support the development of vaccines against infectious diseases. On the basis of our review, however, we believe that describing an ideal challenge model is possible, as is further developing existing and future challenge models.
In this thesis, we aimed to evaluate the immunomodulatory effects of (selected) bovine milk components (and metabolites induced by them) on the immune function of humans. Chapter 1 gives an introduction to the topic and provides an outline of fundamental aspects of the immune system that are referred to in later chapters. In Chapter 2 , we summarized and addressed the primary components of bovine milk that have the potential to induce epigenetic changes to exert their immune-supportive effects during childhood. We reviewed the proposed mechanisms, including innate immune training that induces epigenetic modification. Through these mechanisms, the components may exert an effect on the immune system with implications for allergies and asthma. Living in a farm environment and raw bovine milk bioactive components were addressed as contributing factors that may reduce allergies in infancy and beyond. HMOs and bovine milk oligosaccharides (mainly 3'-Sialyllactose) serve as substrates for the SCFA-producing microbiota. SCFAs are potent immune modulators and have significant roles in maintaining homeostasis and steering the response of the immune cells to the environment. In Chapter 3 , we showed that butyrate and propionate had inhibitory effects on the activation of myeloid cells and lymphocytes, whereas acetate had a more selective impact on the immune cells. Production of inflammatory cytokines was suppressed in monocytes, mDCs, and pDCs, as well as T lymphocytes. SCFAs could not train the monocytes for enhanced response to secondary TLR stimulation in vitro but instead induced a tolerance-like phenotype. We attempted to explain the observed effects according to the differential expression of relevant SCFA receptors and transporters. Bovine milk IgG (bIgG) binds to human pathogens such as RSV and, via the Fc portion, interacts with the FcγRs on human immune cells. The relevance of bIgG-containing immune complexes (ICs) on activation of CD32 was studied in Chapter 4 , where we could establish a method to show the direct binding of bIgG ICs to immune cells. It was demonstrated that ICs containing bIgG are directly bound to human CD14+. Subsequently, we could show the role of bIgG ICs on induction of trained immunity after binding to monocytes while contrary to previous reports, (monomeric) bIgG alone did not have similar effects suggestive of the presence of other contributing factors. Human infection challenge models are used as an alternative to field trials to study the immune-supportive effects of dietary components. In Chapter 5 , we found that ingestion of a dairy product (WPC) in a human challenge model did not influence the responsiveness of myeloid cells from healthy volunteers to ex vivo stimulation with TLR ligands. It also did not change the gene expression pattern of the PBMCs isolated from the same donors. Although the model was utilized successfully before, the study product did not exert beneficial effects. We speculated that the study population might be a critical factor for no apparent impact. In Chapter 6 , we focused on optimizing an E. coli infection challenge model in humans to study the protective effects of dietary components and the correlates of protection at rechallenge. Primary infection with even low doses (1E6 CFU) of E. coli protected the subjects against reinfection with a high dose (1E10 CFU) of the same pathogen. Following the primary infection, serum anti-CFAII IgG levels were raised, and monocytes and mDCs responded more strongly to ex vivo stimulation. The latter may indicate the occurrence of trained immunity in vivo. Throughout Chapter 7 , we reviewed and discussed the most important findings of our research and placed the findings in a broader context by relating them to the most recent published literature. In addition, we identified subjects for future study perspectives to follow the work done in this project. Immunomodulation by nutrition or supplementation of the food with potent immunomodulatory components can provide immune support for the immune system in individuals with an immature or impaired immune system. To substantiate the dietary components' beneficial effects and define the supporting mechanisms involved, we studied bovine IgG and metabolites induced by milk oligosaccharides to substantiate their health-promoting and immune-mediated effects.
Colostrum (or immune milk) is the first milk that mammals produce after childbirth, and its composition differs markedly from milk obtained later in lactation. Colostrum is a rich source of immunoglobulins and other biologically active components. The purpose of this literature review is to systematize research on methods of treatment and prevention of human infectious diseases using immunomodulatory and immunoprotective properties of colostrum. The open sources hosted in PubMed, Researchgate and eLibrary databases were studied. The history of the use of colostrum from hyperimmunized cows as a treatment for human diseases can be traced back to the 1950s. Many studies on the use of colostrum have explored its potential in both the prevention and treatment of various infectious diseases. The data obtained indicate the high efficiency of the use of cow colostrum and its components both for the prevention and treatment of infectious diseases. Colostrum is capable of producing a heterologous transfer of passive immunity. The immunization protocols used in the production of immune milk can be highly variable. This is especially true for the timing of immunization. Working on immunization protocols that expose animals to specific antigens can result in enhanced humoral immune responses in the mammary gland. The most relevant is the search for ways to use immune milk as a means to contain the SARS-CoV-2 pandemic. The literature review provides a description of antimicrobial, immunomodulatory and growth-stimulating factors in bovine colostrum. Examples and descriptions of homologous and heterologous transmission of passive immunity are given.
The controlled human infection model (CHIM) for enterotoxigenic Escherichia coli (ETEC) has been instrumental in defining ETEC as a causative agent of acute watery diarrhea, providing insights into disease pathogenesis and resistance to illness, and enabling preliminary efficacy evaluations for numerous products including vaccines, immunoprophylactics, and drugs. Over a dozen strains have been evaluated to date, with a spectrum of clinical signs and symptoms that appear to replicate the clinical illness seen with naturally occurring ETEC. Recent advancements in the ETEC CHIM have enhanced the characterization of clinical, immunological, and microbiological outcomes. It is anticipated that omics-based technologies applied to ETEC CHIMs will continue to broaden our understanding of host-pathogen interactions and facilitate the development of primary and secondary prevention strategies.
