Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis. A simple flow cytometric method (FCM) for measuring phagocytosis of Staphylococcus aureus by human neutrophils (polymorphonuclear leukocytes [PMNs]) is described. This assay utilizes 100 microliters of EDTA-anticoagulated whole blood and a simplified method of fluorescently labeling bacteria. A commercially available whole-blood lysing reagent allows for the removal of erythrocytes and the exclusion of external free or adherent bacteria. Phagocytized bacteria are unaffected by this reagent, so PMNs containing internalized bacteria can be easily identified by FCM. Advantages of this method include the following: (i) small sample size, (ii) no requirement for PMN separation, (iii) rapid reliable method of labeling the bacteria, (iv) ability to distinguish between adherent bacteria and those which are actually internalized, (v) avoidance of vital dyes as quenching agents, and (vi) ability to fix cells and store for future FCM analysis.


Excretory-secretory antigenic components of Paragonimus heterotremus recognized by infected human sera. Antigenic components of Paragonimus heterotremus metabolic products were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis of sera from patients with P. heterotremus infection, from patients with other illnesses, and from healthy adults. By SDS-PAGE, it was found that the metabolic products comprised more than eight major polypeptides. Immunoblot analysis revealed 11 components which were strongly recognized by paragonimiasis antisera. These antigenic components had molecular masses ranging from less than 12.3 kDa to 144 kDa. One antigenic band of 31.5 kDa was found to give a consistent reaction with paragonimiasis antisera (97% sensitivity). Of the other patient sera, only sera from patients with Fasciola sp. infection reacted with antigenic bands of 56, 38, and 18.5 kDa. The present findings suggest that the 31.5-kDa component is sensitive and specific for the diagnosis of human P. heterotremus paragonimiasis.


Detection of genes in feces by booster polymerase chain reaction. A 321-bp fragment intragenic to the gene ereA carried by Escherichia coli BM2195 was used as a model target to study the conditions under which DNA amplification by booster polymerase chain reaction can be used to detect specific bacterial DNA sequences in fecal specimens. When target E. coli cells were mixed with 41 freshly obtained fecal specimens, the polymerase chain reaction detection limit varied from 4.5 to 7.1 log CFU/g of feces, depending on the individual fecal specimen used to prepare the test sample. These variations were not statistically related to the sex or age of the subject from whom the specimen was obtained. After storage of the samples for 4 weeks at room temperature on swabs or filter papers, no loss in sensitivity was observed.


Detection of Babesia microti by polymerase chain reaction. Human babesiosis, which is caused by infection with the intraerythrocytic malarialike protozoan Babesia microti, has recently been diagnosed with increasing frequency in residents of New England. Diagnosis is difficult because of the small size of the parasite and the sparse parasitemia that is characteristic of most infections with this pathogen. We generated B. microti-specific DNA sequence information by universal primer amplification of a portion of the eukaryotic 16S-like gene; this was followed by direct DNA sequence analysis. Specific primers were synthesized on the basis of this sequence information for use in the polymerase chain reaction (PCR). The PCR-based system demonstrates a strong bias for detection of B. microti as opposed to Babesia gibsoni and does not amplify vertebrate DNA. The analytical sensitivity of the system is approximately three merozoites. Blood specimens from 12 patients with clinically diagnosed and parasitologically confirmed babesiosis from Nantucket Island, Mass., were PCR positive in a blinded test of this procedure. Thus, DNA amplification may provide an adjunct to conventional methods for the diagnosis of human babesiosis and may provide a new means of monitoring therapy or enhancing epidemiological surveillance for this emerging pathogen.


Isolation and characterization of a newly identified Haemophilus species from cats: "Haemophilus felis". A gram-negative coccobacillus was isolated from the lower respiratory tract of a cat with chronic obstructive pulmonary disease. The isolate required CO2 and V factor for growth and was initially identified as Haemophilus paraphrophilus on the basis of its nutritional requirements, colony morphology, and some biochemical tests. Because of the host specificity of Haemophilus species and discrepancies in catalase, oxidase, and hemolytic activities, additional testing was done. Extensive biochemical testing, G+C content, and DNA reassociation studies indicated that the organism was distinct from other Haemophilus species. Therefore, the organism was identified as a previously unrecognized Haemophilus species and was tentatively named "Haemophilus felis." Bacteria identical to the original isolate were isolated from the nasopharynxes of 6 of 28 apparently normal cats, indicating that H. felis or H. felis-like organisms may be common members of the feline upper respiratory tract flora.


Beta-glucuronidase activities of fecal isolates from healthy swine. Research has shown that various percentages of fecal Escherichia coli isolates obtained from healthy subjects may be beta-glucuronidase negative. The ability to detect beta-glucuronidase activity among fecal E. coli isolates from healthy subjects may be affected by assay conditions. A study was conducted in which agar and broth media containing 4-methylumbelliferyl-beta-D-glucuronide (MUG) were used to examine beta-glucuronidase activities of fecal isolates from healthy swine. Rectal swabs were plated on MacConkey agar plus 100 mg of MUG per liter (MAC-MUG) and incubated at 35 degrees C for 24 h. Each of 986 isolates picked from MAC-MUG was inoculated into duplicate tubes of lauryl tryptose broth plus 50 mg of MUG per liter (LT-MUG). One set of tubes was incubated at 35 degrees C and the other set of tubes was incubated at 44.5 degrees C. Gas production and hydrolysis of MUG, indicated by fluorescence when observed with UV light with a wavelength of 360 nm, were determined after incubation for 24 and 48 h. A higher percentage (P less than 0.01) of isolates was MUG positive at 44.5 degrees C than at 35 degrees C after 24 h of incubation in LT-MUG. A higher percentage (P less than 0.01) of isolates was MUG positive after 48 h than after 24 h of incubation at both 35 and 44.5 degrees C. A lower percentage of isolates (P less than 0.05) was observed to be MUG positive on MAC-MUG agar compared with their MUG reactions in LT-MUG at 35 and 44.5 degrees C. Approximately 89% of the isolates identified were beta-glucuronidase-positive E. coli. The largest proportion of MUG-positive E. coli was detected with LT-MUG at 35 degrees C after 48 h of incubation.


Use of polymerase chain reaction for detection of toxigenic Vibrio cholerae O1 strains from the Latin American cholera epidemic. In January 1991, an outbreak of cholera started in Peru and spread throughout most of Latin America within 8 months. As of March 1992, over 450,000 cases and approximately 4,000 deaths have been reported to the Pan American Health Organization. The causative organism is toxigenic Vibrio cholerae O1 of the El Tor biotype and is distinct from the U.S. Gulf Coast strains. A polymerase chain reaction (PCR) that amplifies a 564-bp fragment of the cholera toxin A subunit gene (ctxA) was used to identify toxigenic V. cholerae O1 strains. A total of 150 V. cholerae O1 isolates were tested. They were of unknown toxin status, were associated with recent outbreaks, and were isolated from patients, food, and water. One hundred forty isolates were found to be toxigenic both by PCR and the routine diagnostic enzyme-linked immunosorbent assay. Thirty-eight known toxigenic strains isolated worldwide from 1921 to 1991 were also positive in the PCR. A collection of 18 nontoxigenic V. cholerae O1 strains, 35 Escherichia coli heat-labile-enterotoxin-I-producing strains, 26 Campylobacter strains, and 8 strains of Aeromonas hydrophila, previously reported to produce cholera toxin-like toxin, were all negative in the ctxA PCR. We conclude that this PCR is a diagnostic method that specifically detects toxin genes in V. cholerae O1 strains in a reference laboratory. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.


Development and clinical evaluation of a polymerase chain reaction test for detection of Chlamydia trachomatis. A polymerase chain reaction (PCR) for the detection of Chlamydia trachomatis was developed and evaluated. Two primer-probe sets were designed; one detected a specific sequence of the plasmid, and the other detected the gene encoding the major outer membrane protein. Both sets reacted species specifically and amplified sequences from all human serovars. A simple protocol was used for sample pretreatment. The PCR was optimized by addition of tetramethylammonium chloride and bovine serum albumin. The results of the PCR with the plasmid primer-probe set were compared with those of culture and the Chlamydiazyme and Gen-Probe PACE 2 tests for urogenital specimens from 220 patients. The rates of prevalence of infection with C. trachomatis were 22.7, 16.4, 15.0, and 14.5%, respectively. The sensitivities of the Chlamydiazyme and Gen-Probe PACE 2 assays compared with culture were 66.7 and 61.1%, respectively, and their sensitivities compared with PCR were 60.0 and 60.0%, respectively. The sensitivity of culture compared with PCR was 70.0%. Forty-eight of the 50 specimens positive by PCR with the plasmid primer-probe set could be confirmed by PCR with the major outer membrane protein primer-probe set or culture. It is concluded that the PCR is the most sensitive technique for laboratory detection of C. trachomatis.


Comparison of three methods for detection of group A streptococci in throat swabs. Group A streptococci are generally detected in throat swabs by (i) rapid antigen tests, (ii) conventional culture, or (iii) combinations of both. Direct fluorescent-antibody testing of a 2-h enrichment broth (FA/EN) was an accepted method for same-day results before the advent of rapid antigen tests. We compared FA/EN in Todd-Hewitt Broth (THB) with conventional culture and a rapid antigen test, TestPack Strep A (TPS). Nine hundred seventy specimens were evaluated in this study. Cultures were performed for 48 h on sheep blood agar (SBA) incubated aerobically and on a selective agar for group A streptococci (SSA) incubated in 5 to 10% CO2. Following a 2-h incubation, the fluorescent-antibody test was performed. A subculture of the centrifuged sediment from the THB enrichment was also done. In comparison with a positive culture on SBA or SSA or subculture of the THB pellet, the sensitivities and specificities of the different methods were as follows: SBA, 92 and 100%; SSA, 92 and 100%; TPS, 68 and 99%; FA/EN, 88 and 98%. The FA/EN method offers the potential for definitive finalized reports on the same day as specimen collection with greater sensitivity than TPS. This study included sequential plating and rapid antigen testing of a single swab. In a separate set of experiments to validate this study design, it was shown that recovery of streptococci from swabs plated sequentially on five plates did not vary with the order of plating and the actual proportion of organisms recovered from a swab on a single plate was only 1%.


