Gut dysbiosis, inflammation and type 2 diabetes in mice using synthetic gut microbiota from diabetic humans / Disbiose intestinal, inflamação e diabetes tipo 2 em camundongos usando microbiota intestinal sintética de humanos diabéticos

The study was aimed to assess impact of high fat diet (HFD) and synthetic human gut microbiota (GM) combined with HFD and chow diet (CD) in inducing type-2 diabetes (T2D) using mice model. To our knowledge, this is the first study using selected human GM transplantation via culture based method coupled dietary modulation in mice for in vivo establishment of inflammation leading to T2D and gut dysbiosis. Twenty bacteria (T2D1-T2D20) from stool samples of confirmed T2D subjects were found to be morphologically different and subjected to purification on different media both aerobically and anerobically, which revealed seven bacteria more common among 20 isolates on the basis of biochemical characterization. On the basis of 16S rRNA gene sequencing, these seven isolates were identified as Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenes (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). The seven isolates were subsequently used as synthetic gut microbiome (GM) for their role in inducing T2D in mice. Inbred strains of albino mice were divided into four groups and were fed with CD, HFD, GM+HFD and GM+CD. Mice receiving HFD and GM+modified diet (CD/HFD) showed highly significant (P<0.05) increase in weight and blood glucose concentration as well as elevated level of inflammatory cytokines (TNF-α, IL-6, and MCP-1) compared to mice receiving CD only. The 16S rRNA gene sequencing of 11 fecal bacteria obtained from three randomly selected animals from each group revealed gut dysbiosis in animals receiving GM. Bacterial strains including Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) and Lactobacillus gasseri (MT152635) were isolated from mice [...].

O estudo teve como objetivo avaliar o impacto da dieta rica em gordura (HFD) e da microbiota intestinal humana sintética (GM) combinada com HFD e dieta alimentar (CD) na indução de diabetes tipo 2 (T2D) usando modelo de camundongos. Para nosso conhecimento, este é o primeiro estudo usando transplante de GM humano selecionado através do método baseado em cultura acoplada à modulação dietética em camundongos para o estabelecimento in vivo de inflamação que leva a T2D e disbiose intestinal. Vinte bactérias (T2D1-T2D20) de amostras de fezes de indivíduos T2D confirmados verificaram ser morfologicamente diferentes e foram submetidas à purificação em meios diferentes aerobicamente e anaerobicamente, o que revelou sete bactérias mais comuns entre 20 isolados com base na caracterização bioquímica. Com base no sequenciamento do gene 16S rRNA, esses sete isolados foram identificados como Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenides (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). Esses sete isolados foram, posteriormente, usados como microbioma intestinal sintético (GM) por seu papel na indução de T2D em camundongos. Linhagens consanguíneas de camundongos albinos foram divididas em quatro grupos e foram alimentadas com CD, HFD, GM + HFD e GM + CD. Camundongos que receberam a dieta modificada com HFD e GM + (CD / HFD) mostraram um aumento altamente significativo (P < 0,05) no peso e na concentração de glicose no sangue, bem como um nível elevado de citocinas inflamatórias (TNF-α, IL-6 e MCP-1) em comparação com os ratos que receberam apenas CD. O sequenciamento do gene 16S rRNA de 11 bactérias fecais obtidas de três animais selecionados aleatoriamente de cada grupo revelou disbiose intestinal em animais que receberam GM. Cepas bacterianas, incluindo Bacteroides gallinarum (MT152630), Ruminococcus [...].

Gut dysbiosis, inflammation and type 2 diabetes in mice using synthetic gut microbiota from diabetic humans

Abstract The study was aimed to assess impact of high fat diet (HFD) and synthetic human gut microbiota (GM) combined with HFD and chow diet (CD) in inducing type-2 diabetes (T2D) using mice model. To our knowledge, this is the first study using selected human GM transplantation via culture based method coupled dietary modulation in mice for in vivo establishment of inflammation leading to T2D and gut dysbiosis. Twenty bacteria (T2D1-T2D20) from stool samples of confirmed T2D subjects were found to be morphologically different and subjected to purification on different media both aerobically and anerobically, which revealed seven bacteria more common among 20 isolates on the basis of biochemical characterization. On the basis of 16S rRNA gene sequencing, these seven isolates were identified as Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenes (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). The seven isolates were subsequently used as synthetic gut microbiome (GM) for their role in inducing T2D in mice. Inbred strains of albino mice were divided into four groups and were fed with CD, HFD, GM+HFD and GM+CD. Mice receiving HFD and GM+modified diet (CD/HFD) showed highly significant (P 0.05) increase in weight and blood glucose concentration as well as elevated level of inflammatory cytokines (TNF-, IL-6, and MCP-1) compared to mice receiving CD only. The 16S rRNA gene sequencing of 11 fecal bacteria obtained from three randomly selected animals from each group revealed gut dysbiosis in animals receiving GM. Bacterial strains including Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) and Lactobacillus gasseri (MT152635) were isolated from mice treated with GM+modified diet (HFD/CD) compared to strains Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629) which were isolated from mice receiving CD/HFD. In conclusion, these findings suggest that constitution of GM and diet plays significant role in inflammation leading to onset or/and possibly progression of T2D. .

Resumo O estudo teve como objetivo avaliar o impacto da dieta rica em gordura (HFD) e da microbiota intestinal humana sintética (GM) combinada com HFD e dieta alimentar (CD) na indução de diabetes tipo 2 (T2D) usando modelo de camundongos. Para nosso conhecimento, este é o primeiro estudo usando transplante de GM humano selecionado através do método baseado em cultura acoplada à modulação dietética em camundongos para o estabelecimento in vivo de inflamação que leva a T2D e disbiose intestinal. Vinte bactérias (T2D1-T2D20) de amostras de fezes de indivíduos T2D confirmados verificaram ser morfologicamente diferentes e foram submetidas à purificação em meios diferentes aerobicamente e anaerobicamente, o que revelou sete bactérias mais comuns entre 20 isolados com base na caracterização bioquímica. Com base no sequenciamento do gene 16S rRNA, esses sete isolados foram identificados como Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenides (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). Esses sete isolados foram, posteriormente, usados como microbioma intestinal sintético (GM) por seu papel na indução de T2D em camundongos. Linhagens consanguíneas de camundongos albinos foram divididas em quatro grupos e foram alimentadas com CD, HFD, GM + HFD e GM + CD. Camundongos que receberam a dieta modificada com HFD e GM + (CD / HFD) mostraram um aumento altamente significativo (P 0,05) no peso e na concentração de glicose no sangue, bem como um nível elevado de citocinas inflamatórias (TNF-, IL-6 e MCP-1) em comparação com os ratos que receberam apenas CD. O sequenciamento do gene 16S rRNA de 11 bactérias fecais obtidas de três animais selecionados aleatoriamente de cada grupo revelou disbiose intestinal em animais que receberam GM. Cepas bacterianas, incluindo Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) e Lactobacillus Gasseri (MT152635D), foram tratadas com dieta modificada / CD) em comparação com as linhagens Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629), que foram isoladas de camundongos recebendo CD / HFD. Em conclusão, esses resultados sugerem que a constituição de GM e dieta desempenham papel significativo na inflamação levando ao início ou/e possivelmente à progressão de T2D.

Gut dysbiosis, inflammation and type 2 diabetes in mice using synthetic gut microbiota from diabetic humans / Disbiose intestinal, inflamação e diabetes tipo 2 em camundongos usando microbiota intestinal sintética de humanos diabéticos

The study was aimed to assess impact of high fat diet (HFD) and synthetic human gut microbiota (GM) combined with HFD and chow diet (CD) in inducing type-2 diabetes (T2D) using mice model. To our knowledge, this is the first study using selected human GM transplantation via culture based method coupled dietary modulation in mice for in vivo establishment of inflammation leading to T2D and gut dysbiosis. Twenty bacteria (T2D1-T2D20) from stool samples of confirmed T2D subjects were found to be morphologically different and subjected to purification on different media both aerobically and anerobically, which revealed seven bacteria more common among 20 isolates on the basis of biochemical characterization. On the basis of 16S rRNA gene sequencing, these seven isolates were identified as Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenes (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). The seven isolates were subsequently used as synthetic gut microbiome (GM) for their role in inducing T2D in mice. Inbred strains of albino mice were divided into four groups and were fed with CD, HFD, GM+HFD and GM+CD. Mice receiving HFD and GM+modified diet (CD/HFD) showed highly significant (P<0.05) increase in weight and blood glucose concentration as well as elevated level of inflammatory cytokines (TNF-α, IL-6, and MCP-1) compared to mice receiving CD only. The 16S rRNA gene sequencing of 11 fecal bacteria obtained from three randomly selected animals from each group revealed gut dysbiosis in animals receiving GM. Bacterial strains including Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) and Lactobacillus gasseri (MT152635) were isolated from mice [...].(AU)