Dysbiosis is alterations in the microbial composition compared with a healthy microbiota and often features a reduction in gut microbial diversity and a change in microbial taxa. Dysbiosis, especially in the gut, has also been proposed to play a crucial role in the pathogenesis of a wide variety of diseases, including inflammatory bowel disease, colorectal cancer, cardiovascular disease, obesity, diabetes and multiple sclerosis. A body of evidence has shown that intestinal polymeric immunoglobulin A (IgA) antibodies are important to regulate the gut microbiota as well as to exclude pathogenic bacteria or viral infection such as influenza and SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) at mucosal sites. Since the 1970s, trials for oral administration of therapeutic IgA or IgG have been performed mainly to treat infectious enteritis caused by pathogenic Escherichia coli or Clostridium difficile. However, few of them has been successfully developed as a clinical application up to now. In addition to the protective function against intestinal pathogens, IgA is well known to modulate the gut commensal microbiota leading to symbiosis. Nevertheless, the development of therapeutic IgA drugs to treat dysbiosis is not progressing. In this review, the advantages of therapeutic IgA antibodies and the problems for their development will be discussed.
Over four billion episodes of diarrhea occur annually in developing countries with diarrheagenic Escherichia coli (DEC) outbreaks also being reported, until now bacterial diarrhea is conventionally addressed by the antibiotic treatment regimes. In recent decades, the emergence of antimicrobial-resistant strains has become a major obstacle in diarrheal treatment; hence, novel and ideal therapeutics are needed. Notably, 80% of DEC is resistant to first-class antibiotics. Among the existing strategies, passive immunization is considered as an alternative to combat drug-resistant bacteria. Antibodies specific to an antigen can be used for prophylactic and therapeutic purposes. In this review, we have systematically discussed the effect of passive immunotherapy to combat DEC and explored the types and advancements in antibodies used against antibiotic-resistant DEC.
Background: Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer’s patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection.
Methodology/Principal Findings: In this report we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to protect mice against invasive Salmonella enterica serovar Typhimurium (STm) infection. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer’s patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer’s patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared, as was a second lower avidity O5-specific IgA mAb that we produced from STm immunized mice. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer’s patch tissues.
Conclusions/Significance: Our results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonethless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.
Author Summary: The bacterium Salmonella enterica is responsible for significant morbidity and mortality in the developed and developing worlds. While the pathogen is most renowed as the causative agent of typhoid fever, the emergence of invasive non-typhoid strains like S. enterica serovar Typhimurium (STm) are of great concern because of their propensity to cause severe disease in children under the age of five. In this report, we demonstrate in a mouse model that oral administration of a monoclonal antibody targeting the surface of STm is able to prevent the bacterium from infecting gastrointestinal tissues, the first step in the dissemination process. We show that IgA antibodies (which are normally found in the gut) were far superior than an equivalent IgG antibody (normally found in blood) at protecting the intestine from STm infection. These results lay the foundation for future studies aimed at the development of an orally administered antibody cocktail capable of providing temporary immunity to pathogens like S. enterica.
This chapter focuses on the most common population at risk for travelers' diarrhea (TD), which is a traveler from an industrialized country traveling to a less developed region with higher rates of enteropathogens, particularly bacterial, than the traveler's country of origin. The clinical syndrome consists of both an increased frequency of bowel movements, along with a change in stool consistency to loose and/or liquid form. "Typical" TD represents a spectrum of illness from a fleeting mild diarrhea without associated symptoms or activity limitation to a serious dehydrating and/or febrile dysentery requiring hospitalization. Bacterial enteropathogens are the predominant etiologic agents associated with TD. The American College of Gastroenterology (ACG) guidelines classified pretravel counseling regarding high‐risk food/beverage avoidance to prevent TD as a conditional recommendation, though with very low‐level evidence of efficacy.
Whey protein contains a rich and heterogeneous mixture of secreted proteins. The ß‐lactoglobulin is the predominant component of whey synthesized in mammary gland and secreted in milk. a‐Lactalbumin consists 123 amino acids in a single peptide chain. One of the most important distinctions between the human and bovine milks is the difference in their protein composition. Bovine serum albumin (BSA), one of the major components in plasma protein, is a single‐chain globular nonglycoprotein. As the major soluble protein constituent of the circulatory system, serum albumins have many physiological functions. Lactoferrin (Lf) is present in milk and, to a lesser extent, in exocrine fluids such as bile and tears. Bovine immunoglobulin form an important component of the immunological activity found in colostrum. Whey protein also contains some proteins with low quantity but high activities as growth factors, lactoperoxidase, milk fat globule membrane (MFGM) proteins and vitamin binding proteins.
CS6, a prevalent surface antigen expressed in nearly 20% of clinical enterotoxigenic Escherichia coli (ETEC) isolates, is comprised of two major subunit proteins, CssA and CssB. Using donor strand complementation, we constructed a panel of recombinant proteins of 1-3 subunits, that contained combinations of CssA and/or CssB subunits and a donor strand, a C-terminal extension of sixteen amino acids that was derived from the N-terminus of either CssA or CssB. While the entire panel of recombinant proteins could be obtained as soluble, folded proteins, it was observed that the proteins possessing a heterologous donor strand, derived from the CS6 subunit different from the C-terminal subunit, had the highest degree of physical and thermal stability. Immunological characterization of the proteins, using a murine model, demonstrated that robust anti-CS6 immune responses were generated from fusions containing both CssA and CssB. Proteins containing only CssA were weakly immunogenic. Heterodimers, i.e. CssBA and CssAB, were sufficient to recapitulate the anti-CS6 immune response elicited by immunization with CS6, including generation of functional neutralizing antibodies, as no further enhancement of the response was obtained with the addition of a third CS6 subunit. Our findings here demonstrate the feasibility of including a recombinant CS6-subunit protein into a subunit vaccine strategy against ETEC.
Immunoglobulins are important factors involved in the immunological activity associated with milk and colostrum. Bovine milk contains the immunoglobulins (Ig) IgG, IgA, and IgM and, in contrast to human milk where IgA is the dominant Ig present, IgG is dominant in bovine milk. Bovine colostrum is a rich source of Igs but the concentration decreases rapidly in mature milk, with breed, age, diet, and health status of the cow also influencing Ig concentrations. Igs act as the first defence system against natural infections and diseases and have been used in many forms such as colostrum, milk‐derived products, and concentrated individual antibodies to support human health. Over the past 60 years, a large number of clinical studies have been carried out in animals and humans to establish the efficacy of such products. Cross‐species activity between human and bovine Igs has been reported and bovine milk IgG binds to many gastrointestinal and respiratory pathogens that also infect humans. Advances in separation techniques have made it possible to fractionate and enrich Igs and formulate so‐called hyperimmune colostral or milk preparations. This chapter summarises the evidence supporting the associated health benefits of Igs, gives an overview of the current methods for Ig isolation, and assesses the effects of processing and digestion on Ig functionality.
Eleven published studies of the etiology of travellers' diarrhea (TD) were reviewed define the etiology of TD and to exam newly developed technology such as Real-Time multiplex polymerase chain reaction (PCR) to identify multiple pathogens in one assay to define the cause of TD. Using PCR methods bacterial pathogens were found in 72% of patients acquiring diarrhea in Latin America and in 80% in travellers with illness acquired in Southeast Asia). In these studies, enterotoxigenic Escherichia coli as the predominant pathogen (42% in Latin America and 28% in Southeast Asia). Ciprofloxacin-resistant Campylobacter was commonly associated with TD in Southeast Asia. Multiplex PCR has improved the detection of enteropathogens and allowed better assessment returning travellers hospitalized with TD and those with persistent diarrhea.
In contrast with normal hosts, in which many infectious diseases are usually self-limited, in immunocompromised patients such infections have the potential of becoming serious illnesses characterized by high morbidity and mortality rates. There are also the opportunistic infections that occur almost exclusively in immunocompromised hosts such as patients with HIV disease. Usually widely distributed in the environment, opportunistic pathogens rarely cause serious illness in immunocompetent hosts (1,2). In this context, it is important to note that prompt and correct diagnosis of disseminated opportunistic infections may become crucial as a result of overt differences in the susceptibility to anti-infectious drugs of sometimes closely related opportunistic pathogens. For example, although both Pseudallescheria boydii and Scedosporium prolificans have been recognized as causes of opportunistic hyalohyphomycoses in immunocompromised patients, diagnosis of disseminated disease caused by S. prolificans has been difficult to attain because its signs and symptoms strongly resemble those of pseudallescheriasis and pulmonary aspergillosis. However, early positive identification of S. prolificans may prove to be essential because of its extreme drug tolerance and the related poor prognosis of disseminated disease caused by this fungal pathogen (2).
Enterotoxigenic Escherichia coli (ETEC) can be attributed to around 200 million diarrheal episodes and 380,000 deaths in the developing regions. Travelers' diarrhea occurs in 15–40% of travelers to developing regions with ETEC being the most important etiologic agent. This study aims to describe the distribution of enterotoxins and colonization factor (CF) profiles of ETEC isolates from stool samples of adult travelers acquiring diarrhea in Mexico, Guatemala, and India and a group of children with acute diarrhea in Houston, TX, between 2007 and 2012. The heat-labile/heat-stable (LT/ST) enterotoxins and CFs from 252 patients were determined using polymerase chain reaction assay. Among the 252 ETEC isolates, 15% were LT-only, 58% were ST-only, and 28% produced both LT and ST. The distribution of LT-only (12–15%) and ST-only (55–56%) isolates was similar between Latin American and Indian sites. The most prevalent CF was CS21, expressed in 65% of the isolates followed by CS6 (25%) and CS3 (17%). Among the international travelers, 64% of the ETEC isolates expressed CS21. CS21 was expressed in 46% of isolates from Latin America compared with 96% of isolates from India (P < 0.0001). CS21 was expressed in 85% isolates from Houston children. CS21 was increasingly found in ST-only (P = 0.003) and ST/LT (P = 0.026) ETEC compared with LT-only ETEC. High frequency of finding CS21 among recent isolates of ETEC over a wide geographic distribution warrants additional studies on this CF. Highly conserved CS21 is an important target for potential multivalent ETEC vaccines.
Enterotoxigenic Escherichia coli strains represent the most frequent etiological agent of travelers diarrhea. Challenge studies with several of these strains were undertaken in volunteers to evaluate the mechanisms of disease-induced immunity. Seventeen students and other community volunteers were given 10(6) or 10(8) organisms of E. coli B7A (O148:H28), which produces heat-labile and heat-stable enterotoxins. Ten individuals developed diarrheal illness closely resembling natural travelers diarrhea; of these ten, rises in titer of serum antitoxin and anti-O antibody occurred in eight (80%). Eight of the volunteers who developed diarrhea in the first test agreed to undergo rechallenge 9 weeks later with 10(8) B7A organisms. Only one of these eight "veterans" developed diarrhea versus seven of twelve controls given the same challenge (P = 0.05). Despite clinical protection, all "veterans" excreted B7A after rechallenge. Four controls who developed diarrhea during the homologous B7A rechallenge test were rechallenged 9 weeks later with 10(9) organisms of E. coli strain E2528-C1 (O25:H-), which produces only heat-labile enterotoxin and possesses a different O, H, and pili antigen composition than B7A. Three of four "veterans" and two of six controls developed comparable diarrhea. These studies demonstrate that prior disease due to enterotoxigenic E. coli confers homologous immunity against subsequent challenge, and the operative mechanism apparently is not bactericidal and is not mediated by serum anti-O antibodies. Heterologous protection was not conferred where the only common antigen was heat-labile enterotoxin, indicating that serum infection-derived antitoxin to heat-labile enterotoxin also is not protective.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
Pregnant cows were successfully hyperimmunized with all four human rotavirus serotypes, resulting in a 100-fold increase in neutralizing milk antibody titers over those of controls. Milk antibodies were isolated batchwise from 1,000 kg of pooled milk for the first 10 lactation days, yielding 10 kg of freeze-dried milk immunoglobulin concentrate consisting of 50% bovine milk immunoglobulins. Milk immunoglobulin concentrate showed neutralizing activities against all four human rotavirus serotypes that were 100 times higher than those in pooled human milk samples and 10 times higher than those in a commercial pooled immunoglobulin preparation from pooled human blood serum. In vitro neutralization tests showed that milk immunoglobulin concentrate had powerful antiviral activity, even against very high doses of infectious rotaviruses. Because the technology of the milk immunoglobulin concentrate ensures that it is innocuous and can be used for oral application, it is proposed that milk immunoglobulin concentrate be used to induce passive immunity to infantile rotavirus gastroenteritis.
