V.S. Moustacas

Development and evaluation of a species-specific PCR assay for the detection of Brucella ovis infection in rams

Brucella ovis infection is a major cause of epididymitis and infertility in rams, resulting in reproductive failure and significant economic losses worldwide. The goal of this study was to develop a PCR test targeting specific B. ovis genomic sequences. Specific primer pairs were designed targeting 12 of those ORFs. Samples of blood, serum, semen, urine, and preputial wash were collected from experimentally infected rams (n=9) every other week up to 180 days post infection (dpi), when tissue samples were obtained. Blood, serum, semen, urine, and preputial wash samples were obtained, in weekly intervals for 1 month, from eight rams belonging to a B. ovis-free flock. Semen samples were also obtained from rams belonging to naturally infected flocks (n=40). The limit of detection of this PCR protocol was 100, 10, and 1 CFU/mL for semen, urine and prepucial wash samples, respectively. Sensitivity and specificity values obtained with this PCR method were similar to that of bacteriology when evaluating biological samples. Agreement between PCR and bacteriology results was greater than 90%. These results clearly indicate that this species-specific PCR method is highly efficient for the diagnosis of B. ovis infection in semen, urine, preputial wash and tissue samples from infected rams.

Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen.

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Inês cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10(6) sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST(+) (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI(-)/PSA(-) (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.

Putative ATP-binding cassette transporter is essential for Brucella ovis pathogenesis in mice.

ABSTRACT Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic. Due to the scarcity of studies on B. ovis infection, a murine model of infection was developed. The roles of B. ovis genes encoding a putative hemagglutinin and an ABC transporter were investigated in the mouse model. The kinetics of B. ovis infection were similar in BALB/c and C57BL/6 mice, and both strains of mice developed multifocal microgranulomas in the liver and spleen, but only minimal colonization and histopathological changes were observed in the genital tract. Therefore, the mouse was considered a suitable infection model for B. ovis but not for B. ovis-induced genital disease. Two mutant strains were generated in this study (the ΔabcAB and Δhmg strains). The B. ovis ΔabcAB strain was attenuated in the spleens and livers of BALB/c mice compared to the wild-type (WT) strain (P < 0.001). Conversely, the Δhmg strain infected mice at the same level as WT B. ovis, suggesting that a putative hemagglutinin is not required for B. ovis pathogenesis. Additionally, the ΔabcAB strain did not survive in peritoneal macrophages, extracellularly in the peritoneal cavity, or in RAW 264.7 macrophages. Moreover, infection with the ΔabcAB strain was not lethal for male regulatory factor 1-knockout mice, whereas infection with the B. ovis WT strain was 100% lethal within 14 days postinfection. These results confirm that the predicted ABC transporter is required for the full virulence and survival of B. ovis in vivo.

Use of nonlinear models for describing scrotal circumference growth in Guzerat bulls raised under grazing conditions

The objective was to use various nonlinear models to describe scrotal circumference (SC) growth in Guzerat bulls on three farms in the state of Minas Gerais, Brazil. The nonlinear models were: Brody, Logistic, Gompertz, Richards, Von Bertalanffy, and Tanaka, where parameter A is the estimated testis size at maturity, B is the integration constant, k is a maturating index and, for the Richards and Tanaka models, m determines the inflection point. In Tanaka, A is an indefinite size of the testis, and B and k adjust the shape and inclination of the curve. A total of 7410 SC records were obtained every 3 months from 1034 bulls with ages varying between 2 and 69 months (<240 days of age = 159; 241-365 days = 451; 366-550 days = 1443; 551-730 days = 1705; and >731 days = 3652 SC measurements). Goodness of fit was evaluated by coefficients of determination (R(2)), error sum of squares, average prediction error (APE), and mean absolute deviation. The Richards model did not reach the convergence criterion. The R(2) were similar for all models (0.68-0.69). The error sum of squares was lowest for the Tanaka model. All models fit the SC data poorly in the early and late periods. Logistic was the model which best estimated SC in the early phase (based on APE and mean absolute deviation). The Tanaka and Logistic models had the lowest APE between 300 and 1600 days of age. The Logistic model was chosen for analysis of the environmental influence on parameters A and k. Based on absolute growth rate, SC increased from 0.019 cm/d, peaking at 0.025 cm/d between 318 and 435 days of age. Farm, year, and season of birth significantly affected size of adult SC and SC growth rate. An increase in SC adult size (parameter A) was accompanied by decreased SC growth rate (parameter k). In conclusion, SC growth in Guzerat bulls was characterized by an accelerated growth phase, followed by decreased growth; this was best represented by the Logistic model. The inflection point occurred at approximately 376 days of age (mean SC of 17.9 cm). We inferred that early selection of testicular size might result in smaller testes at maturity.

Extenders containing dimethylformamide associated or not with glycerol are ineffective for ovine sperm cryopreservation.

