Comprehensive pathological insights and molecular detection of caseous lymphadenitis caused by Corynebacterium pseudotuberculosis in slaughtered sheep, goats, and cattle

Caseous lymphadenitis (CLA) or pseudotuberculosis is a chronic zoonotic bacterial disease caused by Corynebacterium pseudotuberculosis, which affects livestock and humans. This study aimed to describe the pathology, bacteriology and confirm the identity of the pathogen by 16S rRNA gene sequencing in Camelus dromedarius. A total of 12 camels with suspected CLA in three regions of Abu Dhabi Emirate (Abu Dhabi, Al Ain and Al Dhafra), United Arab Emirate (UAE) were subjected to clinical and postmortem examinations from January 2015 to December 2020. Clinically, camels were emaciated and showed the presence of external caseous abscesses suggestive of CLA. Postmortem examination showed multiple abscesses of variable sizes with caseous material encapsulated by fibrous tissue in the liver, lungs, muscle, and lymph nodes. Following clinical and postmortem examination, blood, pus and different tissue samples were collected for subsequent analysis. Histopathological examination of all organs stained with Hematoxylin and Eosin (H&E) indicated a central caseo-necrotic core that was admixed with bacterial colonies and infiltration of chronic inflammatory cells, surrounded by a pyogenic membrane, and an outer fibrous connective tissue capsule. Bacterial culture identified the isolates of Corynebacterium pseudotuberculosis biotype ovis strain, and these isolates were shown to be sensitive to all antibiotics tested (penicillin, ampicillin, Co-trimoxazole, enrofloxacin and tetracycline). Moreover, the identity of the isolates was confirmed by partial sequencing of the 16S rRNA gene which showed a 100% identity to Corynebacterium pseudotuberculosis. Phylogenetic analysis based on 16S rRNA gene sequence clearly differentiates Corynebacterium pseudotuberculosis from other species of Corynebacterium. Briefly, this study provided the basic information for infection of Corynebacterium pseudotuberculosis in Camels and will help in controlling of this pathogen in the region.

Caseous lymphadenitis (CLA) presents a significant challenge to the dairy goat industry worldwide, negatively affecting animal health, productivity, and economic sustainability. It is a disease caused by Corynebacterium pseudotuberculosis, and its symptoms include the development of abscesses in lymph nodes and other organs. Generally, animals affected by CLA experience reduced milk output, reproductive problems, and higher morbidity and death rates. Furthermore, CLA could harm the well-being of animals by causing pain, discomfort, and disruption of normal behaviours. This review aims to determine the prevalence of CLA cases affecting dairy goats, map the geographical distribution of the disease in dairy goat herds throughout different locations, determine hotspots of CLA incidence and comprehend regional differences. Additionally, the study included relevant information on CLA prevalence in Asia. This comprehensive review is expected to help develop educational strategies for CLA prevention and control among dairy goat farmers. Moreover, an assessment of the economic impact of CLA in dairy goat operations on a global scale was performed. The losses associated with CLA are reduced milk production, lower meat yield, and increased veterinary costs. Besides, the variability of clinical presentation and the limits of conventional diagnostic methods make diagnosing CLA in dairy goats difficult. However, new developments in diagnostics, such as serological testing and polymerase chain reaction (PCR) assays, present chances for early detection and targeted control strategies. Examples of management strategies for CLA in dairy goat herds include vaccination, strict biosecurity protocols, and targeted treatment of infected animals. To surmise, the dairy goat industry can strive to mitigate the effects of CLA and maintain the well-being of dairy goat populations by tackling these challenges and executing comprehensive management strategies.