In a randomized double-blind study, Swiss adults traveling to tropical countries for 12 to 28 days took a solid formulation
of bismuth subsalicylate (1.05 or 2.1 g/day on a twice-daily regimen) or placebo. Efficacy was evaluated in 231 volunteers.
Diarrheal incidence was reduced by 41% in persons taking the high dose (P = 0.007) and by 35% in those taking the low dose
(P = 0.03) with excellent compliance. No serious adverse reactions occurred, but objectionable taste, constipation, and nausea
were seen more frequently with active medication (P = 0.04). Twenty patients provided stool samples: no bacteria were detected
in the 8 volunteers who were on active medication, but various bacteria were found in 5 of the 12 patients who had taken placebo
(P = 0.04).
Serum immunoglobulin G antibodies to purified heat-labile enterotoxin (LT) from human (LTh) and porcine (LTp) Escherichia coli strains and cholera enterotoxin (CT) were measured by an enzyme-linked immunosorbent assay. Sera from patients with LTh E. coli infection showed a prominent response with LTh, an intermediate response with LTp, and a meager response with CT. Of 47 persons with clinical LTh-producing E. coli (herein shortened to LTh E. coli) infections, significant rises in antitoxin were detected against LTh in 36 (77%), against LTp in 30 (64%), and against CT in only 13 (28%) patients; seroconversions also occurred in 11 of 14 (79%) patients with subclinical LTh E. coli infections. In North Americans with experimental LTh E. coli infection, anti-Lth did not remain at high levels for more than 3 months. Persons with cholera manifested antitoxin responses that were similarly potent against all three toxin antigens; in fact, net optical density values were often slightly higher against LTh than against CT. The ratio of CT/LTh ELISA net optical density in convalescent sera proved to be a sensitive means to differentiate LT E. coli from cholera infection. All 11 cholera patients tested had CT/LTh ratios of greater than 0.70, whereas in only 1 of 47 LTh E. coli infections did the ratio exceed that value (it was 0.71) (P less than 0.0000000001). In single serum specimens, a net optical density of greater than or equal to 0.30 against LTh was shown to be a useful cutoff in screening sera for recent LTh E. coli or past cholera infection. The CT/LTh ratio was then used to differentiate definitively. Sera from healthy 3- to 5-year -olds and 15- to 19-year-olds in Maryland, Chile, and Bangladesh were tested against LTh and CT. The serological results fit known epidemiological observations. (i) LTh infections are rare in the United States (only 2 of 60 sera had LTh net optical density values of >/= 0.30. (ii) In contrast, evidence of recent LTh E. coli infections was very common in Chilean (69%) and Bangladeshi (57%) 3- to 5-year-olds and not uncommon in 15- to 19-year-olds (38 and 31%, respectively) in those countries. (iii) Only Bangladeshi sera showed serological evidence of cholera infections (CT/LTh ratios of > 0.70). The immunoglobulin G enzyme-linked immunosorbent assay measuring antibodies to purified LTh and CT represents a practical and effective tool for the serological study of LTh E. coli and cholera diarrheal infections.
Enterotoxigenic Escherichia coli (ETEC) belonging to serogroups O6 and O8 do not possess the H-10407-type colonization factor antigen (CFA/I). However, these frequently isolated ETEC were found to possess a second and distinct heat-labile surface-associated colonization factor antigen, termed CFA/II. Whereas CFA/I mediates mannose-resistant hemagglutination of human group A erythrocytes, CFA/II does not. CFA/II mediates mannose-resistant hemagglutination of bovine erythrocytes, and mannose-resistant hemagglutination is rapid only at reduced temperature (4 degrees C). Because CFA/II, like CFA/I, is spontaneously lost by many ETEC isolates in the laboratory, it was possible to produce specific anti-CFA/II serum by preparing antiserum against living cells of a prototype strain (PB-176) and adsorbing this serum with living and heat-treated cells of its CFA/II-negative derivative strain PB-176-P. This serum, which neutralized the colonization factor activity of CFA/II-positive strains in infant rabbits, was employed to confirm the presence of CFA/II on ETEC which exhibited mannose-resistant hemagglutination of bovine but not human erythrocytes. CFA/II, like CFA/I, mediates adherence of the bacteria to the mucosal surface of the small intestine, as demonstrated by indirect immunofluorescence. CFA/II appears to be an important virulence factor for humans since CFA/II-positive ETEC are frequently isolated from diarrhea cases, particularly travelers' diarrhea, in Mexico; these ETEC were not uncommon in a collection of isolates from Bangladesh. The O6:H16 strain of ETEC responsible for an outbreak of diarrhea in the United States was also shown to be CFA/II positive. CFA/I and CFA/II were never found on the same serotypes of ETEC, but 98% of the heat-stable and heat-labile enterotoxin-producing ETEC belonging to the frequently isolated serogroups O6, O8, O15, O25, O63, and O78 were positive for either CFA/I or CFA/II.
