Occurrence of Cryptosporidium and other enteropathogens and their association with diarrhea in dairy calves of Buenos Aires province, Argentina.

Cryptosporidiosis of neonatal dairy calves causes diarrhea, resulting in important economic losses. In Argentina, prevalence values of Cryptosporidium spp. and other enteropathogens such as group A rotavirus (RVA), bovine coronavirus (BCoV) and enterotoxigenic Escherichia coli (ETEC, endotoxin STa+), have been independently studied in different regions. However, an integrative epidemiological investigation on large-scale farms has not been carried out. In this study, fecal samples (n = 908) were randomly collected from diarrheic and healthy calves from 42 dairy farms, and analyzed for the presence of Cryptosporidium spp., RVA, BCoV, ETEC (STa+) and Salmonella spp. In all sampled dairy farms, dams had been vaccinated against rotavirus and gram-negative bacteria to protect calves against neonatal diarrhea. The proportion of calves shedding Cryptosporidium spp., RVA, and BCoV in animals younger than 20 days of age were 29.8%, 12.4% and 6.4%, and in calves aged between 21 and 90 days, 5.6%, 3.9%, and 1.8%, respectively. ETEC was absent in the younger, and occurred only sporadically in the older group (0.9%), whereas Salmonella spp. was absent in both. The observed sporadic finding or even absence of bacterial pathogens might be explained by the frequent use of parenteral antibiotics in 25.3% and 6.5% of the younger and the older group of calves, respectively, within 2 days prior to sampling and/or vaccination of dams against gram-negative bacteria. Diarrhea was observed in 28.8% (95% CI, 24.7-32.8%) of the younger calves and 11.7% (95% CI, 9.1-15.5%) of the older calves. Importantly, Cryptosporidium spp. (odds ratio (OR) = 5.7; 95% CI, 3.3-9.9; p < 0.0001) and RVA (OR = 2.5; 95% CI, 1.2-5.1; p < 0.05) were both found to be risk factors for diarrhea in calves younger than 20 days old. Based on its high prevalence and OR, our results strongly suggest that Cryptosporidium spp. is the principal causative factor for diarrhea in the group of neonatal calves, whereas RVA seems to play a secondary role in the etiology of diarrhea in the studied farms, with about three-times lower prevalence and a half as high OR. Furthermore, a coinfection rate of Cryptosporidium spp. and RVA of 3.7% was observed in the group of younger calves, which strengthens the assumption that these events are independent. In contrast, due to a low infection rate of enteropathogens in older calves, mixed infection (<< 1%) was virtually absent in this group.

Early immune innate hallmarks and microbiome changes across the gut during Escherichia coli O157: H7 infection in cattle.

The zoonotic enterohemorrhagic Escherichia coli (EHEC) O157: H7 bacterium causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans. Cattle are primary reservoirs and EHEC O157: H7; the bacteria predominately inhabit the colon and recto-anal junctions (RAJ). The early innate immune reactions in the infected gut are critical in the pathogenesis of EHEC O157: H7. In this study, calves orally inoculated with EHEC O157: H7 showed infiltration of neutrophils in the lamina propria of ileum and RAJ at 7 and 14 days post-infection. Infected calves had altered mucin layer and mast cell populations across small and large intestines. There were differential transcription expressions of key bovine ß defensins, tracheal antimicrobial peptide (TAP) in the ileum, and lingual antimicrobial peptide (LAP) in RAJ. The main Gram-negative bacterial/LPS signaling Toll-Like receptor 4 (TLR4) was downregulated in RAJ. Intestinal infection with EHEC O157: H7 impacted the gut bacterial communities and influenced the relative abundance of Negativibacillus and Erysipelotrichaceae in mucosa-associated bacteria in the rectum. Thus, innate immunity in the gut of calves showed unique characteristics during infection with EHEC O157: H7, which occurred in the absence of major clinical manifestations but denoted an active immunological niche.

Efficacy of a recombinant Intimin, EspB and Shiga toxin 2B vaccine in calves experimentally challenged with Escherichia coli O157:H7.

