Baseline analysis of Mycoplasma mycoides subsp. mycoides antigens as targets for a DIVA assay for use with a subunit vaccine for contagious bovine pleuropneumonia.

BACKGROUND: Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia in cattle. A prototype subunit vaccine is being developed, however, there is currently no diagnostic test that can differentiate between infected cattle and those vaccinated with the prototype subunit vaccine. This study characterized Mmm proteins to identify potential antigens for use in differentiating infected from vaccinated animals. RESULTS: Ten Mmm antigens expressed as recombinant proteins were tested in an indirect ELISA using experimental sera from control groups, infected, and vaccinated animals. Data were imported into R software for analysis and drawing of the box and scatter plots while Cohen's Kappa assessed the level of agreement between the Mmm antigens. Two vaccine antigens (MSC_0499 and MSC_0776) were superior in detecting antibodies in sera of animals vaccinated with the subunit vaccines while two non-vaccine antigens (MSC_0636 and LppB) detected antibodies in sera of infected animals showing all clinical stages of the disease. Sensitivity and specificity of above 87.5% were achieved when the MSC_0499 and MSC_0636 antigens were tested on sera from vaccinated and infected animals. CONCLUSIONS: The MSC_0499 and MSC_0776 antigens were the most promising for detecting vaccinated animals, while MSC_0636 and LppB were the best targets to identify infected animals. Further testing of sera from vaccinated and infected animals collected at different time intervals in the field should help establish how useful a diagnostic test based on a cocktail of these proteins would be.

Capsular polysaccharide from Mycoplasma mycoides subsp. mycoides shows potential for protection against contagious bovine pleuropneumonia.

Contagious Bovine Pleuropneumonia (CBPP) is a severe respiratory disease caused by Mycoplasma mycoides subsp. mycoides (Mmm) which is widespread in Africa. The capsule polysaccharide (CPS) of Mmm is one of the few identified virulence determinants. In a previous study, immunization of mice against CPS generated antibodies, but they were not able to prevent multiplication of Mmm in this model animal. However, mice cannot be considered as a suitable animal model, as Mmm does not induce pathology in this species. Our aim was to induce antibody responses to CPS in cattle, and challenge them when they had specific CPS antibody titres similar or higher than those from cattle vaccinated with the live vaccine. The CPS was linked to the carrier protein ovalbumin via a carbodiimide-mediated condensation with 1-ethyl-3(3-imethylaminopropyl) carbodiimide (EDC). Ten animals were immunized twice and challenged three weeks after the booster inoculation, and compared to a group of challenged non-immunized cattle. When administered subcutaneously to adult cattle, the vaccine elicited CPS-specific antibody responses with the same or a higher titre than animals vaccinated with the live vaccine. Pathology in the group of immunized animals was significantly reduced (57%) after challenge with Mmm strain Afadé compared to the non-immunized group, a figure in the range of the protection provided by the live vaccine.

Recombinant Mycoplasma mycoides proteins elicit protective immune responses against contagious bovine pleuropneumonia.

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a devastating respiratory disease mainly affecting cattle in sub-Saharan Africa. The current vaccines are based on live-attenuated Mmm strains and present problems with temperature stability, duration of immunity and adverse reactions, thus new vaccines are needed to overcome these issues. We used a reverse vaccinology approach to identify 66 Mmm potential vaccine candidates. The selection and grouping of the antigens was based on the presence of specific antibodies in sera from CBPP-positive animals. The antigens were used to immunize male Boran cattle (Bos indicus) followed by a challenge with the Mmm strain Afadé. Two of the groups immunized with five proteins each showed protection after the Mmm challenge (Groups A and C; P<0.05) and in one group (Group C) Mmm could not be cultured from lung specimens. A third group (Group N) showed a reduced number of animals with lesions and the cultures for Mmm were also negative. While immunization with some of the antigens conferred protection, others may have increased immune-related pathology. This is the first report that Mmm recombinant proteins have been successfully used to formulate a prototype vaccine and these results pave the way for the development of a novel commercial vaccine.

Experimental evaluation of inactivated and live attenuated vaccines against Mycoplasma mycoides subsp. mycoides.

