Campilobacteriose genital bovina : interações microbianas e possíveis efeitos na fertilidade bovina

A Campilobacteriose Genital Bovina (CGB) é uma doença venérea, que causa prejuízos econômicos para o setor pecuário, cujo agente etiológico é o Campylobacter fetus subsp. venerealis (Cfv). Essa bactéria é fastidiosa e microaerófila, o que dificulta o transporte de amostras biológicas, o cultivo microbiológico e a identificação bacteriana. Além disso, existe uma lacuna no entendimento da interação microbiana das comunidades bacteriana e fúngica cervicovaginal, em casos de colonização por Cfv e outros agentes infecciosos carreados por touros, que causam problemas reprodutivos nas fêmeas bovinas. Portanto, esta tese objetivou: i) avaliar a viabilidade do congelamento de muco prepucial de touros para identificação de Cfv por ensaio de PCR; ii) screening molecular dos principais agentes infecciosos (Cfv, M. bovis, M. bovigenitalium, U. diversum e T. foetus) careados por touros e vinculados à infertilidade; iii) determinar a diversidade da comunidade fúngica residente na cérvix de vacas, conforme a ordem de paridade (multíparas e nulíparas), e iv) determinar a diversidade bacteriana residente na cérvix de vacas com CGB. Identificamos que os mucos prepuciais de touros podem permanecer congelados a - 20 °C por 10 dias previamente ao diagnóstico molecular de Cfv, sem prejuízo na sensibilidade do método. O rastreio molecular dos agentes infecciosos reprodutivos carreados por touros de rebanhos com baixas taxas reprodutivas evidenciou que 159/210 touros foram positivos para a presença de pelo menos um dos agentes investigados, sendo Cfv, M. bovis, U. diversum e M. bovigenitalium os mais comumente observados. Os resultados dos sequenciamentos da região ITS sugerem que a comunidade fúngica presente na cérvix sofre significativas mudanças nas vacas com infertilidade, bem como nas vacas multíparas. Ademais, o gênero Candida parece estar relacionado aos casos de infertilidade em fêmeas bovinas. O estudo da comunidade bacteriana cervicovaginal bovina evidenciou que a microbiota desse sítio não sofre alterações pela presença de Cfv. O perfil da microbiota bacteriana está intimamente relacionado ao fato do retorno ao cio na última estação, no qual a Cfv pode interagir e se beneficiar da microbiota presente, mas sem causar alterações diretas nela. Em resumo, esta tese caracteriza a microbiota cervicovaginal de vacas com CGB, permitindo o conhecimento sobre o perfil da interação microbiana no trato reprodutivo de fêmeas bovinas. Os resultados permitirão o aprimoramento do desenvolvimento tecnológico de medidas de controle e redução de perdas reprodutivas na bovinocultura.Bovine Genital Campylobacteriosis (BGC) is a venereal disease that causes economic losses to livestock, whose etiological agent is Campylobacter fetus subsp. venerealis (Cfv). This bacterium is demanding and microaerophilic, making it difficult to transport biological samples, microbiological culture, and bacterial identification. There is a gap in understanding the microbial interaction of bacterial and fungal cervicovaginal communities in cases of colonization by Cfv and other infectious agents carried by bulls, which cause reproductive problems in female cattle. Therefore, this thesis aimed to i) evaluate the viability of freezing the preputial mucus of bulls for identification of Cfv by PCR assay; ii) molecular screening of the main infectious agents (Cfv, M. bovis, M. bovigenitalium, U. diversum, and T. foetus) required by bulls and linked to infertility; iii) determine the diversity of the fungal community residing in cows’ cervix, according to the order of parity (multiparous and nulliparous), and iv) determine the bacterial diversity residing in cows’ cervix with BGC. We identified that the preputial mucus of bulls can remain frozen at -20 °C for 10 days before the molecular diagnosis of Cfv without impairing the sensitivity of the method. Molecular screening of reproductive infectious agents carried by bulls of herds with low reproductive rates showed that 159 out of 210 bulls were positive for the presence of at least one of the investigated agents. Cfv, M. bovis, U. diversum, and M. bovigenitalium were the most common detected agents. The results of sequencing of the ITS region suggest that the fungal community present in the cervix undergoes significant changes in cows with infertility, as well as in multiparous cows. In addition, the genus Candida seems to be related to cases of infertility in female cattle. The study of the bovine cervicovaginal bacterial community showed that the microbiota of this site did not change due to the presence of Cfv. The profile of the bacterial microbiota is closely related to the return to estrus in the last season, in which the Cfv can interact and benefit from the microbiota present without causing direct changes in it. This thesis characterizes the cervicovaginal microbiota of cows with BGC, allowing knowledge about the profile of microbial interaction in the reproductive tract of female cattle. The results will allow the improvement of technological development in control measures and reduction of reproductive losses in cattle

