Modelo experimental para restrição do crescimento fetal em ratos: efeito sobre o glicogênio hepático e morfometria intestinal e renal

OBJETIVO: avaliar a eficácia do modelo de RCIU por ligadura da artéria uterina simulando insuficiência placentária em ratos. MÉTODOS: fetos de ratas prenhes Sprague-Dawley foram divididos em três grupos: RCIU (restrição de crescimento intrauterino), com fetos submetidos à ligadura da artéria uterina com 18,5 dias de gestação (termo = 22 dias), C-RCIU (controle da restrição), com fetos do corno contralateral à ligadura, CE (Controle Externo), com fetos de ratas sem manipulação. Com 21,5 dias de gestação, foi realizada cesárea, os fetos foram pesados e dissecados para análise morfométrica e histológica do fígado, intestino e rins. RESULTADOS: os dados morfométricos avaliados mostraram o peso corpóreo (PC), hepático (PH) e intestinal (PI) dos fetos com RCIU menor que C-RCIU e CE (p<0,001). O peso placentário (PP), renal (PR) e as relações PH/PC, PI/PC e PR/PC não se alteraram. A espessura renal foi menor nos fetos com RCIU (p<0,001) e houve diminuição da camada mucosa e submucosa intestinal (p<0,05). A avaliação histológica mostrou diminuição do glicogênio hepático nos fetos com RCIU em relação aos grupos C-RCIU e CE. CONCLUSÕES: o modelo descrito foi eficiente e causou RCIU fetal simétrica com diminuição da maioria dos órgãos, especialmente do peso hepático, e alteração nos depósitos de glicogênio.

Doença inflamatória intestinal - Doença de Crohn e gravidez: relato de caso

A Doença de Crohn, uma forma de doença inflamatória intestinal, é frequente em mulheres em idade reprodutiva. Seu acompanhamento requer maior atenção durante a gravidez. Apresentamos caso de doença de Crohn refratária em paciente grávida que evoluiu com ileocolectomia no puerpério. Foi revista a literatura sobre doença de Crohn na gravidez, incluindo aconselhamento das pacientes e investigação de doença ativa, além de sintetizados os dados existentes sobre a segurança dos medicamentos usados para tratá-la durante a gravidez e amamentação.

Evaluation of an enzyme-linked immunosorbent assay to detect antibodies against Anaplasma marginale

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Anaplasma marginale using a partially soluble antigen prepared from semi-purified initial bodies from erythrocytes with 80.0% of rickettsiaemia. This technique utilized alkaline phosphatase and p-nitrophenyl phosphate as reaction indicators. The high sensitivity (100.0%) was confirmed with sera from 100 calves experimentally-infected with A. marginale. All of these animals showed seroconversion before or at the same time of the first rickettsiaemia or even when it was not detected. Also the elevated specificity (94.0%) was confirmed by the low percentage of cross-reactions with sera from animals experimentally-infected with Babesia bigemina and Babesia bovis (1.4 and 6.6%, respectively). Performances of ELISA and indirect fluorescent antibody test (IFAT) with 324 sera from enzootically stable area did not show statistical difference (P>0.05), since the former showed 96.9% and the latter 97.2% of positive reactions. The advantage of this ELISA is a shorter execution time than others developed until now, allowing more samples to be analyzed.

Relationship of intestinal histology with the resistance to Trichostrongylus colubriformis infection in three breeds of sheep

The study was carried out to evaluate the relationship of inflammatory intestinal cells with the resistance to Trichostrongylus colubriformis infections in three breeds of sheep (Santa Ines, Suffolk and Ile de France), naturally infected. Mast cells, eosinophils, and globule leucocytes were enumerated in intestinal mucosa. Histamine concentration was estimated in intestinal tissue samples and the length of male and female specimens were determined. The three breeds of sheep showed similar cellular response in the small intestine mucosa (P>0.05). There was extensive variation among sheep in the parasitological and inflammatory cell variables, even in lambs of the same breed. In general, animals presenting less inflammatory cells had a larger worm burden, higher fecal egg counts, and larger T. colubriformis worms. The inflammatory cells possibly impaired the parasite's establishment, development, and survival.