In a group of 133 United States students studied for 18 days after arriving in Mexico, diarrhea developed in 38 (29 per cent). Diarrhea rarely began before the fourth day, and the mean onset was 13 days after arrival. Symptoms lasted an average of 3.4 days but persisted in 21 per cent of sick students. Heat-labile enterotoxin-producing Escheria coli was found in the stools of 72 per cent of sick and 15 per cent of healthy students. None had heat-labile Esch. coli when they entered Mexico. The incubation period was short, generally 24 to 48 hours, and the carrier state was five days or less in 82 per cent of students surveyed. Entamoeba histolytica was found in 6 per cent of cases of diarrhea, but not salmonella, shigella or penetrating Esch. coli. These studies suggest that approximately 70 per cent of travelers' diarrhea in Mexico is associated with heat-labile toxigenic strains of Esch. coli.
Bovine serum IgG1, colostral IgG1 and serum IgG2 with anti-ferritin activity were digested with pepsin or trypsin. Their fragments were characterized by immunoelectrophoresis, gel electrophoresis and gel filtration; their ferritin-binding ability was determined. The kinetics of proteolysis were established by measuring the appearance of free amino groups. No differences were observed between serum and colostrum IgG1. IgG1 was more susceptible to pepsin, and IgG2 to trypsin. This became evident from both the amount of intact IgG determined by gel electrophoresis, immunoelectrophoresis or gel filtration, and from the kinetics of the appearance of amino groups. A model is presented to explain the size, mobilities and properties of the obtained fragments.
We performed a randomized double-blind study to determine the efficacy of doxycycline (100 mg daily) in preventing travelers' diarrhea among 39 Peace Corps volunteers during their first five weeks in Kenya. The volunteers took either doxycycline or placebo for three weeks and were observed for an additional two weeks. Nine of 21 taking placebo and one of 18 taking doxycycline had travelers' diarrhea during the treatment period (P = 0.012). The protection seemed to persist for at least one week after the drug was stopped. Enterotoxigenic Escherichia coli was the only pathogen isolated from the placebo group, but was not detected in persons taking doxycycline. None of these organisms were resistant to doxycycline or tetracycline, whereas resistance to tetracyclines and other antibiotics was common among the nonenterotoxigenic Esch. coli. We conclude that doxycycline effectively prevented most episodes of travelers' dirrhea.
1-week-old rats were inoculated orally with a strain of E. coli (serotype 078) isolated from the blood of a newborn baby who had died of septicemia. During the 3 weeks following inoculation, approximately 50% of the animals died of septicemia and 60% of the surviving rats had pathogenic bacteria in their rectum. Some of the surviving rats were severely impaired in their development. Autopsy showed evidence of active intestinal infection localized mainly in the ileum and cecum. A rabbit anti-E. coli (strain 23) serum (agglutinating titer: 1/2,500) afforded 100% protection when as little as 0.03 mg of serum protein per gram of rat body weight was orally administered in a single dose. The immune serum had an effect both on the mortality rate and on the growth of the rats. However, it never affected the survival of pathogenic bacteria in the rectum, even when administered at a daily dose of 1.5 mg of serum protein per gram of rat body weight on 4 consecutive days. The immune rabbit serum had only a weak bactericidal effect in vitro. The hemagglutination test showed the presence in the immune serum of antibodies against the fimbriae of the pathogenic E. coli strain (titer: 1/1,000). The role of antibody in inhibiting the adherence of bacteria to epithelial cells and/or their progression across the mucous layer are discussed as possible immune mechanisms in the intestinal lumen.
A milk immunoglobulin concentrate (MIC) containing antibodies to enteropathogenic E. coli strains was prepared by hyperimmunisation of pregnant cows and using the milk obtained during the first 6 to 8 days of lactation. The sterile concentrate contained 70 to 80% protein and 35 to 40% immunoglobulin. The antibacterial activity was measured by bacterial passive agglutination, bacteriostatic activity in vitro, phagocytic clearance in vivo, and a protection test in mice. Though differences in titers were observed, adequate immunologic activity was demonstrated by these tests. Clinical studies were performed with 60 patients (aged 10 days to 18 months) suffering from diarrhoea with isolation of enteropathogenic E. coli. They were treated for 10 days with MIC and stool cultures were done prior to, during, and 2, 3 and 4 days after termination of treatment. Among 51 patients infected with E. coli strains incorporated in the vaccine, stool cultures became negative in 43 (84.3%) after treatment with MIC and 8 remained positive. Nine patients infected with strains O 78:K80(B-) and O 114:K--(B-)--which were not included in the vaccine used for immunisation--served as controls. Only one patient in this group became negative. If all patients receiving antibiotics for non intestinal infections during the treatment period are omitted the results remained unaltered: MIC was effective in 32 out of 38 patients (84.2%). These differences were highly significant. These results provide evidence that treatment with specific MIC is effective in eliminating enteropathogenic E. coli from the intestine.
PIP
In an effort to determine the relation of breast versus bottle feeding to hospitalization for gastroenteritis, children hospitalized for gastroenteritis at the Kaiser-Permanente Medical Center in California were compared with a larger normal population of children discharged from the Center's nursery, for incidence of breast feeding. All infants under 12 months of age admitted to the hospital with acute gastroenteritis between January 1, 1973 and December 31, 1975 were identified. All infants had an acute onset of diarrhea and weight loss, some with vomiting. Variable degrees of clinical dehydration also occurred. 107 infants were admitted during the period of study. The type of feeding--breast or bottle--was obtained from the patients' admission history, nursery records, outpatient clinic charts, or by direct contact with parents. Of the 197 patients admitted with acute gastroenteritis during the study period, only 1 was being breast fed at the time of admission. 40 infants were under 6 months of age. Breast feeding accounted for 28 percent of the infant-months in the 0-6 month age group. The lower than predicted incidence of acute gastroenteritis in the breast fed infants was statistically significant. Although about 1/3 of the bottle fed infants had started breast feeding at birth, all had been switched to the bottle at least 1 month prior to hospitalization. The study data strongly indicate that breast feeding plays a major role in protection against intestinal infections.