Comparison of a neutralization enzyme immunoassay and an enzyme-linked immunosorbent assay for evaluation of immune status of children vaccinated for mumps. A 50% neutralization enzyme immunoassay (N50-EIA) was compared with an indirect enzyme-linked immunosorbent assay (ELISA) for determining mumps virus antibodies in three consecutive serum samples from 138 children vaccinated with a live mumps vaccine at the age (in years) of 1.5. By the N50-EIA, most (132 of 138) preserum samples did not show neutralizing activity. Eight to 12 weeks after vaccination, 17 of the children were still negative, but only 7 remained so after 2.5 years, resulting in a seroconversion rate of 125 of 132 (95%). Over the same period, the neutralization geometric mean titer rose from 3.6 to 9.9. By an indirect ELISA, 128 of 138 preserum samples were found negative. The early and late postvaccination sera of 8 children were ELISA negative, resulting in a seroconversion rate of 120 of 128 (94%). Only two children remained seronegative by both methods. Seven of the late postvaccination serum samples yielded noncorresponding results, reflecting 95% correlation between both methods. Due to cross-reactivity with parainfluenza viruses, the ELISA proved to be less specific, but on the other hand, it showed a greater sensitivity than the N50-EIA.


Evaluation of the E test by using selected gram-positive bacteria. The E test (AB Biodisk NA Inc.) was compared with standard reference methods using the National Committee for Clinical Laboratory Standards's recommendations for determining the MICs of four selected antibiotics against 208 clinical isolates of gram-positive bacteria. These bacteria included 32 strains of Streptococcus pneumoniae, 25 strains of Enterococcus faecium, 20 strains of oxacillin-sensitive Staphylococcus aureus (OSSA), 96 strains of oxacillin-resistant S. aureus (ORSA), and 35 strains of coagulase-negative staphylococci. Evaluation included MIC accuracy within 1 dilution, reproducibility testing, and cost analysis. There was 94% agreement between the E test and the reference method in testing S. pneumoniae and penicillin G. There was 92% agreement with ampicillin and 100% agreement with vancomycin in testing E. faecium isolates. Accuracy of the oxacillin E test with staphylococci was significantly improved by the use of salt-supplemented Mueller-Hinton agar, for an agreement of 100% with coagulase-negative staphylococci and oxacillin-sensitive S. aureus and that of 85% with oxacillin-resistant S. aureus, with no major discrepancies. The E test with American Type Culture Collection isolates and clinical strains gave excellent reproducibility and was less costly than microdilution panels when used to test fewer than three antibiotics. The E test is a simple, reliable, reproducible, and cost-effective method for MIC determination for gram-positive organisms.


Enterohemorrhagic Escherichia coli associated with hemolytic-uremic syndrome in Chilean children. A clinicoepidemiological study was undertaken to determine if enterohemorrhagic Escherichia coli (EHEC) was associated with hemolytic-uremic syndrome (HUS) in children in Santiago, Valdivia, and Temuco, Chile. Prospective surveillance detected 20 hospitalized cases of HUS in children less than 4 years of age in these cities from March 1988 to March 1989. Each HUS patient was matched (by sex and age) with two control children (hospitalized elective-surgery patients). To detect EHEC, DNA from stool culture isolates of E. coli was detected by hybridization with biotin-labelled DNA probes specific for the EHEC virulence plasmid, Shiga-like toxin I (SLT-I) or SLT-II. Stool cultures from 6 of 20 cases (30%) and from 2 of 38 controls (5.3%) yielded EHEC (P = 0.0158). EHEC isolates from all HUS cases hybridized with the EHEC plasmid probe and with probes for SLT-I or -II (or both). The serogroups of the isolates included O157, O26, and O111. EHEC causes HUS in Chile, and the biotinylated gene probes are practical diagnostic tools for epidemiologic studies.


Retrospective evaluation of two latex agglutination tests for detection of circulating antigens during invasive candidosis. Two latex agglutination tests for the detection of Candida antigens, Pastorex Candida (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) and Cand-Tec (Ramco Laboratories, Inc., Houston, Tex.), were applied to 79 serum samples from 19 patients who were retrospectively selected on the basis of mycological and clinical evidence of C. albicans infection and the availability of serial serum samples taken near the date of a positive culture. The specificity in 60 control individuals was 100% for Pastorex and 98.3% for Cand-Tec. The tests scored positive for 10 (52.6%) and 9 (47.4%) patients, respectively. Pastorex detected antigen in only 3 of 12 patients (25%) with positive antibody detection tests, but was positive for all 7 patients (100%) who produced no or a low antibody response, suggesting that the test performs better in the absence of antibodies. However, the sensitivity of Pastorex also increased with the number of samples available per patient, which was lower for high-antibody-responder patients (2.8 versus 5.7). If the patients who provided only one or two serum samples were eliminated, the sensitivity of Pastorex rose to 76.9%. For the Cand-Tec, the sensitivity was not related to the presence of antibodies, nor was it related to the number of samples per patient. The observed antigenemia was transient with both Pastorex and Cand-Tec. Only 12.5% of the positive reactions occurred on the same serum sample, confirming that the two tests react with different antigens. A positive antigen test preceded other diagnostic indications for 6 of 10 Pastorex-positive patients and 5 of 9 Cand-Tec-positive patients.


Presence of specific immunoglobulin A-secreting cells in peripheral blood after natural infection with Shigella sonnei. The appearance of antigen-specific immunoglobulin A (IgA) antibody-secreting cells (ASCs) following natural infection with Shigella sonnei during a common-source outbreak caused by this organism was evaluated in a modified enzyme-linked immunosorbent assay (ELISPOT). A mean IgA ASC value of 2,131.6/10(6) cells against homologous S. sonnei lipopolysaccharide (LPS) was detected in blood samples obtained from patients with bacteriologically proven S. sonnei shigellosis 5 and 10 days after the onset of disease. In the same blood samples, the level of ASC measured against heterologous antigen (Shigella flexneri serotype 2a LPS) was significantly lower than that of the homologous antigen (mean value, 33.12/10(6) cells). Furthermore, the mean number of activated B cells that secreted anti-S. sonnei LPS antibodies was significantly higher among patients with S. sonnei shigellosis than it was among patients with non-Shigella diarrhea (2.5/10(6) cells; standard error, 1.0) and healthy subjects (5.1/10(6) cells; standard error, 2.3) (P less than 0.05). The anti-LPS IgA ASC activity was easily detected within 5 days of the onset of disease, a point at which the levels of anti-S. sonnei LPS IgG and even IgA antibodies were hardly detectable in serum.


DNA fingerprinting of Escherichia coli O157:H7 strains by pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis of genomic DNA was carried out on Escherichia coli O157:H7 strains from different geographic locations to determine its value in an epidemiological survey of O157 infections. Pulsed-field gel electrophoresis of XbaI-digested DNA fragments clearly separated E. coli O157:H7 strains from nontoxigenic E. coli O157:H19, O157:H43, and O157:H45 strains and from Shiga-like-toxin-producing E. coli strains of other serogroups. However, among the E. coli O157:H7 strains, the restriction patterns either were identical or differed only by a few fragment bands. In some cases, it was therefore impossible to distinguish among epidemiologically unrelated strains. Hybridization experiments with a DNA probe complementary to Shiga-like toxin II sequences revealed that the Shiga-like toxin II genes were located on DNA fragments of different lengths. Our data show that for a single highly conserved clone, such as E. coli O157:H7, other typing techniques may need to be performed in addition to DNA fingerprinting in epidemiological surveys.


Characterization of a DNA probe for detection of Mycobacterium tuberculosis complex in clinical samples by polymerase chain reaction. We cloned and sequenced a DNA fragment from Mycobacterium tuberculosis for use in the identification of members of the M. tuberculosis complex. The DNA probe for culture confirmation had a sensitivity and a specificity of 100%. By using primers developed from this probe, the polymerase chain reaction detected 20 mycobacteria by ethidium bromide staining. This polymerase chain reaction system demonstrated 98% sensitivity and 100% specificity for detection of the M. tuberculosis complex in 200 sputum specimens.


Evaluation of a urease-based confirmatory enzyme-linked immunosorbent assay for diagnosis of Neisseria gonorrhoeae. A new urease-based enzyme-linked immunosorbent assay utilizing novel monoclonal antibodies was evaluated for the culture confirmation of Neisseria gonorrhoeae, with 270 isolates of N. gonorrhoeae, 56 isolates of diverse Neisseria spp., and 29 Moraxella isolates. The test was highly specific (100.00%) and sensitive (97.83%). No cross-reactions were observed with any of the Neisseria or Moraxella isolates tested. Fifty percent (3 of 6) of the false-negative results were obtained with isolates of serovar IA-4, a serovar rarely encountered in North America.


Evaluation of two colored latex kits, the Wellcolex Colour Salmonella Test and the Wellcolex Colour Shigella Test, for serological grouping of Salmonella and Shigella species. Two colored latex kits (the Wellcolex Colour Salmonella Test [WCT-Salmonella] and the Wellcolex Colour Shigella Test [WCT-Shigella]; Division Diagnostics, Laboratories Wellcome S.A., Paris, France), which allow identification of the most frequently encountered Salmonella serogroups and Shigella species, respectively, were evaluated. WCT-Salmonella and WCT-Shigella yielded sensitivities of 98.4 and 98%, respectively, and a specificity of 100% when they were tested on pure cultures received at a reference laboratory.


Further consideration of the clonal nature of Salmonella typhi: evaluation of molecular and clinical characteristics of strains from Indonesia and Peru. We examined envelope protein profiles, chromosomal restriction endonuclease digest patterns, and immune responses to envelope proteins for collections of Salmonella typhi strains isolated in Peru and Indonesia. Only minor differences in envelope protein patterns were apparent among strains. Strains from 7 of 20 Indonesian patients had a distinct chromosomal digest pattern compared with patterns of Peruvian and other Indonesian strains. Strains with this pattern carried the gene for the j flagellar antigen (H1-j); differences in response to envelope proteins of j and d strains were noted on immunoblot analysis. Our data suggest that there are genotypic and phenotypic differences among S. typhi strains. The clinical importance of these differences remains to be fully evaluated; however, in this study it was not possible to show a clear correlation between strain characteristics and disease severity.


Proposed interpretive criteria and quality control parameters for testing susceptibility of Neisseria gonorrhoeae to beta-lactam-clavulanate combinations. To support future clinical studies, in vitro susceptibility tests were examined to determine whether Neisseria gonorrhoeae could be tested reliably against two beta-lactam-clavulanate combinations. All isolates that were tested appeared to be susceptible to amoxicillin and ticarcillin in combination with clavulanic acid. In the absence of resistant isolates, only a breakpoint for a susceptible category could be defined for agar dilution tests with amoxicillin-clavulanic acid (MIC of less than or equal to 2.0/1.0 micrograms/ml is tentatively proposed). For disk diffusion tests, a corresponding breakpoint zone diameter of greater than or equal to 28 mm is suggested. The validity of the breakpoints for penicillinase-negative penicillin-resistant strains awaits clinical data. Proposed quality control limits for testing amoxicillin-clavulanic acid by agar dilution and disk diffusion methods are a MIC of 0.25/0.125 to 1.0/0.5 micrograms/ml and zones of 30 to 40 mm in diameter for N. gonorrhoeae ATCC 49226, a MIC of 0.125/0.06 to 0.5/0.25 micrograms/ml for Staphylococcus aureus ATCC 29213, and zones of 30 to 38 mm for S. aureus ATCC 25923. Ticarcillin-clavulanate is currently tested against other species by preparing doubling dilutions of ticarcillin with a constant 2 micrograms of clavulanate per ml. By that method, all gonococci were susceptible to low concentrations. However, the amount of clavulanic acid that is included (2 micrograms/ml) will, by itself, inhibit many strains of N. gonorrhoeae. Consequently, the role of ticarcillin in the combination cannot be determined, and such tests are not recommended.


