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.

Survival of Corynebacterium pseudotuberculosis within macrophages and induction of phagocytes death.

Since C. pseudotuberculosis is a facultative intracellular pathogen the aim of this study was focused on evaluating mechanisms that allowed these bacteria to survive in macrophages and determining their influence on induction of cell death. The influence of Corynebacteria on the programmed cell death of macrophages was determined on the basis of induction the autophagy and apoptosis in the cultures of murine macrophage cell lines J774 infected with bacteria. Corynebacterium pseudotuberculosis strains could survive within macrophages more than 48 hours. During that time bacteria were released as a result of the process that lead to death of phagocytes. This property varied among studied strains. There was no increase of microtubule-associated protein I light chain 3 (MAP I LC3) activity in macrophages infected with examined strains comparing with uninfected cultures and cultures treated with autophagy inducer (rapamycin) that served as negative and positive controls, respectively. The study with confocal microscopy did not show the increasing of caspase-3 activity in the infected macrophages and their nucleus did not reveal the fragmentation.

The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence.

BACKGROUND: Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated. RESULTS: Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence. CONCLUSION: The functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host.

Characterization of United Kingdom isolates of Corynebacterium pseudotuberculosis using pulsed-field gel electrophoresis.

Caseous lymphadenitis is a chronic suppurative disease caused by Corynebacterium pseudotuberculosis and is responsible for serious economic losses to the sheep and goat industry. Caseous lymphadenitis was first reported for goats in the United Kingdom in 1990 and for sheep in 1991. Recent evidence suggests that the prevalence of the disease within the national flock is increasing. Fifty isolates of C. pseudotuberculosis from the United Kingdom comprising sheep and horse isolates, the original goat outbreak strain, and the type strain were characterized by biotyping, antimicrobial susceptibility, production of phospholipase D, and genotyping by pulsed-field gel electrophoresis using SfiI and SmaI. All of the isolates were confirmed as C. pseudotuberculosis, and all produced phospholipase D but none reduced nitrate. Restriction with SfiI generated 16 to 18 bands between 48.5 and 290 kb and differentiated six pulsotypes. We conclude that 80% of the strains tested were epidemiologically related to the outbreak strain and that the equine profile was distinct both phenotypically and genotypically.

Mastitis in dairy cattle caused by Corynebacterium pseudotuberculosis and the feasibility of transmission by houseflies. I.

Morbidity due to Corynebacterium pseudotuberculosis infection occurred in 29 dairy herds. The disease appeared basically in three clinical forms: cutaneous, mastitic, and visceral. The appearance of the disease showed a marked seasonality: in 23 herds it occurred during the spring and summer months (dry season) (March-October). The mastitic form occurred in only 10 herds and the causative bacterium was isolated from 33 cows (5.8%). All the strains of C. pseudotuberculosis isolated from the milk samples were found not to be nitrate reducers. The bacterium was excreted in the milk of six cows from herd B during a period of 11 months. In the mastitic cows, a decrease in milk production and considerable increases in the somatic cell count were noted. C. pseudotuberculosis was isolated from houseflies collected over a cow lesion. Laboratory-reared houseflies were successfully infected with C. pseudotuberculosis-contaminated milk, broth and sugar cubes. Flies infected with the bacterium from contaminated milk excreted the bacterium in their droppings for up to 4 h and from their saliva for up to 3 h post infection. The bacterium survived on the external organs of houseflies for no longer than 10 min post infection, after the flies had been dipped in contaminated broth.

Local production of tumor necrosis factor-alpha in corynebacterial pulmonary lesions in sheep.

Corynebacterium pseudotuberculosis infection is a common cause of pyogranulomas in ovine lungs and often occurs as a dual infection with lentiviruses. This coinfection usually leads to the development of chronic pneumonia and cachexia that is similar to the clinical syndrome seen in human beings with AIDS-related pneumonias. Recent in vitro studies indicate that monokines such as tumor necrosis factor-alpha (TNF alpha) are induced by C. pseudotuberculosis, suggesting that TNF alpha is involved in the pathogenesis of corynebacterial lesions in vivo. To substantiate in vitro observations concerning bacterial induction of TNF alpha in ovine pulmonary macrophages, immunohistochemical labeling techniques were used in combination with in situ hybridization to identify TNF-producing cells in corynebacterial lesion sites in vivo. TNF alpha message and translation product were found in macrophages comprising pyogranulomas that were induced by naturally acquired and experimental pulmonary C. pseudotuberculosis infections.

Effect of selected cytokines on the replication of Corynebacterium pseudotuberculosis and ovine lentiviruses in pulmonary macrophages.

Opportunistic bacterial pathogens that induce monokine secretion by pulmonary alveolar macrophages (PAM) are frequently encountered complicating factors in lentivirus-associated pneumonias in ungulates and man. We examined the effect of selected cytokines on the replication of Corynebacterium pseudotuberculosis and ovine lentivirus (OvLV) in ovine PAM. Recombinant bovine (rBo) IL 1 beta, rBoIL-2, rBo interferon-gamma (IFN gamma) and rBoTNF alpha, alone and in combination at physiological doses had no apparent effect on the extracellular growth of C. pseudotuberculosis, compared with the growth of the pathogen in medium alone. Untreated ovine PAM, derived from bronchoalveolar lavage, were found to substantially reduce, but not eliminate the growth of C. pseudotuberculosis in culture. This bactericidal effect was neither enhanced nor inhibited by pretreatment of PAM with the recombinant bovine cytokines or low doses of LPS that induce monokines. In contrast, addition of rBoTNF alpha or rBoIL-1 beta, at physiological doses, at the initiation of, or on Day 4, after OvLV infection resulted in a significant increase in viral replication in PAM, as measured in an antigen capture assay for OvLV p25, compared with untreated infected cells. This effect was more pronounced with lower levels of infecting OvLV, and, in the case of TNF alpha, was abrogated by preincubation of the cytokine with specific anti-serum. Conversely, in most instances, inclusion of rBoIFN alpha in OvLV-infected PAM cultures resulted in a significant decrease in viral replication. These results suggest that these soluble mediators that are probably secreted in response to C. pseudotuberculosis infection may have little direct effect on the extra- or intracellular survival of the bacteria in the lung, but may modulate lentiviral replication and, by extension, disease expression, in sheep with dual infection.