We conducted a prospective study of travelers' diarrhea on 73 physicians and 48 family members attending a medical congress in Mexico City, in October, 1974. Fecal and blood specimens were collected before, during and after their visit and examined for enteric bacterial pathogens, viruses and parasites. In 59 (49 per cent) participants travelers' diarrhea developed. Median duration of illness was five days. Onset occurred a median of six days after arrival. An etiologic agent was found in 63 per cent of ill participants. Enterotoxigenic Escherichia coli of different, non-"enteropathogenic" serotypes was the most common cause; other responsible pathogens included salmonellae, invasive Esch. coli., shigellae, Vibrio parahaemolyticus, Giardia lamblia and the human reovirus-like agent. Consumption of salads containing raw vegetables was associated with enterotoxigenic Esch. coli infection (P = 0.014). Travelers' diarrhea in Mexico is a syndrome caused by a variety of pathogens, the most common of which is enterotoxigenic Esch. col.
One-half of the healthy newborn infants at a rural medical center were initially breast fed; the proportion declined to 4% by one year of age. Breast-feeding was associated with significantly less illnes during the first year, especially if continued beyond 41/2 months of age. Breat-fedding was associated with a higher level of parental education and, by inference, higher socioeconomic status. The health advantage of breast-feeding was still evident after controlling for parental educational status. In better educated families the difference in significant illness between infants who were artifically fed and those who were breast fed for prolonged periods of time was two- to threefold.
PIP
A study was conducted into the relative morbidity among breast- and bottle-fed infants in a rural area in New York state. The records of 326 infants born at the Mary Imogene Bassett Hospital in Cooperstown, New York, were reviewed. Of these, 253 were seen regularly at the pediatric clinic during the 1st year of life. 1/2 of the newborns were initially breast fed; the proportion dropped to 4% by the end of 1 year. Breast fed babies suffered significantly less illness during the 1st year, especially when the breastfeeding was prolonged beyond 4 1/2 months. The incidence of breast feeding was significantly associated with higher educational levels for both mothers and fathers and with increased maternal age. The health advantages of breast feeding, however, were independent of educational levels and, by inference, the family's socioeconomic status.
With the advent of rapid and convenient means of transportation, millions of persons travel each year from industrialized
to developing countries in the tropics and subtropics. These travelers are at risk for a variety of infectious diseases that
are endemic in these areas; the most frequently occurring of these is diarrhea. Studies of groups of travelers to Latin America
and Africa have found that approximately one-half develop diarrhea during their stay abroad. Etiologic investigations of these
illnesses have demonstrated that the important agents that cause travelers' diarrhea are similar to those that cause diarrhea
in children in the developing countries. One-third of the cases are associated with enterotoxin-producing strains of Escherichia coli. Smaller proportions appear to be due to rotavirus, Norwalk virus, Shigella, Salmonella, Giardia lamblia, and Entamoeba histolytica. Although they have not been fully evaluated in travelers' illnesses in Latin America or Africa, Campylobacter jejuni, Aeromonas hydrophila, other viruses, and Cryptosporidium probably cause some of the currently unexplained cases of diarrhea.
The use of a concentrate containing milk immunoglobulins prepared from rotavirus-hyperimmunized cows (neutralization titer,
1:6,000 for a 10% solution) to treat infants hospitalized for acute rotavirus gastroenteritis resulted in a significant (P = .008) reduction in the duration of excretion of virus. Stool samples from treated infants showed the presence of bovine
milk immunoglobulins in 47% of cases and of neutralizing activity in 43% (mean neutralization titer, 1:48); stool samples
from control infants showed neutralizing activities in only 3% of cases (neutralization titers, <1:20). Immunoelectrophoresis
of stool extracts revealed fragment A, a bovine analogue of F(ab′)2 or Fab, as the major product of in vitro and in vivo digestion of the immunoglobulins. Cessation of excretion of virus correlated
with the appearance of neutralizing activities in 19 of 25 infants. Only concentrate-treated infants with high neutralizing
activity in stools showed a statistically significant reduction in duration of excretion of virus; this duration in concentrate-treated
infants with low neutralizing activity was comparable with controls.
Antimicrobial agents are commonly used therapeutically and prophylactically for travelers' diarrhea. Resistance of enteric
pathogens to these agents may prevent the success of such therapy, with the result depending upon the level of resistance
and the drug concentrations achieved in the gastrointestinal tract. Data from a number of geographic locations were collected
in order to determine whether consistent trends exist and whether predictions can be made regarding the susceptibility of
various enteric pathogens worldwide. These data showed marked variability in the prevalence of resistance. Among Shigella, the percentage of strains resistant to commonly used agents varied within the following ranges: ampicillin, 7% (Dacca) to
87% (Thailand); tetracycline, 11% (Sri Lanka) to 91% (Mexico); and trimethoprim-sulfamethoxazole, 0 (Dacca, 1980) to 55% (Dacca,
1984). Resistance in Salmonella strains showed a similar marked variability. Few strains of enterotoxigenic Escherichia coli (⩽10%) were resistant to trimethoprim-sulfamethoxazole. Relatively recent isolates of all pathogens examined tended to be
more resistant than earlier isolates to trimethoprim-sulfamethoxazole as well as to other agents.
