Second round of an interlaboratory comparison of SARS-CoV2 molecular detection assays used by 45 veterinary diagnostic laboratories in the United States

The COVID-19 pandemic presents a continued public health challenge. Veterinary diagnostic laboratories in the United States use RT-rtPCR for animal testing, and many laboratories are certified for testing human samples; hence, ensuring that laboratories have sensitive and specific SARS-CoV2 testing methods is a critical component of the pandemic response. In 2020, the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) led an interlaboratory comparison (ILC1) to help laboratories evaluate their existing RT-rtPCR methods for detecting SARS-CoV2. All participating laboratories were able to detect the viral RNA spiked in buffer and PrimeStore molecular transport medium (MTM). With ILC2, Vet-LIRN extended ILC1 by evaluating analytical sensitivity and specificity of the methods used by participating laboratories to detect 3 SARS-CoV2 variants (B.1; B.1.1.7 [Alpha]; B.1.351 [Beta]) at various copy levels. We analyzed 57 sets of results from 45 laboratories qualitatively and quantitatively according to the principles of ISO 16140-2:2016. More than 95% of analysts detected the SARS-CoV2 RNA in MTM at ≥500 copies for all 3 variants. In addition, for nucleocapsid markers N1 and N2, 81% and 92% of the analysts detected ≤20 copies in the assays, respectively. The analytical specificity of the evaluated methods was >99%. Participating laboratories were able to assess their current method performance, identify possible limitations, and recognize method strengths as part of a continuous learning environment to support the critical need for the reliable diagnosis of COVID-19 in potentially infected animals and humans.     

Herbage potassium levels in a permanent pasture under grazing

Herbage potassium levels were measured in 1986 in a permanent pasture under continuous grazing with cattle and receiving 200 kg N ha⁻¹. In April, before grazing started, K concentration in the herbage was relatively uniform across the pasture, with a value of 1·9 ± 0·038% K in the herbage dry matter. In July, a significantly lower concentration of herbage K was found in the grazed areas of the pasture (1·8 ± 0·10%) compared with the level (2·4 ± 0·088%) found in the rejected areas of the sward. The difference between the grazed and rejected areas was similar in September, with 1·6 ± 0·087% and 2·2 ± 0·172% K, respectively, in the herbage dry matter. This result suggests that herbage growth in the grazed areas might have been limited by K supply and highlights the need for more information on the K requirements of grazed grassland.     

In vitro fertilization and cortical granule distribution of bovine oocytes having heterogeneous ooplasm with dark clusters.

In vitro maturation, fertilization and subsequent development of oocytes with homogeneous (category 1), or heterogeneous ooplasm (category 2) were investigated. No significant differences were observed in the nuclear maturation and total fertilization rates between the two categories. However, category 2 oocytes showed a higher normal fertilization rate due to their lower incidence of polyspermy as compared to category 1 oocytes. Electron microscopic study revealed that all category 2 oocytes had cortical granules lined up next to the plasma membrane, and that some category 1 oocytes still had small clusters of cortical granules after maturation. Although the proportion of cleaved zygotes was higher in category 2, the percentages of cleaved zygotes that developed to the blastocyst stage did not differ between the two categories. These results demonstrate that oocytes with heterogeneous ooplasm have a higher capacity for normal fertilization due to the reduction in polyspermy. This can be attributed to the normal distribution of cortical granules in category 2 oocytes after maturation.     

Isolation and characterization of the eighth component of the bovine complement system

OBJECTIVE: To isolate and characterize the eighth component of the complement system (C8) in cattle. SAMPLE POPULATION: Fresh plasma obtained from beef cattle. PROCEDURES: Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The protein was identified throughout the procedure on the basis of its hemolytic function. Electrophoresis in polyacrylamide gels was used to determine molecular weight and composition of polypeptide chains. Reconstitution of classical and alternative complement pathways was used to characterize the hemolytic function of bovine C8. RESULTS: The bovine C8 protein consisted of a disulfide-bonded alpha-gamma heterodimer that was noncovalently associated with a beta chain. Apparent molecular weight of the alpha, beta, and gamma chains under reducing conditions were 66, 61, and 23 kd, respectively. In the classical pathway of activation, bovine C8 and the ninth component of the complement system (C9) had species incompatibility with human C8 and C9 on sheep erythrocyte target cells. CONCLUSIONS: A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemolytic assays to investigate the roles of complement-mediated lysis in the pathogenesis of inflammatory diseases and the killing of susceptible microorganisms.     