Escherichia coli O157:H7 is a zoonotic pathogen of global importance and the serotype of Shiga toxin-producing E.coli (STEC) most frequently associated with Hemolytic Uremic Syndrome (HUS) in humans. The main STEC reservoir is cattle. Vaccination of calves with the carboxy-terminal fraction of Intimin γ (IntC280) and EspB can reduce E.coli O157:H7 fecal shedding after experimental challenge. Shiga toxin (Stx) exerts local immunosuppressive effects in the bovine intestine and Stx2B fused to Brucella lumazine synthase (BLS-Stx2B) induces Stx2-neutralizing antibodies. To determine if an immune response against Stx could improve a vaccine's effect on fecal shedding, groups of calves were immunized with EspB + IntC280, with EspB + IntC280 + BLS-Stx2B, or kept as controls. At 24 days post vaccination calves were challenged with E.coli O157:H7. Shedding of E.coli O157:H7 was assessed in recto-anal mucosal swabs by direct plating and enrichment followed by immunomagnetic separation and multiplex PCR. Calves were euthanized 15 days after the challenge and intestinal segments were obtained to assess mucosal antibodies. Vaccination induced a significant increase of IntC280 and EspB specific antibodies in serum and intestinal mucosa in both vaccinated groups. Antibodies against Stx2B were detected in serum and intestinal mucosa of animals vaccinated with 3 antigens. Sera and intestinal homogenates were able to neutralize Stx2 verocytotoxicity compared to the control and the 2-antigens vaccinated group. Both vaccines reduced E.coli O157:H7 shedding compared to the control group. The addition of Stx2B to the vaccine formulation did not result in a superior level of protection compared to the one conferred by IntC280 and EspB alone. It remains to be determined if the inclusion of Stx2B in the vaccine alters E.coli O157:H7 shedding patterns in the long term and after recurrent low dose exposure as occurring in cattle herds.

Immunization of pregnant cows with Shiga toxin-2 induces high levels of specific colostral antibodies and lactoferrin able to neutralize E. coli O157:H7 pathogenicity.

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications.

Immune Response in Calves Vaccinated with Type Three Secretion System Antigens and Shiga Toxin 2B Subunit of Escherichia coli O157:H7.

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo.

Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes.

The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.

Effect of coiled-coil peptides on the function of the type III secretion system-dependent activity of enterohemorragic Escherichia coli O157:H7 and Citrobacter rodentium.

Many animal and human pathogenic Gram-negative bacteria such as Salmonella, Yersinia, enterohemorrhagic Escherichia coli (EHEC), and enteropathogenic Escherichia coli (EPEC) possess a type III secretion system (TTSS) that is used to deliver virulence proteins directly into the host cell. Recent evidence has suggested that CoilA and CoilB, two synthetic peptides corresponding to coiled-coil domains of the translocator protein EspA, are effective in inhibiting the action of TTSS from EPEC. In the current study, the action of these coiled-coil peptides on the TTSS of EHEC O157:H7 and Citrobacter rodentium was examined. CoilA and CoilB showed to be effective in reducing the red blood cell lysis mediated by EHEC O157:H7 and the in vitro secretion of translocator proteins EspB and EspD by EHEC O157:H7 and EspD by C. rodentium. Treatment of mice with CoilA and CoilB peptides prevented colon damage when the animals were inoculated with C. rodentium. Colon samples of the non-treated group showed areas with loss of superficial epithelium, damaged cells, and endoluminal mononuclear inflammatory infiltrate, consistent with histological lesions induced by C. rodentium, whereas mice treated with the synthetic peptides displayed normal surface epithelium showing a similar structure as the uninfected control group. These encouraging results prompt us to test coiled-coil peptides as treatment or vaccines in other models of bacterial infections in future work.

Reduced faecal shedding of Escherichia coli O157:H7 in cattle following systemic vaccination with γ-intimin C280 and EspB proteins.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is the most prevalent EHEC serotype that has been recovered from patients with haemolytic uremic syndrome (HUS) worldwide. Vaccination of cattle, the main reservoir of EHEC O157:H7, could be a logical strategy to fight infection in humans. This study evaluated a vaccine based on the carboxyl-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C280) and EspB, two key colonization factors of E. coli O157:H7. Intramuscular immunization elicited significantly high levels of serum IgG antibodies against both proteins. Antigen-specific IgA and IgG were also induced in saliva, but only the IgA response was significant. Following experimental challenge with E. coli O157:H7, a significant reduction in bacterial shedding was observed in vaccinated calves, compared to control group. These promising results suggest that systemic immunization of cattle with intimin and EspB could be a feasible strategy to reduce EHEC O157:H7 faecal shedding in cattle.

Necrotic enteritis in young calves.

Non-enterotoxin (CPE)-producing Clostridium perfringens type A has been associated with enteritis in calves. Recent evidence has suggested that a novel toxin, named beta2 (CPB2), is implicated in the pathogenesis of this disease, although there is little evidence supporting this. In the current study, the role of C. perfringens type A in an outbreak of enteritis in calves was studied. Two 20-day-old dairy calves exhibiting apathy and reluctance to eat, with paresis of the anterior limbs, were euthanized for postmortem examination. Gross and histological changes compatible with acute enteritis, rumenitis, meningitis, and pneumonia were seen in both calves. Clostridium perfringens type A non-CPE, non-CPB2 was isolated from the abomasum and the small intestine. Escherichia coli ONTH8 (with cdtBIII and f17 virulence genes detected by polymerase chain reaction) was also isolated from the brain, abomasum, and intestine from both calves. All the samples were negative for Salmonella spp. When the C. perfringens strain was inoculated into bovine ligated small and large intestinal loops, cell detachment, erosion, and hemorrhage of the lamina propria were observed, predominantly in the small intestine. The results suggest that non-CPE, non-CPB2 C. perfringens type A is able to induce pathologic changes in the intestine of calves, probably enhanced by other pathogens, such as some pathogenic E. coli strains.