Each of 433 adults traveling to Guadalajara, Mexico, from the United States during summer months was enrolled in one of four
clinical trials of the protective effect of antimicrobial agents against travelers' diarrhea. Only one (2%) of 57 subjects
taking trimethoprimsulfamethoxazole (160 mg/800 mg daily) experienced diarrhea during a two-week study, whereas eight (14%)
of 58 subjects taking trimethoprim alone (200 mg daily) and 10(33%) of 30 taking placebo developed illness (P < .05 and P < .0001, respectively). Diarrhea occurred significantly less frequently among subjects receiving trimethoprim than among
placebo recipients (P <.05). None of 11 students given bicozamycin (500 mg four times daily) developed diarrhea during a three-week study, whereas
10 (53%) of 19 placebo recipients became ill (P = .003). Four (7%) of 54 subjects receiving norfloxacin (400 mg daily) experienced diarrhea during a two-week study; in contrast,
34 (60%) of 57 placebo recipients developed diarrhea (P < .0001). The various antimicrobial agents prevented illness due to enterotoxigenic Escherichia coli and Shigella as well as that unassociated with a pathogen. The drugs were well tolerated. Current evidence suggests that trimethoprim-sulfamethoxazole
is the optimal antimicrobial agent available for prophylaxis of travelers' diarrhea.
This paper summarizes the published controlled studies of the prophylaxis of travelers' diarrhea in which the following drugs
have been used: neomycin, nonabsorbable sulfonamides, Streptotriad, doxycycline, erythromycin, and mecillinam. These studies
have shown that antimicrobial prophylaxis can be highly effective in preventing episodes of travelers' diarrhea. The protection,
however, lasts only as long as the drugs are being taken; there is no evidence that subclinical infections occur while the
drug is being taken. None of the data suggest that antimicrobial prophylaxis increases the probability of infection with drug-resistant
enteric pathogens. In these studies few subjects have experienced adverse drug reactions; however, the number of subjects
has been too small to allow determination of accurate incidence data. Antimicrobial prophylaxis for travelers' diarrhea can
be effectively and safely used on an individualized basis for persons traveling to areas of high risk.
Travelers' diarrhea in Asia has been studied among Peace Corps volunteers in Thailand, Japanese travelers, foreign residents
in Bangladesh, guests in hotels, and members of various tour groups. Rates of diarrheal attack of >50% during four- to six-week
sojourns were reported for these groups. Among travelers with diarrhea, the most commonly isolated pathogen was enterotoxigenic
Escherichia coli (20%–34%), followed by Salmonella (11%–15%), Shigella (4%–7%), Campylobacter (2%–5%), and Vibrio parahaemolyticus (1%–13%). In 9%–22% of diarrheal episodes, multiple pathogens were recovered. Among Japanese travelers, Salmonella was more commonly acquired in the Far East; Shigella and Campylobacter, in the Indian subcontinent; and V. parahaemolyticus, in Southeast Asia. Aeromonas hydrophila and Plesiomonas shigelloides were commonly isolated from ill travelers in Thailand but less frequently from other travelers. Protozoa and Vibrio species other than V. parahaemolyticus were isolated in <5% of episodes.
A simple and reproducible two-stage method for conjugating antibody globulin to ferritin by means of glutaraldehyde (GA) is described. The antibody activity of the conjugate is significantly higher than that of an immunoferritin conjugate obtained by a one-stage method. A further advantage of the two-stage method is that it does not lead to undesirable large aggregates.
Chicken, sheep and rabbit erythrocytes were stabilized with formaldehyde, pyruvic aldehyde or both, for use in passive hemagglutination reactions. Bovine serum albumin, insoluble BSA, ovalbumin, rabbit γ-globulin, chicken γ-globulin, bovine γ-globulin, E. coli endotoxin, acetylated endotoxin, de-esterified endotoxin, de-esterified and bromacetylated endotoxin, succinylated endotoxin and synthetic polyglucose were found to adsorb firmly on the stabilized cells.
For optimal coating of stabilized cells with antigens, critical factors included pH, antigen concentration and the duration of contact with antigen. These conditions differed somewhat from one antigen to another.
Coated cells could be stored frozen or lyophilized.
Approximately 1 × 10-7 to 1 × 10-8 mg of anti-BSA antibody were detected by using rabbit erythrocytes coated with BSA. This level of sensitivity compares favorably with tannic acid or BDB procedure for cell coating.
Antiglobulin tests were applicable in all of the cases tested and were found to increase the hemagglutination titers 10- to 30-fold.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The usual methods for identifying enterotoxigenic Escherichia coli (ETEC) strains involve testing for production of heat-labile enterotoxins. To simplify the identification of ETEC, antisera against common ETEC O serogropus were used to identify ETEC in the stools from 618 patients with acute diarrhoea and dehydration (greater than or equal to 5% loss of body-weight) receiving treatment at a hospital in Dacca, Bangladesh. Compared with enterotoxin testing the antisera had a sensitivity of 64%, a specificity of 96%, and a predictive accuracy of 89%. These antisera may be useful in the identification ETEC in clinical laboratories which are unable to perform toxin testing and should be evaluated in other geographical areas.
We performed a prospective study to examine whether the IgA antibodies against cholera that are present in breast milk protect breast-fed infants and children against colonization with Vibrio cholerae 01 and disease. Among families of patients with cholera, we collected breast milk from mothers who had not had diarrhea in the previous week and monitored them and their breast-fed children for cholera colonization and diarrhea for 10 days. Breast milk was assayed for IgA antibodies to cholera toxin and lipopolysaccharide. Ninety-three mother--child pairs were studied; 30 infants became colonized with V. cholerae 01 and disease developed in 19. There were no differences between the antibody levels in milk fed to children who became colonized and in milk fed to children who did not. However, among the children who became colonized, those who had diarrhea drank breast milk containing significantly lower levels of both kinds of cholera antibodies than were present in the milk consumed by children who had no symptoms. We conclude that breast-milk antibodies against cholera do not appear to protect children from colonization with V. cholerae 01 but do protect against disease in those who are colonized.
