Lucienne Garcia Pretto-Giordano

Isolation and characterization of Streptococcus spp. group B in Nile tilapias (Oreochromis niloticus) reared in hapas nets and earth nurseries in the northern region of Parana State, Brazil

The objective of this study was to isolate and characterize Streptococcus spp. in Nile tilapias (Oreochromis niloticus) reared in net-pens and earth nurseries. Eight intensive tilapia-rearing farms were investigated in north Parana, Brazil from April 1st 2001 to April 30th 2002. The fish were reared in a system of hapas nets on four farms and in earth nurseries on other four farms. A total of 370 samples were analyzed of material collected from 120 fish (brain, liver, kidney, skin scrapes, ascites liquid and eye) that were sown on BHI agar (Brain Heart Infusion) supplemented with 1% yeast extract and sheep blood. Streptococcus spp. was isolated in 36 of the samples (18 brain, eight liver, eight kidney and two ascites liquid) from 25 fish. Streptococci were isolated in both systems, almost in the same proportion. First the streptococci were characterized by the catalase and esculin test, growth in methylene blue and sodium chloride at 6.5%. They were classified in groups by the Slidex Strepto-Kit (BioMerieux, France). The phenotypic characteristics were determined by the Api 20 Strep microtest system (BioMerieux, France). The 36 Streptococcus spp. samples did not present hemolysis and were classified as Lancefield group B. Further 16 samples were identified as Streptococcus agalactiae and 20 were not identified by the Api 20 Strep, but presented the same biochemical profile described for the reference strain of Streptococcus difficile (ND-2-22).

Evaluation on the Pathogenesis of Streptococcus agalactiae in Nile Tilapia (Oreochromis niloticus)

The pathogenesis of a Streptococcus agalactiae was evaluated in a three-period experiment. Two groups of 40 fishes were intraperitoneally (i.p.) challenged in each experimental period with different infective doses of the pathogen. Doses varied from 1.0 x 106 to 1.5 x 108 CFU/fish. One group of 40 tilapia i.p. injected with tryptic soy broth (TSB) was used as a control group in each period. Mortalities varied from 67.5% in group 8 (infective dose 1.0 x 106 CFU/fish) to 90.0% in group 1 (infective dose 1.5 x 108 CFU/fish). Significant differences in mortalities were found only between group 8 and each of the other groups, except group 5 (infective dose 6.0 x 106 CFU/fish; mortality 75.0%). The highest mortality coefficients were observed in days 1-2 after inoculation (accumulated mortality 44.4%), and a second peak of mortality occurred at days 6-7. Challenged fishes from all the groups showed alterations in behaviour and similar clinical signs. These were anorexia, lethargy, erratic swimming, exophthalmia and ascites. Macroscopically, skin hemorrhage, splenomegaly, hepatomegaly with organ paleness and visceral adherences were observed. S. agalactiae was re-isolated from all the fishes from the experimental groups submitted to bacteriological examination. The illness observed in tilapia naturally infected with S. agalactiae was experimentally reproduced in this study, and the clinical signs produced were similar to those reported from the natural infections.

Cryptococcus neoformans var. grubii-Induced Arthritis with Encephalitic Dissemination in a Dog and Review of Published Literature.

This article describes the clinical, pathological, and immunohistochemical findings associated with Cryptococcus neoformans var. grubii in a 4-year-old female Boxer dog from Uberlândia, Minas Gerais, Southeastern Brazil. Clinically, there was a swelling at the right metatarsal region and the hock joint with enlargement of regional lymph nodes. Radiographical evaluation revealed lysis of the tarsal bone; cytology demonstrated cryptococcal intralesional organisms at the swollen joint. Despite empirical antifungals therapeutic, the animal developed neurological cryptococcosis and died spontaneously. Significant pathological alterations included arthritis, lymphadenitis, and encephalitic cryptococcomas associated with numerous intralesional narrow-necked budding encapsulated yeasts. Immunohistochemistry utilising monoclonal antibodies that label C. neoformans sp. complex capsule, characterised the yeasts as C. neoformans var. grubii. Collectively, the pathological and immunohistochemical findings of this dog indicate that the intralesional organisms observed within the articular surface of the hock joint, lymph nodes, and the brain were C. neoformans var. grubii, confirming the participation of this fungal pathogen in the development of cryptococcal arthritis. In this case, the most likely pathogenesis was percutaneous inoculation with resultant abscess-like lesion, which resulted in the draining sinus, swelling of the right hind limb with progression to the articular disease. Thereafter, the fungal pathogen probably compromised the adjacent lymph nodes with subsequent haematogenous distribution to the brain, terminating with cryptococcal arthritis, lymphadenitis, and encephalitis.

Fatores de risco, etiologia e aspectos clínicos da mastite em ovelhas de corte no Paraná

Mastits is infrequent in meat ewes. However Santa Ines ewes have a high incidence of this disease and it is severe and difficult to treat. The goal of this study was to characterize clinical, epidemiological and etiological aspects of clinical mastitis in meat ewes reared in the north of Parana, Brazil. Fifith four farms were visited from October 2009 to September 2010. The surveyed data included frequency, breeds of sheep affected, lamb mortality rates, main clinical signs, attempts and outcome of treatment, method and period of weaning and management features. Seventy ewes with clinical mastitis were fully examinated and samples of mammary secretion were asseptically taken for bacteriological culture. Mastitis was identified in 39 farms (72.3%) as a relevant problem (mean frequency was 6,74%). Chronic and acute mastitis were observed in 69% and 31% of the examinated ewes, respectively. In both cases, phlegmonous mastitis was the most prevalent form (65.5%). Coagulase negative Staphylococccus (CNS) was the main isolated microorganism (54.5%), followed by Staphylococcus aureus and Arcanobacterium pyogenes (11.5%, each one). Mannheimia haemolytica was found in two cases. The risk factors for clinical mastitis were intensive management system and Santa Ines breed. Weaning after 120 days of lactation and isolation of affected ewes were associated with lower frequency of mastitis. Preventive measures recommended are daily cleaning of facilities and delayed weaning, mainly in Santa Ines flocks.

Cryptococcus gattii -Induced Infections in Dogs from Southern Brazil

Cryptococcus gattii-induced cryptococcosis is an emerging infectious disease of humans and animals worldwide, with rare descriptions of this infection in domestic animals from Brazil. This study presents the findings associated with C. gattii in dogs from Londrina, Paraná, Southern Brazil. Two dogs, a 3-year-old, female German shepherd and a 6-year-old, male Boxer, were evaluated by a combination of pathological, mycological, and molecular diagnostic techniques. Significant pathological alterations included cryptococcal lymphadenitis, meningoencephalitis, tonsillitis, and rhinitis with nasal cryptococcomas in the German shepherd dog, while cryptococcal lymphadenitis and pneumonia were observed in the Boxer; both dogs had pseudocystic cryptococcosis. The mucicarmine histochemical stain readily identified the intralesional cryptococcal budding organisms in all affected tissues. Mycological culture and isolation confirmed the yeasts as C. gattii due to positive reaction with the l-canavanine glycine bromothymol blue agar. A PCR assay using the internal transcribed spacers (ITS)1 and ITS2 primers, which target the ITS1 and 2 regions including the 5.8S rRNA gene, amplified the desired amplicons; direct sequencing confirmed the isolate as C. gattii. ITS nucleotide differentiation demonstrated that the isolate forms part of the ITS type 4 Cryptococcus organisms which corresponds to the C. gattii VGII molecular subtype or the RAPD type 2 Cryptococcus organisms. Collectively, these findings confirmed the participation of C. gattii in the etiopathogenesis of the lesions observed in these dogs and expanded the epidemiological niche of this important mycotic agent to include Southern Brazil. It is noteworthy to mention that previous epidemiological studies have suggested that C. gattii-induced cryptococcosis is more frequently diagnosed in Northern relative to Southern Brazil, so these findings might suggest an expansion of the distribution of this agent within continental Brazil.

Paracoccidioides brasiliensis-associated dermatitis and lymphadenitis in a dog

Paracoccidioidomycosis (PCM) is an endemic disease of humans from Latin America that is caused by Paracoccidioides brasiliensis and P. lutzii, with most cases of PCM in domestic animals being associated with P. brasiliensis. This study presents the clinical, cytological, mycological, serological, and molecular findings associated with P. brasiliensis in a dog from Southern Brazil. Fine needle biopsies were collected from the skin and several lymph nodes of a 5-year-old female Labrador dog that had enlargement of most superficial lymph nodes. Cytology of the skin and lymph nodes revealed pyogranulomatous dermatitis and lymphadenitis associated with fine-necked, budding fungal structures consistent with the Paracoccidioides genus of organisms; mycological culture derived from the lymph node aspirate demonstrated similar budding structures. Serological assays using exoantigens obtained from the fungal culture demonstrated that the fungal organisms derived from the lymph node were antigenically similar to P. brasiliensis by immunodiffusion and Western blot. A PCR assay, using the fungal culture as input, amplified a partial segment of the internal transcribed spacer 1 and 2 regions of P. brasiliensis; direct sequencing and phylogenetic analyses confirmed the PCR product as P. brasiliensis. The combined cytological, mycological, serological, and molecular findings confirmed a diagnosis of fungal dermatitis and lymphadenitis due to P. brasiliensis in this dog. This case represents the third description of clinical PCM in dogs and the first confirmation of mycotic dermatitis associated with P. brasiliensis in this species. The participation of dogs in the possible dissemination of PCM is reviewed, and it is proposed that dogs are probable accidental hosts in the epidemiological cycle associated with P. brasiliensis.

Fatores de risco associados à mastite bovina causada por Prototheca zopfii

This research had as objective the study of risk factors associated with bovine mastitis caused by Prototheca zopfii. Thirteen dairy herds in Parana and Sao Paulo states were analyzed and selected according to the following criteria: previous confirmation of Prototheca spp. mastitis cases, screening of Prototheca spp. in bulk tanks and milk cans, and herds with somatic cells count over 5x105cel mL-1. The samples collected consisted of: milk, water, soil, manure and swabs of teat cup rubbers. Prototheca spp. was isolated from mammary quarters with clinical and subclinical mastitis of milk samples in one herd and from the environment and cows in four herds. Out of 383 cows examined, Prototheca spp. was isolated in 20 (5.2%) cows with mastitis, and 18 of them were characterized as P. zopfii. In four herds when Prototheca spp. was identified from mammary quarters and environment the agent was isolated from the following samples: water in the waterers, puddled water in the stalls and the milking parlour, supply, sewage, cow pen and pasture soil, teat cup rubbers and manure from calves and swines. The risk factors associated with P. zopfii mastitis consisted of: pasture system, pasture and silage feeding, use of milking machine in stalls, cow pen without fresh feed after milking, raising of swines near bovine housing, existence of dogs, cats and rodents, absence of teats hygienization with water, use of pre-immersion devices with return and without change of antiseptic, calves fed with milk of clinical mastitis cases and the Holstein breed.

Streptococcus iniae: an unusual important pathogen fish in Brazil.

The current paper contains a report on the isolation of Streptococcus iniae in Nile tilapia (Oreochromis niloticus) in Brazil and South America. S. iniae is an important pathogen often associated with significant losses in fish production. It is also known for causing invasive infections in humans. An outbreak of infections characterized by exophthalmos, erratic swimming, ascites and melanosis occurred in intensive fish farming. Samples of kidney, brain and liver of the fish seeded into the culture medium yielded beta-hemolytic, gram-positive cocci isolate from the colonies. The partial sequencing of the 16S ribosomal gene was performed to identify the isolate. The sequence obtained showed 99% identity to 16S sequences of S. iniae present in the Genbank. A phylogenetic analysis was accomplished to confirm the species. Although S. iniae is frequently present in almost all continents, this work disclosed the second case of isolation of this pathogen in farmed fish both in Brazil and South America.

Pneumonia due to Talaromyces marneffei in a Dog from Southern Brazil with Concomitant Canine Distemper Virus Infection

The pathological and molecular findings associated with Talaromyces marneffei-induced pneumonia with concomitant infection by canine distemper virus (CDV) are described in a dog. The principal pathological alteration occurred in the lungs. Histopathology confirmed multifocal granulomatous pneumonia associated with numerous intralesional and intracellular septate fission cells consistent with T. marneffei. A molecular assay designed to amplify a partial fragment of the 18S rRNA gene of T. marneffei provided positive results from two fungal cultures derived from the lung. Sequencing and phylogenetic analyses confirmed the results of polymerase chain reaction (PCR). Furthermore, antigens of the CDV N protein were identified within the bronchial epithelium by immunohistochemistry and a PCR assay amplified the CDV N gene from hepatic and pulmonary fragments. Collectively, the pathological and molecular techniques confirmed a diagnosis of T. marneffei-induced pneumonia with concomitant infection by CDV. These findings represent the first description of pulmonary penicilliosis in the dog and extend the geographical niche of this emerging infectious pathogen. In this case, infection by CDV may have induced immunosuppression, which facilitated the development of pulmonary penicilliosis.

Pyogenic Ventriculitis and Ventricular Empyema associated with Staphylococcus pseudintermedius in a Puppy

A 40-day-old male, blue heeler puppy with hindlimb ataxia, nystagmus, apathy, motor incoordination and hyperaesthesia of the forelimbs died 3 days after the onset of clinical signs. Significant gross findings included cerebellar herniation, cerebral oedema and dilation of the third and right lateral cerebral ventricles due to the accumulation of a purulent exudate. Histopathological examination revealed pyogenic ventriculitis and purulent meningoencephalitis. Pure colonies of a coagulase-positive Staphylococcus were isolated from the purulent cerebral exudate. A polymerase chain reaction assay that targeted the 16S rRNA gene of bacteria amplified the desired product from bacterial colonies. Direct sequencing revealed the organism to be Staphylococcus pseudintermedius. Phylogenetic analyses showed that the organism was antigenically similar to Staphylococcus intermedius and Staphylococcus delphini, being part of the S. intermedius group of bacteria. These findings confirmed the participation of S. pseudintermedius in the development of the pathological manifestations and lesions observed in this puppy.