IgA production, coliforms analysis and intestinal mucosa morphology of piglets that received probiotics with viable or inactivated cells

Two types of probiotics were used in piglets. One product is a mixed culture of viable Lactobacillus acidophilus, Enterococcus faecium e Bifidobacterium bifidum. The second product is composed of inactivated Lactobacillus acidophilus cells. The piglets received two weekly oral doses for 30 days while a control group did not receive probiotics. All piglets were euthanized at the 30th day of life and the mesenteric lymph nodes, the small intestine, and blood samples were collected. The tissue samples were studied by light microscopy and the blood serum was analyzed by ELISA method. The treatment with the probiotic with viable cells produced higher serum levels of IgA (P<0.05) and more IgA expressing cells were found in the mesenteric lymph nodes than observed in the inactivated cells treatment or control groups (P<0.05). Also, intestinal villi were longer, crypts were deeper (P<0.05) and fecal coliform count was lower than found in the inactivated product (P<0.05). These results suggest that viable probiotics are more efficient than inactivated probiotics to induce immunostimulation and intestinal modifications in piglets, thus improving their health and development.

Intestinal lesions in pigs affected with postweaning multisystemic wasting syndrome

Samples of mesenteric lymph nodes and intestines from 79 unthrifty 3- to 5-month-old postweaning pigs, confirmed as naturally affected with postweaning multisystemic wasting syndrome (PMWS), were studied. Pigs originated from 12 farms in southern Brazil and were selected on the basis of clinical signs and/or gross lesions suggestive of enteric disorder. Lymphohistiocytic infiltrates of varying intensity were associated with anti-porcine circovirus type 2 (anti-PCV2) immunostaining (IS) in samples of intestines and mesenteric lymph nodes from all pigs. Although most findings were similar to those described in PCV2-associated enteritis, anti-PCV2 IS in association with depletion of the goblet cell mucin stores (24 pigs), diffuse ileal villous atrophy and fusion (18 pigs), and dilatation of the lymphatic vessels (11 pigs) combined or not with lymphangitis were also observed. PCV2 antigen was immunohistochemically demonstrated in the cytoplasm and nuclei from intralesional epithelial cells, histiocytes, and endothelial-like cells in intestinal tissues. Together these findings imply an association with PCV2. The presence of co-infections by Lawsonia intracellularis, Brachyspira spp., Mycobacterium spp., Salmonella spp., rotavirus, parvovirus, coronavirus and enteric calicivirus with PCV2 in the intestinal lesions was investigated.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

Visceral leishmaniasis is an emergent zoonosis with an increasing number of new cases in Brazil where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. An enzyme-linked immunosorbent assay (ELISA), based upon the use of a total soluble antigenic preparation of L. chagasi, was adapted for the detection of IgM antibodies in the serum of infected dogs. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 110 serum samples were taken from dogs in Belo Horizonte, Minas Gerais, Brazil, and examined for anti-L. chagasi IgM antibody by ELISA and indirect fluorescent antibody test (IFAT). About 25% (n=27) of all the dogs tested were found serologically positive for L. chagasi by IFAT, while 89.09% (n=98) were seropositive by ELISA. The results obtained by ELISA and IFAT were significantly different (P<0.01). The combined use of ELISA and IFAT is recommended in order to enable veterinary services to more efficiently detect canine visceral leishmaniasis.

