Oral vitamin A supplementation of porcine epidemic diarrhea virus infected gilts enhances IgA and lactogenic immune protection of nursing piglets.

Vitamin A (VA) has pleiotropic effects on the immune system and is critical for mucosal immune function and intestinal lymphocyte trafficking. We hypothesized that oral VA supplementation of porcine epidemic diarrhea virus (PEDV)-infected pregnant gilts would enhance the gut-mammary gland-secretory IgA axis to boost lactogenic immunity and passive protection of nursing piglets against PEDV challenge. Gilts received daily oral retinyl acetate (30 000 IU) starting at gestation day 76 throughout lactation. At 3-4 weeks pre-partum, VA-supplemented (PEDV + VA) and non-supplemented (PEDV) gilts were PEDV or mock inoculated (mock + VA and mock, respectively). PEDV + VA gilts had decreased mean PEDV RNA shedding titers and diarrhea scores. To determine if lactogenic immunity correlated with protection, all piglets were PEDV-challenged at 3-5 days post-partum. The survival rate of PEDV + VA litters was 74.2% compared with 55.9% in PEDV litters. Mock and mock + VA litter survival rates were 5.7% and 8.3%, respectively. PEDV + VA gilts had increased PEDV IgA antibody secreting cells and PEDV IgA antibodies in serum pre-partum and IgA+ß7+ (gut homing) cells in milk post piglet challenge compared with PEDV gilts. Our findings suggest that oral VA supplementation may act as an adjuvant during pregnancy, enhancing maternal IgA and lactogenic immune protection in nursing piglets.

Impact of nutrition and rotavirus infection on the infant gut microbiota in a humanized pig model.

BACKGROUND: Human rotavirus (HRV) is a major cause of viral gastroenteritis in infants; particularly in developing countries where malnutrition is prevalent. Malnutrition perturbs the infant gut microbiota leading to sub-optimal functioning of the immune system and further predisposing infants to enteric infections. Therefore, we hypothesized that malnutrition exacerbates rotavirus disease severity in infants. METHODS: In the present study, we used a neonatal germ free (GF) piglets transplanted with a two-month-old human infant's fecal microbiota (HIFM) on protein deficient and sufficient diets. We report the effects of malnourishment on the HRV infection and the HIFM pig microbiota in feces, intestinal and systemic tissues, using MiSeq 16S gene sequencing (V4-V5 region). RESULTS: Microbiota analysis indicated that the HIFM transplantation resulted in a microbial composition in pigs similar to that of the original infant feces. This model was then used to understand the interconnections between microbiota diversity, diet, and HRV infection. Post HRV infection, HIFM pigs on the deficient diet had lower body weights, developed more severe diarrhea and increased virus shedding compared to HIFM pigs on sufficient diet. However, HRV induced diarrhea and shedding was more pronounced in non-colonized GF pigs compared to HIFM pigs on either sufficient or deficient diet, suggesting that the microbiota alone moderated HRV infection. HRV infected pigs on sufficient diet showed increased microbiota diversity in intestinal tissues; whereas, greater diversity was observed in systemic tissues of HRV infected pigs fed with deficient diet. CONCLUSIONS: These results suggest that proper nourishment improves the microbiota quality in the intestines, alleviates HRV disease and lower probability of systemic translocation of potential opportunistic pathogens/pathobionts. In conclusion, our findings further support the role for microbiota and proper nutrition in limiting enteric diseases.

Lactogenic immunity and vaccines for porcine epidemic diarrhea virus (PEDV): Historical and current concepts.

Morbidity, mortality, and loss of productivity from enteric diseases in neonatal piglets cost swine producers millions of dollars annually. In 2013-2014, the porcine epidemic diarrhea virus (PEDV) outbreak led to $900 million to $1.8 billion in annual losses to US swine producers. Passive lactogenic immunity remains the most promising and effective way to protect neonatal suckling piglets from enteric diseases like PEDV. Protecting suckling piglets through lactogenic immunity is dependent on trafficking of pathogen-specific IgA plasmablasts to the mammary gland and accumulation of secretory IgA (sIgA) antibodies in milk, defined as the gut-mammary-sIgA axis. Due to an impermeable placenta, piglets are born agammaglobulinic, and are highly susceptible to a plethora of infectious agents. They rely solely on colostrum and milk antibodies for maternal lactogenic immunity. Previous advances in the development of live and attenuated vaccines for another devastating diarrheal virus of pigs, transmissible gastroenteritis virus (TGEV), provide insights into the mechanisms of maternal immunity and piglet protection. In this chapter, we will review previous research on TGEV-induced lactogenic immunity to provide a historical perspective on current efforts for PEDV control and vaccines in the swine industry. Identifying factors that influence lactogenic immunity and the gut-mammary-sIgA axis may lead to improved vaccine regimens for PEDV and other enteric pathogens in gestating swine and improved overall herd immunity, swine health and industry productivity.

