Ricardo Wagner Portela

High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA

We conducted a seroepidemiological survey to determine the prevalence of caseous lymphadenitis (CLA) in goat herds in Minas Gerais state, Brazil. Serum samples were collected from goats (n=676) from 108 rural properties in 2001, covering most of the sub-regions of this ca. 586,500 square kilometer state. Antibodies against Corynebacterium pseudotuberculosis secreted proteins were detected by an indirect enzyme-linked immunosorbent assay (ELISA). Most of the animals (78.9%) tested positive for CLA; 98% of flocks presented at least one seropositive animal. Goats managed under an extensive production system had a significantly higher seroprevalence of CLA than those in intensive and semi-intensive operations. The age distribution of the animals in the flocks affected the prevalence of this disease; however, goat breed did not. We found seropositivity against C. pseudotuberculosis to be highly prevalent in these Brazilian goat herds; consequently, appropriate management practices for the control of CLA should be implemented.

Role of Cytokines and Major Histocompatibility Complex Restriction in Mouse Resistance to Infection with a Natural Recombinant Strain (Type I-III) of Toxoplasma gondii

ABSTRACT Herein we characterized various genetic markers and the biological behavior of a natural recombinant strain of Toxoplasma gondii (P-Br). From nine genetic markers analyzed, three (B1, ROP1, and SAG1) and three (cS10-A6, GRA6, and SAG3) markers belong to parasites from the type I and type III lineages, respectively. The SAG2 and L363 loci were shown to be type I-III chimera alleles. The cB2l-4 microsatellite marker showed a unique haplotype. The P-Br strain presented low virulence in the acute phase of infection and was cystogenic during the chronic infection. The interleukin 12/gamma interferon axis and inducible nitric oxide synthase were main determinants of resistance during the acute infection with the P-Br strain. As opposed to infection with the type II strain of T. gondii (ME-49), peroral infection with the P-Br strain led only to a light inflammatory infiltrate and no major lesions in the intestine of the C57BL/6 mice. In addition, the BALB/c (resistant to ME-49) and C57BL/6 (susceptible to ME-49) mice were shown, respectively, to be more susceptible and more resistant to cyst formation and toxoplasmic encephalitis when infected with the P-Br strain. Further, the C57BL/KsJ and DBA2/J congenic strains containing major histocompatibility complex (MHC) haplotype “d” were more resistant than the parental strains (C57BL/6 and DBA1/J), when infected with the ME-49 but not with the P-Br strain. Together, our results indicate that resistance to cyst formation and toxoplasmic encephalitis induced during infection with P-Br is not primarily controlled by the MHC haplotype d, as previously reported for type II strains of T. gondii.

A Multihousehold Study Reveals a Positive Correlation between Age, Severity of Ocular Toxoplasmosis, and Levels of Glycoinositolphospholipid-Specific Immunoglobulin A

A cross-sectional household study involving 499 individuals was undertaken in an area of Minas Gerais state, Brazil, where infection with Toxoplasma gondii is endemic. Nearly 50% (n=247) of the sample had T. gondii-specific antibodies, even individuals in the 5-9-year-old age group. Approximately 12.5% (n=28) of a random subsample of participants who were positive for T. gondii antibodies had ocular lesions associated with T. gondii infection. The frequency of ocular toxoplasmosis increased significantly with age, with approximately 50% of individuals >60 years of age having lesions. The size of the ocular lesion correlated positively (r=0.85; P=.01) with the serum level of immunoglobulin A specific for tachyzoite-derived glycoinositolphospholipids. We found that sharing the same residence accounted for 30% of the variation in infectivity among residents in the sample, whereas age was the main risk factor for development of ocular toxoplasmosis in patients who were positive for T. gondii antibodies.

Vaccination with recombinant NetB toxin partially protects broiler chickens from necrotic enteritis

NetB toxin from Clostridium perfringens is a major virulence factor in necrotic enteritis in poultry. In this study the efficacy of NetB as a vaccine antigen to protect chickens from necrotic enteritis was examined. Broiler chickens were immunized subcutaneously with purified recombinant NetB (rNetB), formalin treated bacterin and cell free toxoid with or without rNetB supplementation. Intestinal lesion scores and NetB antibody levels were measured to determine protection after mild oral gavage, moderate in-feed and heavy in-feed challenges with virulent C. perfringens isolates. Birds immunized with rNetB were significantly protected against necrotic enteritis when challenged with a mild oral dose of virulent bacteria, but were not protected when a more robust challenge was used. Bacterin and cell free toxoid without rNetB supplementation did not protect birds from moderate and severe in-feed challenge. Only birds immunized with bacterin and cell free toxoid supplemented with rNetB showed significant protection against moderate and severe in-feed challenge, with the later giving the greatest protection. Higher NetB antibody titres were observed in birds immunized with rNetB compared to those vaccinated with bacterin or toxoid, suggesting that the in vitro levels of NetB produced by virulent C. perfringens isolates are too low to induce the development of a strong immune response. These results suggest that vaccination with NetB alone may not be sufficient to protect birds from necrotic enteritis in the field, but that in combination with other cellular or cell-free antigens it can significantly protect chickens from disease.

Antigens of Corynebacterium pseudotuberculosis and prospects for vaccine development

Corynebacterium pseudotuberculosis continues to cause considerable economic losses in ovine and caprine herds worldwide, causing caseous lymphadenitis. Nevertheless, the immunology of this disease is relatively unknown. Novel antigens may provide vaccines that are more effective and improve diagnostic methods for better control of this disease. The available commercial vaccines are not able to fully protect susceptible animals, cannot be used in all host species and are not licensed for use in many countries. Recent studies on the genomics of C. pseudotuberculosis and on its molecular determinants of virulence should bring us new alternatives for more effective vaccine formulations.