Primary isolation of human T-cell leukemia-lymphoma virus types I and II: use for confirming infection in seroindeterminate blood donors. We describe the use of an immunofluorescence assay and coculture to confirm human T-cell leukemia-lymphoma virus (HTLV) infection. Peripheral blood mononuclear cells from 32 of 32 seropositive donors were positive in the immunofluorescence assay, and 63% of their cocultures produced p24 antigen. Specific antibodies distinguished HTLV type I (HTLV-I) from HTLV-II. HTLV-I or HTLV-II was isolated from donors with indeterminate serologic test results.


In vitro antibody secretion by peripheral blood mononuclear cells as an expression of the immune response to Brucella spp. in humans. We developed an assay to detect antibodies spontaneously secreted in vitro by peripheral blood mononuclear cells (PBMC) against Brucella spp. High levels of anti-Brucella immunoglobulin G (IgG) and/or IgM and/or IgA antibodies were detected in the cell supernatant solution of PBMC cultures for 12 patients suffering from acute or focalized brucellosis and for 5 patients recently vaccinated against brucellosis. This spontaneous in vitro antibody production disappeared 5 to 20 months after onset of clinical signs and 20 to 27 days after vaccination. The transient character of this anti-Brucella antibody production by PBMC is consistent with a temporary in vivo stimulation of the immune system by Brucella antigens. Detection of this secretion could improve the diagnosis of evolutive brucellosis.


Comparison of the MEASLESTAT M test kit with the sucrose density gradient centrifugation-hemagglutination inhibition method for detection of measles virus-specific immunoglobulin M. A new commercial kit, MEASLESTAT M (Whittaker Bioproducts, Inc.), was compared with the sucrose density gradient centrifugation-hemagglutination inhibition method for the detection of measles virus-specific immunoglobulin M. Overall agreement between the two procedures was 97.1% for 104 single and paired serum samples tested. The sensitivity and specificity of MEASLESTAT M were 98.4 and 95.2%, respectively.


Identification of Ehrlichia chaffeensis morulae in cerebrospinal fluid mononuclear cells. We report a case of ehrlichiosis in a 72-year-old man who developed extreme lethargy, acute renal failure requiring hemodialysis, and respiratory insufficiency requiring intubation. Lumbar puncture performed on the second day of hospitalization revealed significant cellular pleocytosis. Ehrlichia morulae were tentatively identified in mononuclear cells in routinely processed Wright-stained cytospin preparations of cerebrospinal fluid (CSF). Identification was confirmed by a specific immunocytochemical staining procedure. Subsequent identification specifically as Ehrlichia chaffeensis morulae was established by polymerase chain reaction analysis, which revealed E. chaffeensis-specific DNA in CSF, bone marrow, and blood samples; by indirect fluorescent-antibody analysis, the patient developed an antibody titer of 32,768 against E. chaffeensis antigen. The patient responded to intravenous therapy with doxycycline and dexamethasone. Subsequently, neurologic, hematologic, renal, and pulmonary status had returned to baseline at follow-up 12 weeks after admission. To our knowledge, this is the first identification of E. chaffeensis morulae in CSF cells in an infected patient.


Clamped homogeneous electric field gel electrophoresis typing of Torulopsis glabrata isolates causing nosocomial infections. Thirty isolates of Torulopsis glabrata were examined by pulsed-field gel electrophoresis, which resolved 13 DNA pieces, allowing the identification of 12 types. Bands at 1,400, 1,200, 1,070, 1,025, 681, and 500 kbp were conserved. When applied to 18 isolates from an outbreak, 10 distinct types were identified by this technique. Seven patients had isolates which were identical.


Ontogenesis of the pyramidal cell of the mammalian neocortex and developmental cytoarchitectonics: a unifying theory. The prenatal development of the mammalian neocortex has been analyzed, with the rapid Golgi method, in a variety of experimental animals (hamster, mouse, rat, and cat) and in humans. A new developmental conception of the structural organization of the mammalian neocortex is discussed. Neocortical development begins with the establishment of the primordial plexiform layer (PPL) which precedes and is a prerequisite for the subsequent formation of the cortical plate (CP). The formation of the CP occurs, in its entirety, within the PPL. During its development, three fundamental neuronal events occur: migration, early differentiation, and late maturation. All migrating neurons, travelling on radial glial fibers, reach layer I, develop an apical dendrite, and establish contacts with its elements. These newly differentiated neurons assume similar morphology resembling embryonic pyramidal cells. As such, an early differentiation stage common to all neurons of the CP is established. During the late maturation stage, all CP neurons acquire their specific phenotypic structural and functional features. Only pyramidal neurons retain and expand their original connections with layer I while other neuronal types lose these connections. The pyramidal cell is redefined in developmental terms: the neocortex's pyramidal cell is both structurally and functionally locked into position between layer I and the cortical depth of its soma. During mammalian evolution pyramidal cells are forced to structurally and functionally elongate their apical dendrite outwardly to accommodate an increasing amount of information without losing either their original anchorage to layer I or their cortical depth. This unique property of pyramidal neurons is considered to be a mammalian innovation. Based on these observations, a unifying developmental cytoarchitectonic theory applicable to all mammals is proposed. The theory considers the CP to be a mammalian innovation and to represent a single, stratified, and expanding telencephalic nucleus. The theory envisions the mammalian neocortex as an open biological system capable of progressive expansion by the recruitment and transformation of primitive neurons from upper layer II into pyramidal cells. Hence, the number of pyramidal cell strata increases over the course of mammalian phylogeny. The developmental roles of layer I in the migration of neurons, formation of the CP, unique morphology of pyramidal cells, and overall structural organization of the mammalian neocortex are emphasized.


Heterogeneity of layer II neurons in human entorhinal cortex. Abnormalities in the layer II neurons of human entorhinal cortex have been implicated in the pathophysiology of Alzheimer's disease and schizophrenia. The reported abnormalities are not homogeneously distributed throughout the entorhinal cortex, suggesting that layer II of entorhinal cortex may contain different subpopulations of neurons, each with a different susceptibility to pathological mechanisms. In order to investigate the possible heterogeneity of neurons in layer II of human entorhinal cortex, we first identified distinct subdivisions of human entorhinal cortex by adapting the cytoarchitectonic criteria for subdivisions of monkey entorhinal cortex described by Amaral et al. (J Comp Neurol 264:326, 1987). The morphology and regional distribution of distinct subpopulations of human layer II neurons were determined through the use of immunohistochemical techniques. Multipolar, stellate, and modified pyramidal neurons in the characteristic cell clusters or islands of layer II were immunoreactive for nonphosphorylated neurofilament proteins. The intensity of immunoreactivity for the nonphosphorylated neurofilament proteins gradually increased along the rostrocaudal axis of entorhinal cortex and was primarily due to a similar gradient in the density of labeled neurons per island. The calcium-binding protein calbindin D-28K was found in both pyramidal and nonpyramidal neurons in layers II and superficial III. The distribution of calbindin-immunoreactive neurons also depended upon the region of entorhinal cortex. In rostral entorhinal cortex, labeled neurons were scattered throughout the superficial layers, whereas in caudal entorhinal cortex, distinctive patches of small calbindin-immunoreactive neurons were found among the layer II islands. Another calcium-binding protein, parvalbumin, was present in nonpyramidal neurons in layers II and III that were distinct from those containing calbindin. The regional distribution of parvalbumin-positive neurons was very similar to that of the neurofilament immunoreactive neurons; in rostral entorhinal cortex very few parvalbumin-labeled neurons were present but their frequency gradually increased in the caudal direction. In addition, punctate parvalbumin immunoreactivity was frequently encountered in the location of the nonphosphorylated neurofilament protein-positive layer II islands. These findings demonstrate that layer II of human entorhinal cortex contains distinct subpopulations of neurons, that the relative density of each subpopulation differs across cytoarchitectonic regions, and that the patterns of distribution of these subpopulations are in some cases similar and in other cases complementary. This heterogeneity in the organization of layer II of human entorhinal cortex has important implications for the study of some neuropsychiatric disorders.


Two classes of vesicles are present and change in relative proportion during post-embryonic development of rectifying electrical synapses in the crayfish. The size and shape of vesicles at junctional appositions of the rectifying electrical synapses between the medial giant fibre and motor giant neurone of the crayfish were measured during the first 2 months after hatching. Summed data over this period reveal a bimodal distribution in vesicle diameter. From the day of hatching until about 7 days of age, small vesicles (circa 25 nm diameter) predominate. From day 7 onwards, larger vesicles (circa 55 nm diameter) occur in increasing numbers, until at day 56 they constitute about 85% of the population at any one junctional apposition. At intermediate ages (day 7-28) individual junctional appositions may show the same bimodal distribution in size as does the age group as a whole, indicating that large and small vesicles occur together at the same junction. The larger vesicles are mainly circular, while the small vesicles are pleomorphic, with shapes ranging from almost circular down to a shape factor of about 0.6.


Connections from the lateral vestibular nucleus to the upper cervical spinal cord of the cat: a study with the anterograde tracer PHA-L. The projections of neurons in the lateral vestibular nucleus (LVN) to the upper cervical spinal cord of the cat were investigated by means of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L). At the junction of C1 and C2, axons were distributed bilaterally in the ventromedial funiculi, and ipsilaterally in the ventrolateral and lateral funiculi. The majority of boutons were found ipsilateral to the injection sites and most of these boutons were found at the base of the ventral horn and throughout the medial two-thirds of lamina VIII. A more modest termination zone was found along the ventral border of lamina VII and a small number of boutons were scattered in the dorsal horn. Contralateral termination zones were similar to the ipsilateral projections. There were significant changes in the distribution of vestibulospinal axons and density of boutons at the junction of C3 and C4. At this level, most vestibulospinal axons travelled ipsilaterally and were found along the medial border of the ventromedial funiculus and the ventral margin of the ventrolateral funiculus. The overall distribution of boutons near the border of C3 and C4 was similar to the pattern seen at the junction of C1 and C2. However, bouton density fell by a factor of three. Large zones of the grey matter were devoid of boutons in individual experiments. These results demonstrate that the projections of neurons in the LVN to the upper cervical spinal cord are densest in the regions containing motoneurons supplying suboccipital muscles. This result suggests that monosynaptic connections to those motoneurons may be an important part of the neural circuitry responsible for vestibulocollic reflexes. However, the large number of boutons found in regions dorsal to motoneuron nuclei in all upper cervical segments indicates that the primary path from vestibulospinal axons to neck motoneurons may be indirect and involve relays via spinal interneurons.