The current control method for contagious bovine pleuropneumonia (CBPP) in Africa is vaccination with a live, attenuated strain of Mycoplasma mycoides subsp. mycoides (Mmm). However, this method is not very efficient and often causes serious adverse reactions. Several studies have attempted to induce protection using inactivated mycoplasma, but with widely contradictory results. Therefore, we compared the protective capacity of the live T1/44 vaccine with two inactivated preparations of Mmm strain Afadé, inoculated with an adjuvant. Protection was measured after a challenge with Afadé. The protection levels were 31%, 80.8% and 74.1% for the formalin-inactivated, heat-inactivated and live attenuated preparations, respectively. These findings indicate that low doses of heat-inactivated Mmm can offer protection to a level similar to the current live attenuated (T1/44) vaccine formulation.

Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.

Inverse relationship between heat stable enterotoxin-b induced fluid accumulation and adherence of F4ac-positive enterotoxigenic Escherichia coli in ligated jejunal loops of F4ab/ac fimbria receptor-positive swine.

Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) increases bacterial adherence to porcine enterocytes in vitro and enhances small intestinal colonization in swine. Heat-stable enterotoxin-b (STb) is not known to affect colonization; however, through an induction of net fluid accumulation it might reduce bacterial adherence. The relationship between fluid accumulation and bacterial adherence in jejunal loops inoculated with ETEC strains that produce LT, STb, both, or neither toxin was studied. Ligated jejunal loops were constructed in weaned Yorkshire pigs in two independent experiments (Exp. 1, n=5, 8-week-old; Exp. 2, n=6, 6-8-week-old). Each pig was inoculated with six F4ac(+)E. coli strains: (1) LT(+), STb(+) parent (WAM2317); (2) STb(-) (ΔestB) mutant (MUN297); (3) MUN297 complemented with STb (MUN298); (4) LT(-) STb(-) (ΔeltAB ΔestB) mutant (MUN300); (5) MUN300 complemented with LT (MUN301); and (6) 1836-2 (non-enterotoxigenic, wild-type). Pigs were confirmed to be K88 (F4)ab/ac receptor-positive in Exp. 2 by testing for intestinal mucin-type glycoproteins and inferred to be receptor-positive in both Exp. 1 and 2 based on histopathologic evidence of bacterial adherence. Strains that produced STb induced marked fluid accumulation with the response (ml/cm) to WAM2317 and MUN298 significantly greater than that to the other strains (P<0.0001). Conversely, bacterial adherence scores based on immunohistochemistry and CFU/g of washed mucosa were both lowest in the strains that expressed STb and highest in those that did not. For the two experiments combined, the Pearson correlation coefficient (R) between fluid volume (ml/cm) and log CFU per gram was -0.57021 (P<0.0001); R(2)=0.3521 (n=197). These results support the hypothesis that enterotoxin-induced fluid accumulation flushes progeny organisms into the lumen of the bowel, thereby increasing the likelihood of fecal shedding and transmission of the pathogen to new hosts.

Immunization of chickens with Salmonella enterica subspecies enterica serovar Enteritidis pathogenicity island-2 proteins.

Several structural components of the type III secretion systems (T3SS) encoded by Salmonella pathogenicity island (SPI)-1 and SPI-2 are exposed to the host's immune system prior to/during the infection/invasion process, making them potential vaccine candidates. In this study we evaluated whether chickens vaccinated with SPI-2 T3SS components could mount a significant humoral immune response (as measured by serum IgG titres) and whether these antibodies could be transferred to progeny (as measured by egg yolk IgG titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with SE. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgG that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered Salmonella enterica subspecies enterica serovar Enteritidis (SE) per bird in most situations.

