Induction of potential protective immunity against enterotoxemia in calves by single or multiple recombinant Clostridium perfringens toxoids

Cattle enterotoxemia caused by Clostridium perfringens toxins is a noncontagious, sporadic, and fatal disease characterized by sudden death. Strategies for controlling and preventing cattle enterotoxemia are based on systematic vaccination of herds with toxoids. Because the process of producing conventional clostridial vaccines is dangerous, expensive, and time‐consuming, the prospect of recombinant toxoid vaccines against diseases caused by C. perfringens toxins is promising. In this study, nontoxic recombinant toxoids derived from α‐, β‐ and ε‐toxins of C. perfringens, namely, rCPA_247–370, rCPB and rEtxHP, respectively, were expressed in Escherichia coli. High levels of specific IgG antibodies and neutralizing antibodies against the toxins were detected in sera from calves vaccinated with either a single recombinant toxoid or a mixed cocktail of all three recombinant toxoids, indicating the potential of these recombinant toxoids to provide calves with protective immunity against enterotoxemia caused by C. perfringens.     

The ability of disease and non-disease producing strains of Clostridium perfringens from chickens to adhere to extracellular matrix molecules and Caco-2 cells

Clostridium perfringens is a major enteric pathogen that is responsible for causing necrotic enteritis of poultry. The ability to adhere to the host’s intestinal epithelium and to extracellular matrix molecules (ECMM) in the gut, are strategies used by numerous bacterial enteropathogens, however, C. perfringens has received comparatively little attention in this respect. The present study investigated sixteen type A C. perfringens isolates from chickens, with varying disease producing ability with respect to necrotic enteritis in chickens, for their ability to adhere to nine different extracellular matrix molecules (ECMM) and to the intestinal epithelial cell line Caco-2. C. perfringens strains were able to bind to ECMMs and there was strain variation. Strains of C. perfringens that produced severe disease, were capable of binding to collagen type III, IV and V, fibrinogen, laminin and vitronectin at higher levels than less severe disease producing strains, suggesting that the ability to adhere to ECMMs might enhance virulence with respect to induction of necrotic enteritis. In addition, severe disease producing strains also bound better to collagen type III and IV and fibrinogen, than non-disease producing strains. The present study also showed that some strains of C. perfringens possessed the ability to adhere to Caco-2 cells; however no relationship was found between the ability to adhere to Caco-2 cells and disease producing ability.     

Efficacy of live B1 or Ulster 2C Newcastle disease vaccines simultaneously vaccinated with inactivated oil adjuvant vaccine for protection of Newcastle disease virus in broiler chickens

Two hundred, one-day-old broiler chicks were divided into groups 1, 2 and 3 containing 60, 70 and 70 chicks, respectively. The groups were divided into subgroups of 10 chicks that were vaccinated according to the following scheme: group 1 unvaccinated control, group 2 vaccinated subcutaneously at 1 day old with inactivated oil adjuvant vaccine (IOAV) in combination with live B1 vaccine. Group 3 was vaccinated in the same mode as group 2 with IOAV and live Ulster 2C vaccine. All birds were challenged when they were 28 days old. Mortality rate, body weight gain and feed conversion ratio (FCR) were monitored before and after challenge. All the chickens in group 1 died, indicating that there was no disease resistance of this unvaccinated control group of chickens. Conversely, the monitored disease resistance of chickens in groups 2 and 3 was 68.57% ± 18.64 and 88.57% ± 9.00, respectively (P < 0.05). The morbidity of chickens in groups 2 and 3 was 37.89% ± 14.36 and 14.76% ± 12.40, respectively (P < 0.05). The body weight gain, feed intake and FCR of group 3 were significantly better than those of group 2 (P < 0.05) during 1–42 days old. The simultaneous vaccination with B1 or Ulster 2C and IOAV of 1-day-old chicks gave some protection of 28-day-old broilers without a booster vaccination.     

