Luciana F. Costa

Salmonellosis in cattle: Advantages of being an experimental model

Salmonellosis is an important disease of cattle caused predominantly by Salmonella enterica serotypes Typhimurium (S. typhimurium) and Dublin (S. dublin). S. typhimurium causes acute enteritis and exudative diarrhea in calves. In addition to enteric disease, S. dublin can cause systemic infections, and may cause abortion in pregnant cows. Calves are considered a relevant model for non-typhoidal salmonellosis in humans. Experimental oral infections or inoculation of ligated ileal loops in calves have been extensively studied recently. This article reviews relevant published results regarding bovine salmonellosis as a natural disease or as an animal model.

Tissue distribution of Leishmania chagasi and lesions in transplacentally infected fetuses from symptomatic and asymptomatic naturally infected bitches.

Visceral leishmaniasis (VL) is primarily transmitted by an invertebrate vector, but transmission in the absence of the vector has been reported. Vertical transmission of VL has been described in man and dogs. The aim of this study was to evaluate the distribution of Leishmania amastigotes in fetal organs and histopathologic changes associated with parasitism and to determinate the frequency of transplacental transmission and potential of vertical transmission by symptomatic and asymptomatic pregnant bitches. Symptomatic (n=4) and asymptomatic (n=4) pregnant bitches, serologically and parasitologically positive for Leishmania sp., carrying a total of 53 fetuses (26 from symptomatic and 27 from asymptomatic bitches) were selected at the Veterinary Hospital of the National University of Asuncion, Paraguay. Samples of placenta and fetal organs such as liver, spleen, lymph nodes, bone marrow, kidney and heart were histologically evaluated and processed for immunodetection of amastigotes and PCR. There were no lesions compatible with VL in fetal tissues in spite of the presence of amastigotes, particularly in lymphoreticular tissues. However, fetal hepatocytes had marked degenerative changes that were independent of the presence of amastigotes in liver. Twenty-six out of 53 placentas (13 symptomatic and 13 asymptomatic) and a total of 17 fetuses out of 53 (nine symptomatic and eight asymptomatic) were PCR positive. Together these findings indicate a high frequency of transplacental transmission and no differences in the potential of transmission when symptomatic were compared to asymptomatic pregnant bitches.

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.

Transcription of innate immunity genes and cytokine secretion by canine macrophages resistant or susceptible to intracellular survival of Leishmania infantum

In this study we assessed the basal transcription of genes associated with innate immunity (i.e. Nramp1, NOD1, NOD2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR9) in canine monocyte-derived macrophages from Leishmania-free dogs. Additionally, secretion of cytokines (IL-10, IL-12, TNF-α and IFN-γ) and nitric oxide in culture supernatants of macrophages with higher or lower resistance to intracellular survival of Leishmania infantum was also measured. Constitutive transcription of TLR9 and NOD2 were negligible; NOD1, TLR1, and TLR7 had low levels of transcription, whereas Nramp1 and TLR2, 3, 4, 5, and 6 had higher levels of constitutive transcription in canine monocyte-derived macrophages. There were no significant differences in transcription between macrophages with higher or lower resistance to intracellular survival of L. infantum. Secretion of TNF-α was higher in more resistant macrophages (designated as resistant) at 24h after infection when compared to less resistant macrophages (designated as susceptible), as well as the secretion of IFN-γ at 72 h post infection. Secretion of IL-10 was lower in resistant macrophages at 24h after infection. No detectable production of nitric oxide was observed. Interestingly, there was a negative correlation between NOD2 transcript levels and intracellular survival of L. infantum in resistant macrophages. This study demonstrated that decreased intracellular survival of L. infantum in canine macrophages was associated with increased production of TNF-α and IFN-γ and decreased production of IL-10; and that constitutive transcription of Nramp1, TLR and NLR does not interfere in intracellular survival of L. infantum.

First Identification of Canine Distemper Virus in Hoary Fox (Lycalopex vetulus): Pathologic Aspects and Virus Phylogeny

Canine distemper virus (CDV) has been reported in several wild animal species, but there have been no reports of CDV in hoary fox (Lycalopex vetulus). This paper characterizes the first case of natural CDV infection in hoary fox, including the clinical and pathologic aspects of the disease as well as the viral strain phylogeny.

Brucella ovis lacking a species-specific putative ATP-binding cassette transporter is attenuated but immunogenic in rams.

Ovine brucellosis caused by Brucella ovis is considered one of the most important reproductive diseases of rams worldwide. This study aimed to characterize the kinetics of infection of a ΔabcAB B. ovis mutant strain in rams. Twelve 1-year-old crossbred rams were used. Six rams were challenged with 2 mL of a suspension containing 1.2×10(9) CFU/mL of B. ovis strain ATCC25840 (wild type) by intraprepucial inoculation and additional 50 μL in each conjunctival sac of a suspension containing 1.2×10(10) CFU/mL of the same strain. The other six rams were challenged with an equivalent number of CFU of the mutant strain ΔabcAB B. ovis through the same routes. Serum samples for serology and semen and urine samples for bacteriologic culture and PCR were collected weekly during 24 weeks. At 24 weeks post infection, tissue samples were collected for bacteriologic culture and PCR. All rams inoculated with wild type or the ΔabcAB strain seroconverted at the fourth week post infection, remaining positive up to the 16th week post infection. PCR and bacteriology demonstrated that only rams inoculated with the wild type strain shed the organism in semen and urine. Lymphocytes from rams inoculated with wild type or ΔabcAB B. ovis had significantly higher proliferation in response to B. ovis antigens when compared with unstimulated controls. Tissue bacteriology and PCR detected B. ovis in all rams challenged with the wild type strain, whereas only one ΔabcAB-infected ram had a positive iliac lymph node sample by PCR.

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.

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.

The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages

Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment.

Distribution of caprine arthritis encephalitis virus provirus, RNA, and antigen in the reproductive tract of one naturally and seven experimentally infected bucks.

Caprine arthritis encephalitis is a worldwide, multisystemic disease caused by a small ruminant lentivirus. Although the main route of transmission is oral, detection of proviral DNA of the caprine arthritis encephalitis virus (CAEV) in caprine semen has been previously described. However, the presence of viral antigens in the male reproductive tract has apparently never been reported. The objective was to study lesions in the buck reproductive system and to detect, in these tissues, the presence of proviral DNA, viral RNA and CAEV antigens. Tissues from eight CAEV-infected bucks (one naturally and seven experimentally infected) were analyzed by histopathology, nested polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and immunohistochemistry. Interstitial pneumonia, synovitis, and lesions in the male reproductive tract were detected in some of the bucks. Proviral DNA was detected in the lungs and joints as well as in the reproductive systems of all animals, whereas viral RNA was detected only in the genital tract of the naturally infected buck. Viral antigens were immunostained in most of the organs of the male reproductive tract. This report was apparently the first to clearly demonstrate CAEV antigen expression in the male reproductive tract, which indicates the possibility of venereal transmission of CAEV.