O estudo teve como objetivo avaliar o impacto da dieta rica em gordura (HFD) e da microbiota intestinal humana sintética (GM) combinada com HFD e dieta alimentar (CD) na indução de diabetes tipo 2 (T2D) usando modelo de camundongos. Para nosso conhecimento, este é o primeiro estudo usando transplante de GM humano selecionado através do método baseado em cultura acoplada à modulação dietética em camundongos para o estabelecimento in vivo de inflamação que leva a T2D e disbiose intestinal. Vinte bactérias (T2D1-T2D20) de amostras de fezes de indivíduos T2D confirmados verificaram ser morfologicamente diferentes e foram submetidas à purificação em meios diferentes aerobicamente e anaerobicamente, o que revelou sete bactérias mais comuns entre 20 isolados com base na caracterização bioquímica. Com base no sequenciamento do gene 16S rRNA, esses sete isolados foram identificados como Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenides (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). Esses sete isolados foram, posteriormente, usados como microbioma intestinal sintético (GM) por seu papel na indução de T2D em camundongos. Linhagens consanguíneas de camundongos albinos foram divididas em quatro grupos e foram alimentadas com CD, HFD, GM + HFD e GM + CD. Camundongos que receberam a dieta modificada com HFD e GM + (CD / HFD) mostraram um aumento altamente significativo (P < 0,05) no peso e na concentração de glicose no sangue, bem como um nível elevado de citocinas inflamatórias (TNF-α, IL-6 e MCP-1) em comparação com os ratos que receberam apenas CD. O sequenciamento do gene 16S rRNA de 11 bactérias fecais obtidas de três animais selecionados aleatoriamente de cada grupo revelou disbiose intestinal em animais que receberam GM. Cepas bacterianas, incluindo Bacteroides gallinarum (MT152630), Ruminococcus [...].(AU)

Gut dysbiosis, inflammation and type 2 diabetes in mice using synthetic gut microbiota from diabetic humans / Disbiose intestinal, inflamação e diabetes tipo 2 em camundongos usando microbiota intestinal sintética de humanos diabéticos

Abstract The study was aimed to assess impact of high fat diet (HFD) and synthetic human gut microbiota (GM) combined with HFD and chow diet (CD) in inducing type-2 diabetes (T2D) using mice model. To our knowledge, this is the first study using selected human GM transplantation via culture based method coupled dietary modulation in mice for in vivo establishment of inflammation leading to T2D and gut dysbiosis. Twenty bacteria (T2D1-T2D20) from stool samples of confirmed T2D subjects were found to be morphologically different and subjected to purification on different media both aerobically and anerobically, which revealed seven bacteria more common among 20 isolates on the basis of biochemical characterization. On the basis of 16S rRNA gene sequencing, these seven isolates were identified as Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenes (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). The seven isolates were subsequently used as synthetic gut microbiome (GM) for their role in inducing T2D in mice. Inbred strains of albino mice were divided into four groups and were fed with CD, HFD, GM+HFD and GM+CD. Mice receiving HFD and GM+modified diet (CD/HFD) showed highly significant (P<0.05) increase in weight and blood glucose concentration as well as elevated level of inflammatory cytokines (TNF-α, IL-6, and MCP-1) compared to mice receiving CD only. The 16S rRNA gene sequencing of 11 fecal bacteria obtained from three randomly selected animals from each group revealed gut dysbiosis in animals receiving GM. Bacterial strains including Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) and Lactobacillus gasseri (MT152635) were isolated from mice treated with GM+modified diet (HFD/CD) compared to strains Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629) which were isolated from mice receiving CD/HFD. In conclusion, these findings suggest that constitution of GM and diet plays significant role in inflammation leading to onset or/and possibly progression of T2D. .

Resumo O estudo teve como objetivo avaliar o impacto da dieta rica em gordura (HFD) e da microbiota intestinal humana sintética (GM) combinada com HFD e dieta alimentar (CD) na indução de diabetes tipo 2 (T2D) usando modelo de camundongos. Para nosso conhecimento, este é o primeiro estudo usando transplante de GM humano selecionado através do método baseado em cultura acoplada à modulação dietética em camundongos para o estabelecimento in vivo de inflamação que leva a T2D e disbiose intestinal. Vinte bactérias (T2D1-T2D20) de amostras de fezes de indivíduos T2D confirmados verificaram ser morfologicamente diferentes e foram submetidas à purificação em meios diferentes aerobicamente e anaerobicamente, o que revelou sete bactérias mais comuns entre 20 isolados com base na caracterização bioquímica. Com base no sequenciamento do gene 16S rRNA, esses sete isolados foram identificados como Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenides (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). Esses sete isolados foram, posteriormente, usados ​​como microbioma intestinal sintético (GM) por seu papel na indução de T2D em camundongos. Linhagens consanguíneas de camundongos albinos foram divididas em quatro grupos e foram alimentadas com CD, HFD, GM + HFD e GM + CD. Camundongos que receberam a dieta modificada com HFD e GM + (CD / HFD) mostraram um aumento altamente significativo (P < 0,05) no peso e na concentração de glicose no sangue, bem como um nível elevado de citocinas inflamatórias (TNF-α, IL-6 e MCP-1) em comparação com os ratos que receberam apenas CD. O sequenciamento do gene 16S rRNA de 11 bactérias fecais obtidas de três animais selecionados aleatoriamente de cada grupo revelou disbiose intestinal em animais que receberam GM. Cepas bacterianas, incluindo Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) e Lactobacillus Gasseri (MT152635D), foram tratadas com dieta modificada / CD) em comparação com as linhagens Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629), que foram isoladas de camundongos recebendo CD / HFD. Em conclusão, esses resultados sugerem que a constituição de GM e dieta desempenham papel significativo na inflamação levando ao início ou/e possivelmente à progressão de T2D.

Microbiota intestinal no excesso de peso e no esqueleto de cães: revisão de literatura / Intestinal microbiota in overweight and on the skeleton of dogs: literature review

A microbiota intestinal realiza a homeostase e a regulação de diversos mecanismos fisiológicos, e está associada a múltiplos órgãos e sistemas. Sabe-se que o desequilíbrio da microbiota, denominado disbiose, acarreta e liberação de metabólitos e fatores inflamatórios, que resulta em quadros de obesidade e enfermidades do sistema esquelético em humanos e roedores. Porém, há poucos estudos sobre essa temática nos cães. Assim sendo, esta revisão de literatura pretende destacar a influência da microbiota intestinal no excesso de peso e no sistema esquelético em cães, com o propósito de colaborar com pesquisas futuras e contribuir para melhoria da conduta clínica dos médicos-veterinários.(AU)

The intestinal microbiota consists of many microorganisms such as bacteria, fungi, protozoans and viruses. It plays an important role in homeostasis and in regulating various physiological mechanisms, being directly and indirectly associated with several organs and systems. It is proven that in humans and mice the imbalance in this medium, known as dysbiosis leads to the release of metabolites and inflammatory factors, which translates into the emergence of diseases such as obesity and skeletal pathology, but there's always a lack of studies on the subject of dogs hence, this literature review aims to analyze the influence of the intestinal microbiota on overweight and skeletal system in dogs to collaborate on future research and contribute to the selection of veterinary resolutions.(AU)

Polioencephalomalacia in sheep - clinical and magnetic resonance imaging findings

Background: Polioencephalomalacia (PEM) is a neurological disease in ruminants, which is characterized by malacia of brain gray matter. Thiamine deficiency and sulfur intoxication are the most common causes of PEM in sheep. Affected animals present signs of cerebrocortical syndrome, including amaurosis, ataxia, head pressing, mental depression, seizures, and opisthotonus. The neurological examination aims to determine the neurolocalization of the lesions and advanced imaging techniques are useful for confirming the affected area(s) in the central nervous system. The aim of this study is to describe clinical features and ante-mortem diagnosis using magnetic resonance imaging (MRI) in a sheep with PEM. Case: A 18-month-old male Dorper sheep from a flock started receiving concentrate 7 days before. According to the owner, no clinical signs of abnormality were observed on the previous morning. However, in the afternoon, the animal became selfisolated and did not follow the flock to the sheepfold. The following day, he was found in recumbency. Physical examination revealed lateral recumbency, rectal temperature 39.5ºC, 52 bpm, 120 bpm, congested mucous membranes, capillary refill time 1 s, ruminal (4/5 min) and intestinal hypomotility. The assessment of the central nervous system revealed a decreased level of consciousness, focal seizures, opisthotonus, and absence of menace response. The following differential diagnoses were listed: PEM, head trauma, focal symmetrical encephalomalacia, bacterial encephalitis, and rabies. Treatment was composed of dexamethasone [0.2 mg/kg - i.v., SID (1st-3rd day), 0.1 mg/kg, i.v., SID (4th-6th day), and 0.05 mg/kg, i.v., SID (7th-9th day)]; mannitol [1 g/kg - i.v. and diazepam 0.4 mg/kg, i.v. single dose at admission]; vitamin B1 [10 mg/kg - i.m., SID], furosemide [1 mg/kg - i.v., SID for 3 days] and sulfadoxine/trimethoprim [30 mg/kg - i.m., SID for 10 days]. After the initial treatment, the patient showed mild clinical improvement; however, the amaurosis was still present. Magnetic resonance imaging of the brain was performed on the 2nd day of hospitalization, showing a symmetrical hypersignal in the parietal and occipital cortices, in the axial and sagittal sequences weighted in T2 and FLAIR. Discussion: This study aimed to describe the clinical signs and MRI findings in a sheep with PEM. In this case, the sudden change to the feed composition probably led to ruminal dysbiosis, inhibition of thiamine-producing microorganisms and proliferation of bacteria that synthesize thiaminase. Thiamine therapy proved to be effective and capable of reverting the clinical signs. The decrease in the level of consciousness, cortical blindness, and opisthotonus are due to alterations in the parietal cortex, in the occipital cortex, and in the cerebellum, respectively, which were demonstrated by hypersignal areas in the MRI. Therefore, the neurolocalization of the lesion based on neurologic examination and the MRI findings were related. The physicochemical and cytological evaluations of the cerebrospinal fluid, and dosage of thiamine and the concentration of hydrogen sulphide in the rumen were not performed. However, the response to thiamine treatment associated with the neurologic examination and MRI findings helped in determining the diagnosis. Additionally, MRI can be used as a useful tool for the ante mortem diagnosis of PEM.

