Factors associated with the seroprevalence of caseous lymphadenitis in sheep from Northeastern Brazil.

Caseous lymphadenitis (CL) is one of the main infectious diseases in sheep, responsible for great economic losses, mainly in Northeast region of Brazil, which has the largest sheep flock in the country. Thus, the aim of this survey was to determine the factors associated with the seroprevalence of this disease in sheep from Northeastern Brazil using a planned sample of flocks and animals. Samples were collected from 2312 adult and young sheep of both sex from 196 farms, located in 51 municipalities in five Northeastern Brazilian states (Ceará, Paraíba, Piauí, Rio Grande do Norte, and Sergipe). Serological diagnosis was performed using the indirect ELISA technique. Factors associated with seropositivity were identified by variables extracted from epidemiological questionnaires administered to breeders, using univariable and multivariable analyses. Factors associated with prevalence were purebred sheep (Prevalence ratio - PR = 1.189; P = 0.017), ram acquisition from animal expositions (PR = 1.192; P = 0.020), offspring is separated from ewes (PR = 1.132; P = 0.048), water supplied to sheep from ponds (PR = 1.365; P = 0.002), and delayed disposal of infected animals (PR = 1.263; P = 0.027). Flock-level and animal-level prevalences were 93.88 % (95 % CI = 89.60-96.46%) and 37.46 % (95 % CI = 35.51-39.45%), respectively. In view of the high prevalence found here and based on the associated factors, the implementation of an efficient CL control programme in Northeastern Brazil is recommended.

Genome sequence of a pathogenic Corynebacterium ulcerans strain isolated from a wild boar with necrotizing lymphadenitis.

OBJECTIVES: Corynebacterium ulcerans can colonize a wide variety of animals and also humans are infected, typically by zoonotic transmission. Symptoms range from skin ulcers or systemic infections to diphtheria-like illness. In contrast, Corynebacterium pseudotuberculosis is widely distributed among herds of sheep, goats and other farm animals, where it causes high economic losses due to caseous lymphadenitis. Here we describe the genome sequence of an atypical C. ulcerans strain isolated from a wild boar with necrotizing lymphadenitis. This strain has similarities to C. pseudotuberculosis. DATA DESCRIPTION: Genome sequence data of C. ulcerans isolate W25 were generated, analyzed and taxonomical relationship to other Corynebacterium species as well as growth properties of the isolate were characterized. The genome of C. ulcerans W25 comprises 2,550,924 bp with a G+C content of 54.41% and a total of 2376 genes.

Transcriptome profile of Corynebacterium pseudotuberculosis in response to iron limitation.

BACKGROUND: Iron is an essential micronutrient for the growth and development of virtually all living organisms, playing a pivotal role in the proliferative capability of many bacterial pathogens. The impact that the bioavailability of iron has on the transcriptional response of bacterial species in the CMNR group has been widely reported for some members of the group, but it hasn't yet been as deeply explored in Corynebacterium pseudotuberculosis. Here we describe for the first time a comprehensive RNA-seq whole transcriptome analysis of the T1 wild-type and the Cp13 mutant strains of C. pseudotuberculosis under iron restriction. The Cp13 mutant strain was generated by transposition mutagenesis of the ciuA gene, which encodes a surface siderophore-binding protein involved in the acquisition of iron. Iron-regulated acquisition systems are crucial for the pathogenesis of bacteria and are relevant targets to the design of new effective therapeutic approaches. RESULTS: Transcriptome analyses showed differential expression in 77 genes within the wild-type parental T1 strain and 59 genes in Cp13 mutant under iron restriction. Twenty-five of these genes had similar expression patterns in both strains, including up-regulated genes homologous to the hemin uptake hmu locus and two distinct operons encoding proteins structurally like hemin and Hb-binding surface proteins of C. diphtheriae, which were remarkably expressed at higher levels in the Cp13 mutant than in the T1 wild-type strain. These hemin transport protein genes were found to be located within genomic islands associated with known virulent factors. Down-regulated genes encoding iron and heme-containing components of the respiratory chain (including ctaCEF and qcrCAB genes) and up-regulated known iron/DtxR-regulated transcription factors, namely ripA and hrrA, were also identified differentially expressed in both strains under iron restriction. CONCLUSION: Based on our results, it can be deduced that the transcriptional response of C. pseudotuberculosis under iron restriction involves the control of intracellular utilization of iron and the up-regulation of hemin acquisition systems. These findings provide a comprehensive analysis of the transcriptional response of C. pseudotuberculosis, adding important understanding of the gene regulatory adaptation of this pathogen and revealing target genes that can aid the development of effective therapeutic strategies against this important pathogen.

Comparison of postmortem inspection procedures for detecting caseous lymphadenitis of Australian sheep and goats.

