Fluorescent quantum dots-zika virus hybrid nanoconjugates for biolabeling, bioimaging, and tracking host-cell interactions.

The earliest possible diagnosis and understanding of the infection mechanisms play a crucial role in the outcome of fighting viral diseases. Thus, we designed and developed for the first time, novel bioconjugates made of Ag-In-S@ZnS (ZAIS) fluorescent quantum dots coupled with ZIKA virus via covalent amide bond with carboxymethylcellulose (CMC) biopolymer for labeling and bioimaging the virus-host cell interactions mechanisms through confocal laser scanning microscopy. This work offers relevant insights regarding the profile of the ZIKA virus-nanoparticle conjugates interactions with VERO cells, which can be applied as a nanoplatform to elucidate the infection mechanisms caused by this viral disease.

Short communication: Parapoxvirus and Orthopoxvirus coinfection in milk of naturally infected cows.

Several studies have shown the occurrence of poxvirus infections associated with exanthematic lesions in cattle from many Brazilian states. Coinfection between viruses belonging to 2 genera, Orthopoxvirus (OPXV) and Parapoxvirus (PPV), was already identified from the lesions of affected cows and humans. The DNA and infectious viral particles of Vaccinia virus, an OPXV, have been detected in milk of naturally and experimentally infected cows. However, to date no reports have described the detection of Pseudocowpox virus, a PPV, in milk. Thus, we investigated the presence of PPV and OPXV in milk samples obtained from dairy cows from a Brazilian region with exanthematic disease outbreaks. From 2011 to 2014, 6 dairy farms with exanthematic disease outbreaks involving dairy cows, calves, and humans were visited. Twelve crusts of cows' teat lesions and 60 milk samples were collected. The crusts and milk samples were analyzed by PCR to detect OPXV or PPV DNA. According to the analyzed crusts, we detected PPV infection in 4 of the 6 visited farms, from which we investigated the PPV contamination in milk. From the 40 milk samples tested, PPV DNA was detected in 12 samples. Of these milk samples, 8 were positive for both PPV and OPXV. This is the first report of PPV DNA detection in milk samples from affected cows, indicating that the virus may be present in milk and potentially contaminating dairy products associated or not with OPXV. In addition to the lesions caused by direct contact, the presence of 2 or more poxvirus species in milk showed that the effect of zoonotic exanthematic diseases on public health and animal husbandry is relevant and cannot be overlooked.

Short communication: Survival of Vaccinia virus in inoculated cheeses during 60-day ripening.

Bovine vaccinia is a neglected zoonosis caused by Vaccinia virus (VACV) and has a major economic and public health effect in Brazil. Previous studies showed infectious VACV particles in milk from either experimentally or naturally infected cows and in fresh cheeses prepared with experimentally contaminated milk. Ripening is a process that leads to major changes in the physical and chemical characteristics of cheese, reducing contamination by spoilage, pathogenic microorganisms, or both. However, it is not known if VACV infectious particles persist after the ripening process. To investigate this issue, viral infectivity at different ripening times was studied in cheeses manufactured with milk experimentally contaminated with VACV strain Guarani P2 (GP2). Cheeses were analyzed at 1, 7, 14, 21, 45, and 60 d of ripening at 25°C. Viral DNA was quantified by real-time PCR, and VACV isolation and titration were performed in Vero cells. The whole experiment was repeated 4 times. Analysis of the mean viral DNA quantification and infectivity indicated a reduction of approximately 2 logs along the ripening process; however, infectious viral particles (1.7 × 102 pfu/mL) could still be recovered at d 60 of ripening. These findings indicate that the ripening process reduces VACV infectivity, but it was not able to inactivate completely the viral particles after 60 d.

Salmonella enterica Serovar Typhi conceals the invasion-associated type three secretion system from the innate immune system by gene regulation.