Designed coiled-coil peptides inhibit the type three secretion system of enteropathogenic Escherichia coli.

BACKGROUND: Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are two categories of E. coli strains associated with human disease. A major virulence factor of both pathotypes is the expression of a type three secretion system (TTSS), responsible for their ability to adhere to gut mucosa causing a characteristic attaching and effacing lesion (A/E). The TTSS translocates effector proteins directly into the host cell that subvert mammalian cell biochemistry. METHODS/PRINCIPAL FINDINGS: We examined synthetic peptides designed to inhibit the TTSS. CoilA and CoilB peptides, both representing coiled-coil regions of the translocator protein EspA, and CoilD peptide, corresponding to a coiled-coil region of the needle protein EscF, were effective in inhibiting the TTSS dependent hemolysis of red blood cells by the EPEC E2348/69 strain. CoilA and CoilB peptides also reduced the formation of actin pedestals by the same strain in HEp-2 cells and impaired the TTSS-mediated protein translocation into the epithelial cell. Interestingly, CoilA and CoilB were able to block EspA assembly, destabilizing the TTSS and thereby Tir translocation. This blockage of EspA polymerization by CoilA or CoilB peptides, also inhibited the correct delivery of EspB and EspD as detected by immunoblotting. Interestingly, electron microscopy of bacteria incubated with the CoilA peptide showed a reduction of the length of EspA filaments. CONCLUSIONS: Our data indicate that coiled-coil peptides can prevent the assembly and thus the functionality of the TTSS apparatus and suggest that these peptides could provide an attractive tool to block EPEC and EHEC pathogenesis.

Efficient immune responses against Intimin and EspB of enterohaemorragic Escherichia coli after intranasal vaccination using the TLR2/6 agonist MALP-2 as adjuvant.

Mucosal vaccine formulations based on purified recombinant C280 gamma-Intimin and EspB (Escherichia coli secreted protein B) from enterohaemorragic E. coli co-administered with a pegylated derivative of the TLR2/6 agonist MALP-2 (macrophage-activating lipopeptide) as adjuvant were evaluated in BALB/c mice. After intranasal vaccination, strong humoral and cellular immune responses were observed against C280 gamma-Intimin and EspB. Sera of immunized mice inhibit bacterial haemolytic activity in vitro. Antigen-specific T-cell proliferation, IL-4, IL-2 and IFN-gamma producing cells, and secretory IgA were mostly detected in animals receiving MALP-2 as adjuvant. These results suggest that C280 gamma-Intimin and EspB are good candidate antigens to be incorporated into mucosal vaccines against this important pathogen.

Bovine colostrum contains immunoglobulin G antibodies against intimin, EspA, and EspB and inhibits hemolytic activity mediated by the type three secretion system of attaching and effacing Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is the main cause of hemolytic-uremic syndrome, an endemic disease in Argentina which had an incidence in 2005 of 13.9 cases per 100,000 children younger than 5 years old. Cattle appear to be a major reservoir of EHEC, and a serological response to EHEC antigens has been demonstrated in natural and experimental infections. In the current study, antibodies against proteins implicated in EHEC's ability to form attaching and effacing lesions, some of which are exported to the host cell via a type three secretion system (TTSS), were identified in bovine colostrum by Western blot analysis. Twenty-seven (77.0%) of the 35 samples examined contained immunoglobulin G (IgG) antibodies against the three proteins assayed in this study: EspA, EspB, and the carboxy-terminal 280 amino acids of gamma-intimin, an intimin subtype associated mainly with O157:H7 and O145:H- serotypes. Every colostrum sample was able to inhibit, in a range between 45.9 and 96.7%, the TTSS-mediated hemolytic activity of attaching and effacing E. coli. The inhibitory effect was partially mediated by IgG and lactoferrin. In conclusion, we found that early colostrum from cows contains antibodies, lactoferrin, and other unidentified substances that impair TTSS function in attaching and effacing E. coli strains. Bovine colostrum might act by reducing EHEC colonization in newborn calves and could be used as a prophylactic measure to protect non-breast-fed children against EHEC infection in an area of endemicity.