Absence of intestinal colonization by vancomycin-resistant enterococci in nonhuman primates

The animal reservoirs of vancomycin-resistant enterococci (VRE) have important role in the epidemiology of the bacteria and resistant genes. The present work searched fecal samples taken off nonhuman primates for the presence of VRE. Resistance profiles, virulence traits, and genetic variability among enterococci isolates were also analyzed. The samples included Capuchin monkeys (Cebus apella, n=28) and Common marmoset (Callithrix penicillata, n=37) housed in the Primate Center of the University of Brasília, Brazil. Most individuals were captive monkeys from the Central-West and South-East regions of Brazil (n=48). We collected rectal swabs and carried out selective isolation followed by multiplex Polymerase Chain Reaction (PCR) to identify species and resistance genes. No vanA or vanB-containing enterococci were found. The carriage rates ranged from 1.5% for the VanC-type E. casseliflavus and E. gallinarum until 12.3% (n=8) for Enterococcus faecalis. All E. faecalis isolates showed susceptibility to vancomycin, teicoplanin, ampicillin, gentamicin, and streptomycin. The virulence genes ace and esp were prevalent (100.0%, 87.5%). Multilocus variable number of tandem repeats (MLVA) revealed diversity in the number of repeats among E. faecalis isolates and targets, which was higher for espC, efa5, and efa6. We identified six different MLVA genotypes that were divergent from those described in human beings. Also, they were clustered into two genogroups that showed host-specificity for the species Cebus apella or Callithrix penicillata. In conclusion, no vanA- or vanB-containing enterococci were found colonizing those primate individuals. This finding suggested that the primate individuals investigated in our study are not directly involved in the epidemiological chain of high-level vancomycin-resistant genes vanA or vanB in Brazil. Our study also showed that E. faecalis isolated from nonhuman primates carry virulence traits and have ability to spread their lineages among different individuals.

Effects of infection with Toxoplasma gondii oocysts on the intestinal wall and the myenteric plexus of chicken (Gallus gallus)

This paper aims to analyze the effects of the Toxoplasma gondii infection in the intestinal wall and myenteric plexus of chicken (Gallus gallus). Ten 36-day-old chickens were separated into two groups: control and experimental, orally inoculated with oocysts of the T. gondii strain M7741 genotype III. After 60 days the birds were submitted to euthanasia and had their duodenum removed. Part of the intestinal segments was submitted to histological routine, HE staining, PAS histochemical technique, and Alcian Blue. Qualitative analysis of the intestinal wall and comparative measurements among the groups with respect to total wall thickness, muscle tunic, mucosa, and tunica mucosa were carried out. Caliciform cells were quantified. The other part of the intestinal segments was fixed in formol acetic acid and dissected having the tunica mucosa and the tela submucosa removed. Neurons were stained with Giemsa, counted, and measured. Chickens from the experimental group presented diarrhea and inflammatory infiltrates in the tunica mucosa, thickness reduction of all the parameters assessed in the intestinal wall, and an increase of the number of caliciform cells. There was a ~70% reduction regarding the intensity of myenteric neurons; and the remaining cells presented a reduction of ~2.4% of the perikarion and ~40.5% of the nucleus (p<0.05). Chronic infection induced by T. gondii oocysts resulted in intestinal wall atrophy, mucin secretion increase, death and atrophy of chicken myenteric plexus neurons. Death and atrophy of myenteric plexus neurons may be related with the causes of diarrhea observed in chickens with toxoplasmosis.

The use of enzyme-linked immunosorbent assay and immunoblotting for the detection of Campylobacter fetus immunoglobulins in the cervico-vaginal mucus of female cattle

An indirect enzyme-linked immunosorbent assay was developed to detect antigen-specific secretory IgA antibodies to Campylobacter fetus subsp. venerealis in bovine vaginal mucus with a protein extract of the Campylobacter fetus subsp. venerealis by the acid glycine extraction method. Mean optical density measurement (λ=450 nm) was 0.143±0.9. The most immunoreactive protein bands of the Campylobacter fetus subsp. venerealis or Campylobacter fetus subsp. fetus recognized by IgA in immunoblotting, using bovine vaginal mucus samples, migrate at 42.6 kDa. The protein that migrates at 93 kDa was recognized exclusively for C. fetus subsp. venerealis. A positive vaginal mucus sample of a cow from negative herd recognized antigens of C. jejuni subsp. jejuni e C. fetus subsp. fetus.