Síndrome cólica em equinos induzida por ingestão de cana de açúcar / Equine colic syndrome induced by the ingestion of sugarcane

Background: Equine colic syndrome comprises numerous conditions associated with abdominal pain in horses. Impaction, a common cause of this manifestation, is strongly related to these animals' diet. Highly fibrous diets such as sugarcane can predispose horses to colic. The clinical condition can be worsened by fermentative processes, which lead to dysbiosis, circulatory disorders and even endotoxemia. The aim of this study was to report 4 cases of colic syndrome among 8 horses that underwent an experiment to adapt them to a sugarcane-based diet, and to correlate the animals' clinical conditions to the forage they ingested. Cases: Eight male castrated Mangalarga Marchador horses, between 5.5 and 7 years old, were subjected to an experiment to test the feasibility of sugarcane as forage. Four of these horses were taken to the Large Animal Veterinary Hospital (HVGA) of the Federal Rural University of Rio de Janeiro for treatment of abdominal signs of discomfort a few days after the exclusive consumption of sugarcane, in a proportion of 1.75% of live weight in dry matter. The animals' symptoms ranged from behavioral signs indicative of pain to changes in vital parameters and structure of the feces, as well as changes revealed by transrectal palpation. Three of the 4 cases presented impaction in the small colon, and 1 of the horses also presented impaction in the right dorsal colon and rostral displacement of the pelvic flexure, with accumulation of contents in the right ventral colon and sternal flexure. Two of the cases were treated medically, while the other 2 required surgical intervention. The clinical condition of all the patients evolved favorably and they were discharged between 2 and 18 days. Discussion: Colic originating in the digestive system is a syndrome strongly associated with management, especially with respect to confinement, nutrition, and parasite control. During the experiment, 4 of the 8 horses fed with sugarcane presented with colic syndrome. The low quality of sugarcane fiber is due to the high degree of lignification of the plant cell wall, which favors accumulation of ingesta. The poor digestibility and sweet taste of this roughage favor increased consumption. Furthermore, its high sucrose content, associated with an increased rate of passage in the small intestine, alters the intestinal microbiome, and hence, the fermentation byproducts and pH of the ingesta. High intestinal content, allied to longer retention times in the colon and activation of the renin-angiotensin-aldosterone system, promote greater dryness of the ingesta, predisposing the occurrence of impactions in the most distal portion of the large intestine. Intestinal distension and mesenteric traction caused by the accumulation of contents and gases trigger pain, which can worsen due to displacement of the large colon. Small colon impaction, which is easily identified by transrectal palpation, evolves gradually and its treatment, both clinical and surgical, tends to have a favorable prognosis. The need for alternative food sources for horses is a growing demand; however, sugarcane as an exclusive roughage has been shown to be unsafe for horses. The low quality of the fiber and the high sucrose content of this forage can alter the digestive physiology of horses through changes in the passage rate, microbiome and motility of digesta, predisposing them to intestinal dysfunction, ingesta compaction and displacement of the large colon.

Salmonelose em bezerros pelo sorovar Dublin no estado do Paraná, Brasil - aspectos clínico-patológicos / Salmonellosis in calves by serovar Dublin in Paraná State, Brazil - clinicopathological aspects

Background: Calf diarrhea remains one of the main diseases affecting the cattle industry. Persistence of this significant problem is associated with the complexity of factors that may be involved (infectious, environmental). An accurate diagnosis is essential for confirming the cause and helping clinicians and cattle producers to apply appropriate strategies in a timely manner. This report describes the histological changes according to the degree of salmonellosis severity, which is a contagious infectious disease caused by Salmonella enterica subsp. enterica, a Gram-negative bacterium, in two beef calves in northern Paraná State, southern Brazil. Cases: Two 90-day-old crossbred Angus and Nellore calves from a beef farm in northern Paraná State were referred to a Veterinary Hospital. Animal 1- developed acute clinical signs (enteritis, ataxia, and muscle rigidity) and died the day after the signs began. Gross findings included heavy and non-collapsed lungs, pulmonary oedema, hepatomegaly, enteritis, and severe diffuse typhlitis. Microscopic analysis revealed severe diffuse necrotic enteritis, typhlitis, severe diffuse interstitial pneumonia, moderate centrilobular hepatic necrosis, mild multifocal nephritis, and severe spleen and lymph node necrosis. Paratyphoid nodules were evidenced on the liver, spleen, and mesenteric lymph nodes. Animal 2- presented apathy, green fibrinous diarrhea, and died three days after the onset of clinical signs. The macroscopic examination showed moderate diffuse enteritis and hepatosplenomegaly. At the microscopic examination, many paratyphoid nodules were observed on the liver, spleen, kidneys, and Peyer's patches, which were associated with intralesional and intravascular bacilli. Additional findings included severe diffuse fibrinous necrotic enteritis with intralesional bacilli, diffuse interstitial pneumonia, multifocal splenic necrosis, lymphoid depletion, and moderate multifocal to coalescent lymphocytic nephritis. Histological Gram staining was performed on selected samples, revealing intralesional Gram-negative bacilli in the liver and intestine. Thus, liver and intestine fragments were used for the microbiological examination. Microbiological culture, isolation and biochemical tests identified the genus Salmonella spp. Then, the colonies were subjected to serological tests for serovar identification, according to ISO/TR 6579-3, which determined the serovar Dublin. After identifying the disease etiological agent, the outbreak was controlled by appropriate antibiotic therapy combined with the correction of sanitary measures. Discussion: Enteritis is a frequent disease in calves, posing a diagnostic challenge in identifying the etiological agent. In the present case, the histological, microbiological, and serological results confirmed a disseminated Salmonella spp. infection. The microscopic findings, such as interstitial pneumonia, fibrinous necrotic enteritis markedly in ileum and paratyphoid nodules in various organs, are the most common aspects of the disease. However, fibrinous cholecystitis, which is considered pathognomonic for salmonellosis, was not observed in this study. Therefore, the absence of such a lesion should not exclude the disease in sick animals. A microscopic injury score was used to determine lesion severity by assigning values from 1 to 4, wherein: 1 = no apparent lesions, 2 = mild lesions, 3 = moderate lesions, and 4 = severe lesions. Both calves were scored as 4. Multiple predisposing factors for the condition were identified in this farm such as different age animals in the same paddock and no specific paddock for sick animals, given that the infectious agent remains in feces, saliva, and nasal discharge. The serovar Dublin induces several clinical signs such as septicemic, respiratory, and enteric manifestations, making a clinical diagnosis a challenge

Effect of photoperiod on intestinal microbiota and circadian rhythm, and its influence on performance, the immune system and the welfare of broiler chickens

The intestinal microbiota of broiler chickens still reserves a great amount of secrets and mysteries. However, it is known that it acts directly on the host's health, but that it is also influenced by several factors, among them the light. Due to the importance of the world production of birds, this review of literature had the objective to shelter the most recent studies, which relate the photoperiod and the circadian rhythm, with the intestinal microbiota, the zootechnical performance, the immune system and the welfare of broiler chickens. Describing in a single work the existence of a bilateral relationship between the intestinal microbiota, the immune system and its host, and that can be directly influenced by light management.

Effect of photoperiod on intestinal microbiota and circadian rhythm, and its influence on performance, the immune system and the welfare of broiler chickens

The intestinal microbiota of broiler chickens still reserves a great amount of secrets and mysteries. However, it is known that it acts directly on the host's health, but that it is also influenced by several factors, among them the light. Due to the importance of the world production of birds, this review of literature had the objective to shelter the most recent studies, which relate the photoperiod and the circadian rhythm, with the intestinal microbiota, the zootechnical performance, the immune system and the welfare of broiler chickens. Describing in a single work the existence of a bilateral relationship between the intestinal microbiota, the immune system and its host, and that can be directly influenced by light management.(AU)

Resposta Inflamatória Sistêmica, Disfunção Orgânica e Laminite Induzidas por Oligofrutose em Equinos Suplementados ou não com Lithothamnium Muelleri.