Occurrence of Clostridium perfringens from different cultivated soils

The occurrence of Clostridium perfringens was estimated in 750 samples originated from a variety of soils bearing various bulb crops: Brawnica oderacea (vegetable), Olea europaea, Daucus carota (carote), Solanum tuberosum (potato), Phaseolus vulgaris (green haricot), Beta vulgaris var. rapaceum (beetroot), Cucurbita pepo (squash), Allium cepa (onion), Cucumis sativus (cucumber) and Capsicum annum (pepper). All isolated strains were tested for their antimicrobial activities to amoxicillin, penicillin G, kanamycin, tetracycline, streptomycin, erythromycin, chloramphenicol and metronidazole.When considering the type of the bulb production, it was observed increased number of C. perfringens spore densities in the most undersurface bulb soils. Moreover, C. perfringens spore are likely to occur in particularly large numbers in soil contaminated by fecal matter. Additionally, there is a close relationship between the spore amount and nature of organic content. Presence of C. perfringens was associated with acidic soil. Most of our strains showed resistance to the studied antibiotics applied usually for human and veterinary care. A systematic monitoring of the cultivated soil ecosystems must include bacteriological parameters together with chemical indices of organic pollution in order to obtain information adequate for assessing their overall quality.     

Clostridium perfringens challenge and dietary fat type affect broiler chicken performance and fermentation in the gastrointestinal tract

The aim of the present work was to examine how different fats commonly used in the feed industry affect broiler performance, nutrient digestibility and microbial fermentation in the gastrointestinal tract of broiler chickens challenged with virulent Clostridium perfringens strains. Two experiments were carried out, each including 480-day-old male broilers (Ross 308), which were randomly distributed to eight experimental groups using six replicate pens per treatment and 10 birds per pen. In Experiment 1, birds were fed diets containing soybean oil, palm kernel fatty acid distillers, rendered pork fat and lard. In Experiment 2, birds were fed diets containing rapeseed oil, coconut oil, beef tallow and palm oil. In both experiments, the birds were either not challenged or challenged with a mixture of three C. perfringens type A strains. Irrespective of the fat type present in the diet, C. perfringens did not affect broiler chicken body weight gain (BWG) and mortality in either of the two experiments. The BWG was affected by dietary fat type in both experiments, indicating that the fatty acid composition of the fat source affects broiler growth performance. In particular, the inclusion of animal fats tended to improve final BW to a greater extent compared with the inclusion of unsaturated vegetable oils. In Experiment 2, irrespective of the dietary fat type present in the diet, C. perfringens challenge significantly impaired feed conversion ratio in the period from 14 to 28 days (1.63 v. 1.69) and at 42 days (1.65 v. 1.68). In both experiments apparent metabolizable energy values were affected by dietary fat type. Irrespective of the fat type present in the diet, C. perfringens challenge decreased the digesta pH in the crop and ileum, but had no effect in cecal contents. Moreover, in Experiment 1, total organic acid concentration in the ileum was two to three times lower on soybean oil diets as compared with other treatments, indicating that C. perfringens as well as dietary fat type significantly affects microbiota activity in the broiler chicken gastrointestinal tract.     

Investigating the role of small, acid-soluble spore proteins (SASPs) in the resistance of Clostridium perfringens spores to heat

Background  

Clostridium perfringens type A food poisoning is caused by enterotoxigenic C. perfringens type A isolates that typically possess high spore heat-resistance. The molecular basis for C. perfringens spore heat-resistance remains unknown. In the current study, we investigated the role of small, acid-soluble spore proteins (SASPs) in heat-resistance of spores produced by C. perfringens food poisoning isolates.     

Comparison of alternatives to in‐feed antimicrobials for the prevention of clinical necrotic enteritis

Aims: The capacity for Lactobacillus johnsonii and an organic acid (OA) blend to prevent Clostridium perfringens‐induced clinical necrotic enteritis (NE) in chickens was studied. Methods and Results: Cobb 500 birds were allocated into six groups (n = 25 birds/pen, eight pens/treatment); Unchallenged, Challenged, Antimicrobial (zinc bacitracin (ZnB)/monensin), OA, probiotic Lact. johnsonii and probiotic sham (Phosphate–buffered saline). All birds were challenged with Eimeria spp. and Cl. perfringens except for unchallenged controls. Birds fed antimicrobials were protected from NE development as indicated by maintenance of body weight, low mortality and clostridium levels, and decreased intestinal macroscopic lesion scores compared to challenged controls (P < 0·05). Lactobacillus johnsonii‐fed birds had reduced lesion scores, whilst OA‐fed birds had decreased Cl. perfringens levels. Both Lact. johnsonii and OA‐fed birds had improved feed efficiency between days 0 and 28 compared to challenged controls; however, mortality and body weights were not improved by either treatment. Microbial profiling indicated that the challenge procedure significantly altered the jejunal microbiota. The microbiota of antimicrobial‐fed birds was significantly different from all other groups. Conclusions: Whilst Lact. johnsonii and OA altered specific intestinal parameters, significant protection against NE was not observed. Significance and Impact of the Study: Lactobacillus johnsonii and OA did not prevent NE; however, some improvements were evident. Other related treatments, or combinations of these two treatments, may provide greater protection.     