Proteolytic activity of proteinases on macropeptide isolated from kappa-casein. Proteolytic activities of chymosin, bovine pepsin, Mucor miehei rennet, Cryphonectria parasitica (formerly Endothia parasitica) rennet, trypsin, and chymotrypsin on kappa-casein macropeptide were measured. Macropeptide solutions (10 mg/ml of .05 M, pH 6.6 phosphate buffer) were incubated with the enzymes at 37 degrees C for various times, and their reactions were stopped by adding .025 ml of pepstatin (1 mg/ml of methanol). Peptides released from kappa-casein macropeptide were then fractionated using reverse-phase HPLC. At the pH of milk (pH 6.6), kappa-casein macropeptide was resistant to enzymic action by chymosin, bovine pepsin, and M. miehei and C. parasitica rennets. Bovine pepsin hydrolyzed kappa-casein macropeptide at pH 3. kappa-Casein macropeptide was readily hydrolyzed at pH 6.6 by trypsin and chymotrypsin. Possible physiological functions of the kappa-casein macropeptide are discussed in light of these findings.


Klebsiella pneumoniae as a spoilage organism in mozzarella cheese. A high concentration of total and fecal coliforms (5 x 10(8) and 3 x 10(7) cfu/g, respectively) was observed in samples representative of two production lots of Mozzarella cheese from a local dairy. Contamination was manifested by the swelling of plastic pouches and the formation of gas holes in Mozzarella cheese. Aerobic and anaerobic sporeformers, heterofermentative lactic acid bacteria, propionibacteria, and yeasts were absent. Of the 41 isolates identified, there were 37 Klebsiella pneumoniae, 2 Klebsiella oxytoca, 1 Enterobacter aerogenes, and 1 Escherichia coli. Exposure at 63 degrees C for 15 min caused the death of all total and fecal coliforms.


Survival of Lactobacillus acidophilus and Bifidobacterium bifidum in ice cream for use as a probiotic food. Probiotic ice cream was made by fermenting a standard ice cream mix with Lactobacillus acidophilus and Bifidobacterium bifidum cultures and then freezing the mix in a batch freezer. Survival of the L. acidophilus and B. bifidum, as well as beta-galactosidase activity, was monitored during 17 wk of frozen storage at -29 degrees C. After freezing of the fermented mix, bacterial counts were 1.5 x 10(8) cfu/ml for L. acidophilus and 2.5 x 10(8) cfu/ml for B. bifidum. Seventeen weeks after freezing, these counts had decreased to 4 x 10(6) and 1 x 10(7) cfu/ml, respectively. During the same period, beta-galactosidase activity decreased from 1800 to 1300 units/ml. Probiotic ice cream was prepared at pH 5.0, 5.5, and 6.0 to determine consumer preferences and was compared with standard Utah State University "Aggie" ice cream. All samples were strawberry-flavored and were evaluated by 88 judges. The preferred pH of probiotic ice cream, based on overall acceptance, was pH 5.5. We demonstrated that probiotic ice cream is a suitable vehicle for delivering beneficial microorganisms such as L. acidophilus and B. bifidum to consumers. The bacteria can be grown to high numbers in ice cream mix and remain viable during frozen storage.


Mammary-derived growth inhibitor protein and messenger ribonucleic acid concentrations in different physiological states of the gland. Expression of mammary-derived growth inhibitor in tissue from lactating and involuting bovine mammary glands was investigated. Seventeen lactating, pregnant (220 to 272 d in gestation) cows were divided in two groups of 8 and 9 cows each. Cows of the first group were slaughtered while in lactation. Cows of the second group (9 involuting cows) were slaughtered at 13 to 52 d following sudden cessation of milking. High concentrations of mammary-derived growth inhibitor (.63% of the total protein) were detected in mammary tissue of lactating cows. Mammary-derived growth inhibitor (less than .10% of the total protein) was dramatically reduced during most of the involution period (13 to 45 d following cessation of milking). Mammary-derived growth inhibitor was again detected (.28% of the total protein) during the last stage of the involution (46 to 53 d after cessation of milking), which coincided with colostrum formation. When steady state concentrations of mammary-derived growth inhibitor mRNA were examined, the results obtained mirrored those obtained at the protein concentration. These data suggest that regulation of mammary-derived growth inhibitor occurs via modulation of the steady state concentration of its mRNA. Furthermore, there is a strong correlation between mammary-derived growth inhibitor expression and lactation in dairy cows.


Diagnosis of Staphylococcus aureus intramammary infection by detection of specific antibody titer in milk. The diagnostic value of the determination of Staphylococcus aureus antibody titer in milk as a method for identification of mammary quarters with S. aureus IMI was evaluated. Ten cows with a history of S. aureus IMI and 9 cows with no history of S. aureus IMI were sampled daily for 10 d. Quarter and composite milk samples were collected and processed by standard methods for concentration of S. aureus, S. aureus antibody titer (percentage of positive laboratory control), and SCC (cells per milliliter). Microbiologic culture identified 13 S. aureus-infected quarters from the 10 cows with a history of S. aureus IMI. Only 2 of the 130 samples (1.5%) from these infected quarters had undetectable concentrations of S. aureus. Antibody titers in milk from infected quarters of infected cows were below the previously established level considered to be indicative of IMI in 6 of 130 samples (4.6%). Four samples from infected quarters of infected cows had titers considered to be in the suspect range. The sensitivity of the antibody test was 83% (13% SE) when the suspect samples were included and 86% (12% SE) when they were not. Milk from uninfected quarters of cows with S. aureus IMI tended to have S. aureus antibody titers greater than the test's positive control, which would suggest that the quarters were infected with S. aureus. Antibody titer was below the infection threshold level in all pooled samples of uninfected cows, suggesting that the test correctly identified all cows free of S. aureus IMI.(ABSTRACT TRUNCATED AT 250 WORDS)


Coculture of in vitro fertilized bovine embryos with oviductal epithelial cells originating from different stages of the estrous cycle. Bovine embryos derived from in vitro fertilization procedures were cocultured in vitro with oviductal cells obtained from heifers between d 4 and 6 or d 14 and 16 of the estrous cycle. In addition, proteins secreted by oviductal cells isolated between d 4 and 6 or d 14 and 16 of the cycle were monitored. Embryos (2- to 4-cell) were incubated in Tissue Culture Medium-199 with 10% fetal bovine serum with or without oviductal cells at 39 degrees C for 10 d following in vitro insemination. There were more morulae, blastocysts, and hatched blastocysts following coculture with oviductal cells than with culture in medium alone. However, no differences were noted in embryo development following coculture with oviductal cells obtained between d 4 and 6 or d 14 and 16 of the estrous cycle. Also, no differences were detected in the amount of [35S]methionine-labeled proteins secreted by oviductal cells isolated from different days of the estrous cycle. These results indicate that oviductal epithelial cells isolated from early and late luteal phases of the estrous cycle will effectively support early embryonic development following prolonged in vitro culture.


Effect of progesterone on the expression of estrus at the first postpartum ovulation in dairy cattle. Fifty-two lactating Holstein cows were randomly assigned to receive either a progesterone-releasing (2 g of progesterone) or a control-releasing intravaginal device (0 g of progesterone). Intravaginal devices were inserted on d 10 and removed on d 15 postpartum. Daily blood samples were collected from d 10 to 90 postpartum for subsequent determination of progesterone concentrations. Observations for estrus were conducted three times daily in a dirt paddock containing a testosterone-treated cow. Serum concentrations of progesterone in the progesterone-releasing intravaginal device group were elevated on d 11, 12, 13, 14, and 15 compared with those of the control group. The days to first post-partum ovulation were similar between the treated and control groups, respectively (30.6 vs. 30.5 d). Also similar was the proportion of cows expressing estrus at first, second, and third postpartum ovulations (9/27 vs. 3/24, 14/23 vs. 15/21, and 14/21 vs. 9/15, respectively), length of the first postpartum estrous cycle (17.9 vs. 18.3 d), and peak serum concentrations of progesterone during the first estrous cycle (3.5 vs. 2.9 ng/ml). These data indicate that administration of progesterone early postpartum did not increase the proportion of cows expressing estrus at the first ovulation.


Effect of recombinant bovine somatotropin on estradiol-induced estrous behavior in ovariectomized heifers. To evaluate whether bST administration could affect the intensity of estrous behavior in the absence of the ovary, 17 ovariectomized heifers were assigned to receive a placebo or 500 mg/14 d of recombinant bST and were induced to display behavioral estrus by administration of 2 mg of estradiol cypionate. Estrous behavior was monitored for the following 60 h with a surveillance camera and a video recorder, and mounting activity was quantified. Blood samples were collected before and after bST administration. Administration of bST numerically elevated plasma insulin and NEFA and caused a significant increase in concentration of insulin-like growth factor-I in plasma. Heifers that received bST started to display estrous behavior later (24.5 +/- 1.1 and 21.9 +/- 1.1 h after estradiol administration for bST and placebo treatments, respectively), displayed estrous behavior of shorter duration (bST, 13.2 +/- 1.2 vs. placebo, 15.8 +/- 1.1 h), and instigated fewer mounting events (bST, 45.5 +/- 5.9 vs. placebo, 62.6 +/- 5.7 mounts) than control heifers, although only the number of mounting events was significantly different between the two groups.


Water partitioning and intake prediction in dry and lactating Holstein cows. Data from four energy and N balance trials with lactating Holstein cows (n = 329) and one trial with dry cows (n = 60) were used to predict free water intake and water-related traits. Lactating cows were between 36 and 159 DIM and, individually, were allowed ad libitum water and forage (corn silage without or with wilted haycrop silage) plus concentrates; dry cows accessed ad libitum water and single forages (grass, clover, or alfalfa, as hays or as wilted silages, or corn silage) varying in maturity. Intake of DM per day and dietary DM percentage were significant and positively related predictors of free water intake in dry and lactating cows. Daily milk yield (range 16 to 52 kg/d) was related linearly to water consumption (.60 L/kg of milk), and season effect in lactating cows was curvilinear; peak water intake was in late June and nadir in late December. Ration CP percentage (DM basis) affected free water intake only in dry cows; 1 unit of increase resulted in an increase of about 1 kg/d in water intake between 12 and 13% CP. As ration moisture dropped from 70 to 40%, free water intake increased about 7 L/d in dry cows, but this was accompanied by a drop of 15 L/d in total water intake (free plus feed water). Free water intakes were predicted with R2 of .64 and .69 in dry and lactating Holstein cows, respectively. Fecal water and urine outputs also were predicted. We found no significant relationship between DM content of the diet and the resulting ad libitum intake in either dry or lactating cows.