Background: Caseous Lymphadenitis (CL) is a chronic infectious disease caused by the bacterium Corynebacterium pseudotuberculosis, which is considered the main agent responsible for abscess lesions. In the visceral form it can affect the internal organs of sheep and goats, which could negatively affect animal health and cause large economic losses for producers.Case: This study aims to report a case of intestinal CL in sheep, with suspected diagnosis during physical examination and identification during the performance of the oophorectomy procedure, adopted as a management approach. It is a mixed breed sheep, aged over 5 years; weight 28 kg; emaciated on physical examination; with pale pink and moist eyelid mucosa; heart and respiratory rate: 81 beats/min and 22 movements/min, respectively; body temperature 39.2°C; ruminal movements at 1 movement/min; without identification of lymphadenomegaly on palpation, however, it was observed that the right submandibular lymph node presented tissue retraction compatible with the healing process. For the surgical procedure, an 18-h fast was used and pre-anesthetic medication with 2% xylazine (0.1 mg/kg), 10% ketamine (5 mg/kg) and 50 mg/mL tramadol (2 mg/kg) administrated intramuscularly. The animal was placed in the left lateral decubitus position, then was performed trichotomy and epidural administration of 2% lidocaine (4 mg/kg) and maintenance with propofol 10 mg/mL intravenous dose-effect and oxygen mask 3 liters/min, antibiotic prophylaxis was performed with 10% enrofloxacin (2.5 mg/kg). Flank oophorectomy was performed according to the classic technique and during abdominal inspection, abscess lesions were found in the mesentery and intestinal loops. Incisional biopsy was performed to collect samples in the jejunal segment. At the end of the procedure, 50 mg/mL (2.2 mg/kg) of flunixin meglumine was administered intravenously. A 0.6 cm x 0.2 cm sample, was submitted to histological analysis, which showed the presence of central necrosis areas formed by concentric lamellae, with the presence of large bacterial colonies and foci of mineralization, surrounded by a strip of inflammatory infiltrate with epithelial macrophages and few neutrophils. In the adjacent layer, lymphocytes and plasmocytes were found and the entire lesion was delimited by fibrous conjunctive tissue, compatible with lesions caused by Corynebacterium pseudotuberculosis, the causative agent of CL. During the whole postoperative period the animal was kept isolated from the herd. Due to the unfavorable prognosis and histological confirmation of visceral CL, euthanasia was indicated. The animal was sent for necropsy and no lesions compatible with CL were found in other organs or tissues besides the anatomical structures where the lesions were previously described (intestine and mesentery).Discussion: As it is an infectious disease, isolation and euthanasia should be indicated in cases of CL, in order to not compromise the health of the herd. Thus, justifying the orientation of euthanasia after histological confirmation. The case did not have other possibilities of diagnostic aid, however, histological lesions of CL are characteristic but not pathognomonic, as it could be confused with lesions caused by other pyogenic pathogens. In this way, clinical considerations and complementary exams are relevant to support the diagnostic. To conclude, the observation and physical examination of the herd were fundamental tools for raising the diagnostic hypothesis ante mortem. Complementary tests allowed the confirmation of the disease and prevented the spread in the herd, which could lead to large economic losses for producers and negatively affect animal health.

Caseous lymphadenitis is a chronic infection caused by Corynebacterium pseudotuberculosis that affects the lymphatic system, resulting in abscesses in the lymph nodes and internal organs. This study was conducted to determine the prevalence rate of caseous lymphadenitis among slaughtered sheep in Duhok abattoirs, Duhok, Iraq and to investigate to what extend rabbits can develop caseous lymphadenitis just like that in sheep. A total of 1052 carcasses of sheep were randomly selected (939 males and 113 females) and carefully inspected for detection any suspected caseous lymph adenitis. The isolated Corynebacterium pseudotuberculosis from lymph nodes of sheep carcasses were identified by PCR before used in in vivo infection in rabbits. Data revealed that the prevalence rate of caseous lymphadenitis was at 1.9% among the slaughtered sheep and the prevalence rates were higher in females and older ages than that of males and younger ages, respectively. Experimentally infected rabbits developed various clinical signs were ranging from in inappetence to sudden death, mainly during first week of infection. Histopathologically, the infected rabbits developed caseous lymphadenitis and pyogranuloma after a time point of 10 days post-infection. Liver, spleen and lymph nodes of infected rabbits showed caseo-necrotic foci with multiple micro-granulomas with presence of thick pyogenic membrane infiltrated with leukocytic cells; these findings are strictly resemblance to that reported in sheep. The obtained preliminary data of using rabbits as an animal model is promising which will enhance a better understanding the pathogenesis of caseous lymphadenitis in animals.

Abscess diseases of sheep (caseous lymphadenitis, CLA, caused by Corynebacterium pseudotuberculosis, and Morel disease, caused by Staphylococcus aureus) are endemic in Saudi Arabian sheep farms. The etiology and pathology of visceral abscess disease is poorly documented for Saudi Arabian sheep. This study was carried out to investigate the etiology and pathology of internal (visceral) abscesses of sheep at Qassim region, central Saudi Arabia. One hundred and twenty abscessed internal lymph nodes were collected from sheep with single organ-lymph node abscesses, and slaughtered in Qassim abattoirs. All the surveyed sheep had clinical external abscess disease. Animals with multiple internal abscesses were discarded from survey. Pus samples obtained from the lymph nodes were cultured aerobically and anaerobically. Isolates were obtained from 99 infected lymph nodes (82.5%). The numbers of recovered bacterial isolates were as follows: Staphylococcus aureus (39), Corynebacterium pseudotuberculosis (27), Staphylococcus aureus subsp. anaerobicus (25), Streptococcus spp. (6) and Pseudomonas aerugenosa (2). These organisms were isolated from the following lymph nodes: hepatic (51), mediastinal (42) and renal (6). Identification of C. pseudotuberculosis was confirmed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) targeting the phospholipase D (PLD) gene.

In this study, it was aimed to determine the incidence of Corynebacterium pseudotuberculosis (C. pseudotuberculosis) in caseous lymphadenitis cases in superficial lymph nodes of sheep and to evaluate its effect on oxidative stress factors. Thus, it was aimed to determine the early diagnosability of superficial and visceral caseous lymphadenitis cases and to prevent the spread of diseases and related economic losses. A total of 103 sheep, 50 of which were healthy and 53 of which had caseous lymphadenitis, were evaluated in the study. Microbiological examinations were performed by taking 3-5 ml of pyogenic aspirate from the superficial lymph nodes of sheep with caseous lymphadenitis. Blood samples were taken from sheep with C. pseudotuberculosis isolated in microbiological examinations and the levels of oxidative stress factors were determined. C. pseudotuberculosis was isolated in 23 of the pyogenic aspirates of 53 sheep with caseous lymphadenitis. When sheep isolated from C. pseudotuberculosis were compared with healthy sheep, it was determined that there was a statistically significant decrease in the levels of antioxidant molecules such as GSH-pX (14.62%), GSH (23.81%), SOD (4.70%), CAT (22.23%) (p˂0.001). The level of toxic malondialdehyde (MDA) (18.62%), the end product of lipid peroxidation, was found to be statistically significantly increased in sheep isolated from C. pseudotuberculosis (p˂0.001). As a result, it was determined that oxidative stress factors showed statistically significant variability in cases of superficial caseous lymphadenitis (caused by C.pseudotuberculosis). For this reason, by determining the levels of oxidative stress factors in suspected herds, it was possible to make early diagnosis of the superficial and visceral forms of caseous lymphadenitis, and a basis was established for future studies.

Johne's disease in two Korean black goats (Capra hircus coreanae) with clinical and subclinical infection: A case report

The actual prevalence of CLA (caseous lymphadenitis) in small ruminant flocks is underestimated in many countries, and because it is not a notifiable disease, it will continue to spread without data and information about its real economic impact. The difficulty in the accurate identification of the causative agent in internal subclinical cases allows the disease to spread within and between flocks. This research intends to assess the utility of an ELISA (enzyme‐linked immunosorbent assay) test in the detection of internal subclinical cases of CLA in farms and to simultaneously add data on the seroprevalence of the disease in Portugal. Sera from 756 small ruminants, 70% sheep (528/756) and 30% goats (228/756) were screened for antibodies against Corynebacterium pseudotuberculosis using the ELISA technique based on a recombinant phospholipase D (ELITEST CLA # CK105A®). The animals showing internal lesions (n ꞊ 58) were sampled for the identification of the aetiological agent. In this investigation, the prevalence of CLA was 34% (258/756), with the ELISA test showing a low specificity (78%) and high sensitivity (100%). The proof was able to detect 57% (13/23) of subclinical cases of CLA confirmed by postmortem examination and conventional PCR (polymerase chain reaction). The results also reveal that goats have a higher propensity for the disease, and dairy farms and non‐extensive production units appear to be more susceptible to CLA. This research clarifies an actual problem and pointed out the importance of CLA in small ruminant herds in Portugal. Finally seems to demonstrate that the ELISA test is a good diagnostic tool for use in CLA eradication programmes.

Corynebacterium pseudotuberculosis, a Gram-positive bacterium is the causative agent of caseous lymphadenitis (CLA), a chronic disease of sheep, goats and other warm blooded animals. In the present study, a total of 1,080 sheep reared under semi-intensive system on organized farms situated in the semi arid tropical region of Rajasthan, India, was clinically examined. Pus samples from superficial lymph nodes of 25 (2.31%) adult sheep showing clinical lesions similar to CLA were collected for laboratory analyses. On the basis of morphological, cultural and biochemical characteristics 12 (48%) bacterial isolates from pus identified it as C. pseudotuberculosis. A polymerase chain reaction (PCR) assay targeting Putative oligopeptide/dipeptide ABC transporter, nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase coenzyme F420-dependent and proline iminopeptidase (PIP) genes of C. pseudotuberculosis was developed that showed 14 pus samples as positive. All C. pseudotuberculosis isolates were also found positive for these genes in the PCR. The specificity of the PCR products was confirmed by sequencing of the amplified products that showed 98-100% homology with published sequences available in the NCBI database. The present study shows the incidence of CLA as 2.31%, 1.1% and 1.29% based on clinical, bacterial culture and direct pus PCR assay, respectively. The PCR assay was rapid, specific and as significant as bacterial culture in detecting bacteria directly in the clinical pus samples. The PCR assay developed in the study can be applied for the diagnosis and control of CLA. Furthermore, the assay can also be applied to detect C. pseudotuberculosis in various clinical samples.

Abattoir survey on caseous lymphadenitis in sheep and goats in Tanta, Egypt

BackgroundSheep caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis (Cp), is associated with direct economic losses and presents significant zoonotic potential. Despite the importance of the disease, a satisfactory vaccine model has not been developed. Thus, this study aimed to investigate the association between haptoglobin (Hp) and IgM levels and the clinical progression of CLA in primarily infected sheep and in sheep immunized with Cp- secreted antigens adjuvanted with Quillaja saponaria saponins. These animals were kept with CLA-positive sheep to simulate natural exposure that occurs in field conditions. During the experiment, the Hp and IgM levels were monitored for 21 days, and the development of internal CLA lesions was investigated through necropsies on day182 post-immunization.ResultsPrimarily infected sheep in Group 2 (inoculated with 2x105 Cp virulent strain) had higher Hp values between the first and ninth days post inoculation (PI) than sheep in Group 1 (control; P < 0.05). Immunized animals in Group 3 had significantly higher Hp values between the third and seventh days PI, compared with the control group (P < 0.01). Binary logistic regression (BLR) analysis of primarily infected sheep indicated an association between Hp concentration and CLA clinical progression: animals with high Hp values had 99.9% less risk of having CLA abscesses than animals with low Hp levels (Odds ratio = 0.001, P < 0.05). Both experimental groups had significantly higher IgM titers than the control group around the ninth and eleventh days PI (P < 0.05). The BLR analysis for immunized sheep indicated an association between IgM levels and clinical progression: sheep with high IgM titers had 100.0% less risk of having CLA abscesses than animals with low IgM levels (Odds ratio = 0.000, P < 0.05).ConclusionsResistance to C. pseudotuberculosis infection is supported by the early acute phase response, in which up-regulation of Hp and IgM were predictive of a lower risk of CLA lesion development. Because the immunogen used in this study induced a high production of both Hp and IgM, Q. saponaria saponin should be considered a promising candidate in vaccine formulations against sheep CLA.