The efficacy of a daily dosage regimen of subsalicylate bismuth in preventing or reducing the severity of diarrhea among young healthy adults was evaluated in a double-blind, randomized, placebo-controlled trial. Diarrhea developed in 14 (23%) of 62 students receiving subsalicylate bismuth compared with 40 (61%) of 66 students taking a placebo. The protective effect of subsalicylate bismuth was apparent within a day or two of the study onset and became more obvious as the number of days at risk increased. The students treated with subsalicylate bismuth experienced fewer intestinal complaints and were less likely to pass soft or watery stools of any number. Once diarrhea occurred, enteropathogens were less commonly identified in stools of students receiving subsalicylate bismuth (33%) compared with placebo (71%). Subsalicylate bismuth was well tolerated by students during the 21-day trial.
The scanty epidemiologic evidence available suggests that enterotoxigenic Escherichia coli (ETEC) are usually spread by contaminated food and water vehicles; little is known of the risk of secondary spread by contact transmission. Studies carried out in a 22-bed isolation Ward at the U. of Maryland Hospital gave the opportunity to determine whether individuals excreting ETEC, with and without diarrhea, would transmit the pathogen to controls living in close contact. In one combined study, seven volunteers who had ingested 10(8) virulent ETEC (strain H10407), were housed day and night for two weeks with eight other volunterrs participating in an intranasal attenuated influenza vaccine study. In a second study, four persons ingesting 10(8) ETEC (strain 214-4) lived with 13 who were inoculated with intranasal influenza vaccine. The individuals in the E. coli and influenza groups were randomly mixed in bedrooms and shared bathrooms, dining and recreation areas of the ward. Seven persons who ingested ETEC developed diarrhea; all 11 excreted the pathogen (10(7)-10(9) organisms/gm or ml of stool), and 10 had significant rises in anti-O or antitoxin antibody. In contrast, no influenza vaccinees, despite close sharing of facilities, developed diarrhea, excreted ETEC or had rises in antibody to E. coli antigens. These data suggest that ETEC are not readily transmitted to healthy adults by direct person-to-person contact. Precautions to prevent contamination of shared food sources would appear to be the most rational intervention to avoid secondary cases of ETEC diarrhea.
The effect of daily administration of trimethoprim (TMP), trimethoprim--sulfamethoxazole (TMP--SMX), or placebo on aerobically grown fecal gram-negative bacteria was monitored in 136 students from the United States during a two-week diarrhea-prevention study in Mexico. Unlike patients in other studies with these agents, who had urinary-tract infection or granulocytopenia, most persons in this study had no change in total fecal Enterobacteriaceae and had high-level TMP and SMX resistance in virtually all these strains. Escherichia coli was the predominant TMP-resistant organism isolated; 96 per cent of 165 TMP-resistant Esch. coli isolates were resistant to at least four antimicrobial agents, and 25 per cent were resistant to seven. TMP resistance was transferable in 40 ot 100 strains tested. Despite the lack of TMP resistance in other studies of prophylaxis, our results clearly demonstrate the remarkable capacity for emergence and dissemination of resistance to this agent.
We examined 205 enterotoxigenic strains of Escherichia coli for colonization factor antigens (CFA) I and II, using an immunodiffusion technique with specific antisera. A total of 36 strains of serogroups O63, O78, O114, O128, and O153 and 1 rough strain possessed CFA/I and gave a single precipitin line; 47 strains of serogroups O6, O8, O80, and O115 possessed CFA/II. The latter strains gave a major precipitin line (component 3) when tested with specific antisera prepared against strain E1392 or PB-176 (both E. coli O6.H16; biotype A). However, all 16 strains of E. coli O6.H16 belonging to biotype A gave a second precipitin line (component 1) when tested with both antisera. When CFA/II-positive strains were tested with a specific antiserum prepared against E. coli O6.H16 strains of biotype B or C, all strains gave component 3, but 16 of 17 strains of E. coli O6.H16 belonging to biotype B, C, or F gave a second precipitin line (component 2) not given by strains of biotype A. CFA/II-positive strains of serogroups other than O6 gave only component 3 in tests with all specific antisera. Nine enterotoxigenic strains of serotypes O7, O15, O25, O115, and O128 gave mannose-resistant hemagglutination of human or calf erythrocytes but lacked CFA/I or CFA/II. Although mannose-resistant hemagglutination was common in non-enterotoxigenic strains of E. coli, none of the non-enterotoxigenic strains possessed CFA/I or CFA/II; these strains included fecal strains of serogroups O6, O8, O63, and O78, fecal strains of enteropathogenic serogroups, and strains from extraintestinal sources.
One hundred forty-seven students from the United States were given trimethoprimsulfamethoxazole (TMP-SMZ; 160 mg of TMP and
800 mg of SMZ) twice daily for 21 days after their arrival in Mexico. They were watched for the development of diarrhea during
the 21 days and for an additional eight days after the termination of TMP-SMZ therapy. Diarrheal illness occurred in 11 (16%)
of 67 students taking TMP-SMZ and in 44 (55%) of 80 students receiving a placebo; the differences were significant (P < 0.001).
Milder symptoms not quite satisfying the criteria for illness were also less common in the group receiving the active drug:
23% vs. 69% (P < 0.001). The drug appeared to prevent infection by fully virulent enterotoxigenic Escherichia coli (strains producing heat-stable toxin and those producing heat-labile toxin) and perhaps by shigella strains. During the eight
days of follow-up after drug administration, 14 students (26%) who had taken TMP-SMZ and two (2.5%) who had taken the placebo
experienced diarrhea (P < 0.05). Twelve subjects (14%) in the TMP-SMZ group and one subject (1%) in the placebo group developed
a generalized rash that necessitated discontinuance of the drug. The eruption occurred after 9–16 days of drug administration
(mean, 10 days). This study shows that TMP-SMZ taken twice daily can prevent travelers' diarrhea for up to three weeks. Diarrhea
will develop, however, if the drug is stopped while the risk remains.
Bovine milk immunoglobulins (Ig), their possible utilization in industrially prepared infant's milk formulae
- H Hilpert
- H Gerber
- H Amster
Preparation of a milk immunoglobulin concentrate from cow's milk
- H Hilpert