Em equinos, o desequilíbrio da microbiota intestinal (disbiose) é causa comum de cólica, capaz de desencadear resposta inflamatória (sepse), que se não controlada, pode evoluir para a disfunção dos órgãos, resultando em laminite grave e até óbito. O Lithothamnium spp. é uma alga marinha calcárea, que demonstrou propriedades anti-inflamatórias e antioxidantes em estudos em animais de laboratório e humanos. Com a hipótese de que a ingestão prévia dessa alga reduz e retarda tais consequências, o objetivo foi avaliar os efeitos da suplementação com Lithothamnium muelleri na resposta inflamatória sistêmica, na disfunção orgânica e no casco em equinos submetidos ao modelo de laminite por oligofrutose. Foram utilizados 12 equinos hígidos, divididos em grupos controle e tratado. O grupo tratado recebeu Lithothamnium muelleri (100 mg/kg PO BID), durante seis dias antes e nas 36 horas após indução de disbiose e laminite por sobrecarga de oligofrutose (1g/kg PO durante 3 dias; e indução com 10g/kg PO no quarto dia). Os animais foram avaliados clínica e laboratorialmente antes e nos tempos 0, 6, 12, 18 e 24 horas subsequentes à indução. Biópsias de casco foram realizadas nos tempos 0, 12 e 36 horas após a indução, para avaliação histológica e imunomarcação de calprotectina (CP+). Os dados paramétricos foram submetidos a ANOVA em blocos ao acaso, seguida pelos testes de Tukey e T de Student. Os dados não paramétricos foram analisados pelo teste de Friedman, seguidos pelos testes de Dunns e Mann-Whitney. Os dois grupos apresentaram inflamação sistêmica, disfunção orgânica e consequente laminite. Contudo, no grupo tratado as alterações ocorreram de forma mais amena e tardia. Em conclusão a ingestão prévia de L. muelleri foi capaz de amenizar e retardar o início do quadro séptico, disfunção de órgãos e laminite em equinos submetidos a modelo de sobrecarga de oligofrutose. Apesar de preliminares, os achados demonstram o potencial da suplementação oral com Lithothamnium muelleri para controle da resposta inflamatória sistêmica frente a uma disbiose intestinal em equinos.

In horses, intestinal microbiota imbalance (dysbiosis) is a common cause of colic, capable of triggering an inflammatory response (sepsis), which, if not controlled, can progress to organ dysfunction, resulting in severe laminitis and even death. Lithothamnium spp. is a calcareous kelp, which has demonstrated anti-inflammatory and antioxidant properties in studies in laboratory animals and humans. With the hypothesis that the previous ingestion of this alga reduces and delays such consequences, the objective was to evaluate the effects of supplementation with Lithothamnium muelleri on the systemic inflammatory response, on organic dysfunction and on the hoof in horses submitted to the oligofructose laminitis model. Twelve healthy horses were used, divided into control and treated groups. The treated group received Lithothamnium muelleri (100 mg / kg PO BID) for six days before and in the 36 hours after induction of dysbiosis and laminitis due to oligofructose overload (1g / kg PO for 3 days; and induction with 10g / kg PO in the fourth day). The animals were evaluated clinically and laboratory before and at times 0, 6, 12, 18 and 24 hours after induction. Hoof biopsies were performed at times 0, 12 and 36 hours after induction, for histological evaluation and immunostaining of calprotectin (CP +). The parametric data were evaluated to ANOVA in randomized blocks, followed by the Tukey and Student's T tests. Nonparametric data were analyzed using the Friedman test, followed by the Dunns and Mann-Whitney tests. Both groups had systemic inflammation, organ dysfunction and consequent laminitis. However, in the treated group, the changes occurred more mildly and later. In conclusion, the previous ingestion of L. muelleri was able to alleviate and delay the onset of septic conditions, organ dysfunction and laminitis in horses submitted to an oligofructose overload model. Although preliminary, the findings demonstrate the potential of oral supplementation with Lithothamnium muelleri to control the systemic inflammatory response to intestinal dysbiosis in horses.

Relação entre o aspecto morfológico de fezes de frangos de corte e sua composição bacteriana

Analisar o aspecto fecal de frangos pode revelar de modo precoce, alterações que poderão ser relacionadas a problemas entéricos, que em grande parte das vezes ocorrem devido ao desequilíbrio da microbiota intestinal (disbiose). Deste modo, o presente estudo teve como objetivo utilizar análises de metagenômica para analisar a relação entre o aspecto morfológico de fezes de frangos e sua respectiva composição bacteriana. O estudo foi composto por um total de oito grupos, os grupos 1, 2 e 3 analisaram fezes com passagem de ração (PR), nos escores 1, 2 e 3, respectivamente. Os grupos 4, 5 e 6 analisaram fezes com muco avermelhado (MA), nos escores 1, 2 e 3, respectivamente. O grupo 7 analisou fezes ileais normais (FIN) e o grupo 8 analisou descargas cecais (DC). Cada grupo foi composto por seis amostras, totalizando 48 amostras. As amostras tiveram o DNA total extraído, e na sequência, se amplificou a região V4 do gene 16S rRNA. O sequenciamento foi realizado no sistema MiSeq e as reads produzidas foram de 2x250 pb. O programa DADA2 fez a modelagem e a correção de erros de amplicons sem a construção de OTUs. As diversidades alfa e beta, assim como, a abundância relativa dos filos e gêneros permitiram entender um pouco mais do comportamento das comunidades bacterianas em seus respectivos aspectos fecais. A diversidade alfa mostrou, através de curvas de rarefação, que as amostras atingiram seu máximo potencial de revelação gênica. E o index de Shannon revelou que o grupo DC apresentou maior diversidade, sendo estatisticamente diferente dos grupos PR-2, PR-3 e MA-1. A diversidade beta mostrou que somente as amostras do grupo DC foram homogêneas. A abundância relativa de filos revelou oito diferentes filos, sendo Firmicutes o mais abundante em todos os grupos, Proteobacteria o segundo mais abundante nos grupos que estudaram fezes ileais normais e suas alterações. Já o filo Bacteroidetes foi o segundo mais abundante no grupo DC. A abundância relativa dos gêneros revelou 136 diferentes gêneros bacterianos. Nos grupos que estudaram fezes ileais normais e suas alterações, os gêneros mais abundantes foram Lactobacillus e Escherichia/Shigella, com exceção do grupo PR-1 que teve essa ordem invertida. Diferente dos demais, o grupo DC teve maior abundância dos gêneros Bacteroides e Faecalibacterium. As análises estatísticas foram feitas com Teste t de Studant (P<0,01). Avaliar o aspecto morfológico de fezes de frangos é tarefa fundamental, que possibilita agir de modo rápido evitando perdas de desempenho e econômicas causadas por disbiose. Desse modo, esse trabalho identificou, através de análises de metagenômica, a existência de 61 diferentes gêneros bacterianos entre o grupo 7 (FIN) e o grupo 8 (DC), assim como, uma variação dos gêneros bacterianos conforme o tipo de alteração fecal, sendo essa mais significativa nos grupos 4, 5 e 6, que estudaram muco avermelhado, mostrando existir uma variação da microbiota intestinal ao longo do dia, devido à ausência de alimento e maior quantidade de muco no intestino, causado pelo período de escuro (jejum).

Analyzing the fecal aspect of chickens may reveal early changes that may be related to enteric problems, which in most cases occur due to intestinal microbiota imbalance (dysbiosis). Thus, the current study aimed to use metagenomic analyses to analyze the relationship between the morphological aspect of chicken feces and their respective bacterial composition. The study was composed by a total of eight groups, the groups 1, 2 and 3 analyzed feces with feed passage (FP), in scores 1, 2 and 3, respectively. The groups 4, 5 and 6 analyzed faeces with reddish mucus (RM), in the scores 1, 2 and 3, respectively. Group 7 analyzed normal ileal feces (NIF) and group 8 analyzed caecal discharges (CD). Each group was composed of six samples, totaling 48 samples. The samples had the total DNA extracted and in the sequence the V4 region of the 16S rRNA gene was amplified. The sequencing was performed in the MiSeq system and the produced reads were 2x250 bp. The DADA2 program did the modeling and the correction of amplicons errors without the construction of OTUs. The alpha and beta diversities, as well as the relative abundance of the phylos and bacterial genus allowed to understand a little more about the behavior of bacterial communities in their respective fecal aspects. The alpha diversity showed through the rarefaction curves that the samples reached their maximum potential of gene revelation. And Shannon's index revealed that the CD group showed greater diversity, being statistically different from FP-2, FP-3 and RM-1 groups. The beta diversity showed that only the samples of the CD group were homogeneous. The relative abundance of phylos revealed eight different phylos, being Firmicutes the most abundant in all groups, Proteobacteria the second most abundant in the groups that studied normal ileal feces and their alterations. The phylos Bacteroidetes was the second most abundant in the CD group. The relative abundance of genus revealed 136 different bacterial genus. In the groups that studied normal ileal feces and their alterations, the most abundant genus were Lactobacillus and Escherichia/Shigella, with the exception of the FP-1 group, which had this order reversed. Unlike the others, the CD group had more abundance of the genus Bacteroides and Faecalibacterium. The statistical analyses were done with Studant's t test (P<0.01). To evaluate the morphological aspect of chicken feces is a fundamental task, which makes it possible to act quickly avoiding performance and economic losses caused by dysbiosis. Thus, this work identified through metagenomic analyses the existence of 61 different bacterial genus between group 7 (NIF) and group 8 (CD), as well as, a variation of the bacterial genus according to the type of fecal alteration, being this variation more significant in groups 4, 5 and 6, that studied reddish mucus, showing to exist a variation of the intestinal microbiota throughout the day, due to the absence of feed and greater amount of mucus in the intestine, caused by the period of dark (fasting).