Alternative postmortem inspection procedures for the detection of gross abnormalities due to Caseous Lymphadenitis (CLA) of sheep and goats were compared quantitatively against the current Australian Standard (AS4696). Studies on sheep and goats in Australia during 2016 addressed data gaps regarding current prevalence, combinations of multiple lesions within affected carcases and sensitivity of inspection procedures enabling a comparison of alternative with current procedures. Using these contemporary inspection data from 54 915 sheep and 48 577 goats a desktop study estimated the effect of implementing alternative procedures of reduced palpation from eleven carcase sites to the four sites most commonly affected. Under current procedures it was estimated that 86 sheep and 34 goat carcases with CLA lesions are missed per 10,000 carcases. Under alternative procedures it is estimated that an additional 48.4 sheep and 10.5 goat carcases with CLA lesions would be missed per 10 000 carcases. Of these, 38.2 sheep and 5.6 goat per 10 000 carcases would contain CLA only in routinely discarded, non-edible tissue sites. Hence, only an additional 10.2 sheep and 4.9 goat carcases per 10 000 inspected, with CLA in edible tissue sites are estimated to be missed. These alternative procedures have now been officially implemented in the Australian domestic standard.

Responses of testosterone hormone concentration, semen quality, and its related pro-inflammatory cytokines in bucks following Corynebacterium pseudotuberculosis and its mycolic acid infection.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, a debilitating chronic disease of sheep and goats. Little is known about the buck's reproductive pathophysiology with respect to inoculation with Corynebacterium pseudotuberculois and its immunogen mycolic acid extract. Therefore, this present study was designed to determine the concentration of testosterone hormone, pro-inflammatory cytokines, and semen quality of the experimental animals. A total of 12 bucks, divided into groups 1, 2, and 3 (Negative control group, Positive control group and Mycolic acid group respectively), were enrolled in this study. Following inoculation, all goats were observed for clinical responses and monitored for 60 days post-challenge and were then sacrificed. Blood samples were collected via the jugular once before inoculation and on a weekly basis post-challenge. Semen samples were collected 2 weeks post-challenge and prior to the sacrifice of the experimental animals. During the post inoculation period of 60 days, the concentration of testosterone hormone for group 2 was increased significantly (p < 0.05) in weeks 5, 6, and 9 but decreased in weeks 2 and 7 post inoculation. In group 3, the mean concentration of testosterone was increased significantly (p < 0.05) in weeks 5, 6, 7, and 9 post inoculation but decreased in week 2. The concentration of interleukin 6 (IL 6) in treated group 2 did not show any significant difference (p > 0.05) but increased significantly (p < 0.05) in week 2 post inoculation in group 3. For concentration of interleukin 1ß (IL1ß) in both treated groups 2 and 3 showed significant difference (p < 0.05) in weeks 2 and 3 post inoculation. The tumor necrosis factor-alpha (TNF-α) concentration in both treated groups 2 and 3 did not show any significant difference (p > 0.05) as compared to group 1. The concentration of interferon-γ (IFNγ) significantly increased (p < 0.05) for group 2 for weeks 2, 3, 4, and 5 where else for group 3 was not in significant difference (p > 0.05) compared to group 1. Both group 2 and group 3 showed a reduction in semen qualities as compared to group 1, but the severity was more intense in group 2 if compared to group 3. In conclusion, therefore, the present study concluded that the mycolic acid group revealed significant responses of testosterone hormone concentration, semen quality, and its related pro-inflammatory cytokines in bucks following infection but the severity lesser compared to Corynebacterium pseudotuberculosis group.

Experimental transmission of Corynebacterium pseudotuberculosis biovar equi in horses by house flies.

BACKGROUND: The route of Corynebacterium pseudotuberculosis infection in horses remains undetermined, but transmission by insects is suspected. OBJECTIVES: To investigate house flies (Musca domestica L.) as vectors of C. pseudotuberculosis transmission in horses. ANIMALS: Eight healthy, adult ponies. METHODS: Randomized, controlled, blinded prospective study. Ten wounds were created in the pectoral region where cages for flies were attached. Three ponies were directly inoculated with C. pseudotuberculosis. Four ponies were exposed for 24 hours to 20 hours C. pseudotuberculosis-inoculated flies. One negative control pony was exposed to noninoculated flies. Ponies were examined daily for swelling, heat, pain, and drainage at the inoculation site. Blood was collected weekly for CBC and biochemical analysis, and twice weekly for synergistic hemolysis inhibition titers. RESULTS: Clinical signs of local infection and positive cultures were observed in 7/7 exposed ponies and were absent in the negative control. In exposed ponies, peak serologic titers (1:512 to 1:2,048) were obtained between days 17 and 21. Seroconversion was not observed in the negative control. Neutrophil counts were higher in the positive and fly-exposed groups than in the negative control (P = .002 and P = .005) on day 3 postinoculation. Serum amyloid A concentrations were higher in the positive control than in the negative control and fly-exposed ponies on days 3 (P < .0001) and 7 (P = .0004 and P = .0001). No differences were detected for other biochemical variables. CONCLUSIONS AND CLINICAL IMPORTANCE: House flies can serve as mechanical vectors of C. pseudotuberculosis and can transmit the bacterium to ponies.

Proteomic characterization of a natural host-pathogen interaction: repertoire of in vivo expressed bacterial and host surface-associated proteins.