Evaluation of the humoral and cellular immune response to different antigens of Corynebacterium pseudotuberculosis in Caninde´ goats and their potential protection against caseous lymphadenitis

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a disease that affects goats and sheep, and can cause severe economic losses. In this study, four different antigenic extracts were obtained from the attenuated strain T1, which was isolated in the state of Bahia (Brazil). Forty-four Canindé breed goats were divided in five groups, each receiving a different antigen solution and saline buffer as a control. The humoral response was monitored through the identification of specific IgG by indirect ELISA and Western Blotting, and the production of IFN-gamma was followed in order to observe the activation of cellular response. After twelve weeks of antigen inoculation, the animals were challenged with 2 x 10(5)CFU of a wild strain, also isolated in Bahia, and necropsy was performed on all animals twelve weeks afterwards. It was observed that the attenuated bacteria gave a protection of 33.3%, in addition to the weak humoral response elicited. Animals inoculated with secreted antigen associated with Freunds incomplete adjuvant and oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) showed a strong humoral response, but this inoculation could not prevent the spread of challenge bacteria in the majority of animals. These results demonstrate the immunogenic potential of the attenuated T1 strain in the development of a vaccine against caseous lymphadenitis in goats.

Corynebacterium pseudotuberculosis: Immunological Responses in Animal Models and Zoonotic Potential

Corynebacterium pseudotuberculosis is a member of the Corynebacterium, Mycobacterium and Nocardia (CMN) group that comprises species of medical, veterinary and biotechnological interest. This pathogen mainly affects small ruminants, causing caseous lymphadenitis (CLA), but it also infects bovines, equines, pigs, deer, camels and humans, showing its zoonotic relevance. Phospholipase D (PLD) and the toxic lipid cell wall are the two most wellstudied virulence factors of this bacterium. They are responsible, in part, for the establishment of disease in the host. Current knowledge on the immunity induced by C. pseudotuberculosis indicates that the resistance to infection is a complex process involving components of both the non-specific and specific host responses, in which humoral and cellular immune responses are both operative. Despite this knowledge and the importance of the disease, a satisfactory vaccine model for sheep and goats has not been developed. Moreover, a control program that includes an efficient diagnostic method in addition to vaccination is crucial for avoiding the spread of bacteria inside flocks. Further, because of its zoonotic potential, C. pseudotuberculosis infection of animals can contaminate meat and milk, putting consumers at risk. The ability of C. pseudotuberculosis to infect both animals and humans makes studies on prevention and diagnosis of this pathogen important.

Corynebacterium pseudotuberculosis secreted antigen-induced specific gamma-interferon production by peripheral blood leukocytes: potential diagnostic marker for caseous lymphadenitis in sheep and goats

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.

Acaricides efficiency on Rhipicephalus (Boophilus) microplus from Bahia state North-Central region

The cattle tick Rhipicephalus (Boophilus) microplus is responsible for major losses in the Brazilian livestock, mainly due to reduction in cattle productive performance. Resistance development to major classes of acaricide widely used nowadays has been extensively reported, as well as the occurrence of residues from these compounds in animal products and the environment. This study aimed to evaluate the efficiency of acaricides on R. (B.) microplus collected from rural properties in the North-Central region of Bahia State. Ticks were collected in several cattle farms in the cited region, and an in vitro acaricide efficiency assay was performed based on the immersion of ticks in acaricide solutions made according to manufacturers recommendations. The results obtained in the experiments indicated varying degrees of efficiency of the several bases studied, with the products benzofenilurea, macrocyclic lactone and fipronil showing the highest levels of efficiency, 100%, 100% and 97.34%, respectively. It was possible to conclude that, for the region studied, there are different degrees of commercial acaricides efficiency, and many present less than 95% effectiveness, value determined as acceptable by the Brazilian legislation.

Haptoglobin and fibrinogen concentrations and leukocyte counts in the clinical investigation of caseous lymphadenitis in sheep

BACKGROUNDnCorynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a disease that affects small ruminants and is responsible for economic losses, including condemnation of carcasses and damaged hides.nnnOBJECTIVEnThe goal of this study was to determine if serum haptoglobin and plasma fibrinogen concentrations and peripheral blood leukocyte counts are biologic markers of CLA in sheep.nnnMETHODSnBlood from 38 clinically healthy Santa-Inês ewes selected and segregated from a commercial flock of 2500 sheep in an area endemic for C. pseudotuberculosis was collected every 30 days for 6 months. An indirect ELISA was used to detect IgM and IgG antibodies against C. pseudotuberculosis. Serum haptoglobin concentration was measured using a hemoglobin-binding assay and plasma fibrinogen concentration by refractometry following heat precipitation. Total leukocyte counts were determined using a hemocytometer, and differential leukocyte counts were performed on smears of peripheral blood.nnnRESULTSnTwenty-one sheep were seropositive at the start of the study; 15 became seropositive during the study. Only 2 sheep were seronegative at the conclusion of the study. Haptoglobin and fibrinogen concentrations and WBC counts were not significantly different for seropositive and seronegative animals. Nine sheep, 5 that were seropositive positive at the start and 4 that became seropositive during the study period, developed abscesses in peripheral lymph nodes. There were 15 animals that became seropositive during the study, and their values did not differ significantly among the 3 phases--seronegative, acute (IgM+/IgG±), and chronic (IgM-/IgG+)--of infection. However, 11 of these sheep did not develop peripheral abscesses and had significantly higher haptoglobin concentrations and lower monocyte counts during the acute phase of the disease than did the 4 sheep that later developed abscesses.nnnCONCLUSIONnSerum haptoglobin concentration and monocyte counts may be potential markers for progression of CLA in sheep.