In the current study, the applicability of the quantification of gamma interferon (IFN-γ) levels for the detection of animals infected with Corynebacterium pseudotuberculosis and for determining caseous lymphadenitis (CLA) clinical status was evaluated. Peripheral blood leukocytes were collected from CLA nonendemic areas animals, from CLA seropositive animals without clinical signs of the disease, and from seropositive animals presenting CLA clinical signs. The leukocytes were stimulated with C. pseudotuberculosis-secreted antigens that were concentrated by the three-phase partitioning technique. An ovine IFN-γ enzyme-linked immunosorbent assay was used to quantify IFN-γ production. Goats and sheep with CLA had higher IFN-γ levels than uninfected seronegative animals. Leukocytes from sheep with CLA chronic abscesses produced higher IFN-γ levels when compared with seropositive sheep without CLA clinical signs, but this difference was not significant in goats. The sensitivity of the assay was 55.8% and 56%, whereas the specificity was 100% and 93%, for goats and sheep, respectively. In conclusion, IFN-γ is a potential marker for the determination of CLA infection status in small ruminants; however, further research is needed to improve assay sensitivity.

Caseous lymphadenitis in sheep flocks of the state of Minas Gerais, Brazil: Prevalence and management surveys

Caseous lymphadenitis (CL), a contagious infectious disease caused by Corynebacterium pseudotuberculosis, stands out with great economic relevance for goat breeding in the country because it is one of the main causes of carcass condemnation and intense depreciation of the leather of small ruminants. The objective was to carry out a descriptive study of the clinical and epidemiological aspects of CL in goats in the state of Pernambuco and also to evaluate the detection of C. pseudotuberculosis in milk, blood, and stool samples by real-time PCR. Six hundred and forty-three goats were evaluated, of which 76 (11.8%) presented clinical signs suggestive of CL. And 88.9% (24/27) of the breeding was considered focus. Among symptomatic animals, 25 were selected and submitted to the collection of abscess content for bacteriological isolation. Milk, blood, and stool samples were analyzed for PLD gene detection by real-time polymerase chain reaction (PCR). Out of the 25 samples of abscess content, 72% (18/25) were positive. The PLD gene was detected in the frequencies of 8% (2/25) and 4% (1/25) in milk and blood samples, respectively. Caseous lymphadenitis is possibly widespread in the dairy herds of goats in the state of Pernambuco, especially in adult animals, with a higher occurrence of submandibular lymph node abscesses. The preliminary detection of C. pseudotuberculosis by mammary and hematogenous pathways may contribute to future approaches on the mechanisms of organic dissemination performed by the pathogen. However, it has little relevance to diagnosing the disease.

Caseous lymphadenitis (CLA) is a chronic and subclinical bacterial disease of ruminants caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis) infection. Until 2014, there were no reports of CLA outbreaks in South Korea; however, the prevalence of CLA cases has steadily increased. In this study, we used recently obtained field isolates to develop the first inactivated CLA vaccine in South Korea and evaluated it in various animal models. The inactivated vaccine was evaluated for virulence and effectiveness. Mice were tested for virulence and immunization challenges, and guinea pigs and Korean Native Black Goats (KNBGs) evaluated various vaccine concentrations to determine the optimal dose and effectiveness. In the case of KNBGs, clinical symptoms were not observed after vaccination. In addition, CLA-specific IgG was detected at a significantly (p < 0.05) high level and was maintained. In histopathological evaluations, inflammation was predominantly observed in the prefemoral lymph nodes in the non-vaccinated+CHAL group. The genetic diversity of C. pseudotuberculosis, which has become widespread in South Korea, is less than 0.5% our vaccine is expected to prevent infection by a wide range of strains effectively. In summary, our CLA vaccine can potentially prevent CLA and foster the growth of South Korea's domestic KNBG industry.