Development of a quantitative PCR for the detection of Rangelia vitalii.

The aim of this study was to develop and validate a SYBR Green qPCR assay to detect and quantify a fragment of the 18S rRNA gene of Rangelia vitalii in canine blood. Repeatability of the qPCR was determined by the intra- and inter-assay variations. The qPCR showed efficiency of E=101.30 (r(2)=0.996), detecting as few as one copy of plasmid containing the target DNA. Specificity of the assay was performed using DNA samples of Babesia canis, B. gibsoni, Ehrlichia canis, E. ewingii and Leishmania sp. No cross-reactivity was observed. Field samples consisting of blood from 265 dogs from Porto Alegre, Brazil were also tested. A total of 24 (9.05%) samples were positive for R. vitalii. Amplicons of 50% of positive samples were confirmed to be R. vitalii by Sanger sequencing. The positive samples had an average of 3.5×10(5) organisms/mL of blood (range: 1.27×10(3)-1.88×10(6)) based on the plasmid-generated standard curve. In conclusion, the SYBR Green qPCR assay developed herein is sensitive and specific and can be used as a diagnostic tool for detection and quantification of R. vitalii in canine blood samples.

Role of acute phase proteins in the immune response of rabbits infected with Trypanosoma evansi.

The aim of this study was to characterize the response of acute phase proteins (APP) in rabbits experimentally infected with Trypanosoma evansi (T. evansi), and to relate the findings with serum immunoglobulins levels, in order to verify the relation between APP and the immune response of rabbits. A total of 12 animals were used in this experiment and divided into 2 groups, control and infected, of six rabbits each. The experimental period was 118 days, and blood was collected on days 0, 5, 20, 35, 65, 95 and 118 post-infection (PI). The infection with T. evansi stimulated APP and immunoglobulins production, once the infected animals showed an increase in C-reactive protein, haptoglobin, alpha 2-macroglobulin and IgM levels. The elevation in IgM levels observed in this study, when related to the increase in C-reactive protein and haptoglobin levels, suggests the involvement of these proteins in host defense against flagellated protozoa, with possible participation in the control of the parasitemia in rabbits infected with T. evansi.

Iron metabolism and its relationship to anemia and immune system in Trypanosoma evansi infected rats.

The aim of this study was to evaluate biochemical parameters of iron metabolism in rats experimentally infected with Trypanosoma evansi. To this end, 20 rats (Wistar) were intraperitoneally inoculated with blood containing trypomastigotes 10(6) (Group T) and 12 animals were used as negative control (Group C) and received saline (0.2 mL) through same route. Blood samples were collected by cardiac puncture on day 5 (C5, T5) and 30 (C30, T30) post-inoculation (pi) to perform complete blood count and determination of serum iron, transferrin, ferritin, total and latent iron fixation capacity, transferrin saturation and prohepcidin concentration. Also, bone marrow samples were collected, to perform Pearls staining reaction. Levels of iron, total and latent iron binding capacity and prohepcidin concentration were lower (P<0.05) in infected rats (T5 and T30 groups) compared to controls. On the other hand, levels of transferrin and ferritin were higher when compared to controls (P<0.05). The transferrin saturation increased on day 5 pi, but decreased on day 30 pi. The Pearls reaction showed a higher accumulation of iron in the bone marrow of infected animals in day 5 pi (P<0.01). Infection with T. evansi in rats caused anemia and changes in iron metabolism associated to the peaks of parasitemia. These results suggest that changes in iron metabolism may be related to the host immune response to infection and anemic status of infected animals.

Trypanocidal activity of human plasma on Trypanosoma evansi in mice.

This study aimed to test an alternative protocol with human plasma to control Trypanosoma evansi infection in mice. Plasma from an apparently 27-year-old healthy male, blood type A+, was used in the study. A concentration of 100 mg.dL(-1) apolipoprotein L1 (APOL1) was detected in the plasma. Forty mice were divided into four groups with 10 animals each. Group A comprised uninfected animals. Mice from groups B, C and D were inoculated with a T. evansi isolate. Group B was used as a positive control. At three days post-infection (DPI), the mice were administered intraperitoneally with human plasma. A single dose of 0.2 mL plasma was given to those in group C. The mice from group D were administered five doses of 0.2 mL plasma with a 24 hours interval between the doses. Group B showed high increasing parasitemia that led to their death within 5 DPI. Both treatments eliminated parasites from the blood and increased the longevity of animals. An efficacy of 50 (group C) and 80% (group D) of human plasma trypanocidal activity was found using PCR. This therapeutic success was likely achieved in the group D due to their higher levels of APOL1 compared with group C.

Trypanocidal activity of human plasma on Trypanosoma evansi in mice / Atividade tripanocida do plasma humano sobre Trypanosoma evansi em camundongos

This study aimed to test an alternative protocol with human plasma to control Trypanosoma evansi infection in mice. Plasma from an apparently 27-year-old healthy male, blood type A+, was used in the study. A concentration of 100 mg.dL-1 apolipoprotein L1 (APOL1) was detected in the plasma. Forty mice were divided into four groups with 10 animals each. Group A comprised uninfected animals. Mice from groups B, C and D were inoculated with a T. evansi isolate. Group B was used as a positive control. At three days post-infection (DPI), the mice were administered intraperitoneally with human plasma. A single dose of 0.2 mL plasma was given to those in group C. The mice from group D were administered five doses of 0.2 mL plasma with a 24 hours interval between the doses. Group B showed high increasing parasitemia that led to their death within 5 DPI. Both treatments eliminated parasites from the blood and increased the longevity of animals. An efficacy of 50 (group C) and 80% (group D) of human plasma trypanocidal activity was found using PCR. This therapeutic success was likely achieved in the group D due to their higher levels of APOL1 compared with group C.

Este estudo teve como objetivo testar um protocolo alternativo com plasma humano para controlar a infecção por Trypanosoma evansi em camundongos. O plasma foi oriundo de um homem aparentemente saudável, com idade entre 27 anos e tipo de sangue A+. Foi detectada uma concentração de 100 mg.dL -1 de apolipoproteína L1 (APOL1) no plasma. Quarenta camundongos foram divididos em quatro grupos, contendo dez animais cada. Grupo A, composto de animais não infectados. Os roedores dos grupos B, C e D foram inoculados intraperitonealmente com um isolado de T. evansi. O Grupo B foi usado como um controle positivo. Três dias pós-infecção (DPI), os camundongos foram tratados com plasma humano. Uma dose única de 0,2 mL de plasma foi administrada nos roedores do grupo C. Os ratos do grupo D receberam cinco doses de 0,2 mL de plasma em intervalos de 24 horas. Os ratos do grupo B apresentaram parasitemia crescente, o que ocasionou a morte dos animais em 5 DPI. Ambos os tratamentos foram capazes de eliminar o parasito do sangue e aumentar a longevidade dos animais. O método da PCR detectou uma eficácia de 50% (grupo C) e 80% (grupo D) no tratamento com plasma humano. Este sucesso terapêutico obtido nos animais do grupo D provavelmente foi por receber maiores níveis de APOL1, comparado ao grupo C.

Lipid peroxidation associated with anemia in rats experimentally infected with Trypanosoma evansi.

This study aimed to assess the plasma lipid peroxidation and the susceptibility of erythrocytes to in vitro peroxidation as indicators of oxidative damage in erythrocytes and their roles in the pathogenesis of anemia during the early acute phase of Trypanosoma evansi infection in rats. Fifty male Wistar rats were randomly distributed into seven groups: three trypanosome-infected groups (T(2), T(4) and T(6); n=10 animals per group) and four uninfected controls (C(0), C(2), C(4) and C(6); n=5 animals per group). Animals from trypanosome-infected groups were inoculated intraperitoneally with 10(6) trypanosomes. Blood samples were collected by cardiac puncture before infection (day 0; group C(0)) or on the 2nd (C(2) and T(2)), 4th (C(4) and T(4)) and 6th (C(6) and T(6)) day post-infection (dpi). Samples were analyzed for red blood cell (RBC) count, hemoglobin (Hb) concentration, packed cell volume (PCV), plasma malondialdehyde (MDA) and in vitro peroxidation of erythrocytes. The mean values of the hematological indices gradually decreased in the infected rats compared with the control. MDA was significantly increased (P<0.001) on the 6th dpi in infected versus control animals and was negatively correlated with PCV (P<0.001; R(2)=0.372). The values for erythrocyte in vitro peroxidation were higher for groups T(4) and T(6) than for the control rats (P<0.01). A positive correlation between erythrocyte peroxidation and MDA (P<0.001; R(2)=0.414) was observed. The results of this study indicate that T. evansi infection in rats is associated with oxidative stress, indicated by lipid peroxidation and oxidative damage in erythrocyte membranes, as demonstrated by in vitro peroxidation. This may be one of the causes of anemia in acute trypanosomosis.