Parameter estimation and use of gamma interferon assay for the diagnosis of bovine tuberculosis in Brazil

This study aimed to evaluate the interference of tuberculin test on the gamma-interferon (INFg) assay, to estimate the sensitivity and specificity of the INFg assay in Brazilian conditions, and to simulate multiple testing using the comparative tuberculin test and the INFg assay. Three hundred-fifty cattle from two TB-free and two TB-infected herds were submitted to the comparative tuberculin test and the INFg assay. The comparative tuberculin test was performed using avian and bovine PPD. The INFg assay was performed by the BovigamTM kit (CSL Veterinary, Australia), according to the manufacturer's specifications. Sensitivity and specificity of the INFg assay were assessed by a Bayesian latent class model. These diagnostic parameters were also estimate for multiple testing. The results of INFg assay on D0 and D3 after the comparative tuberculin test were compared by the McNemar's test and kappa statistics. Results of mean optical density from INFg assay on both days were similar. Sensitivity and specificity of the INFg assay showed results varying (95% confidence intervals) from 72 to 100% and 74 to 100% respectively. Sensitivity of parallel testing was over 97.5%, while specificity of serial testing was over 99.7%. The INFg assay proved to be a very useful diagnostic method.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

Período de absorção intestinal de macromoléculas em cabritos recém-nascidos após a ingestão de colostro bovino

Após o nascimento, os cabritos são dependentes das imunoglobulinas colostrais devido às características placentárias que não permitem a passagem de macromoléculas da circulação materna. De acordo com a literatura, os cabritos possuem capacidade absortiva por até quatro dias. Muitos aspectos fisiológicos de outras espécies são aceitos e utilizados para caprinos, mas aqueles relacionados à transferência de imunidade passiva precisam de investigação. Os objetivos do presente estudo foram determinar o período de passagem de macromoléculas da mucosa intestinal para a circulação e a duração da proteção humoral transferida passivamente pela ingestão de colostro bovino e caprino. Sessenta cabritos recém-nascidos foram distribuídos em seis tratamentos: T 0 (n=25), ingestão natural de colostro caprino à vontade; T 1 (n=7), colostro bovino entre o nascimento e duas horas pós-parto; T 2 (n=7), ingestão de colostro bovino entre quatro e seis horas pós-nascimento; T 3 (n=7), leite nas primeiras oito horas e colostro bovino entre 10 e 12 horas pós-parto; T 4 (n=7), ingestão de leite até 18 horas e colostro bovino entre 22 e 24 horas pós-nascimento; T 5 (n=7), leite até 30 horas e ingestão de colostro bovino entre 34 e 36 horas pós-parto. Determinaram-se as concentrações séricas de proteína total (PT), gamaglobulina, imunoglobulina G (IgG) e a atividade sérica de gama glutamiltransferase (GGT). Ao nascimento, todos os neonatos tiveram valores mais baixos das variáveis, com aumento significativo da PT e gamaglobulina, após dois dias, nos grupos T 0, T 1 e T 2; a IgG e GGT aumentaram em todos os grupos. Os tratamentos T 3, T 4 e T 5 foram considerados como indutores de falha de transferência de imunidade passiva. A absorção de macromoléculas pelo trato intestinal dos cabritos ocorreu até 36 horas pós-parto, sendo mais efetiva até 12 horas. Os níveis de anticorpos persistiram até 75 dias após a ingestão de colostro bovino, porém, com concentrações inadequadas.