Response of Holstein cows to corn gluten meal used to increase undegradable protein in early or later lactation. Supplemental corn gluten meal was used to raise CP by 1.1 to 1.5 percentage units and undegradable intake protein from 35 to 39% of CP in the corn-based diet of parity 1 or greater Holstein cows to study effects of undegradability, parity, stage of lactation, and interactions on DMI, milk yield and composition, BW, and related traits during complete lactations. Cows were assigned at calving to treatments (n = 30, 8 primiparous): control, supplement wk 1 to 8 postpartum (early), or supplement wk 9 to 44 postpartum (late). Total lactation means were not affected significantly by treatments. Supplementation with undegradable protein enhanced forage and, thus, total DMI in later lactation by pluriparous cows; it apparently spared BW loss wk 1 to 8 postpartum and enhanced BW recovery thereafter in first lactation cows with no effect in older cows. Effects of supplementation on milk yield were small, and they were negative in early lactation and generally positive in late lactation; effects were positive on fat test in early lactation for both parity categories but distinctly negative for parity 1 cows in late lactation. Supplementation of undegradable protein in late lactation also decreased milk protein content in parity 1 cows and raised it in older cows. Data suggest that Lys may have been first-limiting, followed by Ile in early lactation and Met in late lactation, and that AA adequacy may be more important than undegradability in ration protein balancing. For most traits measured, treatment by parity interactions were significant, indicating that parity 1 cows did not respond in the same way as older ones to protein supplementation.


Infusion of long-chain fatty acids varying in saturation and chain length into the abomasum of lactating dairy cows. Free long-chain fatty acids were infused into the abomasum of lactating dairy cows to determine postruminal effects on feed intake, production and composition of milk, nutrient digestibilities, and metabolites in blood. Four Holstein cows averaging 120 DIM and fitted with ruminal cannulas were used in a 4 x 4 Latin square design with 14-d periods. Treatments were abomasal infusions of 1) control, 168 g/d of meat solubles (carrier for fatty acids), 2) control plus 450 g/d of mostly saturated fatty acids (C16:C18 = .75), 3) control plus 450 g/d of a mixture of saturated and unsaturated fatty acids (C16:C18 = .40), and 4) control plus 450 g/d of mostly unsaturated fatty acids (C16:C18 = .11). Production of milk and milk components, DMI, and intake of digestible energy decreased linearly as unsaturation and chain length of infused fatty acids increased. Percentages of fat, CP, and SNF in milk and total tract apparent digestibilities of DM, OM, ADF, NDF, energy, and fatty acids were not affected significantly by treatments. Infusing fatty acids decreased proportions and yields of short- and medium-chain fatty acids and increased proportions and yields of unsaturated C18 fatty acids in milk fat. Increasing unsaturation and chain length of infused fatty acids linearly decreased proportion and yield of palmitic acid but increased proportions and yields of polyunsaturated C18 fatty acids in milk fat. Infusing fatty acids increased concentrations of NEFA and cholesterol in blood plasma. The profile of fatty acids reaching the intestine may be an important determinant of responses to supplemental fats fed to lactating dairy cows.


Resistance of fatty acyl amides to degradation and hydrogenation by ruminal microorganisms. Two in vitro trials were conducted to determine whether fatty acyl amides are degraded and hydrogenated by ruminal microorganisms. The treatments consisted of ground hay supplemented with either no lipid, linoleoyl Met ethyl ester, or free linoleic acid plus Met ethyl ester. Incubations were carried out in Erlenmeyer flasks at 39 degrees C under CO2. Cultures were sampled at predetermined times and analyzed for long-chain fatty acids, Met, and VFA. In trial 1, the rate of disappearance of linoleic acid was lower for the amide than for the FFA (.004 and -.047/h, respectively). In trial 2, there were no differences in the rate of disappearance of linoleic acid from 0 to 6 h (-.237 and -.357/h for amide and FFA, respectively), but the rates from 6 to 48 h (-.003 and -.027/h for amide and FFA, respectively) were different. Linoleoyl Met cultures also had higher acetate to propionate ratio and lower loss of Met compared with free linoleic acid cultures. There was no loss of radioactivity from [14C]stearoyl Met after 24 h of incubation, indicating its resistance to bacterial breakdown. The results showed that fatty acyl amides resist bacterial breakdown and prevent loss of double bonds by microbial biohydrogenation.


Impact of alfalfa maturity and preservation method on milk production by cows in early lactation. Ninety-six cows in early lactation were used in two experiments to measure the impact of alfalfa maturity (early or midbud vs. early or midbloom) and method of forage preservation (silage vs. hay) on DMI and milk production. Silage diets were fed as TMR, and hay was fed separately from grain. All diets contained 60% alfalfa (dry basis) and were balanced for 19% CP. Maturity had little effect on milk production in either experiment. Adjusted milk production for early cut silage, late cut silage, early cut hay, and late cut hay were 33.6, 33.4, 30.7, and 32.1 kg/d for Experiment 1 and 38.1, 37.0, 35.0 and 35.0 kg/d for Experiment 2. Increased alfalfa maturity tended to reduce DMI. Cows fed the silage diets consumed 1.2 kg more DM and produced an average of 2.1 kg more milk daily in Experiment 1 than those fed the hay diets. All treatment groups consumed similar amounts of DM in Experiment 2; however, cows fed silage produced 2.6 kg/d more milk than those fed hay. Preserving alfalfa as silage and feeding in a TMR to cows in early lactation resulted in greater milk production via increased DMI or improved feed efficiency compared with preserving alfalfa as hay and feeding grain separately.


Impact of stage of maturity and method of preservation of alfalfa on digestion in lactating dairy cows. Four multiparous Holstein cows in midlactation were fed 60:40 forage:grain (dry basis) diets containing first-cutting alfalfa harvested at the early bud or early flower stage of maturity. Each of the two maturities was preserved as silage and hay. A 4 x 4 Latin square design experiment (28-d periods) was conducted to measure the impact of alfalfa maturity and method of preservation on milk production and composition, intake, digestion, digesta passage, and particle size of digesta. Milk production and composition were not affected by treatment. Increases in fiber intake resulted from increased maturity and preservation as hay, but this did not appear to limit DMI or milk production. Both increased maturity and preservation as hay resulted in the following effects in cows: more time spent ruminating, greater total chewing time, greater wet and dry rumen fill, and a greater volume of rumen contents. Rumen retention time of La applied to forage was 6 h less, and 0-h disappearance of DM from dacron bags was greater for silage than hay. In situ DM disappearance rates averaged 15%/h for silage and 9.5%/h for hay. Lag time was related inversely to 0-h disappearance. Masticates and mixed rumen samples from cows fed hay showed a greater percentage of DM as particles greater than or equal to 9.5 mm in length. Gross feed efficiency was greater for cows fed alfalfa silage than for those fed alfalfa hay, probably because of more rapid digestion and passage. The potential for rumen fill to limit intake in high producing cows appears to be greater for alfalfa preserved as hay than that preserved as silage.


Comparative responses of lactating cows to total mixed rations or computerized individual concentrates feeding. A trial was conducted in a commercial dairy herd in which the concentrate part of the ration was fed individually to a group of cows through computerized self-feeders. Performance results were compared with those of a group fed TMR of 65 to 67% concentrates. Rationing of individual concentrates was according to parity, milk yield, milk yield potential, BW changes, and bunk feed-stuffs. Mean intake of concentrates per cow was about 1 kg/d lower in the individually supplemented cows. This was partly compensated for by a higher intake of bunk feedstuffs. Overall daily milk yield per cow was similar to those receiving a TMR in first parity cows, higher in second parity cows, and lower in third and greater parity cows. The higher performance of the second parity cows was achieved in all milk yield potential classes, and the lower yield in subsequent lactations was due to lower performance in low and high potential classes. The individually supplemented cows gained less BW than those in the TMR group. Milk yield per unit of BW was better than milk yield as a variable to refine individual cow supplementation strategy for allocation of concentrates. Results also suggest that the same criteria used for supplementation of concentrates can be beneficial to cows' assignments and movements among different TMR groups. Computerized dispensing of concentrates, when applied properly, can economize on consumption of concentrates when grouping and feeding different TMR are impossible.


Estimating production benefits through simulation of group and individual feeding of dairy cows. Objectives were 1) to develop DMI and milk prediction equations, 2) to use these equations to simulate group and individual feeding of dairy herds, and 3) to estimate effects of group and individual feeding on FCM production. University of New Hampshire data were used to predict DMI from previous DMI and cow and ration characteristics. The same data were used to predict milk production from DMI and previous milk production. Feeding was simulated for 100 cows over 50 4-wk periods in a number of trials. Effects of individual feeding, additional groups, herd calving intervals, and within-herd variation of annual milk production per cow on daily FCM per cow were isolated in average and high producing herds. Changing from one group to individual feeding can increase daily FCM per cow by .5 to 1.1 kg and two groups to individual feeding by 0 to .8 kg without changing total herd nutrient intake. Reallocation of the same amount of nutrients to two groups instead of one can increase daily milk production by .15 to .8 kg of FCM per cow, reallocation to three groups instead of two by 0 to .6 kg of FCM per cow, and reallocation to four groups instead of three by 0 to .35 kg of FCM per cow.


Influence of feeding varying levels of Amaferm on performance of lactating dairy cows. Amaferm, a fermentation extract of Aspergillus oryzae, was fed as a top-dressing to dairy cows at 0, 1.5, 3, and 6 g/d in two lactation trials using 64 cows in 1989. Lactation trial 1 was conducted in the spring (March to May) and used 40 cows averaging 75 DIM for a 70-d treatment period. Lactation trial 2 was during the summer (June to July). Twenty-four cows averaging 140 DIM were employed in a 60-d study. Measurements included milk yield, feed intake, BW, rectal temperatures, respiration rate, and digestibility of CP, NDF, and DM. None of the levels of Amaferm had a significant effect on milk yield or composition, BW changes, or digestion coefficients in either trial. Cows fed 1.5 g/d of Amaferm had a higher DMI than those receiving 0 or 6 g in trial 1, and respiration rates were significantly higher for cows fed 3 g/d of Amaferm in trial 2. Under the conditions of this study, none of the levels of Amaferm affected the performance of lactating cows. Further elucidation of factors influencing response to Amaferm is needed.