Diagnóstico e epidemiologia molecular de reovírus aviário no Brasil

Reovírus aviários (ARV, avian orthoreovirus) pertencem ao gênero Orthoreovirus, família Reoviridae, e podem infectar aves de diferentes espécies em todo o mundo. A artrite viral em galinhas (Gallus gallus) pode ser causada pelo ARV e representa um problema sanitário e econômico, provocando perdas de produtividade nos lotes em produção e até de industrialização do produto final para consumo. O presente estudo objetivou revisar a filogenia e a classificação de ARVs detectadas em galinhas em todo mundo e avaliar a disseminação das principais linhagens presentes no Brasil. Cem amostras foram obtidas de galinhas com suspeita de ARV de granjas de quatro importantes estados produtores de frango do Brasil (RS, SP, PR e SC). Dezessete amostras foram selecionadas para representar todo o banco, cultivadas em células, submetidas à detecção de RNA por RT-PCR do segmento M e genotipadas pelo sequenciamento do gene σC do segmento S do genoma de ARV. Em paralelo, foi construído um dataset de sequências de nucleotídeos do gene σC disponíveis no Genbank para classificação de ARVs circulantes no mundo e identificação taxonômica dos isolados brasileiros sequenciados. A análise filogenética foi realizada pelo método de máxima verossimilhança e o estudo da história evolutiva das linhagens presentes no Brasil por inferência bayesiana. Os dezessete isolados de ARV apresentaram resultado positivo na RT-PCR, sendo sequenciados e incluídos na análise filogenética. Com base na topologia da árvore, suporte filogenético e identidade de nucleotídeos, a análise do dataset com 468 sequências permitiu uma nova classificação taxonômica em cinco principais linhagens de ARV (I a V), muitas das quais com duas ou mais sub-linhagens (I vacinal, Ia, Ib, IIa, IIb, IIc, IVa e IVb). As amostras brasileiras desse estudo foram identificadas como Ib (n= 11), IIb (n= 2), III (n= 1) e V (n= 3); todas associadas a casos de tenossinovite (com ou sem condenação de carcaças de frango em frigoríficos) e/ou síndrome de má absorção em lotes de aves de produção industrial. A única sub-linhagem que apresentou sinal adequado para estudo temporal foi Ib, sendo demonstrado que esta surgiu no mundo em 1968 (1953 a 1982, intervalo de maior densidade posterior [HPD] de 95%) com um tamanho populacional efetivo e constante ao longo do tempo, já no Brasil a introdução ocorreu em 2010 (2007 a 2012, [HPD] de 95%).Avian reoviruses (ARVs) belong to the genus Orthoreovirus, family Reoviridae, and can infect birds of different species worldwide. Viral arthritis in chickens (Gallus gallus) could be caused by ARV. It is an important health and economic problem in the poultry industry, causing high losses in the productivity of the flocks as well as in the chicken processing in the slaughterhouses. The present study aimed to review the phylogeny and classification of ARVs detected in poultry flocks worldwide and to evaluate the dissemination of the lineages occurring in Brazil. One hundred samples were obtained from birds with suspected ARV from the four main Brazilian poultry-producing states (SC, RS, PR and SP). Seventeen samples were selected to represent all and cultured in cells. All of them were analyzed to detect ARV RNA by RT-PCR based on the M segment and genotyped by RT-PCR and sequencing of σC gene. In parallel, a dataset of gene σC nucleotide sequences available from Genbank was organized to classify circulating ARVs in the world and taxonomic identification of the Brazilian sequences. Phylogenetic analysis was performed by the maximum likelihood method and the study of the evolutionary history of the lineages present in Brazil by Bayesian inference. All 17 ARV isolates presented positive results in RT-PCR and could be genotyped to be included in the phylogenetic analysis. Based on the tree topology, phylogenetic support and nucleotide identity, the overall analysis of a dataset with 468 sequences allowed a new taxonomic classification in five main ARV strains (I to V), many of them with two or more sublineages (I vacinne, Ia, Ib, IIa, IIb, IIc, IVa e IVb ). The results showed the occurrence of the lineages Ib (n = 11), IIb (n = 2), III (n = 1) and V (n = 3) in Brazil; all of them associated with clinical cases of tenosynovitis (with or without condemnation of chicken carcasses in the slaughterhouses) or malabsorption syndrome in industrial poultry flocks. The only sublineage that presented temporal signal was Ib. This lineage appeared in the world in 1968 (1953 to 1982, 95% higher posterior density range [HPD]) with an effective and constant population size over time. In Brazil, it was introduced in 2010 (2007 to 2012, [HPD] of 95%)

