Necrotic enteritis potential in a model system using Clostridium perfringens isolated from field outbreaks.

Necrotic enteritis is an enteric disease of avian species caused by the anaerobic bacterium Clostridium perfringens. The disease is regularly controlled in the broiler chicken industry with antimicrobials in feed but is reemerging in areas such as Europe where there is a ban on antimicrobials as growth promoters. To study prospective therapies, researchers must be able to reproduce this disease in a controlled environment, but this is not always possible because of differences in the pathogenicity of C. perfringens strains. Our objective was to test the potential of five isolates (SNECP43, 44, 47, 49, and 50), taken from field cases of necrotic enteritis, at recreating the disease in a controlled challenge experiment. SNECP43 and 50 were derived from a common clone, with SNECP50 passed in vivo and SNECP43 subcultured in vitro. Four hundred birds were divided into 16 pens, with three pens each receiving one of five treatments, with one control pen. Day-old birds were raised on a high wheat-based diet to promote necrotic enteritis development and were challenged with between 3.4 x 10(9) and 3.2 x 10(11) colony-forming units (cfu) of C. perfringens in feed for a period of 24 hr starting on day 13 of the challenge experiment. Lesion scores were assessed on two birds per pen sacrificed on day 17 and on any dead birds during the 25-day study. Growth performance was assessed up to 25 days, and mortality recorded throughout. Only SNECP50 produced necrotic enteritis mortalities significantly different (P < or = 0.05) from the control. The five isolates were also typed using pulsed-field gel electrophoresis to assess their genetic relatedness. All epidemiologically unrelated isolates were deemed genetically unrelated, whereas SNECP43 and 50 differed by only a single minor band. Toxin type was assessed using polymerase chain reaction (PCR), which was also used for the detection of the gene encoding the beta2-toxin.

The efficacy of bacitracin methylene disalicylate when fed in combination with narasin in the management of necrotic enteritis in broiler chickens.

The efficacy of bacitracin methylene disalicylate (BMD) in the management of necrotic enteritis (NE) when fed in combination with narasin was investigated in a floor-pen study of 2,000 broiler chickens using a Clostridium perfringens inoculum challenge model. Treatments consisted of 1) nonchallenged-nonmedicated; 2) challenged-nonmedicated; 3) challenged-narasin (70 ppm); 4) challenged-BMD (55 ppm); 5) challenged-narasin (70 ppm) + BMD (55 ppm). Medication was provided in the feed from Day 0 to trial termination on Day 41. C. perfringens challenge occurred from Day 14 to 16. BMD and narasin, fed alone and in combination, reduced (P < 0.05) mortality due to NE when compared to challenged-nonmedicated birds. NE lesion scores (Days 0 through 41) were lower among birds fed BMD and narasin, alone and in combination, compared to challenged-nonmedicated birds. Improvements in NE mortality and NE lesion scores were greatest for the BMD + narasin-medicated birds, followed by the BMD-alone, and then narasin-alone treated birds. BMD and narasin, alone and in combination, provided improvements (P < 0.05) in average daily gains over the entire study (Days 0 to 41). The results of this study demonstrate the effectiveness of BMD and narasin in the management of NE in broiler chickens.

Efficacy of in-feed tylosin phosphate for the treatment of necrotic enteritis in broiler chickens.

The efficacy of tylosin phosphate for the treatment of necrotic enteritis (NE) was investigated in a floor pen study of 2,000 broiler chickens. A model in which Clostridium perfringens was administered in the feed on Days 14 to 16 was used to initiate an outbreak of NE. Treatments, allocated at the pen level in a randomized complete block design, consisted of five levels of tylosin phosphate (0, 50, 100, 200, or 300 ppm) administered in the feed on Days 15 to 22, following the identification of an outbreak of NE on Day 15. Mortality due to NE was significantly reduced (P < 0.05) for medicated birds at all dose levels of tylosin phosphate compared to unmedicated birds. Mean NE lesion scores on Day 17 were significantly reduced (P < 0.05) by all levels of tylosin treatment compared to those of unmedicated birds, decreasing linearly from 2.66 at 0 ppm to 0.38 at 100 ppm and 0 at higher doses. Tylosin at all levels provided improvement in Day 29 body weight, average daily gain, feed to gain ratio, and average daily feed intake compared to unmedicated birds. The results of this study provide evidence that tylosin phosphate, when administered in feed, is effective in the treatment of clinical outbreaks of NE in broiler chickens and suggest that the optimal dose for this purpose is 100 ppm.

Experimental Staphylococcus aureus intramammary challenge in late lactation dairy cows: quarter and cow effects determining the probability of infection.

The purpose of this study was to identify factors at the quarter and cow level that determine whether a quarter remains infected after an intramammary challenge with Staphylococcus aureus Newbould 305. A total of 135 cows were studied. Information on animal characteristics, cow-conformation, cow somatic cell count (SCC), and bacteriology, blood vitamin E levels, serology for retro-viral infections, bovine leukocyte adhesion deficiency-carrier status, and the presence of bovine lymphocyte antigens class I alleles was collected on each animal. All quarters of all cows were then challenged with Staphylococcus aureus Newbould 305. The challenge with S. aureus Newbould 305 resulted in 28 cows (20.7%) that did not establish infection in any of the quarters, 21 (15.6%) cows had 1 quarter infected, 35 (25.9%) had 2 quarters infected, 24 (17.8%) had 3 quarters infected, and 27 (20.0%) had all quarters infected. A higher prechallenge SCC decreased the risk of infection. An infection with Corynebacterium bovis prior to challenge decreased the risk of S.aureus infection. Of the bovine lymphocyte antigen alleles, the presence of the W20A allele proved to be significantly associated with a decreased risk of infection. No other factors proved to be significant.

Socioeconomic significance of the HAP group.

An overview is presented of some members of the HAP group important in animal disease. An attempt has been made to highlight the economic importance of these diseases and to alert us to the importance of research that will lead to their prevention and control.

Effect of chlorination of drinking water on experimental salmonella infection in poultry.

The type of drinker used for poults influenced the level of free available chlorine (FAC) in chlorinated water as well as the total plate count, fecal coliform count, and number of salmonellae in chlorinated and non-chlorinated drinking water. Nipple drinkers maintained higher levels of FAC in drinking water than Swish-cups, Swish-cups maintained higher levels than MarkIII, and MarkIII maintained higher levels than trough drinkers. The level of FAC retained in the water in trough drinkers was insufficient to exert a bactericidal effect against coliforms and salmonellae. Chlorination of drinking water and the resulting diminished number or absence of salmonellae in the drinking water did not lower the number of salmonellae per gram of cecal contents in challenged or unchallenged but exposed poults. The number of salmonellae per gram of cecal contents decreased significantly (P less than 0.01) in poults between 14 and 21 days of age, irrespective of whether or not the poults drank chlorinated water.

Observations on intramammary infections in first calf heifers in early lactation.

Intramammary infections and mastitis were monitored on four occasions at three-week intervals in 61 first calf heifers in five dairy herds during the first ten weeks of lactation. Of 940 quarter milk samples examined 65% were classed as negative, 10.4% as positive for mastitis (mainly subclinical), 1.8% as group 3 (infection present but no elevation in somatic cell count) and 22.8% as group 4 (elevated somatic cell count). Seventy-seven percent of the infections detected and identified were those due to coagulase-negative staphylococci, the main species being S. hyicus, S. epidermidis, S. simulans and S. hominis. Other infections detected with Corynebacterium pyogenes (three samples), Escherichia coli (one sample), Micrococcus spp. (one sample), S. aureus (two samples) and Streptococci (non-agalactiae) (seven samples).The geometric mean somatic cell count for 23 quarters infected with coagulase-negative staphylococci was 311 x 10(3) cells / mL compared to 134 x 10(3) cells / mL in noninfected adjacent contralateral quarters. The respective figures for% cell volume in Channel 8 (mainly neutrophils) were 10.6% and 3.5%. There was a highly significant association between herd and the proportion of quarter milk samples in the four mastitis categories.

Comparison of the API Staph-Ident and DMS Staph Trac micromethods for the identification of coagulase-negative staphylococci.

Two rapid identification micromethods, the API Staph-Ident and the DMS Staph Trac, were compared for the identification of coagulase-negative staphylococci. Seventy-five isolates, mainly of bovine origin, were used as the test organisms. Species identification and profile numbers assigned to isolates by each system were compared. However, no clear correlation patterns emerged, indicating the two methods were not comparable.

Preliminary observations on the use of latex agglutination test for the detection of mastitis due to Streptococcus agalactiae in cows.

A commercial latex agglutination test for the detection of Group B streptococcal antigens was used to detect infection due to Streptococcus agalactiae in whey of bovine milk samples. Fifteen out of 17 known infections were detected, but it was necessary to incubate the wheys at 37 degrees C for 18 hours in nine of the samples. It was found that the latex agglutination test could detect Group streptococcal carbohydrate antigens in whey samples from artificially infected quarters from one to four days after failure to detect the organism on culture or after antibiotic therapy of the affected quarter.

Haemophilus somnus: investigations of its potential role in bovine mastitis.

Mammary glands of 6 lactating Holstein cows were inoculated with Haemophilus somnus strain 43826. Three cows developed chronic mastitis and shed bacteria for up to 1 year. Three cows developed acute gangrenous mastitis, with evidence of bacteremia and endotoxemia. Cows with gangrenous mastitis had lower somatic cell counts early after inoculation in affected quarter secretions compared with those in cows that developed chronic mastitis. Cows with gangrenous mastitis developed hypocalcemia, hypoalbuminemia, azotemia, hyperbilirubinemia, mildly increased serum aspartate aminotransferase and creatine kinase activity, and a marked neutropenia with a degenerative left shift. Histopathologic examination of gangrenous quarters revealed edema, necrosis, and vascular thrombosis with few inflammatory cells. A limited survey failed to recover H somnus from dairy cows with clinical mastitis or from mammary secretions from 41 beef cattle at an abattoir.

Studies on the immunogenicity of Streptococcus equi vaccines in foals.

The ability of either formalin-treated or heat-inactivated whole Streptococcus equi cell vaccines or partially purified M-protein of S. equi to give rise to protective antibody levels was studied in Standardbred foals by serological means. Two commercial preparations, i.e. a beta-propiolactone killed whole S. equi cell bacterin and a cell-free extract of S. equi cells were included in the study. The mean passive hemagglutination antibody titers (10 X log2) in sera of foals given either four doses of formalin-treated whole cell vaccine or an initial dose of formalin-treated followed by three doses of heat-inactivated vaccine with or without levamisole were significantly higher two weeks after the final dose. These passive hemagglutination antibody titers were higher in foals given formalin-treated whole cell vaccine (6.7 +/- 1.5) than given commercial bacterin (4.5 +/- 2.1). The passive hemagglutination antibody titers in all the groups decreased at 12 to 16 weeks after fourth dose of the vaccine. Foals given a commercial cell-free extract did not show a significant increase in passive hemagglutination antibody titers even up to four weeks after third dose. A group of six pony foals immunized with partially-purified M protein showed mean passive hemagglutination antibody titers lower than those observed in foals given whole cell vaccines. In a challenge experiment with S. equi, two of six foals vaccinated with partially-purified M-protein and all three controls developed clinical disease. The passive hemagglutination antibody of vaccinated foals increased after challenge, while at 28 days postchallenge the passive hemagglutination antibody titers of vaccinates and recovered controls were similar.

Serological cross-reactions between Brucella abortus and Yersinia enterocolitica 0:9.

Yersinia enterocolitica (Ye) 0:9 is an organism of great significance in veterinary medicine largely as a result of its cross-reaction with Brucella abortus (Ba). Boty Ye 0:9 and Ba possess somatic antigens in common; as a result of which animals exposed to Ye 0:9 have an immune response which is distinguishable only with difficulty from that induced by Ba. Cattle were exposed to Ye 0:9 by the oral or intramammary routes. Oral exposure failed to generate significant serologic response. In contrast, intramammary inoculation produced a marked response. Serum antibodies provoked in this manner reacted strongly with Ba. The anti-Brucella response provoked by inoculation of Yersinia was sufficient to render milk and serum Brucella-seropositive as measured by the standard milk ring and serum agglutination tests. While both Ba and Ye 0:9 have 9 antigens in common, they differ significantly with respect to motility. Thus Ba is always non motile while Ye is motile when grown at room temperature. The presence of Yersinia H agglutinins in serum were shown to be evidence of previous exposure to Ye. The H agglutinins were not generated by Brucella infection. A rapid H agglutination test was shown to provide this differentiation without interference from cross-reacting O antigens. Results of Ba O and Ye O and OH antigens used in the agglutination test were found useful to differentiate antibodies against Ba from those induced by Ye 0:9 in cattle sera. The existence of enterobacterial common antigen (ECA) in Ye and its absence in Ba were utilized in an attempt to provide a method to distinguish Brucella infections from those with cross-reacting Yersinia.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and biological properties of M-protein of Streptococcus equi.

The production of M-protein antigen of Streptococcus equi was studied during in vitro growth in equine blood and in various media. Of 11 S equi strains studied, seven which had initially possessed 0.04 mg or less M-protein per 10 mg of streptococcal cell extract showed an increase in M-protein content after successive culture in heparinised horse blood. Maximum proliferation occurred in Todd-Hewitt (TH) medium with added 0.2 per cent w/v glucose when compared with TH medium alone or TH medium with 2 per cent w/v sucrose, starch, neopeptone or normal horse serum. The M-protein of these strains did not change after the addition of either neopeptone or normal horse serum to TH medium but declined with the addition of sugars. In experiments involving phagocytosis of S equi by equine polymorphs, the percentage of polymorphs which engulfed cocci was higher with a capsule-deficient strain (69.0 +/- 11.6 per cent) than with five typical encapsulated strains (21.1 +/- 7.0 per cent to 30.9 +/- 13.3 per cent). Phagocytosis of five typical strains was greater after growth in a trypsin-containing medium than in medium devoid of trypsin. Trypsin-grown cells took longer to kill mice than did normal cells. It was concluded that M-protein was one of the factors involved in the virulence of S equi.

The effects of treatments of cecal contents on their protective properties against Salmonella in poults.

The effect of manipulating adult turkey cecal contents was assessed. Treatment of day-old poults with cecal contents diluted up to 1/3000 protected 90% of the birds against subsequent challenge with 10(3) colony-forming units of Salmonella typhimurium. The addition of penicillin G and streptomycin to adult cecal contents reduced their protective effects by 20% and 50%, respectively. When cecal contents were heat-treated at 65 C for 15 minutes or filter-sterilized, the protective properties were lost. Anaerobic cultures of cecal contents in reinforced clostridial medium afforded protection against challenge, but protection was lost on subsequent subcultures. Thioglycollate broth culture of cecal contents did not protect.

Acridine orange staining for diagnosis of Mycoplasma bovis infection in cow milk.

Mycoplasma organisms were readily recognized in samples of milk or udder secretions from cows with clinical Mycoplasma bovis mastitis when these samples were stained with 0.01% acridine orange at pH 3.0. Samples could be stored at -4 degrees C for several days or subjected to repeated freezing and thawing without loss of staining or fluorescence properties. Use of this procedure in diagnostic laboratories on suspect samples from cows with clinical mastitis could hasten inauguration of control measures against this highly contagious disease by several days; however, definitive diagnosis still requires standard culture methods.

Practical aspects of competitive exclusion for the control of Salmonella in turkeys.

In laboratory trials, a fourth-passage culture of adult chicken cecal contents was protective against challenge with Salmonella typhimurium in turkey poults raised on wood-shavings poultry litter. The culture was not protective against pre-treatment exposure to hatchery-introduced S. bredeney and was inhibited in poults that had received an antibiotic injection at the hatchery. The inhibitory effect of the hatchery antibiotic could be avoided if the cecal-culture treatment was delayed by 3 to 4 days after antibiotic injection. Under field conditions, there was a significant reduction in the salmonella contamination of turkeys and their environment when cecal culture was given to poults raised on wood-shavings litter sprayed with a quaternary ammonium compound. When used alone, both the cecal culture and the litter disinfectant were ineffective in preventing the establishment of S. heidelberg infection. Further studies are required to confirm the possibility of a synergistic effect between the two treatments. For the control of salmonella in turkeys, use of cecal cultures may be limited by the interference of antibiotics and by their failure to protect against pretreatment exposure to salmonella.

The susceptibility of bovine udder quarters colonized with Corynebacterium bovis to experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

Twenty bovine udder quarters colonized with Corynebacterium bovis SR6 and 20 uncolonized quarters were challenged by inoculation of Staphylococcus aureus Newbould 305 (ATCC 29740) into the teat cistern. The percentage of infection in quarters colonized with C. bovis (50%) was significantly lower than that in controls (100%). By similar challenge no significant difference was observed between the percentage of infection with Streptococcus agalactiae ATCC 27956 in 33 quarters colonized with C. bovis (70%) compared to 33 controls (87.9%). A total of 37 quarters colonized with C. bovis and 37 control quarters were challenged with Staph. aureus Newbould 305 (ATCC 29740) and Maxi (ATCC 27543) and Strep. agalactiae (ATCC 27956) by exposure of the teat orifice. The percentage of teat ducts colonized with C. bovis which became infected with either pathogen was not different from that for controls.

Fatal mastitis of dairy cows: a retrospective study.

The necropsy records of dairy cows with mastitis were reviewed from the provincial veterinary laboratory in Guelph (44 cases of mastitis in nine years) and from the Ontario Veterinary College (168 cases in 14 years). Mastitis was considered to be the primary cause of death in 167 of 212 cows (79%). Of these 167 cases of mastitis, Escherichia coli was involved in 107 (64%), Klebsiella sp. in 12 (7%) and Staphylococcus aureus in 11 (7%). Bacteriology was not reported in 22 cases. Coliform mastitis, the most commonly identified type of fatal mastitis, was characterized histologically by the presence of infarcted areas in affected glands and by the lack of demonstrable bacteria, and was thus easily identified from fatal mastitis caused by S. aureus.

Experimental colonization of the bovine teat duct with Corynebacterium bovis and the effect on milk somatic cell counts.

Colonization with Corynebacterium bovis was established in 59 of 64 (92%), 58 of 59 (98%) and 19 of 34 (56%) of uninfected bovine mammary quarters following inoculation of 83.3 X 10(4) colony-forming units (CFU) of the organism into the teat cistern, 4.7 X 10(3) CFU 5 mm into the teat duct or by exposure of the teat orifice to a milk culture containing 1.6 X 10(7) CFU/mL respectively. Mean somatic cell counts for foremilk samples from 122 quarters were significantly higher after colonization with C. bovis (145,900/mL) compared to before exposure (130,900/mL).