The study aimed at testing the effectiveness of dimethylformamide, alone or combined with glycerol, as cryoprotectant for freezing ram semen. Ejaculates from nine rams were cryopreserved in Tris-based extenders, containing 5% of glycerol, association of dimethylformamide with glycerol, in four proportions achieving 5% of cryoprotectors in the media and pure dimethylformamide (2, 3, 4 and 5%) in replacement to glycerol. The samples were diluted to 100 × 10(6) sptz/ml and stored in 0.25-ml straws in liquid nitrogen. After thawing (37 °C for 30 s), motility was preserved better by the extender containing 5% of glycerol (p < 0.05). The extenders containing pure dimethylformamide, or more than 2% in combination with glycerol, provided sperm motilities close to zero. Plasma and acrosomal membrane integrity were preserved better (p < 0.05) in the extender containing 5% glycerol. It can be concluded that dimethylformamide, alone or combined with glycerol, has no beneficial effects on ovine semen cryopreservation.

Species-specific multiplex PCR for the diagnosis of Brucella ovis , Actinobacillus seminis , and Histophilus somni infection in rams

BackgroundInfectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections.ResultsThe multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples.ConclusionsThe species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

Diagnosis of Brucella ovis infection by serology and PCR in urine samples from naturally infected rams in the state of Piauí

A brucelose ovina causada pela Brucella ovis e uma doenca reprodutiva de carneiros caracterizada por epididimite, orquite, com consequente diminuicao da fertilidade e prejuizos economicos significativos. O presente trabalho teve por objetivo avaliar a aplicabilidade da tecnica de PCR como um metodo de diagnostico em campo, comparado-a com a tecnica de IDGA. Foram coletadas amostras de urina e soro de 90 carneiros oriundos de 31 rebanhos localizados no Estado do Piaui. Quatro das 31 (12,9%) propriedades avaliadas apresentaram animais positivos. Dezoito (20%) amostras de urina foram positivas pela PCR, enquanto o metodo de IDGA identificou 16 (17,8%) carneiros soropositivos. Embora os metodos tenham apresentado concordância baixa na estatistica Kappa (k=0,04), nao foi observada diferenca estatistica entre as tecnicas (P>0,05) pelo teste exato de Fisher. A combinacao dos dois testes aumentou significativamente a deteccao de animais positivos para 34,4% (P <0,05), sugerindo que a associacao de metodos de diagnostico como a tecnica de PCR em amostras de urina e sorologia por IDGA e a avaliacao clinica dos animais e necessaria para um diagnostico eficiente na infeccao por B. ovis.

A comparison of two agar gel immunodiffusion methods and a complement fixation test for serologic diagnosis of Brucella ovis infection in experimentally infected rams

A infeccao por Brucella ovis e considerada uma das principais causas de epididimite e infertilidade em carneiros, resultando em falhas reprodutivas e perdas economicas significativas em rebanhos ovinos ao redor do mundo. O estudo teve o objetivo de avaliar tres testes sorologicos disponiveis para o diagnostico da brucelose ovina por B. ovis, utilizando 181 soros ovinos. Amostras de soro provenientes de carneiros experimentalmente infectados foram coletadas ao longo de 192 dias pos-infeccao (n=117) e durante o periodo pre-infeccao (n=9). Adicionalmente, amostras de soro foram obtidas de ovinos provenientes de um rebanho livre para B. ovis (n=55). As tecnicas de imunodifusao em gel de agar (IDGA), utilizando dois antigenos disponiveis comercialmente, e de fixacao de complemento foram comparadas (FC). Foram obtidos resultados de sensibilidade especificidade semelhantes para ambos os metodos de IDGA e ainda, a tecnica de IDGA foi mais eficiente do que a da FC para o diagnostico sorologico da infeccao por B. ovis.

Clinical and pathological changes in rams experimentally infected with Actinobacillus seminis and Histophilus somni.

Infectious epididymitis is considered a major cause of economic losses for the sheep industry worldwide. This study aimed to investigate clinical and pathological changes associated with experimental infections with A. seminis and H. somni in rams. Twenty rams of age 18 to 24 months were infected by intraepididymal inoculation of A. seminis (n = 10) and H. somni (n = 10). Rams were weekly examined and biological samples were collected during six weeks. All rams inoculated with A. seminis and 80% inoculated with H. somni became infected. The recovery of bacteria was possible in semen and urine samples and tissues in both experimental groups. Clinically, there were a decrease in testicular consistency and an increase in measures of the left epididymis tails in both experimental groups. The main gross changes were observed in the reproductive tract. Microscopically, the main lesions were inflammatory changes in the genitourinary tract and testicular degeneration. A. seminis and H. somni were able to colonize several organs of the genitourinary tract in rams, being indistinguishable by clinical exam, necropsy or histopathology. For differential diagnosis, it is important to use diagnostic techniques for direct confirmation of the etiologic agent.

Effect of extender supplementation with various antimicrobial agents on viability of Brucella ovis and Actinobacillus seminis in cryopreserved ovine semen.

The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 10(6) spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P < 0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis.