Prevalence and risk factors for Leptospira spp. in dairy cattle in western Paraná, Brazil

Leptospirosis is caused by spirochete bacteria of the genus Leptospira and is considered the most widespread zoonosis worldwide. It is an important agent that causes animal production to decrease. In cattle, it affects especially the reproductive tract. The objective of this study was to determine the seroprevalence of Leptospira spp., molecularly detect the bacteria in tissues of aborted fetuses, and identify the main risk factors associated with infection in cattle in dairy farms in Western Paraná. For this purpose, 600 bovine serum samples from 60 properties and 17 bovine fetuses from nine properties were collected. Data about the properties were also collected through an epidemiological questionnaire to assess the main risk factors associated with Leptospira spp. infection. The serum samples were analyzed using microscopic agglutination test (MAT), and the fetal tissues using nested polymerase chain reaction (nested PCR). Seroprevalence of Leptospira spp. in dairy cattle in Western Paraná was 39.83% (239/600) and none of the analyzed fetuses were positive for Leptospira spp. The main risk factors identified are related to the production system, reproductive management, and the presence of dogs on the property. Leptospira spp. infection is widely spread in the cattle population in Western Paraná

Functional annotation and distribution overview of RNA families in 27 Streptococcus agalactiae genomes

Background: Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram-positive bacterium that colonizes the gastrointestinal and genitourinary tract of humans. This bacterium has also been isolated from various animals, such as fish and cattle. Non-coding RNAs (ncRNAs) can act as regulators of gene expression in bacteria, such as Streptococcus pneumoniae and Streptococcus pyogenes. However, little is known about the genomic distribution of ncRNAs and RNA families in S. agalactiae. Results: Comparative genome analysis of 27 S. agalactiae strains showed more than 5 thousand genomic regions identified and classified as Core, Exclusive, and Shared genome sequences. We identified 27 to 89 RNA families per genome distributed over these regions, from these, 25 were in Core regions while Shared and Exclusive regions showed variations amongst strains. We propose that the amount and type of ncRNA present in each genome can provide a pattern to contribute in the identification of the clonal types. Conclusions: The identification of RNA families provides an insight over ncRNAs, sRNAs and ribozymes function, that can be further explored as targets for antibiotic development or studied in gene regulation of cellular processes. RNA families could be considered as markers to determine infection capabilities of different strains. Lastly, pan-genome analysis of GBS including the full range of functional transcripts provides a broader approach in the understanding of this pathogen.Fil: Wolf, Ivan Rodrigo. Universidade Estadual de Londrina; BrasilFil: Paschoal, Alexandre Rossi. Universidade Federal do Paraná; BrasilFil: Quiroga, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Domingues, Douglas Silva. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: de Souza, Rogério Fernandes. Universidade Estadual de Londrina; BrasilFil: Pretto Giordano, Lucienne Garcia. Universidade Estadual de Londrina; BrasilFil: Vilas Boas, Laurival Antonio. Universidade Estadual de Londrina; Brasi

PYOMETRA IN BITCHES: RELATIONSHIP BETWEEN CLINICAL PROGNOSIS AND LABORATORY DIAGNOSIS

Pyometra is a disease resulting from bacterial interaction with cystic endometrial hyperplasia. The aim of this study was to identify bacteria under aerobic and anaerobic conditions of intrauterine secretion of bitches with pyometra, Systemic Inflammatory Response Syndrome, status of the cervix, peritonitis, serum biochemical measurements and to compare the prognosis of surgically treated bitches. We used 15 bitches with pyometra, undergoing ovariohysterectomy. Blood count and dosage of serum biochemicals were performed preoperatively. Urine was collected by cystocentesis during surgery. After the uterus was removed, intrauterine secretion was aspirated; the samples were grown aerobically and anaerobically, and urine aerobically. The animals were divided into two groups, G1 (hospital discharge until 48 hours) and G2 (discharged after 48 hours or death). Eleven samples of intrauterine content (73.3%) had bacterial growth in aerobic and anaerobic conditions, and Arizona hinshawii was isolated in a sample. Four urine samples (26.6%) had bacterial growth. There were significant differences in status of the cervix, urea, creatinine, alkaline phosphatase between G1 and G2. Although the etiology diversity found in intrauterine cultures was high, the evaluation of azotemia, status of the cervix, and serum alkaline phosphatase dosage proved to be good prognostics markers in female dogs with pyometra.  Keywords: anaerobic; creatinine; cystic endometrial hyperplasia; microbiology; SIRS

PIOMETRAS EM CADELAS: RELAÇÃO ENTRE O PROGNÓSTICO CLÍNICO E O DIAGNÓSTICO LABORATORIAL

Piometra é uma afecção resultante da interação bacteriana com a hiperplasia cística do endométrio. O objetivo deste estudo foi identificar bactérias em condições de aerobiose e anaerobiose da secreção intrauterina de cadelas com piometra, presença da síndrome da resposta inflamatória sistêmica, status da cérvix, peritonite, dosagens bioquímicas séricas e comparar com o prognóstico de cadelas tratadas cirurgicamente. Utilizaram-se 15 cadelas com piometra, submetidas à ovariohisterectomia. Hemogramas e dosagens séricas de ureia, creatinina, FA e ALT foram realizados no pré-operatório imediato. Coletou-se urina por cistocentese. Após a remoção do útero e ovários, a secreção intrauterina foi aspirada; as amostras foram então cultivadas em aerobiose e anaerobiose, as amostras de urina em aerobiose. Os animais foram divididos em dois grupos, G1 (alta hospitalar em até 48 horas) e G2 (alta hospitalar após 48 horas ou óbito). Onze amostras de conteúdo intrauterino (73,3%) apresentaram crescimento bacteriano em aerobiose e anaerobiose e em uma amostra isolou-se Arizona hinshawii. Quatro amostras de urina (26,6%) tiveram crescimento bacteriano. Houve diferença significativa das variáveis status da cérvix, ureia, creatinina e fosfatase alcalina entre G1 e G2. Embora a diversidade etiológica encontrada nas culturas de conteúdo intrauterino tenha sido alta, a avaliação da azotemia, status da cérvix e dosagem de FA sérica mostraram-se bons marcadores prognósticos em cadelas com piometra.  Palavras-chave: Anaerobiose; Creatinina; Hiperplasia Endometrial Cística; Microbiologia; SIRS

Cross-Sectional Study of Leptospira spp. and Brucela abortus in Goat Herds from Paraná State, Brazil

Background: Leptospirosis and brucellosis are zoonosis worldwide distributed that have great economic importance, especially in goats. This study aimed to carry out a cross-sectional study to determine the prevalence of anti-Leptospira spp. and anti-Brucella abortus antibodies and to determine epidemiological variables associated to infection by these ethiological agents in goats.Materials, Methods & Results: Serum samples were collected from 1055 goats of 95 properties distributed in 18 regional centers of Paraná State from April to August of 2010. Collected samples were submitted to microscopic agglutination test (MAT) for the detection of anti-Leptospira spp. antibodies with 22 reference serovars: Australis, Bratislava, Autumnalis, Butembo, Castellonis, Bataviae, Canicola, Fortbragg, Whitcombi, Cynopteri, Grippotyphosa, Hebdomadis, Copenhageni, Icterohaemorrhagiae, Panama, Pomona, Pyrogenes, Hardjo, Wolffi, Shermani, Tarassovi and Londrina 1 (L1). The samples were also submitted to buffered acidified plate antigen test (BAPA) and 2-mercaptoethanol test (2-ME) to detect anti-Brucella abortus antibodies. The results obtained from epidemiological and serological study were analysed by Epi Info 3.5.3. We observed 9.9% (104/1055) of goats reagents in at least one serovar of Leptospira spp. and no reagent animal to Brucella abortus. The variables that were statistically significant with leptospirosis were: production type meat, sewer destination in dry sump, frequent abortion, shared use of pastures, slaughter of animals on the property, the presence of pigs and wild animals such as deer and capybaras on the property.Discussion: This was the first study about antibodies prevalence against Leptospira spp. and Brucella abortus in goat herds from Paraná State. The presence of both animal species were statiscally associated to risk of leptospirosis. It is necessary avoid the acess of these animals in herds, decreasing the dissemination of the agent. The production type meat is a form of creation in which the animals have no care of zookeepers and continuous veterinary care. The final destination of sewage was statistically associated to risk of leptospirosis in goats and this is a big problem in rural properties, requiring greater attention of authorities for proper disposal of this wastewater, reducing water and food contamination. Among the variables related to management, the herds that used rented pasture or shared pastures also were statistically associated to risk of leptospirosis in goats; with the sharing of pastures a greater flow of animals transiting in the pickets occurs, allowing not only flocks of goats, but other species also, to use the same pastures and become more exposed to certain diseases. The slaughter of pigs, sheep, cattle and goats on the property is a problem for animal and human health due to lack of sanitary inspection. Abortion is one of the main clinical signs of leptospirosis in farm animals, particularly goats. The knowledge of these results will be useful to develop control programs for the disease in Paraná State herds.  The absence of circulating anti-Brucella abortus antibodies in goat herds has also been reported in other Brazilian States

Serological diagnosis and molecular characterization of Leptospira spp. in the blood and urine of bovine females from refrigerated slaughterhouses

Leptospirosis is an important socioeconomic disease in humans, as well as in domestic and wild animals, being caused by Leptospira spp. Bovine animals are considered reservoirs of this disease, because they intermittently disseminate the bacteria into the environment through their urine. In this way, the cattle an important source of Leptospira infection. The objective of this study was to detect Leptospira spp. antibodies and DNA in bovine females from two refrigerated slaughterhouses in the microregion of Umuarama, Paraná, Brazil. In particular, blood and urine samples from 52 crossbred bovine females older than 36 months from the two slaughterhouses were used. The microscopic agglutination test (MAT) was used to detect leptospiral antibodies, and the polymerase chain reaction (PCR) and subsequent sequencing were used to detect Leptospira DNA. The MAT yielded 22 (42.3%) serum samples considered reagent, while the nested PCR test resulted in one amplified sample (1.9%) of 289 bp. This single sample was then amplified again using primers for the SecY gene (549 bp). Sequencing of this gene characterized the bacteria as L. borgpetersenii that were similar to the serovar Hardjo of the genotype Hardjobovis. This is the first molecular confirmation of Hardjobovis-like L. borgpetersenii in the urine of crossbred bovine females older than 36 months from slaughterhouses in the microregion of Umuarama. This study’s results show that it is important to combine serological and molecular diagnosis in the detection of Leptospira spp. Therefore, both methods were used to improve our understanding of the epidemiology of this disease in bovine animals from the microregion of Umuarama. In addition, the analysis informed the subsequent adoption of preventive measures and educational One Health actions to prevent economic losses related to the herd, as well as social losses related to workers and the environment

Effectiveness of homologous inactivated Streptococcus agalactiae vaccine by immersion bath in Nile tilapia (Oreochromis niloticus)<br>Avaliação da eficácia de vacina autóctone de Streptococcus agalactiae inativado aplicada por banho de imersão em tilápia do Nilo (Oreochromis niloticus)

Tilapia is the most important group between cultivated fish. Intensive handling systems are characterized by high rate of stocking of fish making them susceptible to infectious diseases. The streptococcosis is one of the diseases that cause great economic losses. The objective of the study was to test a vaccine administered by immersion bath (b.i.) against Streptococcus agalactiae and challenge with homologous strain. For this study, 421 Nile tilapia (Oreochromis niloticus) with an average weight of 38.38 grams were used. They were divided into two treatments (T1 and T2) and a control group. At T1, the fish were vaccinated with one dose of vaccine by b.i., with a concentration of 5.4 x 108 CFU mL-1. The T2 received two doses of vaccine by b.i., with an interval of 25 days. The control group received a bath of ultrapure water. The fish were challenged by intraperitoneal (i.p.) with a concentration of 3.0 x 108 CFU mL-1 43 days after the first or second dose of vaccine, according to the treatment. The fish were monitored for 16 days after challenge. Compared with the control group results were obtained for T1: p = 0.0805, RR = 0.79 (95% CI: 0.61 – 1.01) and RPS = 21%. For T2 were: p = 0.0296, RR = 0.74 (95% CI 0.56 to 0.96) and RPS = 26%. Mortality after challenge was 57 fish (40.71%) in T1, 51 (38.06%) at T2, and 76 (51.7%) in the control group. There was no significant difference between T1 and T2, p = 0.7445. This result shows that the vaccine tested by b.i. with one and two doses had low efficacy in vaccination of tilapia.O grupo das tilápias é um dos mais importantes entre os peixes cultivados. O sistema de cultivo mais utilizado é o intensivo, caracterizado por alta taxa de estocagem de peixes tornando-os susceptíveis às doenças infecciosas. A estreptococose é uma das enfermidades que causam grandes prejuízos econômicos. O objetivo deste trabalho foi testar uma vacina de Streptococcus agalactiae inativado aplicada por banho de imersão (b.i) e desafio com cepa homóloga. Foram utilizados 421 tilapia do Nilo (Oreochromis niloticus) com peso médio de 38,38 gramas, distribuídas em dois tratamentos (T1 e T2) e um grupo controle. No T1, os peixes foram vacinados com uma dose da vacina por b.i., com concentração de 5,4 x 108 UFC mL-1. O T2 recebeu duas doses da mesma vacina por b.i., com intervalo de 25 dias. O grupo controle recebeu água ultrapura por b.i. Os peixes foram desafiados por via intraperitoneal (i.p.), com concentração de 3,0 x 108 UFC mL-1 43 dias após a primeira ou segunda dose da vacina, de acordo com o tratamento. Os peixes dos tratamentos e controle foram monitorados durante 16 dias após o desafio. Comparativamente ao grupo controle os resultados obtidos para o T1 foram: p = 0,0805, RR = 0,79 (IC 95%: 0,61 – 1,01) e RPS = 21%. Para o T2 foram: p = 0,0296, RR = 0,74 (IC 95%: 0,56 – 0,96) e RPS = 26%. A mortalidade após o desafio foi de 57 peixes (40,71%) no T1, 51 (38,06%) no T2, e 76 (51,7%) no grupo controle. Não houve diferença significativa entre T1 e T2 e p=0,7445. Este resultado permite concluir que a vacina testada por b.i. com uma e duas doses apresentou baixa eficácia na imunização das tilápias

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 10(6) to 1.5 x 10(8) 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 10(6) CFU/fish) to 90.0% in group 1 (infective dose 1.5 x 10(8) 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 10(6) 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