Metabolic changes in dairy cows with ketonemia in response to feed restriction and dietary 1,3-butanediol. The objective was to measure progressive changes in metabolism of cows during a protocol that induced subclinical ketosis. From d 14 to 42 postpartum, 13 Holstein cows were in either a control group (6 cows) or a ketosis induction group (7 cows) that was restricted to 80% of ad libitum intake and fed 1,3-butanediol (7% of DM). Six ketosis induction cows developed ketonemia but not clinical ketosis; cow 7 developed clinical ketosis. Milk production was less, but fat content was greater, for ketonemic cows. Energy balance reached a nadir of -7.2 Mcal/d at d 21 for ketonemic cows, whereas controls reached energy equilibrium at d 28. Concentrations of NEFA in plasma and of beta-hydroxybutyrate in whole blood increased during ketonemia. Dextran sulfate-precipitable cholesterol and triglyceride in serum were increased only at d 21 and not at d 28, 35, 42, or 49. Concentrations of glycogen, total lipid, triglyceride, and cholesterol in liver increased during ketonemia. Oxidation of palmitate to CO2 was greater at d 21 in liver slices from ketonemic cows, whereas oxidation to acid-soluble products remained constant for those cows but decreased for controls. The ketonemic cows had lower weight ratios of triglyceride to glycogen in liver during pretreatment than those that became clinically ketotic in earlier studies (.5 vs. greater than or equal to 1.8). Susceptibility to clinical ketosis, therefore, may be indicated by increased hepatic triglyceride to glycogen ratios during the peripartal period.


Neutrophil and lymphocyte response to supplementation with vitamins C and E in young calves. Calves, beginning at 3 d of age, were bottle-fed milk replacer unsupplemented, or supplemented with 10 g of vitamin C, or supplemented with 10 g of vitamin C plus 57 IU of vitamin E/kg of dry milk replacer (10 calves per treatment). Neutrophils from wk 2 and 4 blood samples were assayed for Staphylococcus aureus phagocytosis and neutrophil-mediated, antibody-dependent cellular cytotoxicity, and lymphocytes from those samples were assayed for concanavalin A-induced, phytohemagglutinin-induced, or pokeweed mitogen-induced proliferation. Ocular and nasal discharges of calves supplemented with vitamins C or C and E were less than those of controls for wk 1 to 8. Calves receiving supplementation with vitamin C and E tended to have higher mitogen-induced lymphocyte proliferative responses at wk 2. Neutrophil-mediated phagocytosis and antibody-dependent cellular cytotoxicity were lower at wk 2 and 4 for calves supplemented with vitamin C than for controls. Neutrophil function of calves supplemented with vitamins C and E together was near, or slightly higher than, that of unsupplemented calves for wk 2 and 4, suggesting that the addition of vitamin E negated the adverse effects that vitamin C alone had on neutrophil functions.


Production performance and physiological responses of Angora goat kids fed acidified milk replacer. Angora kids were blocked by birth weight and sex and assigned randomly to goat milk or acidified milk replacer. Daily milk intake, weekly BW, and heart girth measurements, and blood parameters (packed cell volume, total protein, glucose, and NEFA) were monitored at 3 d (initial) and at 4, 6, 8, and 9 wk of age. Both groups were fed their respective milks for ad libitum intake for 6 wk and then reduced to 75, 50, 25, and 0% of wk-6 intake during wk 7, 8, 9, and 10, respectively. Solid feed (20% CP and 3.1. Mcal of metabolizable energy/kg of DM) was provided for ad libitum intake starting on wk 3. Pretreatment BW (average 2.4 kg) and blood parameters were similar for milk and replacer groups. Packed cell volume (21.8 and 34.2%), total protein (50.3 and 46.6 g/L), and NEFA (.52 and .69 meq/L) for goat milk and acidified milk replacer groups, respectively, were affected by dietary treatment. Final BW (average 10.5 kg) and mean plasma glucose concentration (84 to 88 mg/dl) were similar between treatments; however, kids fed goat milk produced more mohair (13.8%) than those fed acidified milk replacer. Despite physiological differences, acidified milk replacer can be used successfully to raise Angora kids.


Potential improvements in rate of genetic gain from marker-assisted selection in dairy cattle breeding schemes. The value of marker-assisted selection in dairy cattle breeding schemes is predicted by a deterministic model. In these schemes, associations between markers and milk production were assessed from production records of daughters of a grandsire by a multiple regression model with random marker effects. By tracing markers from the grandsire to grandoffspring, deviations of grandoffspring from their full-sib family mean were predicted. Predictions of the within-family variance of the grandoffspring accounted for by markers amounted to up to 13.3%. This figure decreased when the number of daughters of the grandsire analyzed decreased and, less markedly, when the distance between flanking markers increased. Prediction of within-family deviations hardly improved rates of genetic gain in conventional progeny testing schemes; equal numbers of young bulls were born annually. Genetic gain and improvement of genetic gain because of prediction of within-family deviations were much higher in nucleus schemes. In these shemes, with short optimized generation intervals, conventional selection was mainly for pedigree information and did not use the within-family variance. Analysis of highly polymorphic markers in daughters of both grandsires accounted for 4.1 to 13.3% of the within-family variance, which increased rates of gain by 9.5 to 25.8% and 7.7 to 22.4% in open and closed nucleus schemes, respectively. Risk of breeding schemes, measured by the variance of the selection response, was not increased by the use of markers.


Relationship of in vitro immune function with health and production in Holstein cattle. Eighty-seven lactating Holstein cows from the Iowa State University Breeding Research Herd were evaluated for 20 in vitro measures of immune function. Principal component analysis was used to discard redundant assay variables such that the 11 remaining variables were more nearly independent than the original variables. Multiple linear regression in an animal model was used to determine the effects of these 11 variables on lifetime production and on general, under, and reproductive health traits. A significant joint effect of the 11 immune function variables on California mastitis test scores was observed. California mastitis test scores were positively correlated with antibody-dependent neutrophil cytotoxicity and negatively correlated with antibody-independent neutrophil cytotoxicity. Wisconsin mastitis test scores were also positively associated with antibody-dependent neutrophil cytotoxicity. Cytochrome c reduction was negatively associated with mammary and total health costs. A positive relationship between clinical mastitis and discarded milk and IgG2 was observed, and IgG1 was associated with increased quarter California mastitis test scores and increased production. Thus, certain in vitro immune function assays may serve as indicators of susceptibility to health problems in dairy cattle, particularly for traits associated with udder health.


Estimating milk, fat, and protein lactation curves with a test day model. Test day models were used to estimate lactation curves for milk, fat, protein, fat percentage, and protein percentage and to study the influence of age, season, and herd productivity on Holstein lactation curves. Random effects of lactation within herd and fixed effects of herd test date were absorbed. Fixed effects of cow's age on test day and either DIM (57 divisions) by parity (1, 2, greater than or equal to 3) class or season of calving (winter or summer) by DIM by parity class were estimated. Lactation curves for yield traits derived from DIM solutions were flatter for first versus later lactation, even without addition of age effects. Differences between lactation curves for the two seasons were slight, suggesting that most observed seasonal differences are caused by seasonal productivity accounted for by herd test date effects. At peak, winter calving cows yielded slightly more milk of similar fat percentage but of lower protein percentage than those calving in summer. Data were also partitioned into nine subsets based on rolling herd milk and fat percentage. Lactation curves for yield traits, but not percentage traits, varied with rolling herd milk. Lactation curves for fat yield and percentage varied with rolling herd fat percentage.


Estimates of repeatability of reproductive measures in Canadian Holsteins. Repeatabilities and heritabilities of days to first service, days open, and number of services per conception were estimated from 235,589 records on 80,333 Holstein cows, daughters of 306 sires obtained from the Quebec Dairy Herd Analysis Service, by REML. The model for statistical analyses included herd-year-season as a fixed effect and sire, cow (sire's daughter) within sire, and error as random effects. Variance components corresponding to sire, cow within sire, and error were then estimated by REML. Heritability estimates, obtained from the sire variance component, were .03, .05, and .03 for days to first service, days open, and number of services per conception, respectively. Repeatability estimates, obtained from the sire and cow within sire variance components were .08, .10, and .07 for days to first service, days open, and number of services per conception, respectively.


Evaluation of postmilking teat germicides containing Lauricidin, saturated fatty acids, and lactic acid. The efficacies of postmilking teat germicides containing Lauricidin (glyceryl monolaurate), saturated fatty acids, lactic acid, and lauric acid were determined against new IMI caused by Staphylococcus aureus and Streptococcus agalactiae in three controlled infection trials. In trial 1, a germicide was evaluated containing 1% Lauricidin, 5% caprylic and capric acids, 6% lactic acid, and .85% lauric acid. New IMI with Staph. aureus and Strep. agalactiae were reduced 81.3 and 49.6%, respectively. Trial 2 germicide involved an artificially aged sample of the formulation evaluated in trial 1. The germicide was aged at 40 degrees C for 5 mo, which was approximately equal to 2 yr at room temperature (24 degrees C). Reductions in new IMI were 81.2 and 27.5% for Staph. aureus and Strep. agalactiae, respectively. In trial 3, a teat germicide aged at ambient temperature for 33 mo, which was originally formulated to contain 1% Lauricidin, 5% caprylic and capric acids, and 6% lactic acid, was evaluated. Reductions in new IMI were 75.5 and 40.4% for Staph. aureus and Strep. agalactiae, respectively. The formulation evaluated in trial 1 was superior to other formulations in reducing new IMI by the two test organisms.


Extension factors for incomplete lactations of Serra-da-Estrela sheep. Data for 6537 lactations of 2335 Serra-da-Estrela ewes representing 41 flocks (1966 to 1985) were obtained from the Milk Recording Scheme conducted by the Portuguese Ministry of Agriculture. The suckling period averaged 32 d, and ewes were milked for approximately 200 d after the suckling period. Individual test day yields for 1548 lactations of 600 ewes from 32 flocks (1975 to 1985) were used to define the shape of the lactation curve. A third-order polynomial and an exponential function were equally accurate in describing the shape of the lactation curve in the postsuckling period. A considerable number of incompletely recorded lactations occur, and, with extension factors, these incomplete lactations could be used in genetic evaluation. Factors based on both partial lactation yield and last test day yield were developed. Use of these factors should be restricted to partial lactations of at least 75 d with 2 test days. An alternative of recording only postweaning yield was proposed.