Case of Clostridium perfringens bacteremia after routine colonoscopy and polypectomy

Bacteremia is an uncommon complication after polypectomy and colonoscopy. We report one of the first cases of Clostridium perfringens bacteremia after polypectomy. Our patient was a four years old boy with congenital polyposis, who underwent colonoscopy and polypectomy without complication. Approximately 12 h later he developed a fever and tachycardia with no other clinical symptoms. His blood cultures grew out penicillin susceptible C. perfringens and Enterococcus faecalis. He responded to antibiotic therapy and remained clinically asymptomatic for the duration of his course. There are a few reports of bacteremia after routine polypectomy, but no reported cases of C. perfringens bacteremia in the pediatric population. Clostridial sp. bacteremia can be fatal with devastating consequences if appropriate antibiotics and/or surgical debridement are delayed. Polymicrobial infection, as illustrated in our patient, is also common and can be a poor prognostic risk factor. Therefore, for patients with a history of polypectomy and new onset fever, anaerobic infections should be considered and empiric antibiotic therapy should include coverage for these organisms.     

qPCR quantification and genetic characterization of Clostridium perfringens populations in biosolids composted for 2 years

Aim: The ability of Clostridium perfringens to survive for a long time in the environment makes it a suitable indicator of faecal pollution, but its use as a routine indicator organism in biosolids and composted biosolids has not yet been adopted. This study was performed to improve our understanding of C. perfringens persistence in composted biosolids by monitoring its presence and studying its genetic diversity. Methods and Results: A culture‐independent TaqMan qPCR assay targeting the cpn60 gene was adapted to enumerate C. perfringens in composted biosolid samples varying in age from 1 to 24 months. The pathogen was detected in all compost samples under study, but no correlation between composting time and number of cpn60 copies was observed. Rep‐PCR detected 14 different C. perfringens genotypes, all belonging to toxinotype A, which is the most common biotype found in human and animal gastrointestinal tracts. Conclusions: Composting did not significantly decrease the number of C. perfringens cells. High genetic diversity of C. perfringens isolates present in composted biosolids is reported for the first time. Significance and Impact of Study: This study evaluated tools for surveillance of composting processes, source tracking and risk assessment of composted biosolids.     

Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

Background

Control of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, attenuated Salmonella enterica serovar Typhimurium was used for oral co-administration of chicken interferon-?? (chIFN-??) and chicken interleukin-18 (chIL-18) as natural immunomodulators.

Results

Oral co-administration of S. enterica serovar Typhimurium expressing chIFN-?? and chIL-18, prior to vaccination with inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single administration of S. enterica serovar Typhimurium expressing either chIFN-?? or chIL-18. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were intra-tracheally challenged with a high dose of LPAI H9N2 virus. Combined administration of S. enterica serovar Typhimurium expressing chIFN-?? and chIL-18 showed markedly enhanced protection compared to single administration of the construct, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in different tissues of challenged chickens.

Conclusions

Our results indicate the value of combined administration of chIFN-?? and chIL-18 using a Salmonella vaccine strain to generate an effective immunization strategy in chickens against LPAI H9N2.     

