Neonatal Diarrheic Calves Research Articles

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.

Anion gap correlates with serum D- and DL-lactate concentration in diarrheic neonatal calves.

The objective of this study was to investigate the relationship between serum D- and L-lactate concentrations, and anion gap (AG) in neonatal calves. The association of AG with lactic acidosis in diarrheic calves has only been investigated by measurement of L-lactate in calves with experimentally induced diarrhea. D-lactate has recently been reported to be present in high concentrations in the serum of some diarrheic neonatal calves. The contribution of this acid to AG is not reported. The relationship between AG and L- and D-lactate concentrations was examined in 24 healthy calves and 52 calves with naturally occurring infectious diarrhea with metabolic acidosis. AG was calculated as [Na+ + K+] - [Cl- + HCO3-]. D- and L-lactate were quantified using high-performance liquid chromatography. There was no correlation between L-lactate and AG, contrary to previous reports in the literature. Moderate correlations between D-lactate concentration and AG (r = .74, P < .0001), and between DL-lactate and AG (r = .77), P < .0001) were detected. No differences existed due to the age or sex of the calf. This study indicates that AG provides information on the nature of acidosis in the diarrheic, neonatal calf and reinforces the importance of investigating clinical, in addition to experimental, populations.

Anion Gap Correlates with Serum d- and dl-Lactate Concentration in Diarrheic Neonatal Calves

Anion Gap Correlates with Serum d- and dl-Lactate Concentration in Diarrheic Neonatal Calves

Rotavirus and concurrent infections with other enteropathogens in neonatal diarrheic dairy calves in Spain

Faeces samples from 218, one to 30 days old, diarrheic dairy calves in 65 dairy herds were screened for the presence of rotavirus and concurrent infections with coronavirus, Cryptosporidium, F5 + Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1–7, 8–14, 15–21 and 22–30 days. Rotavirus infection was detected in 46.9%, 45.6%, 33.8% and 48.3% of the calves in the respective age-groups. No significant differences in the detection rate of rotavirus were found among calves on the different age-groups. Rotavirus was the only enteropathogen detected in 39 of the 93 (41.9%) diarrheic calves positive to this agent. Concurrent infections with other enteropathogen(s) were detected in 31.3%, 33.3%, 20.6% and 3.4% of the rotavirus infected calves in the age-groups 1–7, 8–14, 15–21 and 22–30 d, respectively. A significant age-associated decrease in the detection rate of mixed infections ( p<0.01) was found. The detection rates of the other enteropathogens considered in calves with rotavirus infection were 20.4% for coronavirus, 85.2% for Cryptosporidium, 16.7% for F5 + E. coli and 1.8% for Salmonella.

Morphology and morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves

The morphology and morphogenesis of virus strain LY-138 recovered from neonatal diarrheic calves were investigated by electron microscopy using negativestaining techniques and ultrathin sectioning. Purified viral particles were spherical in shape and measured 90 nm in average diameter in negatively stained preparations. Pleomorphic forms were also present. The virions had envelopes with petal-shaped projections characteristic of coronaviruses. In ultrathin sections, cores in viral factories were round with a diameter of 50–60 nm. Most of these cores were electron dense but some had an electron-lucent center. In cytoplasmic vacuoles, Golgi vesicles, and on the apical plasmalemma of intestinal epithelial cells, the virions were round or ellipsoidal in shape, measuring 70–80 nm in diameter, and had fine thread-like projections on their surfaces. Uptake of virus occurred through fusion of viral envelopes with the plasmalemma of the microvillous border or by entry into intercellular spaces and interaction with the lateral cell membranes of adjacent intestinal epithelial cells. As a result of this interaction, the lateral cell membranes became altered and ill-defined. During the early stage of infection, the rough andasmooth elements of the endoplasmic reticulum became distended with electron-dense granulofibrillar material. This material accumulated subsequently as well-defined, smooth membrane-bound areas mainly in the apical cytoplasm of infected cells. These structures were considered to be viral factories. The morphogenesis of virus occurred mainly through condensation of the electron-dense, granulo-fibrillar material into viral cores in cytoplasmic viral factories or within the distended cisternes of the rough endoplasmic reticulum. Viral envelopment occurred on membranes of cytoplasmic vacuoles, Golgi vesicles, or in association with membranes of viral factories. Release of virus from infected cells occurred by lysis and fragmentation of the apical plasmalemma and flow of the cytoplasm with its contents into the gut lumen. Release also occurred by digestion and lysis of extruded infected cells or by fusion of virus-containing cytoplasmic vacuoles with the apical plasmalemma and liberation of their contents.

Algorithms for selected blood acid-base and blood gas calculations.

Algorithms for selected blood acid-base and blood gas calculations.