Comparison of the odds of isolation, genotypes, and in vivo production of major toxins by Clostridium perfringens obtained from the gastrointestinal tract of dairy cows with hemorrhagic bowel syndrome or left-displaced abomasum

OBJECTIVE: To compare the frequency of isolation, genotypes, and in vivo production of major lethal toxins of Clostridium perfringens in adult dairy cows affected with hemorrhagic bowel syndrome (HBS) versus left-displaced abomasum (LDA). DESIGN: Case-control study. ANIMALS: 10 adult dairy cattle with HBS (cases) and 10 adult dairy cattle with LDA matched with cases by herd of origin (controls). PROCEDURE: Samples of gastrointestinal contents were obtained from multiple sites during surgery or necropsy examination. Each sample underwent testing for anaerobic bacteria by use of 3 culture methods. The genotype of isolates of C. perfringens was determined via multiplex polymerase chain reaction assay. Major lethal toxins were detected by use of an ELISA. Data were analyzed with multivariable logistic regression and chi2 analysis. RESULTS: C. perfringens type A and type A with the beta2 gene (A + beta2) were the only genotypes isolated. Isolation of C. perfringens type A and type A + beta2 was 6.56 and 3.3 times as likely, respectively, to occur in samples from cattle with HBS than in cattle with LDA. Alpha toxin was detected in 7 of 36 samples from cases and in 0 of 32 samples from controls. Beta2 toxin was detected in 9 of 36 samples from cases and 0 of 36 samples from controls. CONCLUSIONS AND CLINICAL RELEVANCE: C. perfringens type A and type A + beta2 can be isolated from the gastrointestinal tract with significantly greater odds in cattle with HBS than in herdmates with LDA. Alpha and beta2 toxins were detected in samples from cows with HBS but not from cows with LDA.     

Use of enzyme-linked immunoassays for antibody to types C and D botulinum toxins for investigations of botulism in cattle

The development of specific enzyme-linked immunosorbent assays (ELISA) for antibody to types C and D Clostridium botulinum toxins for investigation of botulism in cattle is described. Partially purified type C and D toxins were used as antigens to develop these ELISAs. Specificity of the ELISAs was evaluated on sera from 333 adult beef and dairy cattle from areas with no history or evidence of botulism in animals or water birds. The test was also evaluated on sera from 41 herds that included herds vaccinated against botulism, confirmed botulism cases and herds from areas where the disease is considered endemic. The ELISAs detected the presence of antibody to botulinum toxins in samples from vaccinated cattle and both convalescent and clinically normal animals from unvaccinated herds with outbreaks of botulism. Antibody was also found in unvaccinated animals from herds in which there had been no diagnosed botulism cases in areas where botulism was considered endemic. Sera from some unvaccinated cattle with high ELISA reactivity was shown to be protective for mice in botulinum toxin neutralisation tests. The use of these tests in investigations of botulism in cattle is discussed.     

Two Cases of Equine Grass Sickness with Evidence for Soil‐borne Origin Involving Botulinum Neurotoxin

Botulism is caused by different types of Clostridium botulinum, a soil bacterium. Equine grass sickness (equine dysautonomia) is suspected of being a clinical form of this disease. On a stud where this disease occurred twice within 8 months, grass and soil samples and necropsy specimens of one horse were tested for the presence of bacterial forms and toxin of C. botulinum. Different types and type mixtures (A–E) of C. botulinum and botulinum neurotoxin were found. For the first time, it has been shown that green grass blades contain botulinum toxin. The results support the hypothesis that equine grass sickness is a clinical form of botulism, a soil‐borne disease.     

Prevalence and Species Distribution of Yeast in Mammary Glands of Dairy Cows in Minnesota

A study of the prevalence of yeast-like fungi in the mammary glands of dairy cattle was conducted in Minnesota. Quarter samples from 6,020 cows were cultured for yeast. Growth of organisms was obtained from 3.2% of the quarter milk samples. The rate of yeast infection for Minnesota dairy cattle in this study was 2.0%.

The majority of the yeast isolated belonged to one of four species of the Candida genus. Candida krusei, Candida parakrusei, Candida guilliermundi, and Candida tropicalis, comprised 89% of the yeasts isolated. All of these species have been reported to cause clinical mastitis (1, 7, 9, 10, 12, 13, 15, 16).

It would appear that yeast-like fungi are of sufficient prevalence in mammary glands that yeast infection would be considered in the differential diagnosis in cases of clinical mastitis.

     

Acute Atypical Bovine Pneumonia Caused by Ascaris Lumbricoides

A case of acute atypical pneumonia (bovine asthma, pulmonary emphysema, or pulmonary adenomatosis) occurred in a group of cross-bred beef type yearlings in late autumn. Clinical signs included a forced expiratory grunt, excessive salivation, ruminal stasis and, on auscultation over the lungs, pulmonary emphysema and oedema. The cattle had been brought in from a poor summer pasture and housed in a pig pen heavily contaminated with Ascaris lumbricoides eggs as it had contained unwormed feeder pigs all summer.

Fifteen out of seventeen head were affected ten days following housing and all within twenty-four hours. One steer of the group died and at necropsy fourth stage A. lumbricoides larvae were isolated from lungs showing profuse oedema and some emphysema. Histopathological examinations of the lungs showed a diffuse interstitial pneumonia. All remaining animals appeared clinically normal six days following the outbreak.

     

The Effects of Age and Previous Infection on the Development of Gastrointestinal Parasitism in Cattle

An age resistance in cattle to establishment of infection with Cooperia oncophora was not demonstrated. Cattle exposed to a heavy infection for the first time at approximately 15 months of age were as susceptible to establishment of infection as 3 to 4 month old calves, but stunting of worms and inhibition of ovulation did occur in the older animals, possibly due to a rapid development of resistance as a result of sensitization by a previous extremely light infection.

An age resistance in cattle to infection with Nematordirus helvetianus was not clearly demonstrated. At necropsy, 8 of 9 calves and 2 of 6 yearlings exposed to pasture infections for the first time did harbour Nematodirus worm burdens, while yearlings which were heavily infected previously were completely free of this species.

Under the conditions of this investigation, age and acquired resistance to Ostertagia ostertagi were not demonstrated, since previously non-exposed calves and yearlings and previously infected yearlings had comparable worm burdens.

This study demonstrated the adverse effect that heavy parasitism has on the development of susceptible animals. Animals which had little or no exposure to parasitism were found to be much more susceptible to the effects of parasites than were resistant animals.

     

Establishment of a Salmonella-Free Guinea Pig Colony

Salmonellosis due to Salmonella typhimurium was enzootic in a guinea pig breeding colony for over 25 years. A Salmonella-free auxiliary colony was established by removing weanlings from the infected colony to a clean area, and preventing infection. Examination of agglutinin titers and necropsy specimens indicated that the auxiliary colony was still free from Salmonella 18 months after its establishment while 24% of the guinea pigs dying in the infected colony yielded Salmonella typhimurium.     

Studies on the Contamination of Products Produced by Rendering Plants

Studies on the bacterial contamination in rendered product and the environment of five rendering plants were carried out. From a total of 180 samples examined, total bacterial and anaerobic spore counts were conducted on 135.

Plants with melter systems produced a sterile product which was recontaminated before reaching the finished stage. Two plants with continuous rendering systems did not achieve sterilization of the product during the heating process. Spore forming organisms regularly survived heating in the continuous rendering system.

Salmonellae were isolated from samples collected in four of the five plants under study. Pathogenic Clostridia, especially Cl. novyi, Cl. septicum and Cl. perfringens were present in samples from all plants. Other pathogens found were Staphylococci, Streptococci, Corynebacteria and Pasteurella.

     

Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on T_m values of 85.03 ± 0.54℃ for stx1 and 87.47 ± 0.35℃ for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/µL), and quantifiable (R² = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.     

Tonsils--place of botulinum toxin production: results of routine laboratory diagnosis in farm animals

Since a case of a veterinarian was reported, who was likely to be infected/intoxicated by Clostridium botulinum during the handling of a diseased animal, tonsils in animals were tested for botulinum neurotoxin and bacterial forms of neurotoxic Clostridium botulinum during routine botulism laboratory examinations including standard samples (intestinal tract and liver) from 48 cattle, 11 horses, and 14 goats. Ten out of 60 samples from tonsils contained free botulinum toxin, and 12 out of 59 were positive for live toxin producing bacteria. In 32 out of 162 intestinal samples toxin was detected. Toxin producing bacteria were found in 37 samples. Eight of 56 liver samples contained free toxin, and 15 out of 43 toxigenic bacteria. Samples from 10 slaughter pigs were all negative, whereas from slaughter cattle tonsils had a high incidence of toxin (7 of 10) or toxigenic bacteria (2 of 8). The results are discussed in the context of effects on animal health and botulism as zoonosis.     

Parasites of Dogs from Indian Settlements in Northwestern Canada: A Survey with Public Health Implications

A total of 959 faecal samples were obtained from dogs in 12 native communities in Northern Saskatchewan, Central and Northern Alberta and the Northwest Territories. All samples were examined using a flotation technique. Samples from an area of endemic human amoebic infections were also examined by a formol-ether sedimentation method. Eighteen necropsies were performed.

Entamoeba histolytica cysts were recovered from dog faeces at Loon Lake, Saskatchewan.

Toxocara canis had low incidence in Saskatchewan and Central Alberta, and appeared to be almost non-existent further North. Toxascaris leonina was found in all areas surveyed. Canine hookworm infections were plentiful in all areas, the highest incidence being recorded from Northern Alberta and Northwest Territories. Many Taenia (or Echinococcus) infections were found consistently in all areas. Only one infection with Dipylidium caninum was discovered.

Metorchis conjunctus infections were found to be common in the Saskatchewan reserves. Infections with Diphyllobothrium sp. were found in all communities with access to good fishing. One specimen of Dioctophyma renale was recovered at necropsy.

Infections with parasites of no known zoonotic importance such as Trichuris, Alaria and Isospora species were also recorded.

     

Colonisation by Pasteurella multocida in atrophic rhinitis of pigs and immunity to the osteolytic toxin

Gnotobiotic pig antisera to purified toxoid from a capsule type A or D strain of Pasteurella multocida contained large quantities of antitoxin but comparatively little antibody to a crude lysate of P. multocida. These sera given intraperitoneally to further pigs were almost completely protective against turbinate atrophy after intranasal inoculation of dilute acetic acid and infection with type D toxigenic P. multocida. In contrast, antisera to a crude lysate or bacterin of toxigenic P. multocida which contained large titres of antibody to P. multocida lysate, but no detectable antitoxin, were not protective. Colonisation by toxigenic P. multocida was significantly reduced in protected pigs and was similar to colonisation by nontoxigenic P. multocida in pigs untreated or treated with dilute acetic acid. These results indicated (1) that antitoxin was protective and cross protective between toxins from different capsule types; and (2) that the toxin was the main colonisation factor produced by toxigenic bacteria in the acetic acid model of infection and that immunity to it did not eliminate infection.     

Evaluation of ovine herpesvirus type 2 infections, as detected by competitive inhibition ELISA and polymerase chain reaction assay, in dairy cattle without clinical signs of malignant catarrhal fever

OBJECTIVE: To monitor ovine herpesvirus type 2 (OvHV-2) infection status and the association between OvHV-2 infection and development of clinical signs of malignant catarrhal fever (MCF) in cattle. DESIGN: Longitudinal study. ANIMALS: 30 mature adult cows and 18 cattle submitted for necropsy. PROCEDURE: Blood and milk samples were collected at monthly intervals from 30 adult cows for 20 consecutive months. Nasal and ocular swab specimens were also collected during months 9 through 20. Polymerase chain reaction (PCR) assay for detection of OvHV-2 was performed on blood, milk, nasal swab, and ocular swab specimens. Competitive inhibition ELISA (CI-ELISA) for detection of antibodies against MCF viruses was performed on serum samples obtained prior to study initiation and monthly during the last 12 months. Tissues obtained from herdmates without clinical signs of MCF that were submitted for necropsy were analyzed for OvHV-2 DNA via PCR assay for possible sites of latency. RESULTS: Initially, 8 of 30 cows had positive CI-ELISA results. Seroconversion was detected in 4 cows. Ovine herpesvirus type 2 DNA was intermittently detected in blood, milk, nasal secretions, or ocular secretions from 17 of 30 cows. Twenty-one cows had positive CI-ELISA or PCR assay results. No cattle in the study developed clinical signs of MCF. Results of PCR assays performed on tissue samples from 2 of 18 animals submitted for necropsy were positive for OvHV-2. CONCLUSIONS AND CLINICAL RELEVANCE; OvHV-2 infection can occur in cattle without concurrent development of clinical MCF. Ovine herpesvirus type 2 DNA was detected intermittently, suggesting fluctuating viral DNA loads or reinfection in subclinical cattle. A definitive site of latency was not identified from tissues obtained during necropsy.     

Larval Distribution and Histopathology of Experimental Strongyloides ransomi Infection in Young Swine

Necropsy at various intervals of three week old pigs following experimental infection with larvae of Strongyloides ransomi showed distribution of the larvae through the body during the first 24 hours. Quiescent larvae at the 24 hour necropsy, as indicated by higher recovery before and after this period may be an indication of physiological changes preceding a moult. Whether or not a moult occurs in the lung prior to migration of the larvae to the intestine has not been determined. At the 72 hour necropsy, larvae were largely confined to the lung and juvenile worms were beginning to appear in the small intestine. Migration to the small intestine was completed by the 96 hour necropsy. Lesions were observed in the skin and lungs up to the 96 hour necropsy, and lymphocytic accumulations were observed in the skin and lungs to this point and lymphocytic accumulations were observed in the liver to the 72 hour necropsy. At 28 days post-application lung lesions were still evident and the duodenum and jejunum were heavily parasitized.

Egg passage began on the sixth day post-application and peaked on the 12th day.

     

Comparison of Pathogenicity of Various Isolates of Bordetella Bronchiseptica in Young Pigs

Eight groups of 12-to 24-hour-old pigs were procured from a respiratory disease-free herd of swine and reared in isolation using a box-rearing procedure. They were inoculated intranasally at 3 days of age with different isolates of Bordetella bronchiseptica.

It was found at necropsy 4 weeks post-inoculation that 4 isolates of swine origin, an isolate of rabbit origin and an isolate of cat origin caused mild to moderate turbinate atrophy in 22 of 24 pigs. An isolate of rat origin caused mild turbinate atrophy in 1 of 4 pigs and an isolate of dog origin caused no turbinate atrophy. Pneumonia was present in most of the pigs inoculated with the swine, cat and rabbit isolates.

Bordetella bronchiseptica was recovered in heavy growth from the nasal and tracheal exudate collected at necropsy from pigs inoculated with the 4 isolates of swine origin and the isolate of cat origin. Fewer organisms were isolated from nasal exudate collected from pigs inoculated with the rat, dog and rabbit isolates.

     

The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells.     

Evaluation of five enzyme immunoassays compared with the cytotoxicity assay for diagnosis of Clostridium difficile-associated diarrhea in dogs.

Clostridium difficile-associated-diarrhea (CDAD) is a nosocomial infection in dogs. Diagnosis of this infection is dependent on clinical signs of disease supported by laboratory detection of C. difficile toxins A or B, or both, in fecal specimens via enzyme-linked immunosorbent assay (ELISA). Unfortunately, to the authors' knowledge, commercially available ELISAs have not been validated in dogs to date. We evaluated 5 ELISAs done on 143 canine fecal specimens (100 diarrheic and 43 nondiarrheic dogs) and on 29 C. difficile isolates. The results of each ELISA were compared with the cytotoxin B tissue culture assay (CTA). Clostridium difficile was isolated from 23% of the fecal specimens. Eighteen of the 143 fecal specimens were toxin positive (15 diarrheic and 3 nondiarrheic dogs). On the basis of multiplex polymerase chain reaction (PCR) analysis for toxin-A and -B genes, 72% of the isolates were toxigenic. The carriage rate of toxigenic isolates in diarrheic dogs was higher than that in the nondiarrheic dogs; however, these differences were not statistically significant. A good correlation was found between CTA, PCR, and culture results. The ELISAs done on fecal specimens collected from diarrheic dogs had low sensitivity (7-33%). In contrast, ELISA for toxin A or B, or both, performed on toxigenic isolates had high sensitivity (93%). These results suggest that commercially available human ELISAs are inadequate for the diagnosis of canine C. difficile-associated diarrhea when tested on fecal specimens. In contrast, the Premier ToxinA/B and Techlab ToxinA/B ELISAs may be useful for the diagnosis of canine CDAD when used on toxigenic isolates.     

The detection of Clostridium botulinum in fecal samples of cattle and swine and in the raw material and animal meal of different animal body rendering plants

Fecal samples of cattle and swine and samples of raw material and pulverized dehydrated meat taken from three rendering plants were investigated with special enrichment methods on the presence of Clostridium botulinum to get a view about the hygienic risk by the incidence of C. botulinum in rendering plants. Eight-six specimens were examined: 25 fecal specimens each of swine and cattle, 11 of raw material and 25 of pulverized dehydrated meat of three rendering plants. Twelve specimens contained C. botulinum: 7 fecal specimens, 6 of swine and one of cattle, 4 raw material specimens and one of pulverized dehydrated meat. C. botulinum was detected by its toxin production in culture medium. Six times C. botulinum type E, twice C. botulinum type B and one time C. botulinum type C was identified. C. botulinum could not be typed in other cases because the toxin quantities were too small. C. botulinum type E was detected in raw material and pulverized dehydrated meat in one of the three examined rendering plants.     

Bacteriological and molecular typing of Clostridium perfringens strains isolated in retail beef in Beijing,China

Clostridium perfringens is an important zoonotic pathogen. This study was designed to explore the prevalence and toxin types of C. perfringens in retail beef collected from Beijing, China. Among 221 beef samples collected, 53 samples were positive for C. perfringens, resulting in the average prevalence as 23.98%. By toxin gene-based typing, the most C. perfringens strains belong to type A (96.23%, 51/53), only 2 strains were identified as type D. By a multi-locus sequence typing (MLST)-based analysis, a total of 36 sequence types (STs) were detected, and the most STs (n=30) represented just a single strain. These finding suggested that the prevalence of C. perfringens in retail beef in Beijing was considerably high and these bacteria displayed extreme diversity in genetics.     

Clostridium difficile in faeces from healthy dogs and dogs with diarrhea

Background

This study was conducted to evaluate the faecal occurrence and characterization of Clostridium difficile in clinically healthy dogs (N = 50) and in dogs with diarrhea (N = 20) in the Stockholm-Uppsala region of Sweden.

Findings

Clostridium difficile was isolated from 2/50 healthy dogs and from 2/20 diarrheic dogs. Isolates from healthy dogs were negative for toxin A and B and for the tcdA and tcdB genes. Both isolates from diarrheic dogs were positive for toxin B and for the tcdA and tcdB genes. The C. difficile isolates from healthy dogs had PCR ribotype 009 (SE-type 6) and 010 (SE-type 3) whereas both isolates from dogs with diarrhoea had the toxigenic ribotype 014 (SE-type 21). One of the isolates from healthy dogs was initially resistant to metronidazole.

Conclusions

This study revealed presence of toxigenic C. difficile in faecal samples of diarrheic dogs and low number of non- toxigenic isolates in healthy dogs from Uppsala-Stockholm region in Sweden. However, more comprehensive studies are warranted to investigate the role of C. difficile in gastrointestinal disease in dogs.