Presence of Porphyromonas and Prevotella species in the oral microflora of cattle with periodontitis / Presença de espécies de Porphyromonas e Prevotella na microbiota oral de bovinos com periodontite

Bovine periodontitis is a progressive purulent infectious process associated with the presence of strictly and facultative anaerobic subgingival biofilm and epidemiologically related to soil management in large geographic areas of Brazil. This study aimed to detect species of the genera Porphyromonas and Prevotella, which occurr in periodontal pockets of cattle with lesions deeper than 5mm (n=26) and in gingival sulcus of animals considered periodontally healthy (n=25). Presence of the microorganisms was evaluated by independent-culture medium diagnostic method, using polymerase chain reaction (PCR) with specific primers of Porphyromonas asaccharolytica, P. endodontalis, P. gingivalis, P. gulae, Prevotella buccae, P. intermedia, P. loescheii, P. melaninogenica, P. nigrescens, P. oralis and P. tannerae. The species P. endodontalis (80.7%), P. melaninogenica (73.1%) and P. intermedia (61.5%) were the most predominant in samples of cattle with periodontitis. Regarding non-injured gingival sulcus of cattle, P. endodontalis (40%) and P. loeschei (40%) prevailed. Porphyromonas gingivalis, P. gulae and Prevotella tannerae were not detected in the 51 samples studied. Data evaluation by T test, enabled to verify that ocorrence of Porphyromonas asaccharolytica (p=0.000003), P. endodontalis (p=0.0023), Prevotella buccae (p=0.0017), P. intermedia (p=0.0020), P. melaninogenica (p=0.00006) and P. oralis (p=0.0028) is correlated with bovine periodontitis.(AU)

A periodontite bovina é um processo infeccioso purulento e progressivo associado à presença de biofilme subgengival anaeróbio estrito e facultativo e de incidência em extensas áreas geográficas do Brasil. O trabalho teve por objetivo detectar espécies dos gêneros Porphyromonas e Prevotella presentes na bolsa periodontal de bovinos com lesões de profundidade maior que 5mm (n=26) e do sulco gengival de animais com idade de 6 a 24 meses e considerados periodontalmente sadios (n=25). A presença dos microrganismos foi avaliada pelo método independente de cultivo bacteriano, por meio da reação em cadeia da polimerase (PCR) com iniciadores específicos para Porphyromonas asaccharolytica, P. endodontalis, P. gingivalis, P. gulae, Prevotella buccae, P. intermedia, P. loescheii, P. melalinogenica, P. nigrescens, P. oralis e P. tannerae. P. endodontalis (80,7%), P. melalinogenica (73,1%) e P. intermedia (61,5%) foram os mais prevalentes nas amostras de bovinos com periodontite. Já no sulco gengival de bovinos sem lesões prevaleceram P. endodontalis (40%) e P. loeschei (40%). Porphyromonas gingivalis, P. gulae e Prevotella tannerae não foram detectados nas 51 amostras pesquisadas. A partir da avaliação dos dados pelo teste T, verificou-se que a ocorrência de Porphyromonas asaccharolytica (p=0.000003), P. endodontalis (p=0.0023), Prevotella buccae (p=0.0017), P. intermedia (p=0.0020), P. melalinogenica (p=0.00006) e P. oralis (p=0.0028) está associada à periodontite bovina.(AU)

Microbiota e produtos de fermentação fecal de cães com Doença Inflamatória Intestinal suplementados com -glucanos

A doença inflamatória intestinal (DII) é considerada como a causa mais comum de doença gastrintestinal em cães e a causa mais importante de vômito e diarreia em cães e gatos. Há consenso na literatura acerca da existência de reação imune aberrante aos microrganismos residentes no trato gastrintestinal no indivíduo geneticamente predisposto, associada à disbiose e consequente alteração na concentração de produtos oriundos da fermentação das bactérias intestinais. Os prebióticos são polissacarídeos não amiláceos resistentes à digestão enzimática de animais monogástricos que chegam ao intestino grosso aptos para servirem de substrato ao crescimento e fermentação de bactérias benéficas, em detrimento das patogênicas. Os efeitos imunomodulatórios desempenhados através desta fermentação seletiva apresentam potencial terapêutico interessante em pacientes com DII, os quais são amplamente discutidos na literatura humana, embora pouco discutidos na literatura veterinária. Assim, o presente estudo objetivou avaliar os efeitos da suplementação de -glucanos na microbiota fecal de cães com DII identificada e quantificada por sequenciamento genético de última geração. Para tal, foram selecionados dezoito cães com DII em fase controlada para compor três grupos experimentais: A suplementado com 0,1% de -glucanos, B suplementado com 0,1% de um blend de prebióticos composto por MOS e -glucanos e C - placebo, de modo que todos os grupos receberam a mesma dieta hidrolisada associada ou não aos prebióticos, durante 60 dias. Foram realizados hemogramas e exames bioquímicos de rotina e específicos, quantificação de lactato fecal e, para fins de acompanhamento dos sinais clínicos, foram aplicados dois escores de severidade de enteropatia canina específicos. As abundâncias observadas para cada filo e gênero foram avaliadas por modelo linear generalizado, considerando distribuição binominal e pelo emprego da função de ligação logit. O modelo incluiu efeitos fixos de grupos (A, B e C), tempos de avaliação (T0 e T60), interação entre estes, além do efeito aleatório de resíduo. Estas análises foram realizadas pelo procedimento PROC GLIMMIX do programa Statistical Analysis System. Valores de P<0,05 foram considerados significativos. Ao todo, foram identificados 10 filos, 17 classes, 48 famílias e 101 gêneros diferentes entre os três grupos experimentais. O filo, classe, família e gênero predominante entre os grupos foram, respectivamente, Firmicutes (24,30% - 99,79%), Clostridia (6,64% 97,39%), Lachnospiraceae (0,6% - 89,7%) e Streptococcus (0,02% - 90,19%). O tratamento com prebióticos modulou a população de diversos táxons em função do tempo, mas principalmente, modulou de forma positiva o filo Firmicutes e, somente o tratamento B controlou o crescimento de Collinsella. O tratamento com -glucano melhorou o Shannon index, o que evidencia possível melhora da disbiose. Não foi observada diferença na concentração fecal de ácido lático entre os tratamentos. A suplementação com os prebióticos melhorou o Canine IBD activity index (CIBDAI; p= 0,0084). O presente trabalho verificou que a suplementação de 0,1% de -glucanos na dieta de cães com DII, durante 60 dias, foi capaz de modular de forma positiva os microrganismos do trato gastrointestinal, bem como aumentou sua biodiversidade.

Inflammatory bowel disease (IBD) is the most common cause of gastrointestinal disease in dogs and the most important cause of vomiting and diarrhea in dogs and cats. There is a consensus in literature about the existence of aberrant immune reaction to microorganisms of the gastrointestinal tract in a genetically predisposed individual, associated with dysbiosis and, consequently, alteration in the concentration of fermentation products produced by intestinal bacteria. Prebiotics are non-starch polysaccharides resistant to enzymatic digestion of monogastric animals that reach the large intestine and serve as a substrate for the growth and fermentation of beneficial bacteria to the detriment of the pathogenic. The immunomodulatory effects performed through this selective fermentation have interesting therapeutic potential in patients with IBD, which are widely discussed in the human literature, although poorly discussed in the veterinary literature. Thus, the present study aimed to evaluate the effects of -glucan supplementation on the fecal microbiota of dogs with IBD identified and quantified by next generation genetic sequencing. For this, eighteen dogs with controlled IBD were selected to compose three experimental groups: A - supplemented with 0.1% -glucans, B 0.1% of a prebiotic blend composed by MOS and -glucans and C - placebo, and all of them received the same hydrolyzed diet for 60 days. In addition, routine blood counts and biochemical tests were performed and, for the purpose of monitoring clinical signs, specific canine enteropathy severity scores were applied. The abundances observed were evaluated by Generalized mixed linear model with binomial data, using logit link function. The model included fixed effects of treatments (A, B or C), time (T0 or T60), and interaction between them, beyond animal as random effect. At total, 10 phyla, 17 classes, 48 families and 101 different genera were identified among the 3 experimental groups. The predominant phylum, class, family and gender among the groups were, respectively, Firmicutes (24.30% - 99.79%), Clostridia (6.64% - 97.39%), Lachnospiraceae (0.6% - 89.7%) and Streptococcus (0.02% - 90.19%). The treatment with prebiotics modulated the population of several taxa according to time, but mainly, it positively modulated the phylum Firmicutes and only treatment B controlled the growth of Collinsella. Only treatment with -glucan improved the Shannon index and showed improvement in dysbiosis. There was no difference in the fecal concentration of lactic acid between treatments. Supplementation with prebiotics improved the Canine IBD activity index (CIBDAI; p = 0.0084). The present study found that 0.1% supplementation of -glucans in the diet of dogs with IBD was able to positively modulate microorganisms in the gastrointestinal tract after 60 days through the growth of beneficial bacteria and improvement of biodiversity.

Caracterização da microbiota fecal de cães obesos e após emagrecimento

A obesidade é considerada na medicina veterinária a afecção nutricional e metabólica mais comum da atualidade. Esta ocorre devido ao acúmulo excessivo de tecido adiposo no organismo, resultado do aumento na ingestão de energia associado a falta de atividade física. A obesidade vem sendo correlacionada com disbiose da microbiota intestinal, bem como em desordens imunológicas, alterações ortopédicas, respiratórias e metabólicas, como resistência insulínica e hiperlipidemia. Estudos demonstraram em ratos geneticamente obesos e induzidos pela dieta, que a microbiota cecal de animais acima do peso apresentam tags genéticos ambientais que codificam enzimas envolvidas na degradação de polissacarídeos e no metabolismo de carboidratos simples. Assim, a microbiota de indivíduos obesos pode ser mais eficiente em digerir e metabolizar a energia da dieta que indivíduos magros. Dessa forma, o objetivo deste trabalho foi caracterizar a microbiota fecal de cães obesos e em escore corporal ideal e avaliar os efeitos do programa de perda de peso sobre os filos e gêneros bacterianos. Foram incluídos vinte cães, fêmeas, castradas, de diferentes raças, com idade entre 1 a 9 anos. Os animais foram divididos em dois grupos: grupo experimental de animais obesos (GO), com escore de condição corporal (ECC) 9 e com porcentagem de gordura corporal superior a 30%, determinada pelo método de diluição de isótopos de deutério e um grupo controle (GC), com ECC 4 ou 5 e gordura corporal máxima de 15%. Os animais obesos foram incluídos em um programa de perda de peso e, quando perderam 20% do peso inicial, passaram a compor o grupo de animais emagrecidos (GE). Foram coletadas amostras de fezes dos três grupos experimentais. Nos grupos controle e obesos, a coleta foi realizada após um período de adaptação com dieta de manutenção e, no grupo emagrecido, a coleta de fezes foi realizada no final do programa de perda de peso. Após cada coleta, as amostras foram armazenadas em freezer -80°C. Na sequência, foi extraído o DNA total das fezes e estas foram sequenciadas pela metodologia Illumina. As abundâncias observadas para cada filo e gênero foram avaliadas por modelo linear generalizado, considerando distribuição binominal e pelo emprego da função de ligação logit. O modelo incluiu efeitos fixos de grupos (Controle, Obesos e Emagrecidos), além dos efeitos aleatórios de animal e resíduo. Todas as análises foram realizadas pelo procedimento PROC GLIMMIX do programa Statistical Analysis System. Valores de P<0,05 foram considerados significativos. Foram identificados 7 filos e 51 gêneros diferentes entre os 3 grupos experimentais. O filo e gênero predominante entre os grupos foi respectivamente o Firmicutes (53,01% - 71,98%) e o Clostridium (42,43 25,51%). Foi observada diferença entre animais obesos e magros para o filo Firmicutes (P=0,0189) e gênero Sutturella (P=0,0095). O programa de perda de peso modulou de forma positiva os filos Actinobacteria (P=0,0128), Firmicutes (P=0,0189) e o gênero Dorea (P<,0001) e, negativamente o Faecalibacterium (P=0,0114). O presente trabalho verificou que o programa de perda de peso foi capaz de modular os microrganismos do trato gastrointestinal, de forma que, os cães emagrecidos apresentaram composição microbiana com maior biodiversidade, aumento de Actinobacteria, Firmicutes e Dorea e menor abundancia de Faecalibacterium.

Obesity is considered the most common nutritional and metabolic disorder in veterinary medicine today. This occurred to the excessive accumulation of adipose tissue in the organism, due to increase in energy intake with low physical activity. Obesity has been associated with an intestinal microbiota dysbiosis, as well as in immune disorders, orthopedic, respiratory and metabolic changes, such as insulin resistance and hyperlipidemia. Studies have shown in both genetically obese and diet-induced mice that the cecal microbiota from obese animals is enriched with environmental genetic tags that encode enzymes involved in the degradation of polysaccharides and the metabolism of simple carbohydrates. Thus, the microbiota of obese individuals may be more efficient in digesting and metabolizing dietary energy than lean individuals. So, the present study aims to characterize the microbioma profile of obese dogs and to evaluate the effects of weight loss on the population of microorganisms of dogs included in a weight loss program. Twenty dogs, female, neutered, different breeds and 1 to 9 years old were included. The animals were divided into two groups: experimental group of obese animals (GO) with a body condition score (BCS) 9 and body fat percentage greater than 30% determined by the deuterium isotope dilution method and a control group (GC) with BCS 4 or 5 and maximum body fat of 15%. The obese animals were included in a weight loss program and when they lost 20% of their initial weight, they became part of the group of emaciated animals (GE). Stool samples were collected from the three experimental groups. In the control and obese groups, the collection was performed after an adaptation period with a maintenance diet and in the emaciated group, the collection of feces was done at the end of the weight loss program. After each collection, the samples were stored at -80°C. Total DNA was extracted from the feces and sequenced using the Illumina methodology. The abundances observed for each phylum and gender were evaluated using a generalized linear model, considering binomial distribution and using the logit link function. The model includes fixed effects of Groups (Control, Obese and Weight Loss), in addition to the random effects of animals and residue. All as analyzes were performed using PROC GLIMMIX procedure from the Statistical Analysis System Program. Values of P <0.05 were considered significant. Seven different phyla and 51 different males were identified among the three experimental groups. The most prevalent phylum and genus among the groups was Firmicutes (53.01% - 71.98%) and Clostridium (42.43 25.51%) respectively. It was observed difference between obese and lean animals for phyla Firmicutes (P = 0.0189) and genus Sutturella (P = 0.0095). The weight loss program regulated positively the phyla Actinobacteria (P = 0.0128), Firmicutes (P = 0.0189) and genus Dorea (P<.0001), and negatively genus Faecalibacterium (P = 0.0114). The present study found that the weight loss program is capable of modulating the microorganisms of the gastrointestinal tract, so that the emaciated dogs presented microbial composition with greater biodiversity, greater presence of Actinobacteria, Firmicutes and Dorea and less abundance of Faecalibacterium.

MICROBIOTA FECAL DE BEZERROS LACTENTES: EFEITO DA IDADE, DA RAÇA E DA DIARREIA

Os animais vivem em simbiose com microrganismos, as bacterias exercem várias funções importantes no organismo como digestão de alimentos, desenvolvimento do sistema imune e resistência à patógenos. Agentes infecciosos podem provocar desequilíbrios da microbiota e desenvolver doenças no hospedeiro. Com o objetivo de estudar os efeitos que alteram a microbiota intestinal dos bezerros lactentes, dois estudos foram desenvolvidos. O primeiro, investigou o efeito da idade e genética na microbiota intestinal de bezerros Nelore (n= 10) e Cruzados (Nelore x Raça britânica; n= 10) mantidos a campo com suas mães, sem receber suplementação. Foram realizadas oito colheitas nos respectivos dias de vida, 5 (5d), 14d, 28d, 60d, 90d, 180d, no desmame (245d) e quinze dias após (260d), e foram colhidas amostras de seis vacas (DAM) no desmame para servir como controle. O efeito do peso ao desmame nas populações bacterianas entre Nelore e Cruzados foi avaliado. As regiões V3-V4 do gene 16S rRNA foram sequenciadas (Illumina). No geral, os filos mais abundantes foram Firmicutes (64.68%), Bacteroidetes (16.81%), Fusobacteria (9.10%), Proteobacteria (1.49%) e Actinobacteria (1.28%). A riqueza aumentou de acordo com o crescimento dos bezerros até se tornar similar à da mãe ao desmame. A composição e a estrutura da microbiota intestinal mudaram com a idade. Não há semelhança na composição da microbiota entre bezerros e as mães, mas a estrutura da microbiota foi semelhante à da mãe próximo ao desmame. O grupo genético demonstrou influenciar a composição aos 60d e 90d e a estrutura aos 60d, 90d, 245d e 260d. O gênero Fusobacterium foi abundante na microbiota dos bezerros de corte saudáveis. Os bezerros Cruzados foram mais pesados do que os Nelore ao desmame (240,70 vs. 216,80 kg). O segundo estudo, teve como objetivos avaliar a eliminação de Cryptosporidium durante o primeiro mês de vida de bezerras leiteiras e as mudanças na microbiota intestinal. Foram colhidas amostras de fezes de 30 bezerras durante o primeiro mês de vida, foram anotados dados de colostro, transferência de imunidade passiva, uso de antibióticos e diagnóstico de doenças. A presença do Cryptosporidium foi detectada com SNPCR em 15 amostras de cada bezerra em dias alternados, totalizando 450 amostras. Amostras de 16 bezerras foram utilizadas para a análise da microbiota intestinal de acordo com o primeiro dia de diarreia (D0), anterior a diarreia (D-4 e D-2), durante o curso da diarreia (D+4), ao final (DX) e seis dias após (DX+6). Amostras do segundo dia de vida (n=30), terceiro (n=12) e quarto dia (n=18) também foram analisadas. A região V4 do gene 16S rRNA foi sequenciada (Illumina). Foi observada ocorrência de diarreia em todas as bezerras, com início no quinto dia de vida e duração média de 7,86 dias. Todas as bezerras foram positivas para Cryptosporidium. Os principais filos observados foram Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes e Actinobacteria. O Cryptosporidium spp. alterou a microbiota intestinal e o filo Fusobacteria aumentou durante a diarreia. A efetiva transferência de imunidade passiva diminuiu o uso de antibióticos e foi observado que o colostro e a imunidade têm funções na restauração da microbiota dos animais e necessitam de maiores estudos.

Animals and microorganisms live in symbiosis, bacteria are responsible by many important functions in the body like degradation of food, development of immune system and resistance against pathogens. Infectious agents can cause microbial dysbiosis and develop disease in the host. In order to study the effects that change the intestinal microbiota of nursing calves, two studies were developed. The first one exploited the effect of age and genetic of fecal microbial communities from beef calves rearing at range, twenty calves from two breed group, 100% Nellore pure breed (n= 10) and Crossbreed (50% Nellore x 50% Britannic breed; n= 10), were maintenance at range with dams without supplementation. Fecal samples were collected on days of life, 5 (5d), 14d, 28d, 60d, 90d, 180d, at weaning (245d) and after 15 days (260d) and of six dams (DAM) at weaning to be used as control. In addition, the effect of weight at weaning on bacterial taxa between Nellore straightbred and crossbred were evaluated. The sequencing was performed in Illumina system of V3-V4 regions of 16S rRNA gene. The phylum most abundant during all trial were Firmicutes (64.68%), Bacteroidetes (16.81%), (Fusobacteria (9.10%), Proteobacteria (1.49%) and Actinobacteria (1.28%). Richness increase as calves growth until be similar to DAM near of weaning. Membership and Structure changes over ages, there was no similarities of membership between calves and DAM, but the structure was similar to DAM around of weaning. Genetic group showed differences for membership at 60d and 90d and for structure at 60d, 90d, 245d and 260d. The Fusobacterium genus was abundant in fecal microbiota of healthy calves. The Crossbreed calves were heavier than Nellore at weaning (240.70 vs. 216.80 kg). The second study, the objective was evaluated the Cryptosporidium shedding during the first month of life and the effect of diarrhea in the intestinal microbiota of dairy calves. Fecal samples from 30 calves were collected during the first month of life, records of colostrum, passive immunity transference, antibiotics use and diseases. Cryptosporidium shedding was evaluated by SN-PCR in 15 samples from each calve every other day, totalizing 450 samples. Samples of sixteen calves were choose for intestinal microbial analysis of 16S rRNA gene, accord first day of diarrhea (D0), prior to diarrhea (D-4 and D-2), during diarrhea course (D+4), at finish of diarrhea (DX) and after six days (DX+6). Additionally, samples from second (n=30), third (n=12) and fourth (n=18) day of life were analyzed too. The v4 gene was sequenced with use of Illumina. Diarrhea was observed in all calves (100%), starting at day 5 of life and an average of 7.86 days. All calves were positive for Cryptosporidium spp. The main phyla observed were Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes and Actinobacteria. The Cryptosporidium spp. change the intestinal microbiota and Fusobacteria phylum increase with diarrhea. The effectiveness of immunity transference decrease the antibiotic uses and it was observed that colostrum and immunity play a role in restoring the animals' microbiota and need further studies.

Avaliação de esfregaços corados pelo gram e prevalência de endoparasitas em araras de vida-livre / Evaluation of fecal gram stains and prevalence of endoparasites in free-living macaws

Although Brazil has a great bird diversity, information on the health status of free living psittacine is scarce. The aim of this study was to determine the prevalence of endoparasites and the ratio of gram-negative versus gram-positive bacteria in the feces of free-living adult Hyacinth and Green-winged macaws. Fecal samples were collected placing camouflaged sheet covers on bird foraging areas. Fifty-five swabs were evaluated by gram staining technique and 101 fecal samples were analyzed using flotation and centrifugation methods. None of the samples presented parasites or ova. The presence of gram-negative bacteria was detected in 1.8% of the fecal samples. These results suggest that parasites and gram-negative bacteria may be transient in the gastrointestinal tract of healthy free-living psittacine birds. This situation is quite different from what is observed for birds in captivity, where stressful events and improper husbandry facilitate the dysbiosis and high parasite loads, with an imbalance in the host-parasite relationship.

Embora o Brasil tenha uma grande diversidade de aves, as informações sobre o estado sanitário de psitacídeos de vida livre são escassas. O objetivo deste trabalho foi determinar a prevalência de endoparasitas e a relação entre bactérias gram-negativas e gram-positivas nas fezes de Arara-azuis e Araras-vermelhas-grandes adultas em vida-livre. Amostras de fezes foram coletadas empregando-se lonas camufladas onde as aves se reuniam para forragear. Cinquenta e cinco esfregaços foram avaliados pela técnica de gram e 101 amostras de fezes foram analisadas pelos métodos de flutuação e centrífugo-flutuação.  Nenhuma amostra foi positiva para a presença de ovos ou formas adultas de parasitas. A presença de bactérias gram-negativas foi detectada em 1.8% das amostras fecais. Esses resultados sugerem que parasitas e bactérias gram-negativas podem ser apenas transientes no trato gastrointestinal de psitacídeos saudáveis de vida livre. Essa situação é bastante diversa do que ocorre em cativeiro, onde situações de estresse e manejo inadequado facilitam a disbiose e o aumento das cargas parasitárias, com um desequilíbrio na relação hospedeiro-parasita.

Avaliação de esfregaços corados pelo gram e prevalência de endoparasitas em araras de vida-livre / Evaluation of fecal gram stains and prevalence of endoparasites in free-living macaws

Although Brazil has a great bird diversity, information on the health status of free living psittacine is scarce. The aim of this study was to determine the prevalence of endoparasites and the ratio of gram-negative versus gram-positive bacteria in the feces of free-living adult Hyacinth and Green-winged macaws. Fecal samples were collected placing camouflaged sheet covers on bird foraging areas. Fifty-five swabs were evaluated by gram staining technique and 101 fecal samples were analyzed using flotation and centrifugation methods. None of the samples presented parasites or ova. The presence of gram-negative bacteria was detected in 1.8% of the fecal samples. These results suggest that parasites and gram-negative bacteria may be transient in the gastrointestinal tract of healthy free-living psittacine birds. This situation is quite different from what is observed for birds in captivity, where stressful events and improper husbandry facilitate the dysbiosis and high parasite loads, with an imbalance in the host-parasite relationship.(AU)

Embora o Brasil tenha uma grande diversidade de aves, as informações sobre o estado sanitário de psitacídeos de vida livre são escassas. O objetivo deste trabalho foi determinar a prevalência de endoparasitas e a relação entre bactérias gram-negativas e gram-positivas nas fezes de Arara-azuis e Araras-vermelhas-grandes adultas em vida-livre. Amostras de fezes foram coletadas empregando-se lonas camufladas onde as aves se reuniam para forragear. Cinquenta e cinco esfregaços foram avaliados pela técnica de gram e 101 amostras de fezes foram analisadas pelos métodos de flutuação e centrífugo-flutuação.  Nenhuma amostra foi positiva para a presença de ovos ou formas adultas de parasitas. A presença de bactérias gram-negativas foi detectada em 1.8% das amostras fecais. Esses resultados sugerem que parasitas e bactérias gram-negativas podem ser apenas transientes no trato gastrointestinal de psitacídeos saudáveis de vida livre. Essa situação é bastante diversa do que ocorre em cativeiro, onde situações de estresse e manejo inadequado facilitam a disbiose e o aumento das cargas parasitárias, com um desequilíbrio na relação hospedeiro-parasita.(AU)

AVALIAÇÃO DA INFLUÊNCIA DA DISBIOSE INTESTINAL CAUSADA PELA AMOXICILINA NO CICLO ESTRAL MURINO

A disbiose intestinal se refere ao desequilíbrio da microbiota, quando da proliferação exacerbada ou diminuição de uma população específica de microrganismos, sendo desencadeada por vários fatores, incluindo a utilização de antibióticos, tais como a amoxicilina, uma penicilina semi-sintética de amplo espectro. Uma vez que a disbiose intestinal está associada a uma grande variedade de condições imunopatológicas e reprodutivas, o principal objetivo deste estudo foi avaliar a disbiose intestinal induzida pela amoxicilina e sua influência sobre o ciclo estral de camundongos. Para isso, camundongos fêmeas da linhagem isogênica 129Sv/Ev foram tratados com amoxicilina ou solução salina tamponada de fosfato (PBS) por gavagem durante 15 dias. As fezes dos animais foram coletadas para análise da microbiota bacteriana fecal por metagenômica. Os animais tratados com amoxicilina apresentaram diferentes filos e grupos bacterianos em suas microbiotas fecais, quando comparadas com o grupo tratamento (PBS), mostrando uma maior proliferação de Proteobacterias e uma presença inferior de Bacteroidetes, demonstrando que o uso da amoxicilina induz a disbiose intestinal murina. Os camundongos disbióticos obtiveram um maior ganho de peso corporal quando comparados ao grupo controle PBS, e ambos os grupos não apresentaram morte de animais durante todo o procedimento. Durante os tratamentos, a citologia vaginal foi realizada por microscopia de campo claro, caracterizada por citometria de fluxo e visualização vaginal determinando a fase do ciclo estral em que os animais se encontravam. As análises de citologia vaginal, visualização vaginal e citometria mostraram que a disbiose induzida pelo uso da amoxicilina alterou o ciclo estral dos animais e diminuiu o número de células totais presente nas fases do ciclo. Ademais, para caracterizar os possíveis parâmetros envolvidos na alteração do ciclo estral, amostras de ceco e ovários foram coletados para a análise da expressão diferencial de genes codificadores de citocinas pró ou anti-inflamatória IL-1 e IL-10 por PCR em tempo real. A expressão de IL-1 se apresentou aumentada no ceco e no ovário de animais disbióticos quando comparados aos dos animais controles. Já a expressão de IL-10 se apresentou reduzida no grupo disbiótico em ambos os órgãos quando comparados ao grupo controle. Sendo assim, os resultados indicam que o uso da amoxicilina induz a disbiose bacteriana intestinal influenciando no ciclo estral murino e um estado pró-inflamatório no ceco e ovários dos camundongos.

The intestinal dysbiosis refers to the imbalance of the gut microbiota when a specific microorganism population exacerbates or diminishes its proliferation being triggered by several factors, including the use of antibiotics, as amoxicillin, semisynthetic penicillin of wide spectrum. Since intestinal dysbiosis is associated with a great variety of immunopathological and reproductive conditions, the main goal of this study was to evaluate the intestinal dysbiosis caused by amoxicillin and its influence on the estrous cycle of mice. For this, female mice of the isogenic strain 129Sv/Ev were treated with amoxicillin or tamponade phosphate (PBS) by gavage for 15 days. The feces of the treated animals were collected for fecal bacterial microbiota population by metagenomics. The amoxicillin-treated animals showed different phyla and bacterial groups in their feces microbiota when compared to the PBS-treated group, showing higher Proteobacteria proliferation and lower presence of Bacteroidetes, demonstrating that the use of amoxicillin induces intestinal dysbiosis. The dysbiotic mice gained body weight when compared to the PBS control group, and both groups did not show any death during the treatments. During the treatments, the vaginal cytology was performed by light microscopy, with cells characterization by flow cytometry and physical evaluation of vaginal parameters in order to determine the estrous cycle phase. These analyses showed that the intestinal dysbiosis caused by the use of amoxicillin not only altered the estrous cycle but also decreased the number of cells present in the cycle phases. In addition, to characterize the possible parameters involved in altering the estrous cycle, caecum and ovarian samples were collected to analyze pro- or anti-inflammatory cytokines IL-1 and IL-10 encoding genes by real-time PCR. The IL-1 expression showed up-regulated in both cecum and ovary from dysbiotic animals when compared to control animals. On the other hand, IL-10 showed down-regulated the dysbiotic group samples when compared to the control group. In conclusion, the results indicated that the use of amoxicillin induces intestinal bacterial dysbiosis influencing the murine estrous cycle and a pro-inflammatory pattern in caecum and ovaries of mice under this antibiotic treatment.

EFEITO DO MANANOLIGOSSACARÍDEO (MOS) SOBRE DESEMPENHO, SAÚDE INTESTINAL E RESPOSTA IMUNE EM LEITÕES PÓS-DESMAME DESAFIADOS POR Escherichia coli

Leitões no pós-desmame estão susceptíveis a quedas de desempenho, saúde intestinal e imunidade devido a alterações fisiológicas que ocorrem nesta fase de criação, concomitante com a ocorrência de infecções patogênicas por Escherichia coli. A fim de minimizar as perdas diretas e indiretas causadas por esta soma de fatores, o mananoligossacarídeo (MOS), prebiótico advindo de parede celular de levedura, é utilizado. Este trabalho teve por objetivo analisar o desempenho, saúde intestinal e resposta imunológica de leitões no pós-desmame em delineamento experimental inteiramente casualizado, com e sem inclusão de 0,1% MOS comercial (HyperGen® , Biorigin) na dieta e com e sem inoculação via oral de Escherichia coli K88+ enterotoxigênica. No cultivo microbiológico houve crescimento de E. coli em todos os tratamentos, o tratamento controle negativo (DC-INO) apresentou o menor crescimento e os tratamentos com desafio (DC+INO e MOS+INO) apresentaram os maiores crescimentos, mostrando que a inoculação foi eficaz devido a maior proliferação de E. coli no intestino dos animais com inoculação, além de maior formação de ácidos graxos voláteis (AGV) no ceco destes animais, devido a possível disbiose provocada pela ação do patógeno. Os animais que receberam MOS, independente de desafio sanitário (MOS+INO e MOS-INO), tiveram maior quantidade de células em proliferação nos tecidos linfoides de íleo e linfonodo mesentérico, com vilosidades intestinais maiores, além de melhores índices de desempenho, comparado ao tratamento controle negativo DC-INO. Isso comprova que a inclusão de MOS na concentração de 0,1% foi capaz de modular o sistema imune, principalmente frente a desafio sanitário, de forma controlada e eficaz, contribuindo com os resultados finais de desempenho. A ação da E. coli causou um desequilíbrio da microbiota intestinal, mas não prejudicou no desempenho de animais tratados com MOS. O tratamento com MOS+INO obteve mais ganhos e melhor desempenho, pela ação do MOS sob a saúde intestinal e a contribuição dos AGV, produzidos em maior quantidade devido a disbiose, que mesmo sendo uma alteração maléfica, aumentou a fonte de energia dos enterócitos, que aumentou o tamanho das vilosidades, o que contribuiu com os ganhos finais.

Piglets in postweaning are susceptible to performance declines, intestinal health and immunity due to physiological changes occurring during this breeding phase, concomitant with the occurrence of pathogenic Escherichia coli infections. In order to minimize the direct and indirect losses caused by this sum of factors, the prebiotic mananoligosaccharide (MOS), from yeast cell wall, is used. The objective of this study was to analyze the performance, intestinal health and immune response of piglets in postweaning in a completely randomized experimental design, with and without inclusion of 0.1% commercial MOS (HyperGen®, Biorigin) in the diet and with and without inoculation via oral Escherichia coli K88 + enterotoxigenic. In the microbiological culture there was growth of E. coli in all treatments, the negative control treatment (DC-INO) showed the lowest growth and the treatments with challenge (DC + INO and MOS + INO) showed the highest growth, showing that inoculation was effective due to the greater proliferation of E. coli in the gut of the inoculated animals, as well as a higher formation of volatile fatty acids (VFA) in these animals due to possible dysbiosis caused by the pathogen. Animals that received MOS, regardless of health challenge (MOS + INO and MOS-INO), had higher numbers of proliferating cells in the lymphoid tissues of ileum and mesenteric lymph node, with larger intestinal villi, and better performance indices compared to treatment negative control DC-INO. This proves that the inclusion of MOS in the concentration of 0.1% was able to modulate the immune system, mainly against sanitary challenge, in a controlled and effective way, contributing to the final results of performance. The action of E. coli caused an imbalance of the intestinal microbiota, but did not impair the performance of animals treated with MOS. The treatment with MOS + INO obtained more gains and better performance, due to the action of MOS under intestinal health and the contribution of the VFA, produced in greater quantity due to dysbiosis, that even being a malefic alteration, it increased the energy source of enterocytes, which increased the size of the villi, which contributed to the final gains.