Reliability of mCP method for identification of Clostridium perfringens from faecal polluted aquatic environments

Aims: The purpose of the work was to evaluate the mCP method to correctly identify and enumerate Clostridium perfringens that are present in surface waters impacted by a mixture of faecal pollution sources. Methods: Clostridium perfringens were enumerated and isolated from sewage influent, surface water and suspended sediments using the mCP method. Molecular characterization of isolates was performed using species‐specific PCR, along with full‐length sequencing of the 16S rRNA gene for a subset of isolates. Results: The environmental isolates were presumptively identified as C. perfringens based on utilization of sucrose, inability to ferment cellobiose and a positive action for acid phosphatase activity. All isolates (n = 126) were classified as C. perfringens based on positive results with species‐specific PCR with a subset confirmed as C. perfringens based on the 16S rRNA gene identity. Conclusions: The molecular results indicated all of the presumptive positive isolates were C. perfringens regardless of the source, e.g. sewage influent or environmental water samples. Sequencing revealed that C. perfringens obtained from sewage and the aquatic environment were nearly identical (c. 99·5% similarity). Significance and Impact of the Study: From this study we conclude that the mCP method is a robust approach to enumerate and isolate C. perfringens from aquatic environments that receive diverse sources of faecal pollution.     

Association between variants in the 5′-untranslated region of the bovine <Emphasis Type="Italic">MC4R</Emphasis> gene and two growth traits in Nanyang cattle

Melanocortin-4 receptor (MC4R) is one of five G-protein-coupled receptors binding melanocortins that is implicated in the control of feeding behavior and energy homeostasis. Six cattle populations (n = 594), including four Chinese indigenous breeds, Chinese Holstein, and a meat type breed (Angus), were used to detect single nucleotide polymorphisms in 5′-untranslated region of MC4R gene by means of PCR–SSCP and DNA sequencing. Four linked SNPs (g.[−293C>G; −193A>T; −192T>G; −129A>G]) were identified. The g.−293C>G and g.−129A>G could be genotyped with a PCR–RFLP using TaiI in three combined genotypes (AA, AB and BB). The two linked SNPs were associated with body weight and daily gain in Nanyang aged 6 months (P < 0.05), but they had no significant effect on body weight and daily gain in Nanyang aged 24 months (P > 0.05).     

Hemp seed cake in organic broiler diets

The aim of this study was to examine production and health of fast-growing broilers fed diets with and without hemp seed cake (HSC) in organic broiler production. Two diets, a control diet (C) and a diet including HSC (H), were fed to 1200 Ross 308 chickens which were divided over 8 pens. Birds were housed indoors until 21 d, and thereafter kept in the same groups in two chicken houses with access to outdoor pasture up to slaughter at 70 d. Production performance was registered weekly. The number of Clostridium perfringens (C. perfringens) in caeca, as well as leg and foot health and carcass quality was registered during the study period or at slaughter. Total mortality was high regardless of diet due to a short heat wave extreme for Swedish conditions which predominantly affected the heavy male broilers. The inclusion of HSC did not affect total production performance or mortality. Furthermore, no effect of HSC inclusion in the feed was seen on the number of C. perfringens in the caeca. There were differences in litter condition and foot health of birds, which may have been related to the high crude fibre (CF) content of the H diet.     

Efficacy of dietary supplemental insoluble fibrous materials in ameliorating adverse effects of coccidial challenge in broiler chickens

ABSTRACT

The current experiment was designed to examine effects of dietary supplemental sunflower hulls (SH) and rice hulls (RH) on growth performance, carcass traits, intestinal morphology, lesion score and oocyst shedding in broiler chickens exposed to coccidial challenge. A total of 540 broiler chickens (Ross 308) were assigned to six dietary treatments based on a factorial arrangement (2 × 3) across 1–14, 14–28 and 28-42-d periods. Experimental treatments consisted of broiler chickens without or with coccidial challenge each offered with three different diets: a basal diet or basal diet supplemented with either RH or SH at 40 g/kg diet, respectively. Infection with Eimeria impaired daily weight gain (DWG) and feed conversion ratio (FCR) of broiler chickens during growing period (p < 0.05) while supplementation of SH or RH reduced the adverse effect of coccidiosis so that birds had similar DWG to those fed the basal diet without infection. However, only dietary SH improved the FCR of broilers challenged with coccidiosis. Regardless of coccidial challenge, dietary access to insoluble fibre improved performance of broilers across the growing period (p < 0.05); however, this effect was not observed during the entire rearing period. Relative weights of liver and pancreas were increased in birds subjected to coccidial challenge on d 21 of age (p < 0.05). Moreover, relative weights of the intestinal segments were enhanced (p < 0.05). Furthermore, gizzard weights were higher in birds receiving diets added with fibre (p < 0.05). Infection with coccidiosis decreased villus height and villus height to crypt depth ratio in duodenum of broilers which received the basal diet compared with those fed the same feed without coccidial challenge (p < 0.05). However, supplemental SH could decrease the negative effect of infection on the noted intestinal morphometric attributes. Similarly, a marked reduction was observed for lesion score and faecal oocyst excretion of challenged broilers fed on dietary supplemental fibre (p < 0.05). In conclusion, supplementation of insoluble fibre could ameliorate negative effects of coccidial challenge on DWG of broiler chickens and inclusion of SH in diet of birds exposed to Eimeria infection could be recommended.     