Socio-emotional and cognitive development in intrauterine growth restricted (IUGR) and typical development infants: Early interactive patterns and underlying neural correlates. Rationale and methods of the study

Intrauterine growth restriction (IUGR) is defined as a fetal growth retardation, resulting in an estimated fetal weight less than the 10th centile for gestational age. IUGR developing brain is affected by the atypical fetal growth, presenting altered structure and connectivity and increased risk for neurodevelopmental impairments. Behaviorally, IUGR infants show reduced responsiveness and engagement with human faces during mother-child exchanges. The neural mechanisms of these patterns of interactions remain unexplored, as well as their potential role in shaping socio-cognitive trajectories of development. Aim of this research project will be to longitudinally investigate mother-infant interactions and infant's event-related potential (ERP) components of face processing (infant N170, P400, Negative central) in 4 and 9 months IUGR as potential early markers of expected atypical cognitive and behavioral outcomes observed at 12 months. Thirty IUGR participants will be recruited after receiving the in utero diagnosis (>28th gestational week). Thirty healthy infants will be enrolled as the control group. Maternal environment will be assessed via Emotional Availability Scales (EASs), with child responsiveness and maternal sensitivity as variables of interest. Infants' scalp-recorded cortical activity in response to social and non-social stimuli will be investigated using a high-density EEG system (EGI Geodesic system). Neurodevelopment will be measured at 12 months of child's life, using Bayley Scales for Infant Development (BSID), while the possible presence of emotional-behavioral problems will be rated via Child Behavior Checklist (CBCL). We expect that being IUGR significantly affects cognitive and behavioral outcomes, through mediation effects of both infants' neural and behavioral capacity to respond to social stimuli. Indeed, we expect an altered response to social stimuli in IUGR infants, resulting in smaller ERP components amplitude in response to human faces compared to healthy matched peers. A significant association between neural response to social stimuli and infants' responsiveness to maternal stimulation during interactions is expected, with impoverished performances on the interactive domain in IUGR, compared to healthy peers. This study will enhance understanding on neural mechanisms underpinning the interactive patterns sustaining socio-cognitive development in IUGR and healthy infants. The study will help in clarifying the role of postnatal environment in buffering the vulnerability experienced by children delayed in their fetal growth

Mechanism and clinical evidence of immunotherapy in allergic rhinitis

Allergic rhinitis is a common upper airway disease caused by hypersensitivity to various aeroallergens. It causes increased inflammation throughout the body and may be complicated by other otolaryngological pathologies such as chronic hyperplastic eosinophilic sinusitis, nasal polyposis, and serous otitis media. Allergic rhinitis is an IgE-mediated disease and immunotherapy can be a possible approach for patients to limit the use of antihistamines and corticosteroids. There is evidence that allergen immunotherapy can prevent the development of new sensitizations and reduce the risk of later development of asthma in patients with allergic rhinitis. However, some patients do not benefit from this approach and the efficacy of immunotherapy in reducing the severity and relapse of symptoms is still a matter of debate. This review highlights new aspects of allergic rhinitis with a particular focus on the impact of sexual dimorphism on the disease manifestation and efficacy to the allergen specific immunotherapy

