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.

Summary Caseous lymphadenitis (CLA) is a small ruminant disease characterized by the development of granulomatous lesions in superficial and internal lymph nodes, as well as in some organs, and causes significant economic losses worldwide. The aetiological agent of CLA is the bacterium Corynebacterium pseudotuberculosis; however, the commercially available diagnostic tools present problems with regard to specificity, which can lead to false‐negative results. This study aimed to develop an indirect enzyme‐linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins in goats and sheep using recombinant C. pseudotuberculosis PLD, CP40, PknG, DtxR and Grx proteins. For validation of the ELISAs, 130 goat serum samples and 160 sheep serum samples were used. The best ELISA for goats was developed using a combination of PLD and CP40 as antigens at a 1:1 ratio, which presented 96.9% sensitivity and 98.4% specificity. The most effective ELISA for sheep presented 91% sensitivity and 98.7% specificity when recombinant PLD alone was used as the antigen. These ELISAs can be used as highly accurate tools in epidemiological surveys and for the serodiagnosis of C. pseudotuberculosis infection in goats and sheep.

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN--gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.

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.

The sheep industry in Brazil is an important economic activity, and with the increasing global demand for sheep meat there is a great interest in the monitoring of the herd health, and serum reference ranges are basic tools for veterinary clinical pathology assays. Mineral elements correspond to 2-5.5% of the body of vertebrates, holding different functions in their physiology. The objective of this study was to obtain reference intervals of the electrolytes magnesium, phosphorus, chloride and calcium for the Dorper and Saint Ines sheep breeds. Sera samples were collected from 487 clinically healthy sheep, 146 from Dorper and 341 from Santa Ines breed. Electrolytes were measured using commercial kits. Data were analyzed taking the race, sex and age variables in account, and reference ranges were established. The results revealed significant statistical differences in reference ranges obtained for the electrolytes calcium and magnesium concerning the variable race, and for the electrolyte phosphorus in the variable age and, when compared with reference values already published, proved the existence of significant differences.

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We present here the genome sequence of the attenuated Corynebacterium pseudotuberculosis strain T1. The sequencing was performed with an Ion Torrent Personal Genome Machine platform. The genome is a circular chromosome of 2,337,201 bp, with a G+C content of 52.85% and a total of 2,125 coding sequences (CDSs), 12 rRNAs, 49 tRNAs, and 24 pseudogenes.

Equatorial Guinea (EG) is a country in Central Africa with typical tropical weather. Sheep are an important source of food in EG, but the absence of information regarding infectious diseases that affect the native flocks of small ruminants is a concern. The country is currently implementing several new practices in the sheep industry associated with the importation of sheep from other countries. This study aimed to evaluate the seroprevalence of bacterial infections that are important to the sheep industry in EG sheep flocks. The detection of specific antibodies for the four agents studied was performed using enzyme-linked immunosorbent assay in 1,002 serum samples from EG sheep. The results showed a true prevalence of 13.37% for antibodies against Corynebacterium pseudotuberculosis, 0.59% for Brucella ovis, 19.89% for Chlamydophila abortus and 0.79% for Mycoplasma agalactiae in animals from production flocks. Among a group of 35 samples from isolated native animals, 47.56% were seropositive for antibodies against C. pseudotuberculosis, 42.84% for B. ovis, 54.28% for C. abortus and 11.35% for M. agalactiae. These results comprise the first report of the prevalence of infectious diseases in sheep in EG. They highlight the importance of adopting adequate measures to prevent infection by bacteria endemic to EG native flocks during the development of the sheep industry in the country.