Vitamin B12 deficiency in newly weaned goat kids associated with clinical infection with Eimeria arloingi / Deficiência de vitamina B12 em cabritos recém-desmamados associada à infecção clínica por Eimeria arloingi

Abstract A severe outbreak of diarrhea associated with poor growth was reported in ten newly weaned goat kids that originated from a research farm (Group A). Two of these kids underwent necropsy examination. Five goat kids of the same age maintained in the same pen showed no clinical signs (Group B). The clinical, gross pathological and histopathological features of the clinically sick animals were consistent with severe coccidiosis. Group A animals had significantly lower levels of serum vitamin B12 (<200 pg/ml) compared with group B animals (2000 pg/ml). In addition, kids belonging to group A had significantly higher Eimeria arloingi oocysts per gram (OPG) of faeces (101,400/g) compared with kids of group B (9,154/g). Microscopy and molecular tools (18S rRNA and COI genes) confirmed that the goat kids were infected with the caprine protozoan parasite E. arloingi. This study provides a definitive association between low levels of serum vitamin B12 and clinical E. arloingi infection, and also provides support to our previous studies that demonstrated how low levels of serum vitamin B12 leads to an impairment of neutrophil function and thereby potential lowered immunity to pathogens.

Resumo Um surto grave de diarreia, associado à baixo crescimento, foi relatado em dez cabritos recém-desmamados, originários de uma fazenda de pesquisa (Grupo A). Dois animais foram submetidos a exame necroscópico. Cinco cabritos da mesma idade e mantidos na mesma instalação não apresentaram sinais clínicos (Grupo B). As características clínicas e as lesões macroscópicas e microscópicas dos animais clinicamente doentes eram consistentes com coccidiose grave. Os animais do grupo A apresentaram níveis significativamente mais baixos de vitamina B12 sérica (<200 pg / ml) em comparação com os animais do grupo B (2000 pg/ml). Além disso, os animais pertencentes ao grupo A apresentaram um número de oocistos de Eimeria arloingi por grama (OPG) de fezes (101,400/g) significativamente mais alto do que os animais do grupo B (9,154/g). As análises microscópica e molecular (genes 18S rRNA e COI) confirmaram que os cabritos estavam infectados com o protozoário E. arloingi. Este estudo fornece uma associação definitiva entre baixos níveis de vitamina B12 no soro e infecção clínica por E. arloingi. Também fornece suporte aos estudos anteriores, que demonstraram como baixos níveis de vitamina B12 no soro comprometem a função dos neutrófilos e, consequentemente, a imunidade a patógenos.

Neutrophil extracellular traps in the intestinal mucosa of Eimeria-infected animals

Objective: To investigate the presence of neutrophil extracellular traps (NETs) in vivo by analysing intestinal sections from experimentally Eimeria bovis- and naturally Eimeria arloingi-infected animals. Methods: Intestinal samples of Eimeria arloingi- and Eimeria bovis-infected animals were analysed by using immunohistochemical and fluorescence approach by using monoclonal antibodies. Results: Classical NET components were confirmed by co-localization of extracellular DNA being decorated with neutrophil elastase and histones in Eimeria-infected tissue samples. Here, extrusion of NETs was exclusively detected in intestinal polymorphonuclear neutrophils infiltrating Eimeria-infected sites. In vivo NETs were either found in close proximity or in direct contact to different Eimeria stages suggesting a stage-independent process. NETs were also found within the gut lumen driven by polymorphonuclear neutrophils that were contacting released oocysts. Conclusions: We postulate that NETs might play an important role in innate defence reactions in coccidiosis therefore significantly altering the outcome of infection.

Dynamic Patterns of Systemic Innate Immunity and Inflammatory Associated Factors in Experimental Caprine Coccidiosis

The present study was designed to assess the dynamic patterns of pro-inflammatory cytokines, including IFN-γ, TNF-α, IL-4, IL-6, acute phase protein (α1-acid-glycoprotein, AGP), and an inflammation associated factor (adenosine deaminase; ADA) following experimental caprine coccidiosis. Ten kids aging from 2 to 4 months were infected orally with 5×104 sporulated oocysts and 10 animals served as controls. Blood samples were collected in both groups before infection and at days 3, 7, 14, 21, 28, and 35 post-infection (PI), and the levels of above-mentioned factors were measured. IFN-γ, TNF-α, IL-4, IL-6, AGP, and ADA activities were significantly higher in infected animals from day 7 PI (P<0.05). In conclusion, the circulatory levels of most systemic inflammatory markers, including pro-inflammatory cytokines (IFN-γ, TNF-α, IL-4, IL-6), AGP, and ADA increased significantly starting from day 3 to day 7 PI in caprine coccidiosis.

Changing Patterns of Acute Phase Proteins and Inflammatory Mediators in Experimental Caprine Coccidiosis

This experiment was conducted to assess the changing patterns and relative values of acute phase proteins and inflammatory cytokines in experimental caprine coccidiosis. Eighteen newborn kids were allocated to 3 equal groups. Two groups, A and B, were inoculated with a single dose of 1x10(3) and 1x10(5) sporulated oocysts of Eimeria arloingi, respectively. The third group, C, received distilled water as the control. Blood samples were collected from the jugular vein of each kid in both groups before inoculation and at days 7, 14, 21, 28, 35, and 42 post-inoculation (PI), and the levels of haptoglobin (Hp), serum amyloid A (SAA), TNF-alpha, and IFN-gamma were measured. For histopathological examinations, 2 kids were selected from each group, euthanized, and necropsied on day 42 PI. Mean Hp concentrations in groups A and B (0.34 and 0.68 g/L) at day 7 PI were 3.2 and 6.3 times higher than the levels before inoculation. The mean SAA concentrations in groups A and B (25.6 and 83.5 microg/ml) at day 7 PI were 4.2 and 13.7 times higher than the levels before inoculation. The magnitude and duration of the Hp and SAA responses correlated well with the inoculation doses and the severity of the clinical signs and diarrhea in kids. These results were consistent with the histopathological features, which showed advanced widespread lesions in group B. In both groups, significant correlations were observed for TNF-alpha and IFN-gamma with SAA and Hp, respectively. In conclusion, Hp and SAA can be useful non-specific diagnostic indicators in caprine coccidiosis.