Obstrução intestinal em bovinos associada ao consumo de Stylosanthes sp. (Fabaceae Papilionoideae)

O objetivo deste trabalho é descrever, em três propriedades rurais no Estado de Mato Grosso, a epidemiologia, o quadro clínico e o patológico da obstrução intestinal por fitobezoares em bovinos que pastoreavam em piquetes com predomínio de Stylosanthes sp. pelo menos 60 dias. A morbidade variou de 3,3% a 15% e a mortalidade foi de 100%. O quadro clínico foi de cólica abdominal caracterizado por apatia, anorexia, inquietação, desconforto, sudorese e vocalização, seguido por episódios de diarreia ou diminuição da produção fecal, desidratação, hipomotilidade gastrintestinal, aumento do volume abdominal, decúbito esternal ou lateral com a cabeça voltada para o flanco e morte. O curso clínico foi de 2-7 dias. A principal alteração notada durante a necropsia dos bovinos foi a obstrução intestinal por fitobezoar ovoide de 2-5 cm de diâmetro, frequentemente no duodeno, próximo ao piloro, ocasionalmente em sua porção média. Na área de obstrução notou-se a necrose da parede intestinal que estava enegrecida e intensamente demarcada em relação a segmento adjacente normal, além de intenso espessamento associado a edema, congestão e hemorragia. O abomaso e os segmentos intestinais anteriores a obstrução apresentavam-se repletos de conteúdo alimentar líquido e posteriormente estava com ausência de conteúdo. O rúmen frequentemente tinha grande quantidade de conteúdo liquido a pastoso. Em todos os bovinos necropsiados múltiplas estruturas sólidas, arredondadas ou ovoides, esverdeadas (fitobezoares) foram observadas no abomaso. Estes achados indicam que pastagens com predomínio de Stylosanthes sp. predispõem a formação de fitobezoares, os quais podem levar à obstrução intestinal e morte em bovinos.

Utilização da vitamina D3 e seus metabólitos na alimentação de frangos de corte sobre parâmetros imunológicos e morfometria intestinal

Os efeitos da suplementação da vitamina D3 e de seus diferentes metabólitos foram avaliados na resposta imune e na morfometria intestinal de frangos de corte. Foram utilizados 952 frangos de corte de um dia de idade, distribuídos em um delineamento inteiramente casualizado, com quatro tratamentos, sete repetições e 34 aves por unidade experimental. Os tratamentos foram constituídos por quatros diferentes fontes de vitamina D3: colecalciferol (D3), 25-hidroxicolecalciferol (25(OH)D3), 1,25-dihidroxicolecalciferol (1,25(OH)2D3) e 1α-hidroxicolecalciferol (1α(OH)D3). As diferentes fontes foram incluídas na dieta, fornecendo 2000 e 1600 UI de vitamina D, nas fases pré-inicial, inicial e de crescimento, respectivamente. O peso relativo do intestino delgado diferiu entre os tratamentos aos 7, 21 e 42 dias e o peso relativo do fígado somente aos 42 dias de idade. Os demais órgãos e parâmetros imunológicos avaliados (peso dos órgãos linfóides, reação de hipersensibilidade cutânea basofílica, avaliação da atividade de macrófagos, dosagem de nitrito e perfil heterofilo: linfócito foram similares entre os animais alimentados com os diferentes metabólitos. Houve efeito (P<0,05) dos diferentes metabólitos da vitamina D3 sobre o comprimento dos vilos de jejuno e íleo aos sete dias, diferindo entre os animais alimentados com 1,25(OH)2D3 e 1α(OH)D3 para jejuno e 1,25(OH)2D3 e vitamina D3 para íleo (P<0,05). Para as demais fases não foi observado influência (P>0,05) dos tratamentos. Os parâmetros imunológicos não foram afetados pelos diferentes metabólitos de vitamina D. Os diferentes metabólitos de vitamina D afetaram positivamente a morfometria intestinal na altura de vilo na fase inicial, sendo os melhores resultados obtido pelos animais alimentados com 1,25(OH)2D3, contudo os parâmetros imunológicos foram similares entre os metabólitos estudados.