Interactions between a host and a bacterial pathogen are mediated by cross-talk between molecules present on, or secreted by, pathogens and host binding-molecules. Identifying proteins involved at this interface would provide substantial insights into this interaction. Although numerous studies have examined in vitro models of infection at the level of transcriptional change and proteomic profiling, there is virtually no information available on naturally occurring host-pathogen interactions in vivo. We employed membrane shaving to identify peptide fragments cleaved from surface-expressed bacterial proteins and also detected proteins originating from the infected host. We optimized this technique for media-cultured Corynebacterium pseudotuberculosis, a sheep pathogen, revealing a set of 247 surface proteins. We then studied a natural host-pathogen interaction by performing membrane shaving on C. pseudotuberculosis harvested directly from naturally infected sheep lymph nodes. Thirty-one bacterial surface proteins were identified, including 13 not identified in culture media, suggesting that a different surface protein repertoire is expressed in this hostile environment. Forty-nine host proteins were identified, including immune mediators and antimicrobial peptides such as cathelicidin. This novel application of proteolytic shaving has documented sets of host and pathogen proteins present at the bacterial surface in an infection of the native host.

Clinicopathological investigation on caseous lymphadenitis in local breed sheep in Algeria.

This study was carried out of 4,419 animals raised in an extensive system on local breed sheep of the Tiaret region during 2 years (2010-2011) to determine some clinical and pathological aspects associated to caseous lymphadenitis (CL). Clinical examination revealed a prevalence rate of 5.7 %. CL prevalence rate was significantly higher in females (6 %) than in males (4.8 %). The most affected superficial lymph nodes were mandibular lymph nodes (46.1 %). Gross examination of 1,679 carcasses of sheep at the abattoir revealed the presence of abscesses on 250 carcasses (14.9 %). Out of 105 cases showing lesions of CL, Corynebacterium pseudotuberculosis (53.6 %) was isolated as mixed infection with Staphylococcus aureus (43 %) and Escherechia coli (17.9 %). CL prevalence was significantly higher in adult than in young sheep and in female than in male ones. Visceral form of CL was associated to "thin ewe syndrome" responsible for significant economic losses in Algeria.

Interaction of Corynebacterium pseudotuberculosis with ovine cells in vitro.

Caseous lymphadenitis is an infectious and contagious disease caused by Corynebacterium pseudotuberculosis, with a worldwide distribution and high prevalence in small ruminant populations. This disease causes significant economic loss in small ruminants through reduced meat, wool, and milk production. C. pseudotuberculosis can also affect horses, domestic and wild large ruminants, swine, and man. It is considered an occupational zoonosis for humans. As part of in vitro investigations of the pathogenesis of C. pseudotuberculosis, this study analyzed its capacity to adhere to and invade the FLK-BLV-044 cell line, derived from ovine embryonic kidney cells. C. pseudotuberculosis showed a measurable capacity to adhere to and invade this cell line with no significant differences between the four strains assessed. The incubation of the cell line at 4ºC, pre-incubation with sugars, complete and heat inactivated antiserum, and heat-killed and ultraviolet-killed bacteria produced a significant (P < 0.05) decrease in the invasion efficiency or inability to invade the cell line. Plate counting and fluorescence studies showed intracellular bacteria for up to 6 days. Non-phagocytic cells may therefore act as a suitable environment for C. pseudotuberculosis survival and play a role in the spread of infection and/or maintenance of a carrier state.

Mixed infections of Corynebacterium pseudotuberculosis and non-tuberculous mycobacteria in South African antelopes presenting with tuberculosis-like lesions.

Routine meat inspection of antelope carcasses from a South African game reserve revealed a high prevalence of tuberculosis-like lesions. This study aimed to identify the causative agent of this disease and to describe its pathological features. In total, 139 antelopes were randomly harvested from the game reserve and subjected to meat inspection. Of these animals, 46 (33%) showed gross visible, tuberculosis-like lesions. Histopathological examination revealed the presence of encapsulated necrogranulomas in organs and/or lymph nodes of 22 of 27 animals tested. Tissue samples from lesions were processed for both non-selective bacterial culture and mycobacterial culture following decontamination. In non-selective cultures of lesions from 25 of 31 animals tested, Corynebacterium pseudotuberculosis was detected. Isolation of C. pseudotuberculosis was closely associated with the presence of necrogranulomas. In mycobacterial cultures of lesions from 9 of 41 animals tested, different species of non-tuberculous mycobacteria (NTMs) were detected. In 5 instances, depending on the culture procedure that was applied, either C. pseudotuberculosis or NTMs were isolated from the same tissue sample. Our results suggest that the disease has been caused by infections with C. pseudotuberculosis. In sub-Saharan Africa, the role of pathogens other than Mycobacterium bovis may be underestimated in causing tuberculosis-like lesions. In cases where potentially pathogenic NTMs are isolated from mycobacterial cultures of tuberculosis-like lesions, the non-use of additional non-selective culture techniques could lead to misinterpretations of the diagnostic test results.