Effect of direct-fed microbials on rumen microbial fermentation. Nonbacterial, direct-fed microbials added to ruminant diets generally consist of Aspergillus oryzae fermentation extract, or Saccharomyces cerevisiae cultures, or both. Results from in vivo research have been variable regarding effects of direct-fed microbials on ruminant feedstuff utilization and performance. Some research has shown increased weight gains, milk production, and total tract digestibility of feed components, but others have shown little influence of direct-fed microbials on these parameters. In vitro research with mixed ruminal microorganisms likewise has been inconsistent regarding the effects of direct-fed microbials. Several researchers observed that direct-fed microbials increased cellulolytic bacterial numbers in the rumen and stimulated the production of some fermentation end products. This suggests that direct-fed microbials may be providing growth factors for the ruminal microbes. However, other researchers have reported no effect of direct-fed microbials on in vitro fiber digestion. Recent research demonstrated that growth of the predominant ruminal bacterium Selenomonas ruminantium in lactate medium as well as lactate uptake by whole cells of Sel. ruminantium were markedly increased by an A. oryzae fermentation extract and an S. cerevisiae culture. In addition, both products increased the production of acetate, propionate, succinate, total VFA, and cell yield (grams of cells per mole of lactate). Therefore, it appears that these direct-fed microbials provide soluble factors that stimulate lactate utilization by Sel. ruminantium. Evidence is presented indicating that the malate content of the A. oryzae fermentation extract and S. cerevisiae culture may be involved in this stimulation.


Bacteriophage resistance in Lactococcus lactis ssp. lactis using antisense ribonucleic acid. Antisense RNA against a conserved bacteriophage gene when expressed in a Lactococcus lactis ssp. lactis strain renders it resistant to bacteriophage infection. Two open reading frames have been identified in a L. lactis ssp. lactis bacteriophage that are conserved in a majority of isolates. They code for an 18-kDa (designated GP18C) protein and a 24-kDa (GP24C) protein, respectively, which are arranged along with previously identified open reading frames in a tandem motif similar to other bacteriophages. The presence of gp18C and gp24C in a number of bacteriophage isolates was confirmed by polymerase chain reaction using primers specific for these regions. Plasmids bearing various fragments of gp18C, gp24C, or both were constructed such that the respective open reading frames were positioned in the antisense direction relative to the Lactococcus lactis ssp. cremoris Wg2 promoter, p59. These antisense RNA-producing vectors inhibited the efficiency of plaquing of L. lactis ssp. lactis bacteriophage phi 7-9 up to 50%; the resulting plaques were extremely small and irregular in shape. The replication of the bacteriophage was severely inhibited, and the total number decreased over the first 3 h during infection in strains expressing antisense RNA compared with the host strain alone, in which the bacteriophage number increased 10(4)-fold.


Variations and distributions of O-glycosidically linked sugar chains in bovine kappa-casein. The variation and distribution of O-glycosidically linked sugar chains in kappa-casein from bovine mature milk were analyzed by HPLC, fast atom bombardment mass spectrometry, and 13C nuclear magnetic resonance. A monosaccharide alditol (N-acetylgalactosaminitol) and four oligosaccharide alditols [a neutral disaccharide (galactosyl-beta 1-3N-acetylgalactosaminitol), two acidic trisaccharides (straight chain: N-acetylneuraminyl alpha 2-3galactosyl-beta 1-3N-acetylgalactosaminitol and branch chain: galactosyl beta 1-3(N-acetylneuraminyl alpha 2-6)N-acetylgalactosaminitol), and an acidic tetrasaccharide(N-acetylneuraminyl alpha 2-3galactosyl-beta 1-3(N-acetylgalactosaminyl alpha 2-6)N-acetylgalactosaminitol] were identified as sugar chains linking on normal bovine kappa-casein. The most dominant sugar chain was an acidic tetrasaccharide. The four oligosaccharide chains were identical to those already found, but N-acetylgalactosamine was a newly identified sugar chain linking on kappa-casein. The distribution of monosaccharide, disaccharide, trisaccharide (straight), trisaccharide (branched), and tetrasaccharide chains were determined by HPLC to be .8, 6.3, 18.4, 18.5, and 56.0%, respectively (means of five kappa-caseins).


Effect of a prolonged-release formation of N-methionyl bovine somatotropin (sometribove) on milk composition. Forty Holstein cows received bi-weekly injections of prolonged-release formulation of bST, and 39 received bi-weekly injections of excipient, in a study to evaluate the effects of long-term bST administration on milk composition and component production. Injections began at 60 +/- 3 d postpartum. Administration of bST increased production of milk and of all measured components. Concentrations of lactose (4.85 and 4.81%), fat (3.76 and 3.67%), total solids (12.57 and 12.44%), SNF (8.83 and 8.75%), casein (2.56 and 2.53%), and true protein (3.13 and 3.08%) were similar in milks from cows receiving bST and excipient, respectively. Percentages of NPN (times 6.38) and total protein were greater in milk from bST-treated cows (.179% NPN and 3.32% total protein) compared with milk from cows injected with excipient (.172% NPN and 3.24% total protein). Use of bST did not change the relative percentages of alpha s-casein, beta-casein, kappa-casein, beta-lactoglobulin, alpha-lactalbumin, or casein proteolysis products. A cyclical pattern of milk production, component production, and composition within each 14-d injection interval was observed. This suggests that a diminishing amount of bST was delivered during the latter third of each injection interval. There were no effects of bST on milk composition that would be of any practical significance to dairy product manufacturers or consumers.


Effect of a prolonged-release formulation of N-methionyl bovine somatotropin (sometribove) on milk fat. Nine Holstein cows were injected bi-weekly with a prolonged-release formulation of N-methionyl bST, and 9 cows were injected with excipient. Intramuscular injections began at 60 +/- 3 d postpartum and continued at 14-d intervals for the full lactation. Administration of bST increased production of milk, total fat, and all milk fat components measured. Average fatty acid composition of milk fat was not influenced by bST treatment. Stage of lactation had a large influence on production and percentage of individual fatty acids in milk fat from both bST-treated and control cows. The stage of lactation impact on the fatty acid composition of milk fat reflected changes in the relative contributions of body fat mobilization and de novo synthesis of milk fat components in response to changes in energy balance. Initiation of bST treatment caused some transient changes in milk fatty acid composition that were related to energy balance. These changes were small compared with the normal changes because of stage of lactation in all cows. Phospholipid and cholesterol content of milk also changed with stage of lactation but were not influenced by bST treatment. Melting properties of milk fat were influenced greatly by stage of lactation. Bovine somatotropin did not cause any changes in composition or physical properties of milk fat that were outside the range of normal variation.


Nonspecific antibacterial factors in milk from cows immunized with human oral bacterial pathogens. Both the immunoglobulins and non-specific antibacterial factors in milk from cows immunized with pathogenic oral bacteria have the potential to influence the oral microflora during passive immunization studies. The first six milks after calving were collected from 2 cows immunized with adjuvant and from 14 cows immunized with adjuvant and heat-killed strains of periodontopathic Actinomyces, Porphyromonas, Prevotella, and Fusobacterium. Analysis of the products from the first to the sixth milks revealed that the protein and lysozyme content decreased approximately 66 and 72%, respectively; the mean specific activity of the enzyme remained relatively constant. In contrast, the mean lactoperoxidase activity increased 2.3-fold in the second milking and increased further in the fourth and sixth milkings. The mean iron-binding activity increased 1.2-fold from the first to the second milkings and then decreased 3.6-fold through the sixth milking. Cows immunized with adjuvant alone showed similar responses. Per unit volume, the milk contained approximately 150 times less lysozyme than whole human saliva obtained from six subjects but higher concentrations of lactoperoxidase and iron-binding components. Purified bovine nonspecific factors prevented the growth of the bacteria used for immunization when bacteria were tested at concentrations similar to those found in saliva and milk. Because bovine nonspecific antibacterial factors could influence both the pathogenic target bacteria and the indigenous microflora in oral passive immunization studies with bovine immunoglobulins, the presence of these proteins should be considered.


Antigenic homology of endotoxin with a coliform mastitis vaccine strain, Escherichia coli O111:B4 (J5). This study examined recognition of heterologous Gram-negative endotoxin by antibodies recognizing common lipopolysaccharide core antigens. Gram-negative endotoxins from 11 heterologous bacterial strains were tested for recognition by antibodies against common lipopolysaccharide core antigens. Serum was harvested from a calf immunized with the Rc mutant, Escherichia coli O111:B4 (J5), and affinity purified against endotoxin derived from an Ra mutant, Salmonella typhimurium, producing an antibody reagent recognizing homologous Gram-negative core antigens present in the Rc mutant vaccinal antigen. This reagent demonstrated reactivity against 11 chemically purified Gram-negative endotoxins. Included were endotoxins derived from 3 smooth E. coli species, 2 Salmonella spp., Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, and lipid A. Endotoxin derived from K. pneumoniae had significantly higher ELISA reactivity with core antigen specific antibodies than did endotoxin derived from either E. coli O111:B4 (J5) or P. aeruginosa. These results suggest immunization with R mutant bacterins may have utility in the prevention of Gram-negative mastitis even when whole bacteria react poorly with antibodies recognizing common core antigens.


Kinetics and characteristics of bovine neutrophil alkaline phosphatase during acute Escherichia coli mastitis. Alkaline phosphatase activity of isolated bovine blood neutrophils was investigated before and during experimentally induced Escherichia coli mastitis. Activities markedly increased 1 wk after infection in neutrophils of cows suffering from moderate or from severe disease. Elevated neutrophil alkaline phosphatase activity did not correlate with a specific stage of maturating postmitotic neutrophils appearing in circulation during mastitis. Neutrophil alkaline phosphatase from healthy cows before infection and from mastitic cows was characterized by means of thermostability, specific inhibitor patterns, slab gel electrophoresis, and kinetic parameter analysis. The leukocyte enzyme from healthy and mastitic cows displayed very similar characteristics, suggesting that the increase in activity during mastitis is most probably related to the enhanced expression of the normal alkaline phosphatase enzyme.


Prevalence of Staphylococcus species during the periparturient period in primiparous and multiparous cows. During a 14-mo period, 77 multiparous and 36 primiparous cows were sampled to determine the prevalence of staphylococci during the periparturient period. Distal streak canal swabs were taken at 14 d prepartum, and foremilk was sampled the first 5 consecutive wk of lactation. Staphylococcus aureus was isolated from 7.6% of quarters of primiparous cows but from only .6% of quarters of multiparous cows at parturition. Prevalence in primiparous cows declined to 3.5% by the wk-1 sampling. Quarter prevalence of coagulase-negative Staphylococcus species prepartum, at parturition, and wk 1 to 5 in primiparous cows was 38.9, 27.8, 15.3, 14.6, 13.2, 15.3, and 14.6%, respectively. In multiparous cows, prevalence at these times was 50.3, 12.3, 6.2, 8.1, 10.7, 7.1, and 8.1%. Staphylococcus chromogenes was the predominant species isolated, accounting for over 50% of the staphylococci isolated at each sampling time. Results suggest that high prevalence of staphylococci isolated prepartum is a reflection of natural skin flora and that a higher postpartum prevalence of these organisms was observed in primiparous cows than in multiparous cows. These data suggest also that the peripartum heifer could be a source of Staphylococcus aureus in the lactating herd.


