Juliana Torres Tomazi Fritzen

Frequency of virulence genes in Escherichia coli strains isolated from piglets with diarrhea in the North Parana State, Brazil

Identification of Escherichia coli causing porcine postweaning diarrhea requires knowledge regarding the prevalent pathotypes within a given region. A total of 100 Escherichia coli isolates from piglets with diarrhea in Londrina city, Parana State, South Brazil, were screened for the presence of genes for F4, F5, F6, F18, F41 fimbrial antigens by specific probes and for enterotoxins (STa, STb, LT and STx2e) by polymerase chain reaction (PCR). The results showed that 60% of the isolates were positive for one or more of the fimbrial antigens and 92% were positive at least for one of the virulence factors examined. Virulence factor genes detected were F4 (44%), F18 (38%), F5 (30%), F41 (32%), F6 (25%), LTp-I (71%), STa (40%), STb (47%) and STx2e (3%). Twenty four patterns of virulence factor according to the different virulence genes form were found and the most frequent virulence gene pattern was F4, F18, F41, STa, STb and LT. Most of the isolates that carried genes for adhesins also harboured genes for toxins.

Concomitant canine distemper, infectious canine hepatitis, canine parvoviral enteritis, canine infectious tracheobronchitis, and toxoplasmosis in a puppy

The concomitant infections of Canine distemper virus (CDV), Canine adenovirus A types 1 (CAdV-1) and 2 (CAdV-2), Canine parvovirus type 2 (CPV-2), and Toxoplasma gondii are described in a 43-day-old mixed-breed puppy. Clinically, there were convulsions and blindness with spontaneous death; 14 siblings of this puppy, born to a 10-month-old dam, which was seropositive (titer: 1,024) for T. gondii, also died. Necropsy revealed unilateral corneal edema (blue eye), depletion of intestinal lymphoid tissue, non-collapsible lungs, congestion of meningeal vessels, and a pale area in the myocardium. Histopathology demonstrated necrotizing myocarditis associated with intralesional apicomplexan protozoa; necrotizing and chronic hepatitis associated with rare intranuclear inclusion bodies within hepatocytes; necrotizing bronchitis and bronchiolitis; interstitial pneumonia associated with eosinophilic intracytoplasmic inclusion bodies within epithelial cells; atrophy and fusion of intestinal villi with cryptal necrosis; and white matter demyelination of the cerebrum and cerebellum associated with intranuclear inclusion bodies within astrocytes. Polymerase chain reaction (PCR) amplified the partial fragments (bp) of the CDV N gene (290 bp), CPV-2c VP2 capsid protein gene (583 bp), and CAdV-1 (508 bp) and CAdV-2 (1,030 bp) E gene from urine and tissue samples. The PCR assays demonstrated that the apicomplexan protozoa observed within several organs contained DNA specific for T. gondii; genotyping revealed T. gondii type III. The findings support the characterization of concomitant infections of CDV, CAdV-1, CAdV-2, CPV-2, and T. gondii in this puppy. Further, seroreactivity to T. gondii of the dam in association with the systemic disease observed in the puppy described herein is suggestive of congenital toxoplasmosis.

Molecular characterization of encephalitic bovine listeriosis from southern Brazil.

Reports of bovine listeriosis in Brazil are uncommon, being restricted to citations within retrospective studies, resulting in scarce documented information of this important disease of cattle. This manuscript describes the molecular findings associated with spontaneous encephalitic listeriosis in two steers from distinct herds within the state of Paraná, southern Brazil. Both animals demonstrated altered consciousness suggestive of brain stem dysfunctions and died a few days after the initial onset of disease. Polymerase chain reaction (PCR) assays were designed to target specific genes of infectious neurological agents of cattle. These included bovine herpesvirus 1 and 5 (BoHV-1 and BoHV-5), ovine herpesvirus 2 (OvHV-2), Listeria monocytogenes, and Histophilus somni. Rabies virus was discarded in evaluations done at the official state diagnostic laboratory. Gross alterations were insignificant; histopathology demonstrated rhombencephalitis associated with macrophage-predominant, multifocal to coalescing microabscesses and extensive perivascular cuffings in both steers. The L. monocytogenes PCR assay amplified the 172-bp amplicon of the listeriolysin gene from the brain stem of both animals and from the telencephalon, thalamus, and cerebellum of one of them. Phylogenetic analyses demonstrated that the strains derived from this study clustered with known strains of L. monocytogenes lineage I. The BoHV-1 and BoHV-5, OvHV-2, and H. somni PCR assays were negative. These results confirm the participation of L. monocytogenes lineage I in the etiopathogenesis of the neurological disease herein described and represent the first complete description of encephalitic listeriosis in cattle from Brazil.

Histopathological and molecular characterization of encephalitic listeriosis in small ruminants from northern Paraná, Brazil

Listeriosis is a disease primarily of ruminants caused by the Gram-positive bacterium Listeria monocytogenes. Ruminants either demonstrate manifestations of the encephalitic, septicemic, or reproductive form of listeriosis. The pathological and molecular findings with encephalitic listeriosis in a 5.5-month-old, male, mixed-breed goat and a 3-year-old Texel-crossed sheep from northern Paraná, Brazil are described. Clinically, the kid demonstrated circling, lateral protrusion of the tongue, head tilt, and convulsions; the ewe presented ataxia, motor incoordination, and lateral decumbency. Brainstem dysfunctions were diagnosed clinically and listeriosis was suspected. Necropsy performed on both animals did not reveal remarkable gross lesions; significant histopathological alterations were restricted to the brainstem (medulla oblongata; rhombencephalitis) and were characterized as meningoencephalitis that consisted of extensive mononuclear perivascular cuffings, neutrophilic and macrophagic microabscesses, and neuroparenchymal necrosis. PCR assay and direct sequencing, using genomic bacterial DNA derived from the brainstem of both animals, amplified the desired 174 base pairs length amplicon of the listeriolysin O gene of L. monocytogenes. Phylogenetic analyses demonstrated that the strains associated with rhombencephalitis during this study clustered with known strains of L. monocytogenes lineage I from diverse geographical locations and from cattle of the state of Paraná with encephalitic listeriosis. Consequently, these strains should be classified as L. monocytogenes lineage I. These results confirm the active participation of lineage I strains of L. monocytogenes in the etiopathogenesis of the brainstem dysfunctions observed during this study, probably represent the first characterization of small ruminant listeriosis by molecular techniques in Latin America, and suggest that ruminants within the state of Paraná were infected by the strains of the same lineage of L. monocytogenes.

Virus neutralization technique as a tool to evaluate the virological profile for bovine viral diarrhea virus infection in dairy water buffalo (Bubalus bubalis) herds

Serological evidence shows that the bovine viral diarrhea virus (BVDV) infections are present in Brazilian dairy and beef water buffalo herds. As few reports describe the BVDV infection profile the aim of this study was to evaluate the dynamics of BVDV circulation in Brazilian dairy water buffalo herds through analysis of the seropositivity rate and the titer range of anti-BVDV neutralizing antibodies in a group of animals that are considered sentinels. Blood samples (n = 305) were obtained from unvaccinated, asymptomatic young water buffalos from four dairy herds randomly identified as A (n = 106), B (n = 62), C (n = 119), and D (n = 18). The detection and titration of anti-BVDV neutralizing antibodies were evaluated by the virus neutralization test according to the World Organization for Animal Health. Analysis of the results revealed two distinct epidemiological conditions. The first is represented by herds A and C where high rates of seropositive animals (A = 39.6%; C = 51.3%) and high and very variable antibodies titers suggested active BVDV infection. The other condition is represented by herds B and D with low rates of seropositive animals (B = 8.1%; D = 11.1%) and low and little variable antibodies titers suggesting an epidemiological condition of infection stability. Some variables were observed in herds with a distinct BVDV infection profile. Herds with active infection were big, open herds, and had more management practices. In contrast, the herds with infection stability were small, closed herds with few management practices. These results highlight the importance of evaluation, monitoring, and control of BVDV infection also in dairy water buffalo herds.