Delivery of microbial products into the mammalian cell cytosol by bacterial secretion systems is a strong stimulus for triggering pro-inflammatory host responses. Here we show that Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, tightly regulates expression of the invasion-associated type III secretion system (T3SS-1) and thus fails to activate these innate immune signaling pathways. The S. Typhi regulatory protein TviA rapidly repressed T3SS-1 expression, thereby preventing RAC1-dependent, RIP2-dependent activation of NF-κB in epithelial cells. Heterologous expression of TviA in S. enterica serovar Typhimurium (S. Typhimurium) suppressed T3SS-1-dependent inflammatory responses generated early after infection in animal models of gastroenteritis. These results suggest that S. Typhi reduces intestinal inflammation by limiting the induction of pathogen-induced processes through regulation of virulence gene expression.

Identification of sialic acid receptors for influenza A virus in snakes.

The tissue tropism and the wide host range of influenza A viruses are determined by the presence of sialic acid (SA) α2,3-Gal and SA α2,6-Gal receptors. Recent studies have shown that animals possessing both receptors allow for the rearrangement and emergence of new viral strains of public health importance. This study aimed to evaluate the expression and distribution of human and avian influenza A receptors in nine Neotropical snake species using lectin immunohistochemistry. We selected 17 snakes that were examined post mortem at the Veterinary Pathology Sector of the Universidade Federal de Minas Gerais between 2019 and 2023. Sections of nasal turbinate, trachea, lung, oral mucosa, stomach and intestine were subjected to immunohistochemical analysis using the lectins Maackia amurensis and Sambucus nigra. This research detected, for the first time, co-expression of SA α2,3-Gal and SA α2,6-Gal receptors in the respiratory and digestive tracts of snakes, indicating the possible susceptibility of these species to influenza A virus of avian and human origin. Consequently, snakes can be considered important species for monitoring influenza A in wild, urban and peri-urban environments. More studies should be conducted to investigate the role of snakes in influenza A epidemiology.

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

BACKGROUND: Infectious 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. RESULTS: The 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. CONCLUSIONS: The 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.

Carboxymethylcellulose biofunctionalized ternary quantum dots for subcellular-targeted brain cancer nanotheranostics.

Among the most lethal forms of cancer, malignant brain tumors persist as one of the greatest challenges faced by oncologists, where nanotechnology-driven theranostics can play a critical role in developing novel polymer-based supramolecular nanoarchitectures with multifunctional and multi-modal characteristics to fight cancer. However, it is virtually a consensus that, besides the complexity of active delivering anticancer drugs by the nanocarriers to the tumor site, the current evaluation methods primarily relying on in vitro assays and in vivo animal models have been accounted for the low translational effectiveness to clinical applications. In this view, the chick chorioallantoic membrane (CAM) assay has been increasingly recognized as one of the best preclinical models to study the effects of anticancer drugs on the tumor microenvironment (TME). Thus, in this study, we designed, characterized, and developed novel hybrid nanostructures encompassing chemically functionalized carboxymethylcellulose (CMC) with mitochondria-targeting pro-apoptotic peptide (KLA) and cell-penetrating moiety (cysteine, CYS) with fluorescent inorganic semiconductor (Ag-In-S, AIS) for simultaneously bioimaging and inducing glioblastoma cancer cell (U-87 MG, GBM) death. The results demonstrated that the CMC-peptide macromolecules produced supramolecular vesicle-like nanostructures with aqueous colloidal stability suitable as nanocarriers for passive and active targeting of cancer tumors. The optical properties and physicochemical features of the nanoconjugates confirmed their suitability as photoluminescent nanoprobes for cell bioimaging and intracellular tracking. Moreover, the results in vitro demonstrated a notable killing activity towards GBM cells of cysteine-bearing CMC conjugates coupled with pro-apoptotic KLA peptides. More importantly, compared to doxorubicin (DOX), a model anticancer drug in chemotherapy that is highly toxic, these innovative nanohybrids nanoconjugates displayed higher lethality against U-87 MG cancer cells. In vivo CAM assays validated these findings where the nanohybrids demonstrated a significant reduction of GBM tumor progression (41% area) and evidenced an antiangiogenic activity. These results pave the way for developing polymer-based hybrid nanoarchitectonics applied as targeted multifunctional theranostics for simultaneous imaging and therapy against glioblastoma while possibly reducing the systemic toxicity and side-effects of conventional anticancer chemotherapeutic agents.