Melanin biosynthesis in Pyricularia oryzae: Site of tricyclazole inhibition and pathogenicity of melanin-deficient mutants

Tricyclazole (5-methyl-1,2,4-triazolo[3,4-b]benzothiazole) inhibits melanin synthesis in Pyricularia oryzae at concentrations less than 0.01 μg/ml. The primary site of inhibition in the biosynthetic pathway occurs between scytalone and vermelone. Accumulation of several metabolites derived from melanin precursors along branch pathways is associated with inhibition of melanin biosynthesis. At low tricyclazole concentrations (0.01–1 μg/ml), predominant accumulation of 2-hydroxyjuglone and 3,4-dihydro-3,4,8-trihydroxy-1-(2H)-naphthalenone (3,4,8-DTN) occurs as a result of the primary block between 1,3,8-trihydroxynaphthalene and vermelone. As the concentration of tricyclazole is increased from 1 to 10 μg/ml, flaviolin accumulation is markedly enhanced, whereas that of 3,4,8-DTN and 3,4-dihydro-4,8-dihydroxy-1-(2H)-naphthalenone is depressed, indicating possible secondary sites of inhibition in the main and branch pathways. Five melanin-deficient mutants of P. oryzae that phenotypically resemble the tricyclazole-treated wild-type strain were nonpathogenic or rarely infected two rice varieties. Three of the mutants studied were genetically defective in the melanin biosynthetic pathway at the site blocked by tricyclazole in the wild type. The wild-type strain converted both scytalone and vermelone to melanin; whereas the three mutants and the tricyclazole-treated wild type converted only vermelone to melanin. The data suggest a relationship between melanin biosynthesis and pathogenicity in P. oryzae.     

Clearance of ovine isolates of Bordetella parapertussis from murine trachea and lungs

Thirty-six mice were inoculated intranasally with Bordetella parapertussis organisms (isolated from sheep with chronic non-progressive pneumonia) to study their deposition and clearance in the lower respiratory tract. The deposition of the organism was greater in the trachea than in the lungs. At 48 hours after inoculation, almost 100% of organisms were cleared from the trachea but only 55% of organisms were cleared from the lungs. This result correlates well with the morphological changes seen previously in the pulmonary parenchyma and airways of mice and lambs experimentally infected with this organism.     

Secretion of dopamine and norepinephrine in hypophyseal portal blood and prolactin in peripheral blood of Holstein cattle

The objective was to test the hypothesis that dopamine regulates prolactin (PRL) secretion by determining acute changes in catecholamine concentrations in hypophyseal portal blood of cattle, and their relation to peripheral blood concentration of PRL in hypophyseal stalk-transected (HST) and sham-operated controls (SOC). Holstein heifers (606 +/- 21 kg BW; mean +/- SE) were subjected to neurosurgery for 8 h to collect hypophyseal portal blood with a stainless steel cannula designed with a cuff placed under the pituitary stalk and peripheral blood via a jugular vein catheter. PRL plasma concentration was measured by radioimmunoassay, and dopamine and norepinephrine in portal plasma by radioenzymatic assay. During anesthesia before HST or SOC, PRL plasma concentration ranged from 20-40 ng/ml throughout 255 min. PRL abruptly increased and remained above 90 ng/ml after HST compared with a steady decrease to <20 ng/ml in SOC heifers throughout 440 min. Within 5 min after severing the hypophyseal stalk, dopamine in portal blood (>8 ng/ml) was significantly increased (P < 0.05) compared with peripheral blood (<2 ng/ml). Norepinephrine concentration in portal blood was significantly greater (P < 0.05) than in peripheral blood during the first 60 min. The sustained high PRL level in peripheral plasma after severing the hypophyseal stalk stimulated hypothalamic dopamine secretion from hypophyseal portal vessels during the prolonged period of blood collection. Norepinephrine concentration in these cattle was greater in hypophyseal portal than in peripheral blood, implicating both an important hypothalamic source of the catecholamine as well as an adrenal gland contribution during anesthesia.