Presence of BoHV-5 genome in cerebrospinal fluid of cattle with herpetic meningoencephalitis

The diagnosis of bovine herpesvirus 5 (BoHV-5) encephalitis is confirmed after death by laboratory methods applied to brain fragments. Alternative methods to confirm ante-mortem diagnosis are important because the disease is not always lethal. The aim of this study was to investigate whether the presence of the virus genome in the cerebrospinal fluid (CSF) detected by polymerase chain reaction (PCR) might be admitted as a method for ante-mortem diagnosis. CSF samples were taken from 14 animals suffering from BoHV-5 encephalitis, diagnosed by characteristic histopathological lesions in the brain and by identification of the virus genome by PCR in different portions of the brain. Virus DNA was detected in the CSF of 21.42% (3/14) of the evaluated animals. Ante-mortem detection of the virus genome in the CSF showed low sensitivity to confirm the diagnosis. The diagnosis is confirmed by a positive result but a negative one does not discard the disease.

IMMUNOHISTOCHEMICAL AND MOLECULAR EVIDENCE OF PUTATIVE NEORICKETTSIA INFECTION IN COATIS (NASUA NASUA) FROM SOUTHERN BRAZIL

Abstract The pathologic, molecular, and immunohistochemical findings associated with Neorickettsia helminthoeca are described in coatis (Nasua nasua). Tissue sections (small intestine, lungs, kidney, liver, and spleen) of coatis (n = 3) that died at the Bela Vista Biological Refuge, Foz do Iguaçu, Paraná, southern Brazil were routinely processed from histopathology. Selected formalin-fixed paraffin-embedded (FFPE) tissue sections of the small intestine, lungs, and spleen were used in an immunohistochemical (IHC) assay designed to identify the antigens of N. helminthoeca. Additionally, FFPE tissue sections of the small intestine were used to demonstrate antigens of canine parvovirus-2 (CPV-2) by IHC. Histopathology revealed chronic enteritis in all coatis. Parasitic enteritis was diagnosed in two coatis; one of these contained examples of a trematode within the lumen of the small intestine and the ovum of a trematode encysted in the intestinal mucosa. Other significant pathologic findings included interstitial pneumonia (n = 2) and pyogranulomatous splenitis (n = 1). Positive immunolabeling for N. helminthoeca was identified within macrophages of the small intestine and reticuloendothelial cells within the germinal centers of the spleen of all coatis; the intestinal trematode was N. helminthoeca IHC-positive. All pulmonary sections revealed negative immunolabeling for N. helminthoeca. Furthermore, the antigens of CPV-2 were not identified in the intestine of any coati. These findings indicate that these coatis were infected by N. helminthoeca, but since clinical and gross pathological findings were not recorded, it is uncertain if this pathogen produced clinical disease in this canid host; therefore, coatis may be asymptomatic or dead-end hosts for this organism.

Investigação sorológica e molecular de agentes virais em onças-pintadas de vida livre no Pantanal de Mato Grosso, Brasil

This study investigates the exposure of free-living jaguars from two federal protected areas in the Pantanal of Mato Grosso, Brazil, to a variety viral agents. These viral agents, particularly causing zoonotic diseases, were analyzed using serological and molecular methods. None of the jaguars was positive by RT-PCR for the molecular detection of avian influenza and West Nile Fever (WNF). Only one animal was serologically positive for Eastern Equine Encephalitis (EEE) by virus neutralization test in VERO cell cultures, representing the first reported case of jaguar exposure to EEE virus. However, all the animals were negative for Western Equine Encephalitis (WEE) virus and Venezuelan Equine Encephalitis (VEE) virus. Eleven jaguars were tested by two tests for the detection of antibodies against rabies virus (Simplified Fluorescent Inhibition Microtest – SFIMT and Rapid Fluorescent Focus Inhibition Test – RFFIT), resulting in five positive animals, two animals in each test and one in both serological tests. Furthermore, three out of 14 samples subjected to the neutralization test were positive for antibodies against canine distemper virus (CDV), and 15 out of 17 samples subjected to the hemagglutination-inhibition test (HI) were positive for antibodies against canine parvovirus (CPV). In view of the findings of this study, it is unlikely that the viruses examined here represent a threat to the jaguar populations in this region.