Outbreak of canine distemper and coinfections in a maned wolf (Chrysocyon brachyurus) and in three giant anteaters (Myrmecophaga tridactyla).

Canine distemper outbreak and coinfections in three giant anteaters and in a maned wolf has been described. Three giant anteaters developed respiratory and digestive clinical signs after the introduction of a maned wolf to a Wildlife Rehabilitation Center. The maned wolf and two anteaters died, and one anteater was euthanized. Post mortem and histopathologic exams revealed lesions associated with numerous intraepithelial inclusion bodies, mainly in the respiratory and digestive systems. Infection by distemper virus was confirmed in all animals by RT-PCR and gene sequencing, which revealed the Europe 1/ South America 1 strain, closely related to the strain from Canis familiaris. In addition to distemper, the animals had other comorbidities, such as toxoplasmosis and salmonellosis in the maned wolf and cutaneous candidiasis in an anteater. Considering the chronology of clinical manifestation in both species and the viral characterization, it is possible that the maned wolf was the source of infection to the anteaters. This study demonstrates the importance of implementing biosecurity measures in enclosures that house animals of different species, highlighting the importance of quarantine before introduction of new animals into the same environment.

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

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.

Laboratory animal models for brucellosis research.

Brucellosis is a chronic infectious disease caused by Brucella spp., a gram-negative facultative intracellular pathogen that affects humans and animals, leading to significant impact on public health and animal industry. Human brucellosis is considered the most prevalent bacterial zoonosis in the world and is characterized by fever, weight loss, depression, hepato/splenomegaly, osteoarticular, and genital infections. Relevant aspects of Brucella pathogenesis have been intensively investigated in culture cells and animal models. The mouse is the animal model more commonly used to study chronic infection caused by Brucella. This model is most frequently used to investigate specific pathogenic factors of Brucella spp., to characterize the host immune response, and to evaluate therapeutics and vaccines. Other animal species have been used as models for brucellosis including rats, guinea pigs, and monkeys. This paper discusses the murine and other laboratory animal models for human and animal brucellosis.

Retrospective and prospective studies of transmissible viral proventriculitis in broiler chickens in Brazil.

We investigated the occurrence and pathologic findings of transmissible viral proventriculitis (TVP) associated with the chicken proventricular necrosis virus (CPNV) in commercial broiler chickens in southeastern Brazil. Seventy-three broilers, 25-36 d old, with a history of reduced growth, were referred to our veterinary pathology services from 2013 to 2017. Broilers were clinically examined, weighed, and euthanized for postmortem examination. Broilers of different ages with proventricular histologic lesions were positive for CPNV by RT-PCR; however, the intensity of histologic lesions was higher among 33-d-old animals, and viral RNA detection was more frequent among those that were 28 d old. In the proventriculi of 35 of 73 (48%) broilers, lesions were characterized by glandular epithelial necrosis, lymphoplasmacytic and histiocytic infiltrates, and metaplasia of glandular epithelium to ductal epithelium. In 24 of 73 (36%) broilers with histologic TVP-compatible lesions, CPNV was detected by RT-PCR for the viral protein 1 (VP1) gene. Broilers with histologic lesions were lighter than expected compared to the Cobb 500 standard weight. TVP has not been reported previously in broiler chickens in Brazil, to our knowledge.

Active Circulation of Madariaga Virus, a Member of the Eastern Equine Encephalitis Virus Complex, in Northeast Brazil.