Pet pyometra : correlating bacteria pathogenicity to endometrial histological changes

Pyometra is a life-threatening infectious disease that frequently affects bitches and queens. Although histopathological patterns of pyometra have been extensively explored, the microbiological aspects, such as bacteria pathogenicity, have not been correlated to microscopy endometrial lesions so far. In this study, these two pathological aspects of pyometra were analysed and correlated. Uterus fragments and intrauterine content samples were collected from pets diagnosed with pyometra (30) and submitted to histopathology analysis and bacterial culture, respectively. The degree of endometrial histopathological lesions in pyometra cases were classified as mild, moderate and severe. Thirty different bacteria isolates were identified from intrauterine content culture. Escherichia coli(E. coli) was pure isolated in 57.7% and highly related to severe endometrial lesions. Immunohistochemistry assay revealed the adhesion and invasion of this bacteria agent to the injured endometrium. Virulence aspects of these E. coli strains were explored, demonstrating biofilm formation ability and a set of virulence genes in most isolates. These results support the adaptive genetic and phenotypic advantages of E. coli for uterus infection, and justify the high frequency of this agent involved in pyometra cases

Aerossacolitis and pneumonia in an indian peafowl caused by Lactobacillus agilis

Background: The peafowl is an ornamental bird that has the habit of eating directly from the earthy soil, which makes this bird more susceptible to endoparasites. One important endoparasite is Eucoleus contortus, which leads to inflammatory processes that alter the local microbiota, potentializing disease. By the other way, a member of the bird’s microbiota there is the genus Lactobacillus, but when occurs some imbalance, these bacteria can overgrowth and even cause some infection. This report describes the pathological and microbiological findings of chronic necrotizing pneumonia and aerossacolitis caused by Lactobacillus agilis in a peafowl, associated with parasitism by E. contortus. Case: A peafowl (Pavo cristatus), adult, male, who lived on a farm with contact with other species of animal, was submitted to post-mortem examination due to sudden death. This animal lived in an extensive system on the property and was the only one of its species. During the gross evaluation, the air sacs were filled with solid yellowish crumbly material. The same material was observed forming well-defined nodules that occupied > 50% of the lung parenchyma. Histological analysis showed multiple parabronchi dilated and filled with caseous necrosis, characterized by abundant cellular debris and fibrin deposition. These areas were surrounded by the proliferation of fibrous connective tissue and inflammatory infiltrate of macrophages, giant cells, lymphocytes, and plasma cells. The air sacs parenchyma showed fibrin deposition and mixed inflammatory infiltrate. Multiple gram-positive bacilli were observed within the caseous foci in Gram-stained slides. In the crop and esophageal mucosa, cross-sections of filiform nematodes morphologically compatible with E. contortus were associated with chronic inflammatory infiltrate and epidermal hyperkeratosis. A lung section was submitted to Gram-Brown-Hopps and Ziehl-Neelsen (ZN) stains for bacterial investigation, and Grocott’s methenamine silver (GMS) stain for fungal investigation. Short gram-positive bacilli bacteria are observed within the caseous foci in Gram-stained slides. By the other way, no agents were identified on the ZN and GMS stains. Following the analysis, lung fragments were cultivated at aerobic and microaerophilic conditions on sheep blood agar and McConkey agar. All the microbial cultures were incubated at 37°C to 48 h. Pure culture, in microaerophilic condition, of Gram-positive bacilli, was observed. The isolated bacterium was identified by MALDI-TOF MS as L. agilis. Discussion: Although uncommon, L. agilis was the single bacterium identified and therefore, associated as a primary cause of necrotic pneumonia and aerossacolitis in the studied peafowl. The presence of E. contortus could induce the aspiration of regurgitated of little amount of material from the gastrointestinal tract with sufficient bacterial load to initiate an infection, but not enough to smother the animal. The gradual aspiration can induce a chronic inflammatory condition. Infections by bacteria from the host microbiota have been observed in animals and humans with immunodeficiency. In summary, both the parasitosis and the inflammation could be resulted by the parasite and the aspiration of gastric product, which probably interfered in the immune response and allowed the overgrowth of L. agilis. In the current case report, based on macroscopic, microscopic and bacteriological results, we have provided insights to understand how the parasitosis made possible a pneumonia from a bacterium from the host’s microbiota. Finally, to the best of our knowledge, this is the first report of Lactobacillus agilis as causal agent of fatal necrotic pneumonia and aerossacolitis in peafowl

Aerossacolitis and Pneumonia in an Indian Peafowl Caused by Lactobacillus agilis

Background: The peafowl is an ornamental bird that has the habit of eating directly from the earthy soil, which makes this bird more susceptible to endoparasites. One important endoparasite is Eucoleus contortus, which leads to inflammatory processes that alter the local microbiota, potentializing disease. By the other way, a member of the bird’s microbiota there is the genus Lactobacillus, but when occurs some imbalance, these bacteria can overgrowth and even cause some infection. This report describes the pathological and microbiological findings of chronic necrotizing pneumonia and aerossacolitis caused by Lactobacillus agilis in a peafowl, associated with parasitism by E. contortus. Case: A peafowl (Pavo cristatus), adult, male, who lived on a farm with contact with other species of animal, was submitted to post-mortem examination due to sudden death. This animal lived in an extensive system on the property and was the only one of its species. During the gross evaluation, the air sacs were filled with solid yellowish crumbly material. The same material was observed forming well-defined nodules that occupied > 50% of the lung parenchyma. Histological analysis showed multiple parabronchi dilated and filled with caseous necrosis, characterized by abundant cellular debris and fibrin deposition. These areas were surrounded by the proliferation of fibrous connective tissue and inflammatory infiltrate of macrophages, giant cells, lymphocytes, and plasma cells. The air sacs parenchyma showed fibrin deposition and mixed inflammatory infiltrate. Multiple gram-positive bacilli were observed within the caseous foci in Gram-stained slides. In the crop and esophageal mucosa, cross-sections of filiform nematodes morphologically compatible with E. contortus were associated with chronic inflammatory infiltrate and epidermal hyperkeratosis. A lung section was submitted to Gram-Brown-Hopps and Ziehl-Neelsen (ZN) stains for bacterial investigation, and Grocott's methenamine silver (GMS) stain for fungal investigation. Short gram-positive bacilli bacteria are observed within the caseous foci in Gram-stained slides. By the other way, no agents were identified on the ZN and GMS stains. Following the analysis, lung fragments were cultivated at aerobic and microaerophilic conditions on sheep blood agar and McConkey agar. All the microbial cultures were incubated at 37°C to 48 h. Pure culture, in microaerophilic condition, of Gram-positive bacilli, was observed. The isolated bacterium was identified by MALDI-TOF MS as L. agilis.Discussion: Although uncommon, L. agilis was the single bacterium identified and therefore, associated as a primary cause of necrotic pneumonia and aerossacolitis in the studied peafowl. The presence of E. contortus could induce the aspiration of regurgitated of little amount of material from the gastrointestinal tract with sufficient bacterial load to initiate an infection, but not enough to smother the animal. The gradual aspiration can induce a chronic inflammatory condition. Infections by bacteria from the host microbiota have been observed in animals and humans with immunodeficiency. In summary, both the parasitosis and the inflammation could be resulted by the parasite and the aspiration of gastric product, which probably interfered in the immune response and allowed the overgrowth of L. agilis. In the current case report, based on macroscopic, microscopic and bacteriological results, we have provided insights to understand how the parasitosis made possible a pneumonia from a bacterium from the host's microbiota. Finally, to the best of our knowledge, this is the first report of L. agilis as causal agent of fatal necrotic pneumonia and aerossacolitis in peafowl.Keywords: respiratory infection, ornamental birds, secondary infection, parasitosis