Colonization of broilers by <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> internalized within <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis>

Although Campylobacter survives within amoeba in-vitro, it is unknown if intra-amoeba Campylobacter jejuni can colonize broilers. Five groups of 28 day-of-hatch chicks were placed into separate isolators. Groups (1) and (2) were challenged with page’s amoeba saline (PAS), and disinfected planktonic C. jejuni NCTC 11168, respectively. Groups (3), (4) and (5) were challenged with a C. jejuni positive control, C. jejuni in PAS, and intra-amoeba C. jejuni, respectively. After 1, 3, 7 and 14 days post challenge, seven birds from each unit were examined for C. jejuni colonization. For the first time we report that intra-amoeba C. jejuni colonized broilers.     

Screening Escherichia coli, Enterococcus faecalis, and Clostridium perfringens as Indicator Organisms in Evaluating Pathogen-Reducing Capacity in Biogas Plants

This study was conducted to identify an indicator organism(s) in evaluating the pathogen-reducing capacity of biogas plants. Fresh cow manure containing 10⁴ to 10⁵ colony forming unit (CFU) per milliliter of Escherichia coli and Enterococcus faecalis along with an inoculated Clostridium perfringens strain were exposed to 37°C for 15 days, 55°C for 48 h, and 70°C for 24 h. C. perfringens was the most heat-resistant organism followed by E. faecalis, while E. coli was the most heat-sensitive organism. E. coli was reduced below detection limit at all temperatures with log₁₀ reductions of 4.94 (10 s), 4.37 (40 min), and 2.6 (5 days) at 70°C, 55°C, and 37°C, respectively. Maximum log₁₀ reductions for E. faecalis were 1.77 at 70°C (1 day), 1.7 at 55°C (2 days) and 3.13 at 37°C (15 days). For C. perfringens, maximum log₁₀ reduction at 37°C was 1.35 log₁₀ units (15 days) compared to less than 1 unit at 55 and 70°C. Modeling results showed that E. faecalis and C. perfringens had higher amount of heat-resistant fraction than E. coli. Thus, E. faecalis and C. perfringens can be used as indicator organisms to evaluate pathogen-reducing capacity in biogas plants at high temperatures of 55°C and 70°C while at 37°C E. coli could also be included as indicator organism.     

Nucleotide distribution in bacterial DNA's differing in G + C content

Summary The DNA's ofMicrococcus lysodeikticus andClostridium perfringens were fragmented to about 7 000 nucleotide pairs long by shear and fractionated with respect to buoyant density of mercury complexes in Cs₂SO₄. The distribution of G + C content in both DNA's was characteristically asymmetric. InM. lysodeikticus DNA, low G + C fragments were more numerous than high G + C fragments, whereas inC. perfringens DNA, high G + C fragments were more numerous than low G + C fragments. The G + C content of fragments ofM. lysodeikticus DNA varied from 70 to 77%, with a mean and standard deviation of 73.7 ± 1.92% G + C and that ofC. perfringens DNA varied from 27 to 34%, with a mean and standard deviation of 29.8 ± 1.34% G + C. The standard deviation was smaller than that ofEscherichia coli DNA fragments of similar size. Biological meanings of relatively low heterogeneity in nucleotide composition inM. lysodeikticus andC. perfringens are discussed.     

Clostridium perfringens in retail chicken

Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% “atypical” genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken.