Financial impact of an outbreak of clinically diagnosed blackleg - a case study from Lao PDR.

Blackleg (BL) is an acute to peracute highly fatal infectious disease of mainly large ruminants characterised by lesions of myonecrosis caused by Clostridium chauvoei, usually presenting as a sudden onset of sporadic mortalities. In Southeast Asia, 'BL' is considered a cause of occasional outbreaks of a subacute febrile illness, although there are few published reports available. Investigation of a major outbreak of clinically diagnosed BL occurring in large ruminants (cattle and buffalo) in three neighbouring villages in central Laos in mid-2017, was conducted to determine the financial impacts of BL on smallholder livelihoods. Owners of BL-infected large ruminants in the three affected villages were interviewed (n = 27) and financial losses including losses due to mortality, morbidity and costs of treatments, were determined. The reports of clinical signs of subcutaneous swelling with palpable crepitus in febrile animals were considered consistent with a diagnosis of BL. The outbreak occurred in 47 of a total 449 households with large ruminants across the three villages, affecting 147 of a total population of 3505 'at risk' large ruminants with 71 deaths reported. At the household level, the mean morbidity and mortality rates were 5 ± 4(95% CI: 3-6) and 3 ± 2(95% CI: 1-4) heads per household, respectively. The estimated financial losses due to BL per affected household was USD 822 ± 692(95% CI: 518-1125), being 122% of their annual household income from large ruminant sales. The comparison between the estimated losses due to BL per village and cost of annual BL vaccination programmes, indicated a potential economic benefit of USD3.09 and USD12.37 for every dollar invested in the vaccination programme, if BL outbreaks occur every 20 and 5 years, respectively. This study indicates that clinically diagnosed BL can cause significant losses to smallholder households, and requires Lao animal health authorities to consider vaccination interventions to prevent losses from re-emergence of BL in the known endemically affected areas.

Characterization of quorum sensing system in Clostridium chauvoei.

Clostridium chauvoei causes fatal black quarter infection in cattle and buffaloes. The quorum sensing (QS) system, a bacterial cell to cell communication process, of the pathogen was characterized in the current study. The results indicated that C. chauvoei lacked luxS (autoinducer-2) based quorum sensing as detected by the sensor strain Vibrio harveyi BB170. This was supported by absence of luxS gene in C. chauvoei genome. However, the genomic analysis indicated the presence of agrBD system in all three genomes of C. chauvoei available at the NCBI database. The AgrD, which synthesizes QS messenger auto-inducing peptide, was a 44 amino acid protein which shared 59% identity and 75% similarity with AgrD of C. perfringens strain 13 and 56% identity (20% coverage) with Staphylococcus aureus N315. The functional cysteine amino acid was conserved in all the strains. The genomic organisation further suggests the presence of diguanylate cyclase, a gene responsible for synthesis of secondary messenger cyclic di-GMP, at 3' immediate downstream of agrD gene. The real time expression analysis for agrD gene indicated that expression was better at 37 °C (1.9-3.7 fold increase) compared to a higher temperature of 40 °C. However, stable expression was observed at different growth stages (log and early stationary phase) with 0.8-1.4 fold changes in expression pattern. The results indicate the presence of a constitutively expressed agrBD quorum sensing system in C. chauvoei.

Intracellular survival of Clostridium chauvoei in bovine macrophages.

Clostridium chauvoei is the etiological agent of blackleg, a severe disease of domestic ruminants, causing myonecrosis and serious toxemia with high mortality. Despite the known importance of this agent, studies evaluating its pathogenesis of blackleg are scarce, and many are based on an unproven hypothesis that states that macrophages are responsible for carrying C. chauvoei spores from the intestines to muscles in the early stages of blackleg. Therefore, the present study aimed to investigate the survival of C. chauvoei vegetative cells or spores after phagocytosis by a murine macrophage cell line (RAW 264.7) and bovine monocyte-derived macrophages and to profile inflammatory and anti-inflammatory cytokine transcripts of bovine macrophages infected with C. chauvoei vegetative cells or spores. Both vegetative cells and spores of C. chauvoei remain viable after internalization by murine and bovine macrophages. Bovine macrophages infected with vegetative cells showed a pro-inflammatory profile, while those infected with spores displayed an anti-inflammatory profile. Together, these results corroborate the classical hypothesis that macrophages may play a role in the early pathogenesis of blackleg. Moreover, this is the first study to evaluate the infection kinetics and cytokine profile of bovine monocyte-derived macrophages infected with a Clostridium species.

Detection and identification by PCR of Clostridium chauvoei in clinical isolates, bovine faeces and substrates from biogas plant.

BACKGROUND: Clostridium chauvoei causes blackleg, an acute disease associated with high mortality in ruminants. The apparent primary port of entry is oral, during grazing on pasture contaminated by spores. Cases of blackleg can occur year after year on contaminated pastures. A method to determine the prevalence of C. chauvoei spores on pasture would be useful.The standard method for C. chauvoei detection is culture and biochemical identification, which requires a pure culture. In most muscle samples from cattle dead from blackleg the amount of C. chauvoei in samples is high and the bacterium can easily be cultured, although some samples may be contaminated. Detection by PCR would be faster and independent of contaminating flora.Digested residues from biogas plants provide an excellent fertiliser, but it is known that spore-forming baeria such as Clostridium spp. are not reduced by pasteurisation. The use of digested residues as fertiliser may contribute to the spread of C. chauvoei. Soil, manure and substrate from biogas plants are contaminated with other anaerobic bacteria which outgrow C. chauvoei. Therefore, detection by PCR is would be useful. This study applied a PCR-based method to detect of C. chauvoei in 25 muscle and blood samples, 114 manure samples, 84 soil samples and 33 samples from the biogas process. METHODS: Muscle tissues from suspected cases of blackleg were analysed both by the standard culture method followed by biochemical identification and by PCR, with and without preculture. To investigate whether muscle tissue samples are necessary, samples taken by swabs were also investigated. Samples from a biogas plant and manure and soil from farms were analysed by culture followed by PCR. The farms had proven cases of blackleg. For detection of C. chauvoei in the samples, a specific PCR primer pair complementary to the spacer region of the 16S-23S rRNA gene was used. RESULTS: Clostridium chauvoei was detected in 32% of muscle samples analysed by culture with identification by biochemical methods and in 56% of cases by culture in combination with PCR. Clostridium chauvoei was detected in 3 (out of 11) samples from the biogas plants collected before pasteurisation, but samples taken after pasteurisation and after digestion all tested negative. Clostridium chauvoei was not detected in any soil or silage samples and only one manure samples tested positive. CONCLUSION: The diagnostic method used for C. chauvoei was not applicable in estimating the risk of blackleg on particular pastures from manure or soil samples, but found to be highly useful for clinical samples.