Madariaga virus (MADV) is a member of the eastern equine encephalitis virus (EEEV) complex that circulates in Central and South America. It is a zoonotic, mosquito-borne pathogen, belonging to the family Togaviridae. Disturbances in the natural transmission cycle of this virus result in outbreaks in equines and humans, leading to high case fatality in the former and acute febrile illness or neurological disease in the latter. Although a considerable amount of knowledge exists on the eco-epidemiology of North American EEEV strains, little is known about MADV. In Brazil, the most recent isolations of MADV occurred in 2009 in the States of Paraíba and Ceará, northeast Brazil. Because of that, health authorities have recommended vaccination of animals in these regions. However, in 2019 an equine encephalitis outbreak was reported in a municipality in Ceará. Here, we present the isolation of MADV from two horses that died in this outbreak. The full-length genome of these viruses was sequenced, and phylogenetic analyses performed. Pathological findings from postmortem examination are also discussed. We conclude that MADV is actively circulating in northeast Brazil despite vaccination programs, and call attention to this arbovirus that likely represents an emerging pathogen in Latin America.

Equine coital exanthema caused by equid alphaherpesvirus 3: a report of an outbreak in northeastern Brazil / Exantema coital equino causado pelo alphaherpesvirus equídeo tipo 3: relato de um surto no nordeste do Brasil

Equine coital exanthema is a venereal infectious disease poorly reported in horses in Brazil and was never described in the northeastern region of the country. This work aims to describe the clinical and pathological aspects of an outbreak of equine coital exanthema caused by equid alphaherpesvirus 3, occurred in a herd of horses at the semiarid region of the State of Rio Grande do Norte. Main clinical signs consisted of anorexia, hiporexia, fibrinous or purulent secretion in the penis mucosa and vagina. Two mares presented mild to minimal lesions that consisted of scars in the mucosa of the vagina and in the perivulvar region. In a stallion the disease consisted of severe, multifocal, umbilicated-exanthematous ulcers of approximately 1cm in diameter on the penis mucosa. Other areas where ulcers and crusts were focally observed included the skin of the scrotum and on the lips and mucocutaneous junctions of the oral cavity. Histologically, the main lesion consisted of multifocal severe ulcerative and fibrinous necrotizing balanoposthitis and mild multifocal necrotizing, lymphocytic dermatitis in the lips and scrotum. The equide alphaherpesvirus 3 DNA was amplified in blood samples and penis mucosa using the PCR technique. This is the first report of molecular diagnosis of equine coital exanthema affecting horses in northeastern Brazil. Further studies should be carried out in order to investigate the epidemiology and the importance of this herpetic disease in the country.(AU)

O exantema coital equino é uma doença infecciosa venérea pouco relatada em equinos no Brasil e nunca descrita na região Nordeste do país. Este trabalho tem como objetivo descrever os aspectos clínicos e patológicos de um surto de exantema coital equino causado pelo alphaherpesvirus equídeo 3, que ocorreu em um haras na região semiárida do Estado do Rio Grande do Norte. Os principais sinais clínicos consistiram em anorexia, hiporexia, secreção fibrinosa ou purulenta na mucosa do pênis e vagina. Duas éguas apresentavam lesões discretas que consistiam em cicatrizes na mucosa da vagina e na região perivulvar. Em um garanhão, a doença consistia em úlceras umbilicadas-exantematosas severas, multifocais, de aproximadamente 1 cm de diâmetro na mucosa do pênis. Outras áreas onde úlceras e crostas foram observadas focalmente incluíram a pele do escroto, lábios e junções mucocutâneas da cavidade oral. Histologicamente, as principais lesões consistiam em balanopostite multifocal ulcerativa e necrosante fibrinosa grave e dermatite linfocítica necrosante multifocal leve nos lábios e escroto. O DNA do alphaherpesvirus equídeo tipo 3 foi amplificado em amostras de sangue e mucosa do pênis pela técnica de PCR. Este é o primeiro relato de diagnóstico molecular de exantema coital equino afetando cavalos no nordeste do Brasil. Novos estudos devem ser realizados a fim de investigar a epidemiologia e a importância dessa doença herpética no país.(AU)

