Molecular analysis of the virulence determinants ofClostridium perfringens associated with foal diarrhoea

During an epidemiological study of foal diarrhoea, over half of the cases yielded Clostridium perfringens whichwas significantly associated with disease (Netherwood el al., 1996b). However, the association could not be accounted for by enterotoxigenic isolates which had a low prevalence (Netherwood et al., 1997). Nonetheless, we have hypothesized that the association may be caused by a pathogenic sub-population which would be significantly more common amongst C. perfringens-positive cases compared with C. perfringens-positive healthy controls if it acted as a pathogen when present. Conversely, if foal diarrhoea caused by C. perfringens was dependent on a predisposing factor, then such an association might not be evident. As a first step to determine if a molecular marker was more frequently to be found in C. perfringens-positive cases than controls, we have genotyped the study isolates (up to five per foal) by polymerase chain reaction (PCR) based on the published gene sequences for the major lethal toxins alpha, beta, epsilon and iota as well as for theta toxin, large and small sialiclases, hyaluronidase and virulence regulation. Isolates of major toxin types B, C, D and E, or isolates which were untypeable, were isolated from less than 15% of C. perfringens-positive foals and these were not associated with diarrhoea nor were they more commonly found in C. perfringens-positive cases. Isolates of type A were found in more than 90% of all C. perfringens-positive foals. A number of different genotypes were identified by their different patterns of gene possession but types without any of the genes for theta toxin, large and small sialidases, hyaluronidase and virulence regulation were found in only 10% of positive foals. Only type A isolates with all of these genes were associated with diarrhoea overall but they were not more commonly isolated from C. perfringens-positive cases than controls. In conclusion, genotyping by the sequenced virulence genes did not identify a marker for a sub-population of C. perfringens which may be acting more frequently as a pathogen when present.     

Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study

Antimicrobial therapy is a useful tool to control bovine mastitis caused by Staphylococcus aureus, as consequence an increase in staphylococci resistant cases has been registered. Alternative strategies are desirable and bacteriocins represent attractive control agents to prevent bovine mastitis. The aim of this work was to evaluate the activity of five bacteriocins synthesized by Bacillus thuringiensis against S. aureus isolates associated to bovine mastitis. Fifty S. aureus isolates were recovered from milk composite samples of 26 Holstein lactating cows from one herd during September 2007 to February 2008 in México and susceptibility of those isolates to 12 antibiotics and 5 bacteriocins from B. thuringiensis was evaluated. S. aureus isolates were mainly resistant to penicillin (92%), dicloxacillin (86%), ampicillin (74%) and erythromycin (74%); whereas susceptibility to gentamicin, trimethoprim and tetracycline was detected at, respectively, 92%, 88%, and 72%. All S. aureus isolates showed susceptibility to the five bacteriocins synthesized by B. thuringiensis, mainly to morricin 269 and kurstacin 287 followed by kenyacin 404, entomocin 420 and tolworthcin 524. Our results showed that S. aureus isolates had differences in the antimicrobial resistance patterns and were susceptible to bacteriocins produced by B. thuringiensis, which could be useful as an alternative method to control bovine mastitis.     

Bovine Mastitis in Selected Areas of Southern Ethiopia

A study on bovine mastitis, designed to determine the causal agents, prevalence of infection and impact of risk factors in three cattle breeds, was conducted in selected areas of southern Ethiopia. A total of 307 lactating and non-lactating cows, of which 162 were indigenous Zebu, 85 Jersey and 60 Holstein-Friesian, were examined by clinical examination and the California mastitis (CMT) test. Of these, 40.4% were positive by CMT and bacteriology for clinical or subclinical mastitis, with prevalence rates of 37.1% and 62.9%, respectively. Out of 1133 quarters examined, 212 (18.7%) were found to be infected, 83 (39.2%) clinically and 129 (60.8%) subclinically. The prevalence of mastitis was significantly higher in Holstein-Friesian than in indigenous Zebu, in non-lactating cows than in lactating cows, in the early lactation stage than in the mid-lactation stage, in cows with lesions and/or tick infestation on skin of udder and/or teats than in cows without this factor, and in the wet season than in the dry season. Mastitis increased with parity number (R = 0.9). Of 248 CMT and clinically positive udder quarter samples analysed microbiologically, 212 were culturally positive for known mastitis pathogens and 36 were negative. Of the 199 positive samples, Staphylococcus accounted for 39.2%, Streptococcus for 23.6%, coliforms for 14.1%, Micrococcus and Bacillus species for 8.0% each and Actinomyces or Arcanobacterium (Corynebacterium) for 7.0%. It was concluded that there was a high prevalence of clinical and subclinical mastitis, mainly caused by Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli, in this study area.     

利用科学免疫方式防控育肥猪胀气猝死试验报告

诺维氏梭菌导致的猪只猝死是集团规模猪场母猪和育肥猪猝死的重要原因。试验在华北地区某集团各选择1 200头育肥猪，应用某厂家猪大肠杆菌-C型产气荚膜梭菌-诺维氏梭菌三联灭活疫苗H和另一厂家产气荚膜梭菌二价疫苗（A型、C型）Z进行预防猪只胀气猝死的效果比较。结果表明：免疫三联灭活疫苗H组死亡率下降3.25个百分点，免疫二价疫苗Z组死亡率下降2个百分点，猪只因诺维氏梭菌导致的猝死比例显著下降，有效提高了猪场经济效益。     

ermB-mediated erythromycin resistance in Streptococcus uberis from bovine mastitis

The ermB gene was identified in 111 erythromycin resistant isolates of Streptococcus uberis from cases of bovine mastitis associated either with a constitutive (47/111) or an inducible (64/111) phenotype, as well as a phenotypic resistance to all macrolides tested. Resistance to lincosamides was identified in 14 other isolates of S. uberis from bovine mastitis cases and was mainly mediated by the linB gene; resistance conferred by a combination of two genes (linB–lnuD, ermB–linB) was also detected.     

Prevalence of Clostridium perfringens in faeces and ileal contents from grass sickness affected horses: Comparisons with 3 control populations

Reasons for performing study: While previous studies have demonstrated an association between equine grass sickness (EGS) and the presence of Clostridium botulinum within ileal contents and faeces, no such associations with other intestinal‐derived anaerobic bacteria have been extensively investigated. Hypothesis: The prevalence of C. perfringens in the ileal contents and faeces of EGS horses is greater than control horses; the detection of C. perfringens in faeces by ELISA could be diagnostically beneficial in a clinical setting. Methods: The prevalence of C. perfringens in faeces from EGS horses and healthy grazing control horses was determined by both selective culture and ELISA to permit both validation of the ELISA and inter‐group comparisons. Additionally, the prevalence of C. perfringens (ELISA) in ileal contents from EGS horses was compared with that for control horses with nongastrointestinal disease. Finally, the prevalence of C. perfringens (ELISA) in faeces from EGS cases was compared with that from both horses with which they shared pasture at the time of disease onset and non‐EGS colic horses. Results: When compared with culture, the ELISA had a sensitivity and specificity of 86 and 98%, respectively. The prevalence of C. perfringens in faeces as determined by both culture and ELISA was significantly higher (P<0.001) for EGS horses (7/9 and 15/37, respectively) than for healthy grazing controls (0/60 and 1/74, respectively). The prevalence of C. perfringens in ileal contents from EGS horses (5/10) was greater than that for horses with nongastrointestinal disease (1/12) at a level that approached significance (P = 0.056). EGS cases had a significantly greater prevalence of C. perfringens in faeces (15/37) than co‐grazing horses (1/18) and colic (1/16) horses. The specificity (93%) and PPV (94%) of the detection of C. perfringens by ELISA on faecal samples in relation to disease status (EGS compared with colic horses) was good. Sensitivity (41%) and NPV (39%) were poor. Conclusions and potential relevance: The use of a commercial ELISA to detect faecal C. perfringens may be diagnostically beneficial when differentiating EGS cases from colic cases, although further work is required to fully evaluate its potential.     

Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.     

Streptococcus uberis: A Permanent Barrier to the Control of Bovine Mastitis?

The prevalence of bovine mastitis has been reduced over the past 25 years due to the implementation of a five-point control plan aimed at reducing exposure, duration and transmission of intramammary infections by bacteria. This has markedly reduced the incidence of bovine mastitis caused by bacteria which show a contagious route of transmission, but has had little effect on the incidence of mastitis due to bacteria which infect the gland from an environmental reservoir. Streptococcus uberis is one such bacterium which is responsible for a significant proportion of clinical mastitis worldwide.The inadequacies of the current methods of mastitis control have led to the search for additional measures, particularly vaccines to prevent intramammary infection by this bacterium. Such an approach requires detailed knowledge of the pathogenesis of intramammary infection. Our understanding of this area has grown in recent years but a lack of information still hampers disease control. Both live vaccines and, recently, crude sub-unit vaccines have shown promise against bovine mastitis due to S. uberis. Vaccines against mastitis must, however, be able to control infection without the participation of a marked inflammatory response. This review provides an overview of the recent advances which have been made in our understanding of host-pathogen interactions which promote infection and disease and highlights areas for strategic research aimed at controlling this bacterial infection.     

Inclusion of Bacillus amyloliquefaciens strain TOA5001 in the diet of broilers suppresses the symptoms of coccidiosis by modulating intestinal microbiota

Coccidiosis is an intestinal parasitic infection and one of the most prevalent and economically damaging diseases of chickens. Furthermore, coccidia‐induced mucogenesis promotes secondary colonization by Clostridium perfringens, a major pathogen of chickens that causes necrotic enteritis. Our previous work found that supernatant of a culture of Bacillus amyloliquefaciens strain TOA5001 (BA) inhibited the growth of C. perfringens on Gifu anaerobic broth medium. Accordingly, we evaluated the effectiveness of dietary BA administration in inhibiting C. perfringens colonization of the intestine in broilers that were experimentally infected with coccidia. Ten healthy broilers from a BA‐supplemented (2 × 10⁵ colony‐forming units/g of feed) broiler group and 10 from a non‐treated group were challenged with Eimeria tenella and E. maxima (5000 oocysts of each species/chick) at 28 days old. At 36 days old, five chicks from each group were slaughtered, whereas the remaining five in each group were killed at 49 days old. Dietary BA administration into Eimeria‐challenged birds reduced coccidial symptoms such as intestinal lesions. It also modified the cecal microbiota through suppressing C. perfringens and E. coli colonization, and inducing domination of Faecalibacterium prausnitzii, the Lactobacillus group and unknown Lachnospiraceae genera by bacterial DNA‐based metagenome analyses. B. amyloliquefaciens TOA5001 supplementation suppressed the symptoms of coccidiosis by modulating cecal microbiota in Eimeria‐challenged broilers.     

Bacteria of Pathogenic Importance in Faeces from Cadavers of Free-ranging or Corralled Semi-domesticated Reindeer in Northern Norway

There is little information on bacteria that have the potential to cause disease in reindeer husbandry. In this project, faecal samples from 35 free-ranging or corralled reindeer, adults and calves, that died in the winter of 2000 in northern Norway, were examined for the occurrence of Campylobacter spp., Clostridium perfringens, Listeria spp., Salmonella spp. and Yersinia spp. to evaluate the role of these microrganisms in loss and mortality in reindeer husbandry. In addition, 31 of these samples were examined for the occurrence of bacteria producing shigatoxin-1 and 2. C. perfringens was isolated in 20 (57.1%) of the faecal samples. In the free-ranging reindeer, 44% were positive carriers of C. perfringens and 90% of the corralled ones were positive for C. perfringens. In addition, the gene encoding for shigatoxin-1 was detected in one of the samples derived from a corralled reindeer. The other bacteria investigated were not found. Shigatoxin-1-producing bacteria were isolated for the first time from reindeer in Norway. However, no correlation between C. perfringens or shigatoxin-1-producing bacteria and mortality in the reindeer could be established.     

Prevalence and Aetiology of Mastitis in Cows from Two Major Ethiopian Dairies

The study was undertaken to determine the aetiology and prevalence of mastitis in hand-milked cows (n = 186) in two major Ethiopian dairies. The California Mastitis Test and culturing for bacteria revealed that 21.5% of the cows were clinically infected and 38.2% had subclinical mastitis. Most mastitis pathogens isolated from milk samples testing positive by the California Mastitis Test were Gram-positive cocci. Staphylococci constituted 57% of the isolates, of which the predominant cause of bovine mastitis was Staphylococcus aureus (40.5%). Other mastitis pathogens isolated include streptococci (16.5%), coliforms (9%) and corynebacteria (5%). Retrospective analysis of farm records indicated that mastitis was the second most important cause of culling and accounted for 27% of the cows removed from these two dairies.     

Chlamydiaceae in cattle: commensals, trigger organisms, or pathogens?