Prediction of mastitis using milk somatic cell count, N-acetyl-beta-D-glucosaminidase, and lactose. The objectives of this work were 1) to examine the responsiveness of SCC, lactose concentration, and NAGase activity in milk to changes in bacteriological status and 2) to develop models for predicting bacteriological status of mammary glands. Data included 550 cows in 10 commercial herds. Natural logarithm NAGase and log cell count were most responsive to changes in bacterial status. The log NAGase was relatively more effective in identifying major from minor pathogen infections, whereas log SCC was better able to differentiate between infected and uninfected classes. Non-transformed NAGase, SCC, and lactose were considerably less responsive to infection status. Logistic regression of bacterial status on herd, lactation number, milk, log SCC, log NAGase, and stage of lactation was performed. The least significant variables were removed in a stepwise process. Final predictors of infection status were herd, log SCC, and log NAGase. The role of log SCC was to discriminate infection from no infection, whereas log NAGase discriminated major from minor pathogens. The log NAGase, alone or in combination with log SCC, added substantially to the detection power of the model. Chi-square goodness of fit tests found no significant differences between observed and predicted infection probabilities. Substitution of herd averages for log SCC and log NAGase for the herd variables resulted in significant differences between predicted and observed herd infection probabilities.


Influence of involution on intramammary phagocytic defense mechanisms. Mammary secretions (n = 34 cows) and mammary phagocytes (n = 18 cows) were collected throughout the nonlactating (dry) period to determine changes in intramammary phagocytic defense mechanisms. Mammary secretions were evaluated for their ability to support phagocytosis of Staphylococcus aureus by neutrophils from donor cows and mammary phagocytes for phagocytic and chemiluminescence activity. Ability of secretions to support phagocytosis decreased with advancing length of the dry period. This effect was more pronounced when dry cow secretions constituted 50% of the phagocytic mixture. Phagocytic activity of mammary phagocytes decreased with advancing dry period when autologous secretion was used in the incubation mixture. With homologous secretion, the percentage of phagocytosis increased 5 to 6 d after drying off compared with before drying off and then gradually decreased throughout the remainder of the dry period. Chemiluminescence activity (log10 counts per minute) of mammary phagocytes was lower during the dry period and decreased with advancing dry period. Results indicated diminishing ability of secretions to support phagocytosis and diminished phagocytic and bactericidal mechanisms during the dry period.


Casein, actin, and tubulin expression during early involution in bovine and murine mammary tissue. Hybridization methods and in vitro translation were used to examine the expression and functional condition of messenger RNA encoding caseins and cytoskeletal proteins in the mammary gland during early involution. In the mouse, steady state mRNA levels for alpha-, beta-, and gamma-caseins coordinately decreased to 20% of initial levels between 12 and 72 h after pup removal. In vitro translatability of mouse casein mRNA, as determined by immunoprecipitation, electrophoresis, and gel slice counting, revealed a pattern that closely paralleled mRNA expression. In contrast, bovine casein mRNA levels were only slightly reduced by 72 h postmilking, whereas in vitro translatability decreased by about one-half. Northern blot analysis of total mouse mammary RNA that were hybridized with probes to cytoskeletal proteins showed a gradual decrease of alpha-tubulin mRNA, but an increase in beta-actin mRNA during early involution. Two-dimensional gel analysis of in vitro translated products indicated a concordant increase in beta-gamma-actin. In the cow, beta-actin mRNA at 72 h of involution was equal to or greater than that during lactation. These results demonstrate the generally slower involution response in the cow and suggest that differing regulations are involved. Early events of cellular involution may be related to a reorganization of the cytoskeleton.


Depletion of intramuscularly injected ceftiofur from the milk of dairy cattle. Ceftiofur sodium, a new broad-spectrum cephalosporin, has been approved in the US, Canada, and several other countries throughout the world to treat bovine respiratory disease in cattle and dairy cows. In Experiment 1, 6 lactating cows were intramuscularly treated with 2.29 mg of [14C]ceftiofur/kg of BW daily for 5 d. In Experiment 2, 30 additional cows at three locations were similarly treated with 2.2 mg of ceftiofur (unlabeled)/kg of BW. Milk was collected every 12 and 24 h after each dose and every 12 h up to 5 d after the last dose. The majority of milk samples, both during treatment (12 and 24 h after each dose) and after the last dose (up to 5 d following ceftiofur treatment), were negative by screening procedures based on microbial inhibition (Delvotest-P, Bacillus stearothermophilus disk assay, and cylinder plate assays). The receptor-binding Charm Test II assay, which has a limit of detection of .005 ppm of ceftiofur, gave positive tests for milk samples up to 48 h following treatment. When the Charm Test II assay is used with .008 IU/ml of penicillin as a positive control, 44% of the samples from individual cows were negative at 12 h posttreatment. Ninety percent of the samples from individual cows were negative at 24 h after the last treatment. The use of ceftiofur in dairy cattle in accordance with the label directions does not result in total residues in milk higher than the FDA-calculated safe concentration of 1-ppm ceftiofur equivalents. The milk from individual cows did not test positive by the commercial screening assays examined in this study, except for the Charm Test II. The Charm Test II was 90% negative using the Charm Sciences criteria at 24 h after the last treatment.


Effects of late gestation heat stress on postpartum milk production and reproduction in dairy cattle. Carry-over effects of late gestation heat stress on postpartum productive and reproductive traits were estimated from DHI records using 341 lactations from six sites in Mississippi. Climatological data were gathered from records of weather stations near the sites. Using multiple linear regression analyses, predictor variables for lactations were age at calving, lactation number, maximum degree-days (above 32.2 degrees C) during the periods 30 and 60 d prepartum, and precipitation 30 and 60 d prepartum. Months and sites were indicator variables. Dependent variables included milk and fat production during early, mid, and late lactation; days to peak lactation; days open; services per conception; and body weight. Age at calving affected milk and fat production in mid and late lactation and services per conception. Degree-days for 60 d prepartum had the greatest negative influence on production variables; its statistical significance was shown in predictions of milk and fat production in early and midlactation. Days open were higher for July than for cows calving in August or September. Sites had effects on many milk and fat measurements and some reproductive traits. These results indicate that heat stress in the last 60 d of gestation has negative effects on some production variables.


Milk yields and hormone concentrations of Holstein cows in response to sometribove (somatotropin) treatment during the dry period. Holstein cows (n = 135) under commercial management were used to determine whether sometribove (recombinant methionyl bST, 25 mg/d) administered during the dry period affected milk yield during the ensuing lactation. Cows scheduled to begin lactations (greater than or equal to 2) during January to March were assigned randomly to treatments of sodium bicarbonate excipient (n = 67) or bST (25 mg/d, n = 68). Subcutaneous injections were given for 14 d, corresponding to d -21 to -7 relative to expected calving date. Days dry prior to first injection (64.0, 60.2) and number of injections received (13.9, 13.8) were similar for control and treatment groups, but days from last injection to calving (8.8, 7.1) differed. No differences in incidence of dystocia or udder edema were detected. Previous lactation yields were 8251 and 7952 kg, and yields for lactations following treatment were 8328 and 7852 kg, based on complete lactation data. Mean test date 3.5% FCM yields for control and treated groups during experimental lactation differed before (30.3 vs. 28.1 kg) but not after (29.5 vs. 28.4 kg) covariance adjustment for previous total lactation milk yield. Test of heterogeneity of regression provided no evidence that respective curves for FCM yield during lactation were not parallel or of different magnitude. Elevation of serum bST during 2 wk of the dry period resulted in no apparent increase in extent of mammogenesis or lactogenesis that was translated into an increase in milk yield.


Time series cross-correlation analysis of postparturient relationships among serum metabolites and yield variables in Holstein cows. Time series cross-correlation analysis was used to determine relationships among serum beta-hydroxybutyrate, glucose, FFA, cholesterol, milk yield, DMI, and estimated net energy balance for 42 d postpartum for 14 multiparous cows. Milk yield was positively associated with beta-hydroxybutyrate concentrations on the same day, and milk yield was a primary determinant of metabolic change. Dry matter intake was negatively correlated with beta-hydroxybutyrate concentrations 3 d later and on the same day. The data supported in vitro studies showing that FFA are positively associated with beta-hydroxybutyrate production, showed that glucose concentrations are negatively cross-correlated with beta-hydroxybutyrate concentrations, and found that estimated net energy balance is negatively cross-correlated with beta-hydroxybutyrate. Analyses suggested that serum glucose and cholesterol concentrations significantly decreased with increased milk yield; analyses also suggested that elevated beta-hydroxybutyrate concentrations were associated with decreases in milk yield 6 d later. beta-Hydroxybutyrate and FFA concentrations 3 to 9 d before parturition were positively related to cholesterol concentrations, and relationships were positive between estimated net energy balance and cholesterol, suggesting that cholesterol concentrations increased as precursors for cholesterol synthesis were available. Time series cross-correlation analysis was a useful tool in examining relationships among variables when repeated samples were obtained from the same individuals.


Effect of prepartum dry matter intake on liver triglyceride concentration and early lactation. Depression in feed intake during the final week before calving was hypothesized to be a major factor in the etiology of fatty liver development near parturition. Eleven cows were allowed to eat for ad libitum intake prior to calving (control), and 11 cows were maintained at the same level of DMI recorded during d 21 to 17 prior to calving by force feeding the feed refusals via rumen cannulas. Feed intake by control cows decreased 28% during the final 17 d prior to calving. Lipid triglyceride increased 227 and 75% for control and force-fed cows between d 17 prior to parturition and d 1 following calving. Dry matter intake prior to calving was correlated negatively with liver triglyceride immediately after calving (r = -.80). Plasma glucose concentrations for control and force-fed cows were 63 and 76 mg/dl 2 d prior to calving and also were related closely to liver triglyceride immediately after calving (r = -.50). By d 28 after calving, there were no differences in liver triglyceride between treatments. Cows that were force-fed prior to calving tended to yield milk with greater fat percentage (4.22 vs. 3.88%) and to yield more 3.5% FCM (46.1 vs. 41.7 kg/d) during the first 28 d postpartum.