Equine coital exanthema caused by equid alphaherpesvirus 3: a report of an outbreak in northeastern Brazil

ABSTRACT: Equine coital exanthema is a venereal infectious disease poorly reported in horses in Brazil and was never described in the northeastern region of the country. This work aims to describe the clinical and pathological aspects of an outbreak of equine coital exanthema caused by equid alphaherpesvirus 3, occurred in a herd of horses at the semiarid region of the State of Rio Grande do Norte. Main clinical signs consisted of anorexia, hiporexia, fibrinous or purulent secretion in the penis mucosa and vagina. Two mares presented mild to minimal lesions that consisted of scars in the mucosa of the vagina and in the perivulvar region. In a stallion the disease consisted of severe, multifocal, umbilicated-exanthematous ulcers of approximately 1cm in diameter on the penis mucosa. Other areas where ulcers and crusts were focally observed included the skin of the scrotum and on the lips and mucocutaneous junctions of the oral cavity. Histologically, the main lesion consisted of multifocal severe ulcerative and fibrinous necrotizing balanoposthitis and mild multifocal necrotizing, lymphocytic dermatitis in the lips and scrotum. The equide alphaherpesvirus 3 DNA was amplified in blood samples and penis mucosa using the PCR technique. This is the first report of molecular diagnosis of equine coital exanthema affecting horses in northeastern Brazil. Further studies should be carried out in order to investigate the epidemiology and the importance of this herpetic disease in the country.

RESUMO: O exantema coital equino é uma doença infecciosa venérea pouco relatada em equinos no Brasil e nunca descrita na região Nordeste do país. Este trabalho tem como objetivo descrever os aspectos clínicos e patológicos de um surto de exantema coital equino causado pelo alphaherpesvirus equídeo 3, que ocorreu em um haras na região semiárida do Estado do Rio Grande do Norte. Os principais sinais clínicos consistiram em anorexia, hiporexia, secreção fibrinosa ou purulenta na mucosa do pênis e vagina. Duas éguas apresentavam lesões discretas que consistiam em cicatrizes na mucosa da vagina e na região perivulvar. Em um garanhão, a doença consistia em úlceras umbilicadas-exantematosas severas, multifocais, de aproximadamente 1 cm de diâmetro na mucosa do pênis. Outras áreas onde úlceras e crostas foram observadas focalmente incluíram a pele do escroto, lábios e junções mucocutâneas da cavidade oral. Histologicamente, as principais lesões consistiam em balanopostite multifocal ulcerativa e necrosante fibrinosa grave e dermatite linfocítica necrosante multifocal leve nos lábios e escroto. O DNA do alphaherpesvirus equídeo tipo 3 foi amplificado em amostras de sangue e mucosa do pênis pela técnica de PCR. Este é o primeiro relato de diagnóstico molecular de exantema coital equino afetando cavalos no nordeste do Brasil. Novos estudos devem ser realizados a fim de investigar a epidemiologia e a importância dessa doença herpética no país.

Distribution of antibodies against influenza virus in pigs from farrow-to-finish farms in Minas Gerais state, Brazil.