Epidemiological data indicate that infection of cattle with chlamydiae such as Chlamydophila (C.) pecorum, C. abortus, C. psittaci and Chlamydia suis, is ubiquitous with mixed infections occurring frequently. The apparent lack of association between infection and clinical disease has resulted in debate as to the pathogenic significance of these organisms, and their tendency to sub-clinical and/or persistent infection presents a challenge to the study of their potential effects. However, recent evidence indicates that chlamydial infections have a substantial and quantifiable impact on livestock productivity with chronic, recurrent infections associated with pulmonary disease in calves and with infertility and sub-clinical mastitis in dairy cows. Data also suggest these infections manifest clinically when they coincide with a number of epidemiological risk factors. Future research should: (1) use relevant animal models to clarify the pathogenesis of bovine chlamydioses; (2) quantify the impact of chlamydial infection at a herd level and identify strategies for its control, including sub-unit vaccine development; and (3) evaluate the zoonotic risk of bovine chlamydial infections which will require the development of species-specific serodiagnostics.     

Enterotoxaemia in goats

Enterotoxaemia of sheep and goats occurs worldwide, but the condition in goats is poorly understood. The disease in goats is mostly caused by Clostridium perfringens type D, although the role of the toxins of this microorganism in the pathogenesis of the disease is not fully understood. The disease occurs in three forms, peracute, acute and chronic, the cardinal clinical sign of the acute and chronic forms being diarrhoea. The main biochemical alterations are hyperglycaemia and glycosuria, while at necropsy the disease is often characterized by haemorrhagic colitis. The typical histological changes observed in the brain of sheep with enterotoxaemia are not considered to be a common feature of enterotoxaemia in goats. Although the pathogenesis of caprine enterotoxaemia has not yet been properly defined, it is usually accepted that the presence of C. perfringens type D in the small bowel, together with a sudden change to a diet rich in carbohydrates, is the main predisposing factor for the disease. Vaccination seems to be poorly effective in preventing caprine enterotoxaemia, which might be due to the fact that the enteric form of the disease is partially independent of circulating C. perfringens toxin. More studies are needed on caprine enterotoxaemia, especially of its pathogenesis and immunity, in order to develop more efficient control measures for this disease.     

Milk protein profiles in response to Streptococcus agalactiae subclinical mastitis in dairy cows

The objective of this study was to investigate the milk protein profiles of normal milk and those of milk during the course of subclinical mastitis, caused by natural Streptococcus agalactiae infection. Two‐dimensional gel electrophoresis and liquid chromatography mass spectrometry were used to assess protein profiles and to identify the proteins. The results showed that S. agalactiae subclinical mastitis altered the protein profiles of milk. Following Mascot database matching, 11 and 12 protein types were identified in the milk collected from healthy and S. agalactiae subclinical mastitic udders, respectively. The distinct presence of the antibacterial protein cathelicidin‐1 was detected in infected milk samples, which in turn was highly correlated to the severity of subclinical mastitis as represented by the milk somatic cell count (r = 0.616), but not the bacterial count. The protein profile of milk reveals changes in the host response to S. agalactiae intramammary infection; cathelicidin‐1 could therefore serve as a biomarker for the detection of subclinical mastitis in dairy cows.     

Bovine Mastitis due to Prototheca zopfii: Clinical,Epidemiological and Pathological Aspects in a Brazilian Dairy Herd

The clinical, epidemiological and pathological aspects of protothecal mastitis in a Brazilian dairy herd are described. Prototheca zopfii infection was diagnosed in 11 of 121 milking cows. Clinical mastitis refractory to usual therapy was observed in 7 cows. Several environmental conditions conducive to the growth of Prototheca spp., such as wetness, muddiness and the presence of organic material, were present in the dairy. Improper milking practices and insanitary infusion of the intramammary antibiotics were also observed. Six cows with protothecal mastitis were slaughtered and the affected quarters of each cow were examined by histology and immunohistochemical staining for bovine keratin and P. zopfii. The histological lesions were characterized by interstitial infiltrates of macrophages, plasma cells and lymphocytes; algae were seen in the alveolar lumen and interstitium. The lack of a positive reaction with an antiserum against bovine keratin in the mammary alveolar epithelial layer in some affected areas suggests destruction of milk-producing tissues, which may be related to the low milk production observed. The algal organisms stained positively with a polyclonal antibody against P. zopfii.     

Clostridium Infection (Jisizheng) in Yaks in Qinghai,China

Since the mid-1980s, outbreaks of a disease characterized by a sudden onset, acute deaths and extensive haemorrhages in the viscera and digestive tract of yaks have been prevalent in Qilian, Qinghai, China. The disease is known as jisizheng by local people. Virulent Clostridium perfringens type A and Clostridium haemolytica were isolated from yaks that had died of jisizheng. In 1996 and 1997, yaks were immunized with a polyvalent inactivated vaccine against C. perfringens and with an inactivated vaccine against C. haemolyticum, and this prevented the occurrence of jisizheng.     

Expression of cytokines and respiratory burst activity of milk cells in response to Azadirachta indica during bovine mastitis

The immuno therapeutic potential of hydro-methanolic extract of Azadirachta indica (A. indica) was studied during bovine clinical mastitis (CM). The somatic cell count (SCC), total bacterial count (TBC), milk differential leukocyte count (DLC), hydrogen peroxide (H₂O₂), superoxide anion (O₂ ⁻) production and interleukin- 2 (IL-2) and gamma interferon (IFN-γ) cytokines expression were studied before and after intramammary infusion of A. indica extract in diseased cows. The results revealed that A. indica treatment significantly (P < 0.05) decreased the SCC, TBC, milk neutrophil percent and significantly (P < 0.05) enhanced milk lymphocyte percent, H₂O₂ and O₂ ⁻ production by milk cells. The IL-2 and IFN-γ were expressed in normal healthy cows and diseased cows after A. indica treatment, whereas both the cytokines could not be expressed in cows treated with antibiotic and in untreated diseased cows. The results of the present study indicated anti inflammatory, antibacterial and immunomodulatory potential of the herb, these activities could be due to the presence of bioactive principle in the extract. This is a preliminary trial indicated beneficial effect of the herb against bovine mastitis it can be developed as an alternative therapy where the use of antibiotics is normally restricted.     

Vaccination against staphylococcal mastitis

Extract

Sir:- Immunisation with staphylococcal vaccines is a potential means of increasing the resistance to bacterial invasion of the udder. However, the development of an effective vaccine for control of staphylococcal mastitis in ruminants has proved to be an elusive goal. There have been numerous attempts to develop vaccines against coagulase-positive Staphylococcus aureus mastitis, but very few attempts to develop vaccines using coagulase-negative staphylococci. Development of a vaccine against coagulase-negative staphylococci may help control the sub-clinical mastitis caused by these organisms and assist in the search for an effective vaccine against mastitis caused by the more pathogenic coagulase-positive S. aureus.We describe here an experiment in which ewes were immunised with live coagulase-negative staphylococcal vaccine and challenged during lactation by intramammary infusion of a homologous strain.     

Adherence and colonization by bacterial pathogens in explant cultures of bovine mammary tissue

Explant cultures of bovine mammary tissue taken from virgin heifers were used to examine adherence, colonization and cytopathogenesis ofStreptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Staphylococcus aureus andEscherichia coli in the putative target tissue. None of the five bacteria was able to adhere to healthy ductular epithelium but all showed a marked tropism for exposed connective tissue.S. aureus andE. coli induced a marked cytopathic effect in ductular epithelium after 6 hours in culture but the bacteria were not in close association with the affected tissue. No evidence could be found to support the hypothesis that adherence to epithelium might be the first stage in the pathogenesis of mastitis caused by these organisms.     

Intracellular fate of strains of <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from dairy cows with acute or chronic mastitis

Research on mastitis in dairy cows caused by Escherichia coli has reported the emergence of strains capable of inducing chronic mastitis and that these strains adhered to and internalized into bovine mammary epithelial cells better than strains of E. coli isolated from acute mastitis. To understand mechanisms and strategies used by chronic E. coli strains to survive intracellularly internalization studies using bovine mammary epithelial cells treated with inhibitors of caveolae-mediated endocytosis (CME) and receptor-mediated endocytosis (RME), double immunofluorescence labeling confocal laser and fluorescence microscopy were conducted. Internalization studies showed that strains chronic E. coli strains persisted intracellularly longer than acute E. coli strains. Treatment of bovine mammary epithelial cells CME or RME inhibitors resulted in lower numbers of intracellular E. coli strains associated with chronic or acute mastitis than untreated controls. In addition, when selective CME inhibitors were used significantly fewer chronic E. coli were detected intracellularly than acute E. coli or untreated controls. Confocal laser microscopy showed that chronic E. coli strains colocalized preferentially with caveolae whereas acute strains did so with early endosomes, an early step of RME. These results suggest that strains of E. coli associated with chronic mastitis exploit lipid rafts/CME to internalize into and move through mammary epithelial cells. By exploiting this endocytosis pathway, chronic E. coli strains avoid bactericidal mechanisms such as endosome acidification and endosome-lysosome fusion, thus allowing intracellular survival. Data from this study helps to explain how these strains are capable of causing chronic E. coli mastitis.