Evaluation of Salmonella enterica serovar Enteritidis pathogenicity island-1 proteins as vaccine candidates against S. Enteritidis challenge in chickens.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of gastrointestinal disease in humans worldwide, which mainly results from the consumption of contaminated poultry meat and eggs. Vaccination of chickens is an important strategy to lower the prevalence of Salmonella in poultry flocks. The S. Enteritidis type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island-1 (SPI-1) is an important virulence factor that plays a role in invasion and systemic spread in chickens. In this manuscript, we evaluated the efficacy of SPI-1 proteins as vaccine candidates for protection against S. Enteritidis oral challenge. Our results demonstrate for the first time that SPI-1 T3SS proteins elicit antigen specific IgG antibody responses in chickens. In one study we show that vaccination with the aforementioned proteins reduces the levels of S. Enteritidis in the liver, but not in the spleen and cecal contents of chickens. However, a second study shows that vaccination of hens with SPI-1 proteins using a seeder model of infection does not affect the levels of S. Enteritidis in the cecal contents or internal organs of progeny obtained from these hens. Hence, the SPI-1 proteins, in conjunction with other proteins, may form important components of subunit vaccines used for protection against colonization by S. Enteritidis in poultry.

Salmonella enterica serovar Enteritidis tatB and tatC mutants are impaired in Caco-2 cell invasion in vitro and show reduced systemic spread in chickens.

Salmonella enterica subsp. enterica serovar Enteritidis is a leading causative agent of gastroenteritis in humans. This pathogen also colonizes the intestinal tracts of poultry and can spread systemically in chickens. Transfer to humans usually occurs through undercooked or improperly handled poultry meat or eggs. The bacterial twin-arginine transport (Tat) pathway is responsible for the translocation of folded proteins across the cytoplasmic membrane. In order to study the role of the Tat system in the infection and colonization of chickens by Salmonella Enteritidis, we constructed chromosomal deletion mutants of the tatB and tatC genes, which are essential components of the Tat translocon. We observed that the tat mutations affected bacterial cell morphology, motility, and sensitivity to albomycin, sodium dodecyl sulfate (SDS), and EDTA. In addition, the mutant strains showed reduced invasion of polarized Caco-2 cells. The wild-type phenotype was restored in all our Salmonella Enteritidis tat mutants by introducing episomal copies of the tatABC genes. When tested in chickens by use of a Salmonella Enteritidis Delta tatB strain, the Tat system inactivation did not substantially affect cecal colonization, but it delayed systemic infection. Taken together, our data demonstrated that the Tat system plays a role in Salmonella Enteritidis pathogenesis.

Salmonella enterica subspecies enterica serovar Enteritidis Salmonella pathogenicity island 2 type III secretion system: role in intestinal colonization of chickens and systemic spread.

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. For the purposes of our studies we used a chicken isolate of S. Enteritidis (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen-free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type strain Sal18: Sal18 attTn7 : : tet and Sal18 attTn7 : : cat, while the other two groups received the wild-type strain (Sal18 attTn7 : : tet) and one of two mutant strains. From this study, we concluded that S. Enteritidis strains deficient in the SPI-1 and SPI-2 systems were outcompeted by the wild-type strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains: the wild-type strain, and three other strains lacking either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge, we observed a reduced systemic spread of the SPI-2 mutants, but by day 3, the systemic distribution levels of the mutants matched that of the wild-type strain. Based on these two studies, we conclude that the S. Enteritidis SPI-2 T3SS facilitates invasion and systemic spread in chickens, although alternative mechanisms for these processes appear to exist.

Salmonella enterica serovar enteritidis pathogenicity island 1 is not essential for but facilitates rapid systemic spread in chickens.

Salmonella enterica subsp. enterica serovar Enteritidis is a leading cause of human food-borne illness that is mainly associated with the consumption of contaminated poultry meat and eggs. To cause infection, S. Enteritidis is known to use two type III secretion systems, which are encoded on two salmonella pathogenicity islands, SPI-1 and SPI-2, the first of which is thought to play a major role in invasion and bacterial uptake. In order to study the role of SPI-1 in the colonization of chicken, we constructed deletion mutants affecting the complete SPI-1 region (40 kb) and the invG gene. Both DeltaSPI-1 and DeltainvG mutant strains were impaired in the secretion of SipD, a SPI-1 effector protein. In vitro analysis using polarized human intestinal epithelial cells (Caco-2) revealed that both mutant strains were less invasive than the wild-type strain. A similar observation was made when chicken cecal and small intestinal explants were coinfected with the wild-type and DeltaSPI-1 mutant strains. Oral challenge of 1-week-old chicken with the wild-type or DeltaSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection of the liver and spleen was delayed in birds that were challenged with the DeltaSPI-1 strain. These data demonstrate that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of the organism in chickens.

A two-dose regimen of a vaccine against type III secreted proteins reduced Escherichia coli O157:H7 colonization of the terminal rectum in beef cattle in commercial feedlots.

A large-scale clinical vaccine trial of commercially fed cattle was conducted to test the efficacy of a two-dose regimen of a vaccine product against type III secreted proteins of enterohemorrhagic Escherichia coli O157:H7 on the probability to detect the same organism from terminal rectal mucosa (TRM) as a measure of gut colonization. Vaccine was administered to all cattle within treated pens at arrival processing and at reimplant processing. At harvest, TRM was collected from a sample of cattle from within vaccinated and nonvaccinated pens. The TRM were collected by scraping the mucosa of the terminal rectum 3-5 cm proximal to the rectoanal juncture. E. coli O157:H7 was isolated and identified from TRM using standard culture methods involving selective enrichment, immunomagnetic separation, and PCR confirmation. The probability to detect E. coli O157:H7 from TRM was modeled using a generalized linear mixed model with a logit link function and accounting for random effects of pen within feedlot. Seven hundred eighteen cattle were tested from within 21 pens of cattle (11 vaccinated and 10 not vaccinated) representing 3683 cattle. E. coli O157:H7 was cultured from 68 of 718 (9.5%) TRM samples. Eleven of 382 (2.9%) vaccinated cattle and 57 of 336 (17.0%) nonvaccinated cattle were TRM culture positive. From the multilevel logistic model, vaccinated cattle were 92% less likely to be colonized with E. coli O157:H7 than nonvaccinated cattle (odds ratio [OR] = 0.07, p = 0.0008). Additional explanatory variables were region of the state (OR = 7.4, p = 0.04), and pens with fewer cattle (OR = 0.22, p = 0.05). We concluded that the two-dose vaccine regimen effectively reduced the probability for E. coli O157:H7 colonization of the terminal rectum of commercially fed cattle at harvest.

Comparison of the contributions of heat-labile enterotoxin and heat-stable enterotoxin b to the virulence of enterotoxigenic Escherichia coli in F4ac receptor-positive young pigs.

In swine, the most common and severe enterotoxigenic Escherichia coli (ETEC) infections are caused by strains that express K88 (F4)(+) fimbriae, heat-labile enterotoxin (LT), heat-stable enterotoxin b (STb), and enteroaggregative E. coli heat-stable toxin 1. Previous studies based on a design that involved enterotoxin genes cloned into a nontoxigenic fimbriated strain have suggested that LT but not STb plays an important role in dehydrating diarrheal disease in piglets <1 week old and also enhances bacterial colonization of the intestine. In the present study, we compared these two toxins in terms of importance for piglets >1 week old with a study design that involved construction of isogenic single- and double-deletion mutants and inoculation of 9-day-old F4ac receptor-positive gnotobiotic piglets. Based on the postinoculation percent weight change per h and serum bicarbonate concentrations, the virulence of the STb(-) mutant (Delta estB) did not significantly differ from that of the parent. However, deletion of the LT genes (Delta eltAB) in the STb(-) mutant resulted in a complete abrogation of weight loss, dehydration, and metabolic acidosis in inoculated pigs, and LT complementation restored the virulence of this strain. These results support the hypothesis that LT is a more significant contributor than STb to the virulence of F4(+) ETEC infections in young F4ac receptor-positive pigs less than 2 weeks old. However, in contrast to previous studies with gnotobiotic piglets, there was no evidence that the expression of LT enhanced the ability of the F4(+) ETEC strain to colonize the small intestine.

Reduced intestinal colonization of adult beef cattle by Escherichia coli O157:H7 tir deletion and nalidixic-acid-resistant mutants lacking flagellar expression.

The importance of the Escherichia coli O157:H7 translocated intimin receptor (Tir) protein in intestinal colonization and the effect of infection with Tir(+) strains on protection against subsequent challenge was studied in adult beef cattle. Cattle were orally inoculated (C1) with a Shiga toxin-2(+)E. coli O157:H7 strain that was Tir(+) or Tir(-), and 42 days later were re-challenged (C2) with the nalidixic acid (Nal)(R) parent strain to test whether prior infection had any effect on fecal shedding. During the first 14 days post-C1, the Nal(S) wildtype (WT) strain was shed at significantly higher levels and for a longer period than the other strains; however, the mean levels of shedding of the Nal(R) and Deltatir complemented strains were not significantly different from that of the Tir(-) strains. The Deltatir, Tir complemented mutant, and Deltatir vector control strains inadvertently did not express flagellin, and did not effectively colonize the intestine. We were unable to determine whether Tir exposure at C1 had any effect on protection. Further, those given an initial inoculation with a non-flagellated variant of E. coli O157:H7 were more susceptible to a second challenge with motile E. coli O157:H7 than those originally inoculated with motile strains. The cause of the loss of expression of flagellin was not addressed. We suggest that either the flagellum or a factor that regulates both its production and that of some other effector has a significant function in colonization.

Significance of heat-stable and heat-labile enterotoxins in porcine colibacillosis in an additive model for pathogenicity studies.

Although heat-stable (ST) and heat-labile (LT) enterotoxins produced by enterotoxigenic Escherichia coli (ETEC) have been documented as important factors associated with diarrheal diseases, investigations assessing the contributions of individual enterotoxins to the pathogenesis of E. coli infection have been limited. To address the individual roles of enterotoxins in the diarrheal disease caused by K88-positive ETEC in young pigs, enterotoxin-positive and -negative isogenic E. coli strains were constructed by using pBR322 to clone and express LT and STb. Four strains, K88+ astA, K88+ astA/pBR322, K88+ astA STb+, and K88+ astA LT+, were constructed and subsequently included in gnotobiotic piglet challenge studies, and their pathogenesis was assessed. The results indicated that all K88+ isogenic strains were able to colonize the small intestines of piglets exhibiting the K88 receptor. However, only LT- and STb-positive strains caused appreciable diarrhea. Piglets inoculated with the K88+ astA LT+ strain became dehydrated within 18 h, while those inoculated with the K88+ astA STb+ strain did not, although diarrhea developed in several piglets. The changes in the blood packed-cell volume and plasma total protein of gnotobiotic piglets inoculated with the LT-positive strains were significantly greater than those of pigs inoculated with the K88 astA/pBR322 strain (P = 0.012, P = 0.002). Immunochemistry image analysis also suggested that LT enhanced bacterial colonization in a gnotobiotic piglet model. This investigation suggested that LT is a major contributor to the virulence of K88+ ETEC and that isogenic constructs are a useful tool for studying the pathogenesis of ETEC infection.

Relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces multiple enterotoxins.

Enterotoxigenic Escherichia coli (ETEC) strains that produce multiple enterotoxins are important causes of severe dehydrating diarrhea in human beings and animals, but the relative importance of these enterotoxins in the pathogenesis is poorly understood. Gnotobiotic piglets were used to study the importance of heat-labile enterotoxin (LT) in infection with an ETEC strain that produces multiple enterotoxins. LT(-) (DeltaeltAB) and complemented mutants of an F4(+) LT(+) STb(+) EAST1(+) ETEC strain were constructed, and the virulence of these strains was compared in gnotobiotic piglets expressing receptors for F4(+) fimbria. Sixty percent of the piglets inoculated with the LT(-) mutant developed severe dehydrating diarrhea and septicemia compared to 100% of those inoculated with the nalidixic acid-resistant (Nal(r)) parent and 100% of those inoculated with the complemented mutant strain. Compared to piglets inoculated with the Nal(r) parent, the mean rate of weight loss (percent per hour) of those inoculated with the LT(-) mutant was 67% lower (P < 0.05) and that of those inoculated with the complemented strain was 36% higher (P < 0.001). Similarly, piglets inoculated with the LT(-) mutant had significant reductions in the mean bacterial colony count (CFU per gram) in the ileum; bacterial colonization scores (square millimeters) in the jejunum and ileum; and clinical pathology parameters of dehydration, electrolyte imbalance, and metabolic acidosis (P < 0.05). These results indicate the significance of LT to the development of severe dehydrating diarrhea and postdiarrheal septicemia in ETEC infections of swine and demonstrate that EAST1, LT, and STb may be concurrently expressed by porcine ETEC strains.