BACKGROUND: Swine influenza virus (SIV) is the cause of an acute respiratory disease that affects swine worldwide. In Brazil, SIV has been identified in pigs since 1978. After the emergence of pandemic H1N1 in 2009 (H1N1pdm09), few studies reported the presence of influenza virus in Brazilian herds. OBJECTIVES: The objective of this study was to evaluate the serological profile for influenza virus in farrow-to-finish pig farms in Minas Gerais state, Brazil. METHODS: Thirty farms with no SIV vaccination history were selected from the four larger pig production areas in Minas Gerais state (Zona da Mata, Triângulo Mineiro/Alto Paranaíba, South/Southwest and the Belo Horizonte metropolitan area). At each farm, blood samples were randomly collected from 20 animals in each production cycle category: breeding animals (sows and gilts), farrowing crate (2-3 weeks), nursery (4-7 weeks), grower pigs (8-14 weeks), and finishing pigs (15-16 weeks), with 100 samples per farm and a total of 3000 animals in this study. The samples were tested for hemagglutination inhibition activity against H1N1 pandemic strain (A/swine/Brazil/11/2009) and H3N2 SIV (A/swine/Iowa/8548-2/98) reference strain. RESULTS: The percentages of seropositive animals for H1N1pdm09 and H3N2 were 26.23% and 1.57%, respectively, and the percentages of seropositive herds for both viruses were 96.6% and 13.2%, respectively. CONCLUSIONS: The serological profiles differed for both viruses and among the studied areas, suggesting a high variety of virus circulation around the state, as well as the presence of seronegative animals susceptible to influenza infection and, consequently, new respiratory disease outbreaks.

Early transcriptional responses of bovine chorioallantoic membrane explants to wild type, ΔvirB2 or ΔbtpB Brucella abortus infection.

The pathogenesis of the Brucella-induced inflammatory response in the bovine placenta is not completely understood. In this study we evaluated the role of the B. abortus Type IV secretion system and the anti-inflammatory factor BtpB in early interactions with bovine placental tissues. Transcription profiles of chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), ΔvirB2 or ΔbtpB Brucella abortus were compared by microarray analysis at 4 hours post infection. Transcripts with significant variation (>2 fold change; P<0.05) were functionally classified, and transcripts related to defense and inflammation were assessed by quantitative real time RT-PCR. Infection with wild type B. abortus resulted in slightly more genes with decreased than increased transcription levels. Conversely, infection of trophoblastic cells with the ΔvirB2 or the ΔbtpB mutant strains, that lack a functional T4SS or that has impaired inhibition of TLR signaling, respectively, induced more upregulated than downregulated genes. Wild type Brucella abortus impaired transcription of host genes related to immune response when compared to ΔvirB and ΔbtpB mutants. Our findings suggest that proinflammatory genes are negatively modulated in bovine trophoblastic cells at early stages of infection. The virB operon and btpB are directly or indirectly related to modulation of these host genes. These results shed light on the early interactions between B. abortus and placental tissue that ultimately culminate in inflammatory pathology and abortion.

The virB-encoded type IV secretion system is critical for establishment of infection and persistence of Brucella ovis infection in mice.

Brucella spp. are gram-negative intracellular bacterial pathogens that cause chronic infections. Brucella virulence factors include a type IV secretion system (T4SS) and its lipopolysaccharide (LPS), which are essential for persistence. However, the role of the virB-encoded T4SS has not been investigated in naturally rough Brucella species such as Brucella ovis. In this study, male 6-week old BALBc mice were infected with B. ovis, Brucella abortus, and their respective ΔvirB2 mutant strains. During early infection, B. ovis and B. abortus wild type strains were similarly recovered from spleen. Interestingly, in contrast to ΔvirB2 B. abortus that was recovered at similar levels when compared to the wild type strain, the ΔvirB2 B. ovis was markedly attenuated as early as 24h post infection (hpi). The ΔvirB2 B. ovis was unable to survive and multiply in murine peritoneal macrophages and extracellularly within the peritoneal cavity at 12 and 24 hpi with lower splenic colonization than the parental strain at 6, 12 and 24 hpi. In contrast, wild type B. abortus and ΔvirB2 B. abortus had a similar kinetics of infection in this model. As expected, the T4SS was essential for intracellular replication of smooth and rough strains in RAW macrophages at 48 hpi. These results suggest that T4SS is important for survival of B. ovis in murine model, and that a T4SS deficient B. ovis strain is cleared at earlier stages of infection when compared to a similar B. abortus mutant.

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.